key: cord-0008683-sb5c1xko
authors: Winkler, H.; Fischer-Colbrie, R.
title: The chromogranins A and B: The first 25 years and future perspectives
date: 2003-03-19
journal: Neuroscience
DOI: 10.1016/0306-4522(92)90222-n
sha: dc966c31c20501efb81076971d21e754bc539453
doc_id: 8683
cord_uid: sb5c1xko

nan

Schneider, Silver and Smithz7 named the soluble for their major component (see Fig. 1 ). When this Journal, Neuroscience, was started in 1976, the first proteins of bovine chromaffin granules chrome-co_entary~~ in volume 1 dealt with the early investixations on the chromotzranins. It now avvears *To whom correspondence should be addressed. have already revealed that the chromogranins were a complex mixture of proteins3"" (see Fig. I ). This is due to the presence of a large number of distinct proteins, but is also caused by the fact that these proteins are processed within the chroma~n granules by endogenous proteases (see below). Thus in addition to CgA several breakdown products of this proprotein are present149 (see Fig. 1 ).

In I 98479,366 we described a second group of acidic proteins, i.e. the chromogranins B (CgB) which could be immunologically differentiated from CgA. These proteins were subsequently also called se~etogra~n I, *"' however, since they are related to CgA (see below) in some respects this name is less appropriate (see nomenclature agreement).@ In addition (see Fig. 1 ) chromaffin granules contain secretogranin 118',274 (originalty discovered in the pituitary gland).276 This protein has also been called chromogranin C 61 but a nomenclature proposal agreed on the name secretogranin II.69 Furthermore, another protein isolated from pituitary, 782,**' is also present in chromaffin granules223 and so are numerous neuropeptides and their precursors. Higher concentrations are found for the enkephalins and neuropeptide Y.3M,371 Finally the secretory proteins include dopamine /I-hydroxylase, glycoprotein III,3M carboxypeptidase H93,'93 and endoproteases of the kex2 t~pe.~.~.'*~~ The recent proposalrs4 to call the acidic proteins of secretory organelles "granins" would at present add a further, quite unnecessary name, since the original name chromogranins already covered the same proteins. Further nomenciature proposals should await a better understanding of the function of all these proteins.

In the present commentary we will concentrate on the two closely related proteins, CgA and CgB, and will not deal with the other secretory proteins. Several reviews on this topic have been published. 57 Dibasic cleavage sites are marked by two lines for those conserved in the five species (the single-letter amino acid code is given for these sites).

Cleavage sites not conserved are marked by double broken lines. The species comparison demonstrates which features of the sequence are conserved. A high degree of conservation is found in the N-(aa, amino acid l-76) and C-terminals (aa, amino acid 316431) whereas in the middle part of the molecule, where the defined peptides are located, the conservation is rather limited.

For the isolation of chromogranins several methods have employed chromatography on molecular sieve, ionic exchange or butyl Sepharose columns. 49~1%180~31'J1s~323 core recent purification schemes are based on high pressure liquid chromatography with molecular sieve, ionic exchange and reverse phase columns, w*~**% chromatography on calmodulin columns,"' immunoaffinity chromatog-raphy359 and preparative one-or two-dimensional electrophoresis.25'*252*274 The heat stability274 of these proteins is very useful for preventing proteolysis during isolation and also provides a very efficient step of purification since many other proteins can simply be removed by boiling tissue.286 Since clones for both CgA and CgB are available, pure chromogranins can now also be produced by recombinant techniques.'" products but one misses smaller peptides (< 10,000 mol. wt) formed from them. An ideal assay would depend on an antiserum against a synthetic peptide (flanked by basic pairs in the CgA sequence) used in a radioimmunoassay before and after digestion of all larger CgA molecules to smaller peptides (cf. enkephalin assays). 337 For the chromogranins this approach has apparently only been used omxU5

In the following we will only deal with some general features related to the primary amino acid sequence of these proteins. Further properties acquired by post-translational modification (e.g. glycosylation) and the calcium binding ability of CgA will be discussed below. Radioimmunoassays70~232~~7*253 and enzyme as-

The primary amino acid sequences of these pro-says64*'S0 have been developed with antisera for CgA, teins have been determined for several species (see but also for the @A-derived polypeptide pancrea-Figs 2 and 3). For bovine CgA the mRNA has a statin3'. 53,99,178,255*284,345 and the GAWK peptides delength of 2100 bases, for CgB 2600. This message rived from CgB. 279,29( These assays are very sensitive codes for mature proteins of 43 1 amino acids (48,000 (100 pg for radioimmunoassay, 1 pg for enzyme mol. wt) for bovine CgA and 626 amino acids (71,500 assay); however, it is not clear which peptide moieties mol. wt) for CgB. As already recognized previously are actually measured, since the antisera react both from amino acid analyses (see Ref. 362) common with intact CgA and its breakdown products irrespecfeatures of these proteins are the high glutamic acid tive of whether they were originally raised against (25%) and proline (about 10%) content yielding an CgA or peptides derived from it. With quantitative acidic p1 of about 4.5-5.0. 8*362 The homology between immunoblotting79*3M one can measure the relative CgA and CgB is rather limited, in agreement with the concentrations of CgA or its larger breakdown obvious lack of immunological cross-reaction;79T274 however, there is a potential disulfide loop present in both molecules and in this N-terminal region (coded by exon 3 of both proteins) a homology of 42% is found. In the C-terminal end a homology of 44% (exons 5 and 8: see Figs 2 and 3) is present (see Fig. 2 ). Further properties of these molecules are discussed in the subsequent sections. Previous estimates of the molecular weight of bovine CgA by analytical ultracentrifugation'*"~"' indicated a value of about 80,000. On the other hand, in the presence of guanidine chloride a molecular weight of 53,000 was obtainedin4" and for parathyroid CgA a molecular weight of 51,900 was determined in a sedimentation equilibrium study."" The reasons for these discrepancies are obscure. It is easier to explain the discrepancies between the molecular size found in sodium dodecylsulfate (SDS) electrophoresis (about 75,000) vs the real molecular mass of 48,000. This abnormal electrophoretic behavior is probably due to the acidic and hydrophilic nature of this protein causing reduced SDS binding, since a protein with a similar high glutamic acid content of 25%, DARP-32, exhibits an analogous anomaly between migration in SDS and molecular mass.357 Similarly, bovine CgB moves in SDS electrophoresis like a protein of 100,000 mol. wt79 but actually has a molecular mass of 71,461."

The molecular size of CgA as determined by gel-filtration or by analytical ultracentrifugation depends very much on the ionic strength of the solvent. Based on these findings, Smith and Winkler3" suggested that CgA has a configuration approaching that of a random coil polypeptide. This concept was confirmed by further in vitro studies'84 and also by nuclear magnetic resonance experiments on intact chromatEn granulesHx298 indicating that this property of CgA was not due to denaturing conditions during isolation. A detailed recent study employing circular dichroism measurements found that "native" CgA had a dominant random coil (>60%) but also some alpha-helix structure (25540%) with no beta-sheets.375 Such a model is also in agreement with predictions based on the primary structure of CgA."4 Data on the secondary structure of CgB have apparently not been obtained; however, due to the similar amino acid composition "random coil" properties are likely to be present.

CgA is a hydrophilic protein without any hydrophobic stretches (see Ref. 114) . Despite these properties the question of whether this molecule exists also in a membrane bound form has remained a controversial one (for a discussion of early work see Ref. 362) . More recently Settleman claimed that CgA is the major integral membrane protein of chromaffin granules which could not be removed by high or low salt washes; however, the most efficient procedure, i.e. washing in 0.1 M Na, CO,'% was apparently not used. In immunelectron microscopy of the adrenal medulla, positive immunostaining is confined to the content of chromaffin granules whereas the membrane separated from the content in these micrographs is clearly unlabeled.'0~370 On the other hand, for dopamine /i-hydroxylase, which is present in the membrane, a positive immunostaining of the membrane was indeed obtained." During isolation of membranes in vitro, small amounts of CgA might become enclosed in small vesicles formed during the lysis procedure. This might explain the difficulty in removing these traces by washing procedures. An analysis of isolated membranes by immunoelectron microscopy might finally settle this question. The hydrophilic nature of the chromogranins and the random coil properties are probably responsible for the heat stability of these proteins.274~28"

A Southern blot analysis of genomic DNA digested with various restriction enzymes revealed that both CgA 1~'5*~'86~230~240~2M and CgB263 are present as single genes in the haploid genome. However, it has also been claimed that there are genomic differences in the amino acid sequences of CgA which may have arisen from a duplicated CgA gene.Ms The gene for human CgA was assigned to chromosome 14q32230,2m and that of mouse CgB to chromosome 2."' In the human genome a two-allele restriction fragment length polymorphism (RFLP) was reported for CgA. Nine out of 10 restriction enzymes did not detect a RFLP; however, Bgl II digests of genomic DNA from 56 Caucasians did identify two alleles of 24.0 and 8.0 kb with frequencies of 0.34 and 0.66, respectively, and a constant band of 6 kb. 230 The bovine and mouse CgA gene comprise eight exons separated by seven introns (Fig. 2) which span approximately 13.6 kb16* and 11 kb.374 The mouse CgB gene consists of five exons and four introns2'j3 and the coding region spans 12.1 kb (Fig. 3) . A comparison of the mouse CgA gene with the mouse CgB gene reveals homologies in the structural organization of three exons. These three exons code for the N-terminal and C-terminal domains which share significant sequence homologies between both proteins. Exon 2 of mouse CgA and CgB encodes the last three amino acids of the signal peptide plus the first 13 amino acids of the N-terminus of mature CgA and CgB; exon 3 encodes for the next 33 amino acids containing the disulfide loop motif present in both CgA and CgB. Exon 8 of CgA and exon 5 of CgB code for the 27 and 25 C-terminal amino acids of both proteins. The variable middle domain of CgA and CgB that differs substantially between species and shares no significant homology between CgA and CgB is reflected also by a different genomic organization.

The region upstream of the Cg promoter contains one cyclic AMP response element in both CgA and CgB. With the exception of two Sph 1 sites for CgA and one Sph 1 site for CgB no further consensus matches for other known transcription factors were found.'62*263,374 The elements responsible for tissuespecific expression in multiple neuroendocrine cells await identification; for CgA the region containing this information was shown to reside within 1.2 kb upstream of the coding region.374

Originally CgA was isolated from the chromaffin granules'34~3'7~3'* of adrenal medulla. It was also found in the large dense-core vesicles of sympathetic nerve'2,'5*56 and in the brainstem rich in adrenergic neurons.14' Based on these studies it was assumed that CgA was an "adrenergic" protein confined to sympathetic neurons and to the adrenal medulla. In 1982 we discovered% that CgA and secretory protein I (also called parathyroid secretory protein, PSP, Ref. 323) independently characterized (see Ref. 47) in the parathyroid gland were apparently identical proteins. Lloyd and Wilson2'6 had raised a monoclonal antibody against a protein present in pheochromocytoma tissue which reacted with a large number of endocrine tissues and their tumors and the responsible antigen was then identified as CgA.358 Within a short time several groups firmly established that CgA had a widespread distribution in endocrine tissues46@,248~35* (for further references see Table 1 ). When CgB was characterized in adrenal medulla in 198479,366 it was clear from the beginning that CgB shared this property with CgA*4*274 (for further references see Table 1 ).

The occurrence of immunoreactive CgA and CgB, of peptides derived from them and of their mRNAs in most endocrine tissues is well established and we will only comment on a few points (for references for the presence of CgA and CgB in tumor tissue see Refs 210, 217, 348, and an extensive review, 355) .

Chromogranin immunoreactivity as defined by immunohistochemistry exhibits great variations depending on the fixation method"6~203~237~2"~273~277 and dilution of antisera."' This capricious behavior makes it rather difficult to draw definite conclusions on the presence or absence of chromogranins in various cell types and therefore explains discrepancies found in the literature. For Table 1 we have not included negative results when positive immunostaining was reported by other groups, since a positive result would appear more reliable. In any case, for many tissues independent methods (immunohistochemistry, immunoblotting, in situ hybridization and radioimmunoassay) provide us with concordant results.

Immunohistology does not reveal the molecular properties of the detected antigens, but for several tissues immunoblotting (see Table 1 ) has established that these endocrine tissues contain CgA and CgB of the same size as in the adrenal medulla but also a variable amount (see below) of breakdown products.84~'22*'59~2'0~244*277 Furthermore, for two tissues (pituitary and endocrine pancreas) the cDNA clone for CgA was found to be practically identical to the adrenal one. ','58

It is interesting to consider the relative size of the CgA stores in neuroendocrine tissues.327 Thus in human beings the largest store of CgA is found in the adrenal medulla; the pituitary contains 25% of this amount (which appears surprisingly high), the pancreas and stomach plus intestine 5% each, whereas all other endocrine glands represent less than 1%. In addition to the classical endocrine tissues, CgA has also been found in scattered neuroendocrine cells in several organs like the breast, the lung or the prostate (see Table 1 ). Its presence in the storage granules of the myoendocrine cells of the heart storing the atria1 natriuretic factorJ2' raises the question whether it is also found in the renin granules of the kidney. One might also ask whether the erythropoietin cells of the same organ contain CgA. Thus CgA presents a general neuroendocrine marker and that has made it useful for a ~stopatholo~~l classification of neuroendocrine tumo~.~,35s However, recent publi~tions for the first time also reported an ~munos~~ng for CgA in exocrine cells, i.e. alveolar type II cells of rat lung'73 and for CgA and CgB in duct cells of the submandibular gland whose nature was, however, not well derined.ZM305~24

As far as the immune system is concerned, one study reported the presence of scarcely distributed C&A-positive cells in spleen, lymph node, thymus and fetal liver.14' By Western blotting small amounts of immunoreactive CgA were also found in these organs. Unfortunately this interesting observation has not been followed up. Are these special cells natural killer cells which are known to store and secrete enzymes and peptides by exocytosis?324

The relative concentration of CgA and CgB varies from tissue to tissue; however, in general, both proteins are found to be co-stored, but there is also evidence for a dissociation of their storage. Examples are the high concentration of CgA in the parathyroid gland (see Table 1 ) but the presence of only small amounts of CgB2i0,2'2 if any33~322*195,273 or the presence of CgA in ovine and bovine intermediate pituitary but the absence of CgB'95*277 or the differential staining of some enterochromaii'in cells for A, but not for B in the stomach;33J82 however, a staining of at least some of these cells for the GAWK protein derived from CgB has also been reported.2s Analogous results, i.e. a differential localization of CgA and CgB, have been obtained for brain (see below).

The presence of CgA in the central nervous system was already established in early studies.147J44~2s'*3'9 A detailed immunohist~he~~l mapping of ovine brain and spinal cord319 revealed a widespread distribution of the antigen. The immunoreactivity partly overlapped with those of several neuropeptides and other markers like tyrosine hydroxylase and dopamine /I-hydroxylase. However, the conclusion reached was that CgA is present in neurons using many different transmitters. This protein, originally thought to be "adrenergic" is also found in cholinergic neurons like the motoneurons of the spinal cord.3'9 In agreement, CgA was also found in cholinergic nerve terminals in the muscles of the diaphragm. y2 For human brain, Munoz and collaborators demonstrated that if methodical difficulties are overcome, a ~desp~d dist~bution of CgA can be found.23"37 Interestin~y, a high proportion of senile plaques in Alzheimer's disease,32J%J33*z~,S9 and also of Pick's bodies in another brain disease leading to dementia, consistently immunostain for CgAqM9 Furthermore, the CgA/synaptophysin ratio in such diseased brains is bigh.1"~u9

In porcine brain, CgA and a peptide derived from it, i.e. pancreastatin, have an identical, widespread dis~bution.'~ CgA ~unore~tivity has also been found in bovine2*4 and pore&?

retina. Quantitative analyses of CgA by ~~oi~unoas~y in bovine brain'80*2" and by immunoblotting in rat brain3m revealed that the concentration of CgA was only about O.Ol-0.2% of that in the respective adrenal medulla. The claimlw that the rat olfactory bulb as measured by immunoblots contains CgA in amounts comparable to that of adrenal medulla and the pituitary is likely to be wrong since at least in ox the pituitary gland has only 3% of the CgA concentration found in adrenal medulla.'"~251

Bovine and rat brain contain CgA mRNA which has been measured by Northern blotting.'~,1~,2i9,3~ With tissue hyb~d~ation CgA mRNA was found in the caudate nucleus of bovine brain.M2 A detailed mapping analysis of rat brain2'9 revealed the highest concentration in the pyramidal cell layer of the hippocampus, in the subiculum and the septum, with lower concentrations in many other brain areas, in agreement with the immunohistochemical data. For CgB a first comprehensive study measured the distribution of GAWK, a peptide derived from CgB, in the brain of several species, by radioimmunoassay. In human brain the highest concentrations were found in the hypothalamus, ~p~ampus, amygdala, nucleus accumbens, the basal ganglia and the cerebellum; much lower con~nt~tions were present in rat and guinea-pig brain. Immunoblotting measuring the proprotein CgB in rat brain revealed the highest concentration in the rat cerebellum, followed by the hypothalamus and amygdala.3s0 Only very limited immunohistochemical data are available for human brain with a positive staining found in the Purkinje cells of the cerebellumz8* and for rat brain.274

Two studies92,219 used in situ hybridization to study the distribution of CgB mRNA in rat brain. In agreement with the immunochemical data, a wide ~s~bution was reported with high concentrations in parts of the cortex, pyramidal cell layer of the hippocampus, in the dorsal raphe, pontine nucleus and several other regions. A comparison of the distribution of the mRNA of CgA and CgB2r9 revealed that some nuclei apparently synthesized only CgA but not CgB, e.g. the subiculum, whereas the dorsal raphe and the granule cell layer of the cerebellum were only positive for the CgB-message. Immunoblotting indicated that CgA and CgB found in brain are identical to the adrenal proteins in size and migration behavior (ovine CgA;'" rat CgA and CgB). 274,350 However, a relatively higher con~ntration of the proteoglycan form of CgA was present when compared with adrenal medulla and especially with endocrine tissue like the anterior pituitary.'= 7. SUBCELLULAR LOCALIZATION Two methods are particularly useful to establish the subcellular localization of a component, i.e. subcellular fractionation and immunohistochemistry at the ultrastructural level. With the first method the specific localization of CgA in bovine79~25'~278 and humanZ5* and of CgB in bovine79 chromaffin granules has been established. The same approach was used for localizing CgA in the storage vesicles of anterior pituitary24s and of betagranin, a peptide derived from CgA, in the insulin granules of pancreas's7 and of CgA 56*242,2*9 and CgB in the large dense-core vesicles of splenic nerve.'2',2s9 One of these studies242 also revealed that small dense-core vesicles of adrenergic nerves were devoid of CgA. What is the concentration of CgA in these vesicles relative to other peptide constituents? In bovine chromaffin granules the CgA family represents the major component. although the values obtained rather differ (70% as calculated from densitometric scans and the degree of degradation'* vs 46% as determined by radioimmunoassay).'4 We have calculated that in a single bovine chromaffin granule there are about 5000 molecules of the proprotein CgA present.37' A quite similar value (2763 molecules) was found by quantitative immunelectron microscopy.224 In the anterior pituitary granules, CgA is only a minor (4.5%) component'4 and this is also true for the endocrine pancreas where the insulin/CgA ratio is about 2000.('* Even considering that in this tissue, most of the CgA is processed (see below) this would make CgA a minor component in insulin granules (taking 90% breakdown into account: only 3 mg total CgA/ 100mg insulin). The observation25' that after subcellular fractionation of the bovine brain the cytosol contained the majority (69%) of CgA apparently suggested that in this tissue CgA is not localized within the secretory compartment. However, immunelectron-microscopy3'9 revealed that CgA reactivity is confined to Golgi stacks and large dense-core vesicles, but absent from translucent synaptic vesicles and from the cytosol. Most likely during homogenization of brain a considerable part of the Golgi stacks and large dense-core vesicles release their content including CgA.

In 1974 we published the first data on the immunelectron microscopic localization of CgA in adrenal medulla.'" Immunoreactivity was confined to the content of the vesicles; there was no staining of their membranes. Several studies'0~4'~'9'~224*320 have confirmed this finding and extended it to CgB4',320 and to other endocrine tissues (Table 1 ). By double immunolabeling or labeling of serial sections not only the presence of CgA and CgB in vesicles but also their co-localization with transmitters or neuropeptides within the same vesicles was established. Combinations of co-localization were: CgA/CgB/ secretogranin II/neuropeptide Y,320 CgA/parathyroid hormone,'

CgA/insulin6' CgA/glucagon,67 CgA/ atria1 natriuretic factor,'*' CgA/calcitonin,3"4 thyroid stimulating hormone/CgA/CgB/secretogranin IIih and luteinizing hormone/CgA/CgB/secretogranin II. 16.344 Within the same cell the respective hormones and the chromogranins are not always stored together in the same vesicles. This was first demonstrated for secretogranin II, prolactin and growth hormone for bovine somatomammotroph cells"' and has now been extended to CgA and CgB.16 In the somatomammotroph cells, two types of granules are present, i.e. small (150-300 nm) and larger (400-550 nm) ones. Only the small ones apparently contained CgA, CgB (and also secretogranin II, thyroid stimulating hormone and luteinizing hormone), whereas only the large ones contained growth hormone and prolactin. Apparently, in this special type of cell with multihormone storage, quite distinct sorting takes place. An analogous result was obtained for gonadotrophs of the anterior pituitary, in which large-sized granules reacted for follicle stimulating hormone, luteinizing hormone and CgA, whereas smaller ones were only positive for luteinizing hormone and secretogranin II.344 It will be interesting to see whether detailed studies on other cells will reveal further subpopulations of granules exhibiting a differential storage of the various hormones and chromogranins.

Immunelectron microscopy has also revealed some uneven labeling for CgA within the vesicles. This was particularly marked for the glucagon-storing vesicles of endocrine pancreas where CgA or pancreastatin labeling was preferentially localized on a less electron-dense halo surrounding a dense core67~'3"y'~33y whereas for insulin granules a peripheral immunostaining was reported only by one group6' but not by others.'3'a'9' A labeling of the peripheral part of the vesicles has also been reported for Merkel cells"' and for secretin-storing cells of the intestine,"8 and for CgB in small granules of the somatomammotroph cellsI and in the granules of gastrin cells4' but apparently not for all the other secretory granules where CgA and CgB have been localized by immunostaining (see Table 1 ).

The initial studies on CgA were performed with bovine tissues.'39~3'7,3'8 However, it was soon recognized that analogous components to CgA as shown by electrophoresis or by immunoreaction were present in horse, pig and sheep adrenals'33~2M~372 and in human adrenals and pheochromocytoma.322 CgA was then isolated and characterized from human252,359 and also rat materia1.86S274 In immunohistochemical studies a cross-reaction with antigens in several species including pig, cat, guinea-pig, dog and rabbit was observed.42,"8.273 For CgB in the first study79 the occurrence of similar (electrophoresis, immunoreaction) components in several species (pig, horse, cat, guinea-pig and rat) was recognized and this was soon extended to human material.'Ob~'"

The cloning of CgA and CgB from several species provides now the molecular basis for these immunological results. In some parts of the molecules (especially the N-and C-terminals) there is a high degree of homology between the individual mammalian species in agreement with the immunological cross-reaction (see Fig. 2 ). Unfortunately, only clones from mammalian species have been analysed. However, it is already clear that analogous proteins are present much earlier in phylogeny. Rieker et ~1.~'~ found immunoreactive components comparable in size to CgA and CgB in chicken, frog, fish and lobster. In chicken ultimobranchial gland,"' in toad urinary bladderSS and in several fish species, CgA immunoreactivity (with antimammalian CgA) could also be demonstrated by immunohistology and radioimmunoassay.58~333 In the ostrich pituitary a large peptide (see Fig. 2 ) with an 80% homology to the N-terminal of bovine CgA has been described'99 and in Drosophila heads an immunoreactive band for CgA could be detected by immunoblotting (Kapelari and Winkler, unpublished observation). A detailed immunohistochemical study on CgA and pancreastatin in the gastro-entero-pancreatic system revealed positive reaction apart from mammalian species in birds, reptiles and fish but not in deuterostomian invertebrates?"

A molecular cloning of chromogranins from nonmammalian species would seem extremely useful for defining regions with a high degree of conservation. Peptides in these regions would be good candidates for a relevant function which has evolved during phylogeny. Finally, even in the protozoan, Paramecium tetraurelia, peptides immunologically crossreacting with antisera against CgA and synthetic peptides derived from it have been found.260 However, in contrast to all other species only small peptides of 20,000 mol. wt were present. On the other hand, the mRNA hybridizing with a CgA probe suggests a much larger molecular weight at least for an unprocessed proprotein. Since the CgA antisera immunostained components which represented the major soluble proteins of the trichocysts of Puramecium an artefact is very difficult to exclude. Therefore these data should be confirmed by an independent approach, e.g. sequencing of some of these peptides.

As compiled by Fischer-Colbrie et al.,82 the relative amounts of CgA vs CgB in the different species vary. In ox adrenals, CgA is by far the dominating component, whereas in rat and human material CgB is prominent. 86~1u Thus the CgA/CgB ratio in ox is 9, whereas in human material it is about 0.8.'06

Several immunohistochemical studies investigated the ontogenic development of CgA. In rat fetuses, CgA immunoreactivity appeared at day 18 of the gestation period and at 15 days of postnatal age was similar to the adult staining pattern."' In human fetuses a positive reaction for CgA23' and CgA-mRNA138 was found in adrenal primordia at six to eight weeks of the gestational period, slightly proceeded by immunostaining for tyrosine hydroxylase. At nine weeks, positive staining was confined to "large cells" considered to be progenitors of chro-maIlin 1~11s.~~' In the endocrine cells of the human intestinal tract, CgA immunoreactivity appeared together with peptides typical to these cells, e.g. somatostatin, at eight weeks of gestation" and also in the ultimobranchial gland of the chicken'7s and in the carotid body'76 there was a concomitant appearance with other peptides and with tyrosine hydroxylase. For CgB the appearance of the mRNA has been studied in developing rat brain.g2 It was 6rst detected at embryonic day 14 and then increases till three weeks postnatally, when it starts to drop again to a level close to that found at birth.

A detailed study by immunoblotting of the development of CgA and CgB in rat adrenal medulla revealed two additional interesting features.2*7 Both chromogranins were detected on the 17th prenatal day. Their levels increased together with that of the catecholamines. However, the relative concentration of CgA vs CgB showed a significant change. At the end of the gestational period the ratio of CgA to CgB increases strongly at exactly the same time when the adrenaline/noradrenaline ratio becomes elevated. This latter effect is known to be due to the induction of methylation caused by the increase in cortisone production at this gestational time point.45 For CgA we have shown83*303 that its biosynthesis is increased by corticosteroids. The observation of an increase of CgA biosynthesis together with that of adrenaline establishes the ontogenic significance of this concept. A second finding, described by Schober et a1.,287 concerns the proteoglycan form of CgA. Whereas fetal adrenal contains a relatively high concentration of this CgA form, in adult animals it is much less prominent. Since rat brain also contains a prominent proteoglycan form of CgASM this result was interpreted as a sign of the developmental relationship between early chromaffin cells and neurons.

Secretion of catecholamines from the adrenal medulla and chromaffin cell culture is accompanied by the release of all soluble constituents of chromaffin granules 13.363 including ~g~14~70~~85~285~30696 and CgB.79 This is also true for noradrenaline release from the sympathetic nerve."'j From the parathyroid gland, CgA is released with the parathormonea and betagranin, derived from CgA, is co-released with insulin from endocrine pancreas"' and so is another CgAderived peptide, pancreastatin.3~255 This latter peptide is also secreted from a human pancreastatin-producing cell line.99 GAWK, a peptide derived from CgB, is released when rat anterior pituitary or bovine chromaffin cells are depolarized.'""79 CgB is also released from PC12 cells274 and a medullary thyroid carcinoma cell.'"

Since secretion from all endocrine tissues and neurons occurs by exocytosis, it is obvious that in all these tissues chromogranins must be co-secreted with the respective hormone. After secretion from endocrine organs, CgA reaches the bloodstream via the capillary or the lymph. For the adrenal medulla it has been shown that, in contrast to the catecholamines, most of CgA is first directed into the lymph vessels? In human serum, O'Connor and collaborators found by radioimmunoassay about 30-50 ng/ml CgA in normal controls,2s0~253 whereas in patients with endocrine tumors these levels were significantly elevated.250 Differential stimulation of various endocrine organs revealed that only stimulation of the adrenal gland led to measurable increases in plasma CgA levels.327 On the other hand, in adrenalectomized patients, CgA levels in serum are in the normal range.32s Furthermore, blockade of the sympathetic system by a ganglion blocker and non-selective blockade of neuroendocrine secretion by somatostatin led to a decline of the basal levels by 25 and 48%, respectively.325 Thus basal CgA levels in plasma are mainly derived from these sources and not from the adrenal medulla (see also Ref. 52) . The half-life of CgA in plasma is about 18 min.247 Plasma levels of pancreastatin-like immunoreactive material, mainly representing high molecular weight peptides with only small amounts of free pancreastatin have also been determined.3,3'

For GAWK (20-38), a peptide derived from CgB, a level of 150 pmol/l was found for human plasma.'94 Also, these levels are elevated in patients with neuroendocrine tumors. This concentration corresponds to 10 ng/ml of the proprotein CgB which is in the same range as the values for CgA. This seems reasonable considering that equal amounts of CgA and CgB are present at least in human adrenal medulla.

Human cerebrospinal fluid contains 170 ng/ml CgA3" which is higher than the serum values (40 ng). This finding indicates either significant release of CgA from brain or a longer half-life of CgA in the cerebrospinal fluid. For GAWK (20-38) a value of 1.1 pmol/ml has been reported (85 ng CgB/m1).279

The studies discussed above were based on radioimmunoassays which do not define the molecular size of the measured molecules. However, in a recent study with immunoblotting the presence of the unprocessed proprotein CgA could be demonstrated in bovine serum.352 Apparently, neuroendocrine tissues release unprocessed CgA which is not quickly degraded in serum. O'Connor and collaborators'5'~2s3~379 reported that patients with renal failure had high CgA levels in serum. This indicated that the kidney might be essential for the removal of plasma CgA.

Kidney tissue contains CgA, but no CgA-mRNA."' Immunelectron microscopy revealed that CgA immunoreactivity is found in proximal tubule cells. Within the cells it was present in small vesicles with or in close proximity to the brush border and closer to the nucleus in typical lysosomal structures. These results make it likely that CgA reaches kidney tubule cells by glomerular filtration and is taken up into the endocytotic lysosomal pathway followed by degradation. On the other hand, smaller peptides derived from CgA may reach the urine. At least, pancreastatin-like immunoreactivity has been detected in it.'"'

After in vitro translation of adrenal medullary mRNA, two translation products of very similar molecular weight (apparent molecular weight in SDS electrophoresis of about 70,000) can be immunoprecipitated with antisera against bovine CgA.74,'*' In analogous findings, four products of very similar size were reported for parathyroid mRNA.220.221 When the in vitro translation for adrenal medulla was performed in the presence of dog pancreas microsomes only one immunoprecipitable translation product is found.74 The two or more spots Seen after in vitro translation could be due to different lengths of the signal peptides, but they may represent in vitro artefacts, since there is no evidence that signal peptides of different length are present in the bovine CgA sequence.20T160 In any case, in agreement with the fact that there is apparently only one gene for CgA, only one in vitro translated product is found after the removal of the signal peptide. The claim295 that several translation products are produced in such a system is therefore disproven. For CgB, cell-free systems yielded one translation product in bovine adrenal medulla.74 For rat adrenal (PC12 cells) two products of very similar size are apparently produced.274

Early studies on bovine adrenal medulla had established that after labeling with tritiated amino acids the chromogranins were the major labeled products. Only 30 min after a radioactive pulse could the secretion of labeled CgA be induced by stimulation of the adrenal gland. 2**367~369 Apparently, the synthesis rate of the secretory protein CgA was much higher than that of the membrane proteins which led to the suggestion that the membranes of chromaffin granules are re-used for several secretory cycles from the Golgi region to the plasma membrane.36'.369 These data were recently extended in a careful study3b0 comparing the synthesis rates of CgA, enkephalins and dopamine fl-hydroxylase in bovine chromaffin cell cultures. Whereas CgA and enkephalin had about the same rate of synthesis, that of dopamine flhydroxylase was lower by a factor of 10. The most likely explanation is that this enzyme which is partly membrane bound3" can be replenished at a lower rate since the membranes are reused.

Further studies investigated the rate of amino acid incorporation in the presence of various secretagogues. In one series of experiments a direct correlation of this synthesis to the rate of secretion, as induced by several secretagogues, was found.'07 In contrast, Wilson et al." did not discover any changes in CgA synthesis under very similar conditions. There was also no increase in total (cells and medium) CgA levels after stimulation of chromaffin cells in culture by nicotine treatment.70 Apparently, there are some discrepancies and we will discuss this topic further in the section on the regulation of biosynthesis.

Pulse-chase labeling of chromaffin cells of bovine adrenal medulla followed by two-dimensional electrophoresis revealed that the early labeled CgA and CgB spots behaved identically to the in vitro translation products formed in the presence of microsomes.74 However, at later intervals significant changes in size and p1 occurred which were especially marked for CgB. These modifications were obviously due to the post-translational processes which we will now discuss.

12.2.1. Glycosylation. Bovine CgA of adrenal medulla is a glycoprotein (5.4% carbohydrate) with galactose, N-acetylgalactosamine and sialic acid as major components. 8s~'05~3'7 The sugars are mainly present as 0-glycosidically linked tri-and tetrasaccharides composed of N-acetylgalactosamine, galactose and sialic acid. "* The primary amino acid sequence does not contain potential sites for N-glycosylation in this species; such sites are present in human, rat and mouse sequences (see Fig. 2 ), but it is not known whether N-glycosidically linked sugars are actually added in these species. For CgA of the parathyroid gland a very similar sugar composition has been reported;m however, with one exception, i.e. a much lower content of galactosamine. It seems unlikely that this low value is correct since the tri-and tetrasaccharides require about equal amounts of galactose and galactosamine. It would therefore seem premature to suggest that CgA outside the adrenal medulla is differently glycosylated, but further studies should settle this question.

The sugar composition of rat chromogranins has been investigated but a separation of CgA and CgB was not performed at that time.**' Bovine CgB has a sugar composition similar to that of CgA but with more fucose and mannose (see Ref. 85 ; CgB in this early paper was called Al). In contrast to CgA it reacts with Pisum sativum lectin, but like CgA it binds peanut agglutinin after neuraminidase treatment.7 Sites for N-glycosylation are present in the amino acid sequences of ovine, mouse and human CgB. The presence of fucose and mannose in bovine CgB may indicate that this molecule is actually N-glycosylated. In other tissues the glycosylation of CgB has apparently not been investigated. CgA of porcine parathyroid gland it was originally reported that it contained sulfate linked to tyrosine;'89 however, in a more recent study both bovine and porcine parathyroid CgA were found to be sulfated on oligosaccharides.'Og Chromaffin granules also contain proteoglycans in which the glycosaminoglycan component consists of dermatan sulfate, chondroitin 4-and Gsulfate and heparan sulfate. "~26~'05~'*2 When bovine chromaffin cells are labeled with [35S]sulfate and analysed by two-dimensional electrophoresis a spot moving slower and to a more acidic pH than CgA (see Fig. 1 ) becomes labeled.73x274 This spot was identified as a proteoglycan form of CgA by its sensibility to chondroitinase treatment73 and by its immunostaining for CgA.'83,274 This proteoglycan form of CgA represents only a minor component of the total proteoglycans in bovine chromaffin granules and only about l-2% of CgA is present in this form."* Proteoglycan CgA contains probably only a single chondroitin sulfate or dermatan sulfate chain of approximately 15,000 mol. wt.'12 In rat PC12 chromaffin cells CgA represents a significant portion of the larger proteoglycans. There is no evidence that bovine CgB is present in a proteoglycan form."* In the parathyroid gland a proteoglycan form of CgA was only found in bovine, but not in porcine tissue."' On the other hand, in rat tissues the relative amounts of proteoglycan CgA varied from tissue to tissue, being low or absent in anterior pituitary, with intermediate amounts in the adrenal and a higher concentration in brain.350 During ontogeny of the rat adrenal medulla the relative amount of the proteoglycan form of CgA starts from a high level and then declines.287 12.24. Pyroglutamylation. This post-translational modification of peptides leads to the formation of pyroglutamyl residues from N-terminal glutamic acid36 and the responsible enzyme, glutamic cyclase, is also present in adrenal medulla. A first analysis of bovine chromaffin granules for the presence of pyroglutamyl peptides revealed a 14-amino acid peptide derived from CgB. 9o It will be interesting to see whether this post-translational modification is an essential prerequisite for a possible function of this peptide.

12.25. Carboxymethylation. CgA is a carboxymethylated protein.243 Since carboxymethylase activity in adrenal medulla is cytosolic,6' the modification of the CgA molecules as suggested'02r243 would have to occur co-translationally which, however, is difficult to imagine.

12.2.6. cc-Amidation. Pancreastatin, a peptide derived from CgA has been shown to possess a C-terminal glycine amide structure in pig,33' bovine2" and human'00,284,293 tissues.

12.2.7. Formation of disulfide bonds. Based on reduction studies it has been proposed2' that the two N-terminal cysteines are present in vivo in a disulfidebonded loop structure. This probably requires the action of a disulfide isomerase.

In 1974 we observed'49 that in bovine chromaffin granules there were several smaller proteins immunologically cross-reacting with CgA.'83~251*296 Based on these findings we suggested362 "that these smaller proteins are formed in chromaffin granules in viuo due to the presence of proteases. One is of course reminded of the fact that in insulin-containing secretory granules, proteases are present which are essential for forming insulin from proinsulin". By now this concept is well established not only for CgA but also for additional components of chromaffin granules, e.g. the enkephalins337 and CgB.79 Several defined small peptides proteolytically derived from CgA and CgB have been shown to occur in neuroendocrine secretory granules: pancreastatin33' and betagranin'56 from CgA; and GAWK and CCB,22" BAM 174S90 and OA-8207 from CgB.

Apparently, the processing of the chromogranins in different neuroendocrine vesicles does not occur at the same rate. Thus in adrenal medulla a relatively slow processing is obvious. In pulse-label experiments newly synthesized CgA appeared unprocessed after two74 and even after 18 h,14,'08 whereas for CgB a limited proteolysis occurred earlier74 with some large processed peptides being present after 2 h. On the other hand, in endocrine pancreas (insulinoma cells) a significant breakdown of CgA was already seen within much shorter times. Thus the half-time of conversion of CgA to betagranin (representing the N-terminal end of CgA) was only about 3Omin."

To what degree the chromogranins are processed in various tissues depends both on the rate of processing and on the average life-span of the vesicles before they release their content, since even a low rate of processing may lead to considerable breakdown if vesicles do not secrete their content. In adrenal medulla the half-life of the secretory content (catecholamines) of chromaffin granules has been estimated to be close to seven days.336 Despite this long half-life, CgA in bovine adrenal medulla is only processed on average (in total adrenal medulla) to about 50% whereas for CgB a value of 85% has been reported.'4,82 Obviously the rate of processing might be very low (in agreement with the pulse-labeling data discussed above), but one cannot exclude that processing does not occur throughout the whole life-span of chromaffin granules. For bovine neuroendocrine tissues it is only in the anterior pituitary that the degree of CgA processing is apparently less than in adrenal medulla,14*82 whereas in the intestinal mucosa (enterochromaffin cells) and especially in the endocrine pancreas, proteolytical degradation of CgA is extensive.345 This conclusion was established by a radioimmunoassay with antisera against synthetic peptides and measurements before and after trypsin digestion. The ratio of free peptide to total (after trypsin) peptide was 0.11 in adrenal medulla, 0.52-0.76 for intestinal mucosa and 1.09 for pancreas. Similar results were obtained for other species: in the porcine adrenal, CgA is little processed, whereas in the pancreas considerable breakdown has occurred as measured by pancreastatin radioimmunoassays and gel permeation chromatography,284 while in hypothalamus and especially in anterior pituitary, processing is limited."' For rat tissues the degree of breakdown increases from the adrenal medulla to the pituitary and finally to the endocrine pancreas.lsY In these studies, antibodies against betagranin and the intact CgA molecule were used to probe the degree of proteolysis. In another study5' only an antibody against pancreastatin was employed for this purpose. In this case most immunoreactive material eluted close to the void volume when extracts of adrenal, pituitary, brain but also pancreas were subjected to gel permeation chromatography. Apparently, depending on what antisera are used the results may differ; however, the general conclusion seems valid that the degree of proteolytic processing of CgA in adrenal medulla and anterior pituitary is low; in other tissues especially the endocrine pancreas it is high. For CgB a study using antibodies against the GAWK peptides indicated a more extensive proteolysis in pituitary when compared with adrenals.'"' Proteolytic processing of CgA may also occur after strated, that in rat pancreas the formation of betatheir release from the neuroendocrine cells.% In granin can also occur by first removing the primary cultures of bovine chromaffin cells such a N-terminal end of CgA from the intact molecule by processing was observed in two studies;N**3" but not cleavage at a dibasic site which represents in this in another one." In any case it is not known whether species the first cleavage site from the N-terminal such processing can also occur in vivo. The first end. For CgB of several species, processing of evidence that the rate of proteolytic processing of the intact molecule starting either from the N-or CgA can be upregulated was recently obtained for C-terminal site has also been established for several chromatfin cells treated with reserpine.'& neuroendocrine tissues.106

One of the best-defined cleavage points in the processing of propeptides is at dibasic sites and in fact as shown in Figs 2 and 3 , several of such cleavage sites are found in the CgA and CgB sequences and several of them are phylogenetically conserved at least in mammalian species. A detailed study using microsequencing and region-specific antibodies established actual processing at such sites for bovine CgA at residue 79 (preceded by KK) and 116 (by KR) and for human CgA at the second dibasic (KR) site counting from the N-terminus" (for the latter cleavage see also Ref. 328) . For CgA of the latter species (isolated from carcinoid tumors) there was also processing at position 210 (preceded by KR) and at position 340 (by KR). 283 For the endocrine pancreas cleavage at dibasic site 13 1 (KR) in rat CgA has been established leading to the release of the N-terminal peptide called betagranin.'" An analogous peptide (22,000 mol. wt) seems to be present in chromal%n granules.136

We have already pointed out above that in the various endocrine tissues the degree of proteolytic processing of chromogranins varies. This may lead to an accumulation of peptides of different sizes within the various tissues as, for example, shown in a detailed study on bovine tissues" or in endocrine tumor cells lines.59 These authors concluded that the "pattern of processing was qualitatively similar across tissues". However, we cannot yet exclude that in one of these tissues, CgA is specifically processed to a particular peptide (see Ref. 53a) . For CgB significant differences between proteolysis in adrenal medulla vs pituitary have been found106s165 with the appearance of a unique 40,000 mol. wt C-terminal fragment in the anterior pituitary.'%

For the well-characterized peptide, pancreastatin, which has been originally isolated from porcine pan-creas331 but is also found in human293*328 and bovine tissue,"' processing at the C-terminal end requires cleavage (see Fig. 2 ) at a Glys-Lys site and on the N-terminal site cleavage at a monobasic amino acid at least for the porcineJ3' and the bovine peptide."' A peptide found in bovine adrenal medulla30 which is now known to be derived from CgA, also requires processing at monobasic site 16 (lysine) for its formation In contrast, it is noteworthy that human C&A-derived peptides require for their formation cleavage at Asp-Pro and Trp.2*3 For CgB, processing at dibasic sites is well established, since several peptides detected in various tissues depend on such a cleavage, i.e. human GAWK (420-493) and CCB (597-657)," bovine BAM-1745 (547-560),w; OA 21 207 ovine OA 8207 and OA 60. 226 Proteolytic processing of large proteins like the chromogranins could occur in successive steps either from the C-or N-terminal end or also independently from both sites. For CgA this latter process is apparently operating in bovine chromaffin granules.14*373 In fact, a preferential cleavage site is the dibasic site at the N-terminal end which leads to the formation of a smaller peptide (l-77) representing a significant spot in ZD-electrophoresis (see Fig. 1 ) and a larger size residue of the CgA molecule.'4,373 It seems likely, although it has not been definitely demon-

The first study on secretory granule enzymes responsible for the processing of CgA were performed by Hutton et al. '~6 They found in insulin granules of the endocrine pancreas a calciumactivated enzyme which together with a carboxypeptidase H produced betagranin from CgA. In bovine chromaffin granules we described an analogous enzyme activity which produced a small peptide (22,000 mol. wt) from CgA probably also presenting betagranin.'92 In the mean time two kexZrelated proteases have been cloned for which an involvement in prohormone processing has been established.23,290,3'4*332 The mRNA of both PC1 (high concentration) and PC2 (low concentration) have been found in rat adrenal medulla2!"' and both PC1 and PC2 sequences have been shown to be present in glycoprotein H of bovine chromaffin granule membrane." By using antisera against synthetic peptides derived from the primary amino acid sequence we could immunostain glycoprotein H for PC2, but the PCl-reactive spot corresponded to a larger (85,000 mol. wt) glycoprotein."'" Thus it seems quite likely that the enzymes processing CgA in insulin and chromaffin granules as discussed above are in fact PC1 and PC2. These enzymes are capable of processing prohormones at pairs of basic residues and we have already pointed out that such cleavage occurs for both CgA and CgB. The involvement of those enzymes in CgA processing does not exclude the action of other enzymes which have been described previously in chromaffin granules (see Ref. 364 for references). Thus CgA can apparently also be processed by a trypsin-like enzyme,192 first described by Lindberg et al. 299 This enzyme was reported to have a molecular weight of 3 1 ,000.2'y Furthermore, a thiol an analogous influence of cortisone couid also be protease of 33,000 mol. wt splitting at dibasic sites demonstrated;**~"5 on the other hand, in brain has also been found in chromaffin granules.rVR A dexamethasone treatment or adrenalectomy did not possible action of this enzyme on chromogranins has change CgA levels or that of its mRNA."" In the not been established. As already pointed out, CgA corticotrophic cell line, AtT-20, cortisone treatment processing does not only occur at dibasic sites, thus increases the CgA mRNA levels to 250% whereas proteases acting at monobasic sites or in other pos-those for POMC peptides declines (to 40%)'4' and an itions might also be involved. An endopeptidase with analogous regulation was observed for the CgA such a specificity (monobasic sites) has recently been message in pituitary tumor cell cultures""" and in an purified from pituitary.6' When all these proteases are insulinoma cell line.'62 It has not been established by defined in molecular terms it will be interesting to see what mechanism cortisone induces the biosynthesis whether a concerted action of them is necessary to of CgA. The induction by cortisone of the CgA produce all the peptides formed from chromogranins message in the pituitary occurred relatively late, i.e. and whether different activities of these enzymes in with a time-lag of 24 h after the injection, interpreted various tissues will explain the varying degrees of as the action of cortisone being indirect via protein processing and possibly various ways of peptide synthesis.88 However, a direct effect of the cortisone formation.

receptor on the gene cannot be excluded, although Finally, the unusual possibility has to be con-gene analysis did not reveal a perfect match to a sidered, that the enzyme acetylcholinesterase may be glucocorticoid responsive element in the promoter involved in CgA breakdown. It has been claimed that region.'62.374 highly purified acetylcholinesterase has proteolytic Another example of steroids regulating CgA syn-activity3" and can also process CgA.313 However, this thesis has been found in the parathyroid cell cultures. concept is still controversial3'2 and evidence against 1,25-Dihydroxycalciferol-treatment induced a sevsuch proteolytic properties has also been presented.43 eral-fold increase of the CgA mRNA23','77a via an Whether acetylcholinesterase is present in chromaffin effect on CgA gene transcription, whereas CgA granules and therefore in a position to attack CgA mRNA stability was not affected. Finally oestrogen has also been controversial (see Ref. 364) ; however, steroids are also involved in the regulation of CgA at least this topic now seems settled, since a sub-synthesis in the pituitary gland. In female rat pituspecies of this enzyme appears to be specifically itaries, CgA drops with maturation of the animals.6 localized in chromaffin granules.'* After ovarectomy CgA and its mRNA rises and

The chromogranins are constituents of the secretory content of neuroendocrine vesicles. In principle the biosynthesis of the chromogranins could be regulated en bloc together with other secretory components like the neuropeptides or in differential patterns allowing modifications of the secretory cocktail. We here present evidence that the latter mechanism is operating.

is suppressed again by oestrogen treatment4,' In male rats oestrogen treatment markedly lowered the pituitary mRNA for CgA." Furthermore, male rats had higher CgA mRNA levels in the pituitary than female rats which are of course under the constant influence of oestrogens, whereas CgB message was equal for both male and female. Thus the biosynthesis of CgA in the pituitary is suppressed by endogenous or exogenous oestrogen. These changes take apparent place in the gonadotrophic cells as indicated by immunohistochemistry.87 However, for these oestrogen effects it has not yet been demonstrated that they are due to a direct action on these 14.1.1. Chromogranin A-a peptide regulated by cells. They may also be indirect via hypothalamic steroids. In 1987 we provided the first evidence that regulation. In adrenal chromaffin cells, oestrogen is the biosynthesis of CgA in adrenal medulla was without influence on CgA mRNA levels.4~8' specifically regulated by corticosteroids.303 After 14.1.2. Regulation qf chromogranin B. When hypophysectomy in rats the CgA but not the CgB GH,C, cells (a rat pituitary tumor cell line) are content of adrenal medulla declined markedly, treated with humoral factors (E,, insulin and epiderapparently due to the atrophy of the adrenal cortex ma1 growth factor, EGF) the number of secretory leading to a lack of corticosteroids. This concept was granules increases several-fold. This leads to inconfirmed when it was demonstrated that after hypo-creased levels of prolactin, but also of CgB and physectomy the lowered mRNA levels of CgA (but secretogranin II. 28' In GH,B, pituitary cell lines, not that of CgB) in adrenal medulla could be restored CgB-mRNA is reduced by dexamethasone, whereas to control levels by cortisone treatment.83 The specifi-oestrogen increased it."' city of the action of cortisone could be seen by its lack of effect on the CgB message. Similarly, in 14.2. Regulation of synthesis by stimulation of' cells PC12 cells and in bovine chromaffin cells in culture, When chromaffin cells of the rat adrenal medulla cortisone treatment specifically increases the levels are stimulated reflexly in viva by insulin-induced of the CgA message. '62*266 For the anterior pituitary hypoglycemia, the levels of enkephalins,'77~303 but also of several other neuropeptides, i.e. calcitonin generelated peptide, neuropeptide Y, neurotensin and substance P,'**194 increase several-fold. On the other hand, those of CgA and CgB remain unchangedM3 clearly demonstrating an apparent dissociation of the biosynthesis of chromogranins and neuropeptides. This conclusion was confirmed at the mRNA levels3 Whereas in these experiments with insulin-induced hypoglycemia the biosynthesis of chromogranins was apparently unchanged, treatment of rats with reserpine induced such effects. 142 A high dose of reserpine leads to a prolonged stimulation of the adrenal medulla. This treatment induced a significant increase of the levels of CgB and its mRNA (600%), whereas for CgA mRNA a smaller increase (230%) was observed. No definite explanation can be offered as to why indirect stimulation of the adrenal by insulin or by reserpine affects chromogranin biosynthesis differently; however, it has been suggested'" that the time of stimulation may be important with only a prolonged stimulation as provided by reserpine leading to such biosynthesis changes. For the chromogranin biosynthesis in brain the first data are already available. Thus in rats in which seizures have been induced by the injection of kainic acid, changes in the mRNA levels for CgA3% and for CgB21ga have been observed. Apparently, in this model intensive stimulation of certain neurons in brain does not only change the levels of neuropeptide Y but also those of the chromogranins.

For bovine chromathn cells in culture, results on the biosynthesis of CgA are controversial. Two groups of authors measured the incorporation of radioactively labeled amino acids into CgA. Whereas one groupM7 reported an increased incorporation after depolarization (nicotine, K+), another one found no change 360 for CgA, but the expected change for the enkephalins. Possibly, in oitro factors like the length or intensity of stimulation (see above) may lead to different results. In any case, in pancreatic endocrine islets, amino acid incorporation into both insulin and CgA is increased several-fold in the presence of glucose which was interpreted as an effect at the translational level;n9 however, in all these studies, in vitro mRNA levels of CgA were not yet determined.

In cultured chromaffin cells, amino acid incorporation into CgA is increased in the presence of phorbolesters (short incubation), but not by forskolin.307 At the mRNA level, forskolin has been shown to induce an insignificant rise after 36-48 h,80 but after more than two days of treatment a doubling of the mRNA levels was apparent."j2 This result is consistent with the finding of a CAMP-responsive element in the CgA-gene. '62,374 Varying effects of phorbolester on mRNA levels have been reported in different cells. In a calcitonin-producing cell line, phorbolester but not forskolin treatment for 48 h led to a doubling of the CgA mRNA level.2383239 On the other hand, in bovine chromaflln cells, treatment for two days with the same agent significantly decreased the CgA message80*'62 and an analogous finding has been obtained for human neuroblastoma cell~.~~' Since phorbolesters, depending on the time of incubation, can activate or down-regulate protein kinase C it is not clear by what mechanisms the CgA gene is regulated. Gene analysis has not revealed an established AP-I site for CgA.'62,374

In conclusion, the chromogranin genes can apparently be activated in a specific way. Thus the biosynthesis of the secretory content of neuroendocrine vesicles is not regulated en bloc (see also Ref. 103a) . Much further work is required before we will understand the detailed mechanisms at the cellular and genetic level which regulate the biosynthesis of this secretory cocktail. Certainly, such sophisticated mechanisms argue for specific functions of these regulated peptides, but are we yet in a position to understand them?

When CgA was still considered to be a specific component of chromaffin granules which also contain a high concentration of catecholamines, their possible involvement in the storage of these molecules was a much discussed topic. As outlined above, the chromogranins are now known to be present in many neuroendocrine granules which store only small amounts if any of biogenic amines. This finding indicates that chromogranins cannot be proteins with an exclusive function for amine storage, but it does not exclude such a function in some vesicles. As discussed in detail previously371 (see also Ref. 132) the chromogranins cannot be involved in a stoichiometric binding of catecholamines or ATP and there is also, as shown by nuclear magnetic resonance studies, no stable storage complex made up of chromogranins, nucleotides and catecholamines. However, a certain interaction between all these molecules apparently takes place (see Ref. 371 ) and could help to reduce the high osmolar pressure inside chromaffin granules,'37 although catecholamine/ATP complexes fulfil this purpose. In7 Direct evidence for an interaction of nucleotides with purified CgA has recently been obtained with a fluorescence probe.376 It was concluded that adenosine bases play an important role in this interaction and if this interaction also occurs in vivo it might provide a basis for some involvement in catecholamine storage. From solutions containing CgA, catecholamines and ATP in stoichiometric concentrations comparable to the granule content, an opaque precipitate can be sedimented which also indicates some interaction.94 Finally, calcium, which interacts with the chromogranins (see below), increases the binding of catecholamines to the chromogranins.339"v354

CgA was shown to inhibit the proteolytic cleavage of prohormones by trypsin or serine proteasesz9' It would be interesting to see whether such inhibition can also be observed with the newly defined endoproteases PC1 and PC2. However, to prove whether chromogranins have a role as regulators of posttranslational processing of prohormones*" would require more direct experiments, e.g. by introducing the chromogranin genes into secretory cells originally lacking it and subsequently defining their effects on proteolytic cleavage rates.

speculate that during "the condensation process" calcium enters newly formed chromaffin granules In 1977 we reported before the other small molecules, i.e. ATP and cat-36' echolamines are accumulated. However, there is no the following unpublished observation and some speculations based on it: "In this context it is interesting to note that in vitro direct evidence to support such a sequence of events." calcium can precipitate the acidic proteins of the granule content, i.e. the chromogranins (Winkler, In the meantime, detailed studies on the relationship unpublished observations), which is reminiscent of the fact that calcium precipitates casein, an acidic of calcium and chromogranins have appeared. Gratzl protein of the milk secretion granules." Furthermore, the condensation process, at least in exocrine pancreas, is apparently not energy dependent."' This would be consistent with the involvement of a calcium uptake which does not require energy as found for chromaffin granules. It therefore seems justified to proteins "function as helper proteins in the packaging of various hormones".274 Although it has been granule constituents, i.e. the respective hormones, for demonstrated275 that antibodies against CgB when co-expressed in secretory cells became co-packed with the granule formation process.

CgB in the regulated pathway this does not provide direct evidence for co-packing of hormones and chromogranins which do not interact specifically like

antigen/antibodies. Furthermore, the fact that the concentration of chromogranins/secretogranins in Two features of chromogranins discussed above secretory granules other than chromaffin granules is low makes it difficult to envisage a stoichiometric are consistent with a function of chromogranins as "helper process". The suggestion that the tyrosine sulfation of these proteins274 may act as a sorting precursors of active peptides: chromogranins, like mechanism has not been supported by subsequent studies.14' In a more detailed modification of these proposals it has been suggested that chromogranins are partly membrane bound in the trans-Golgi network and that these molecules together with calcium, aggregate with further chromogranins forming together with other secretory peptides the "granule core" for vesicles which subsequently pinch ~ff.'~~,"'~ This scheme assumes that sorting occurs concomitant with condensation as proposed previously;"' however, evidence to the contrary, i.e. sorting precedes condensation has also been obtained."' One can only re-emphasize that many neurosecretory granules contain only small amounts of chromogranins, thus a general hypothesis which concentrates on these minor components apparently neglects the role of the major et a1.268.26q have established that CgA can bind considerable amounts of calcium (152 nmol/mg protein with a dissociation constant of 54 PM). This property explains why in intact chromaffin granules despite the high concentration of total calcium (40mM) the free concentration is low being about 20)~M.'~ Not surprisingly, parathyroid secretory protein I which is in fact CgA was also found to bind calcium.108*20~ In the presence of this cation, CgA binding to membranes was also enhanced.'08~202 CgB is apparently also able to bind calcium."' The binding of calcium to CgA induces pH-dependent conformational changes.375,377 In these studies, CgA was found to bind up to 1150 nmol of Ca*+/mg protein (dissociation constant 2.7-4 mM).

What are the functional implications of this cal-prohormones or proneuropeptides, are proteolytitally processed in vivo and their biosynthesis can be regulated by humoral factors or by stimulation of the cells storing them. Direct support for this concept was provided when pancreastatin, a 49-amino acid peptide derived from CgA, was isolated from porcine pancreas and found to have a defined function in inhibiting the stimulated secretion of insulin from endocrine pancreas. 66,33' In the mean time pancreastatin peptides were isolated from other species including human'0'~283*293 and bovine24' material and pancreastatin immunoreactivity was found to be widespread in the neuroendocrine system (see Table l ), although this does not prove the presence of the free peptides in all these organs since the antisera against pancreastatin can also react with cium-binding by chromogranins? Several groups CgA. However, in addition to the pancreas the free suggested'08~'i0~367~37' that the aggregation of CgA by peptide has also been found in the pituitary calcium may be involved in the condensation process gland24',284 and chromatographic analysis suggests in the early phase of granule formation in agreement its presence also in porcine thyroid gland and with the speculation quoted above. However, more duodenum." complicated schemes for the function of chromo-Many subsequent studies, but not all of them (see granins (and also secretogranin II) have been pro-below and Ref. 288), have confirmed the original posed: thus it has been suggested that these acidic finding, that the 49-amino acid porcine pancreastatin and also its 16-amino acid C-terminal peptide can inhibit glucose-stimulated insulin release from perfused rat pancreas or isolated acini66*331 and from a pancreas tumor ccl1 line?'s Apparently, porcine pancreastatin is not only effective in the rat pancreas,66~1L3~167~258~305~331 but also in mouse,z")9 whereas in canine pancreas an effect"' or no effects*" have been reported.

For porcine pancreas "no effect at all on endocrine secretion of insulin, glucagon and somatostatin" was found.146 Inhibiting effects on rat pancreas were also exhibited by human pancreastatin95,292 and not surprisingly also by rat pancreastatin and its 26-residue C-terminal peptide. 96*228*257 For glucagon-mediated insulin release, pancreastatin was reported either as inhibitory*" or as stimulatory.'" For glucagon release itself, increases caused by pancreastatin were reported in two studies'16s271 whereas another one, even with the homologous pancreastatin in rat, was unable to find any effect. 257 Effects of pancreastatin on exocrine pancreatic (e.g. amylase release) secretion have also been reported;w227 however, on isolated acini no effect could be seen65*98*u9 and it has been suggested that pancreastatin acts on the secretion by inhibiting vagal nerve activity.229 On the other hand, for guinea-pig, isolated acini pancreastatin inhibited cholecystokinin-induced amylase release.lti Several further tissues have been tested for effects of pancreastatin. In rabbit isolated parietal cells of the stomach, pancreastatin had a direct inhibitory effect on secretion through interference with multiple second messenger pathways;206 on the other hand, in dogs, gastric acid secretion was increased."' Secretion is also inhibited by this peptide in the parathyroid gland.76*77 Interestingly, antibodies against pancreastatin potentiate the secretion from these cells at different calcium concentrations, apparently by neutralizing CgA peptides released into the medium from these ccll~.~~ This appears to be the first evidence that @A-related peptides when secreted have a "physiological" function.

Some actions on the central nervous system are also already indicated. Peripherally administered pancreastatin modulates memory processing in tests on mice9' and intracranical microinfusion'*' leads to changes in blood glucose and free fatty acids.

A second peptide derived from CgA, i.e. chromostatin, has been shown to be an effective inhibitor of catecholamine secretion from chromaffin ccll~.'~~~~ In a first studym '

It was established that CgA inhibits catecholamine secretion induced by nicotine, but not that by high K + , only after pre-incubation with the cells or after trypsin treatment indicating an involvement of a breakdown product. In a second study CgA was processed by proteases and the peptides inhibiting catecholamine secretion were analysed. A shorter peptide present in these sequences was synthesized and found to be a potent inhibitor of catecholamine secretion induced both by carbamoylcholine or by depolarizing K + concentration.

The pcptide, i.e.

chromostatin, inhibited secretagogue-induced calcium intIux.103*'03b It has not been demonstrated that chromostatin is actually formed in vivo or whether a larger molecule has this function on catecholamine release. Chromostatin is not flanked by basic pairs of amino acids (Fig. 1) ; thus the actual processing of this or larger peptides has still to be elucidated.

Finally, some actions of further peptides derived from CgA are likely. The N-terminal peptide (l-40) stimulated the secretion of CGRP and inhibited the secretion from calcitonin and of parathyroid hormone-related protein from a cell line."),6'*65a CgA, or a peptide formed from it during incubation, inhibits the secretion of POMC from the AtT-20 mouse cell line. Furthermore, CgA antisera increased basal secretion indicating a physiological inhibitory feedback of CgA secreted from these cells as discussed above."3 Two recent findings raise interesting new possibilities for the function of chromogranins. CgA, or peptides derived from it, were shown to have neuronotrophic activity for embryonic chick dorsal root ganglia.'" With the availability of synthetic peptides it should be possible to define this observation in more detail. In endocrine pancreas, pancreastatin inhibited beta-cell DNA synthesis and lowered the content of spermidine in these cells which has been implicated in beta-cell replication.3'0 An interaction with other peptides has also been indicated, since the N-terminal segment (l-46) of CgA significantly suppressed contraction of vascular smooth muscle caused by endothelin.' 35 We can conclude that there seems to be a good indication that peptides derived from CgA are functional. Thus the CgA molecule, which is proteolytitally processed in vivo and whose biosynthesis is regulated (see above) seems to belong to the evergrowing group of pro-peptides like those giving rise to neuropeptides. In apparent contrast to many of the neuropeptides, the phylogenetic conservation of the functional peptides (pancreastatin and especially chromostatin) is rather limited (see Fig. 2 ). If these peptides have specific receptors, then it is difficult to see how the lack of conservation of the peptide can be correlated in phylogeny with such a specific receptor. Furthermore, we will still need more data on the actual formation of these peptides (especially chromostatin) in the various tissues in vivo.

The indications that these peptides have trophic functions, or influence cell replication or can interact with other peptides should stimulate more research. Finally, regulatory functions, e.g. on synapse formation during development and on the regulation of postsynaptic receptors, should be considered.

Twenty-five years of research on the chromogranins have led to many established facts on their physico-chemical and molecular properties and on their widespread distribution in the neuroendocrine system. The function of these proteins is most likely to be as precursors for active peptides. In this context, future research to yield the most rewarding results should answer the following questions. Which functional peptides are actually formed in viva? Which proteases are involved? Is the function of the peptides, at present only defined by their exogenous addition, also of physiological significance? Are functional peptides (and their yet undefined receptors) conserved during phylogeny going beyond the mam-malian system? Do changes in the biosynthesis ot these proteins control the functions of peptides formed from them'? Is the proteolytic processing of the chromogranins regulated?

original research of the authors quoted in this paper was supported by the Dr Legerlotz-Stiftung and by the Fonds zur Forderung der wissenschaftlichen Forschung (Austria). We are grateful to Mrs M. Viehweider for typing the manuscript and to Mr Ch. Trawoger for preparing the photographs. I.

3.

28.

53a. Curry W. J., Johnston C. F., Hutton J. C., Arden S. D., Rutherford N. G., Shaw C. and Buchanan K. D. (19Y 1) The tissue distribution of rat chromogranin A-derived peptides: evidence for differential tissue processing from sequence specific antisera. Histochemisfry 96, 531-538. 54. Daniels A. J., Williams R. J. P. and Wright P. E. (1978) The character of the stored molecules in chromaffin granules of the adrenal medulla: a nuclear magnetic resonance study. Neuroscience 3, 573-585. 55. Davis W. L., Schmid K. O., Huettner J. W., Farmer G. R., Jacoby B. H. and Goodman D. B. P. (1990) Immunolocalization of secretory protein-I or chromogranin A in amphibian urinary bladder granular cell granules. Cell Biol. int. Rep. 14, 601-612. 56. De Potter W. P., Smith A. D. and De Schaepdryver A. F. (1970) 

Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

Pancreastatin inhibits insulin secretion and stimulates glucagon secretion in mice

Pancreatic release of pancreastatin in the pig

Estradiol negatively regulates secretogranin 11 and chromogranin A messenger ribonucleic acid levels in the female rat pituitary but not in the adrenal

Regulatory effects of estrogens on the pituitary content of secretogranin II and chromogranin A

Differential expression of secretogranin II and chromogranin A genes in the female rat pituitary through sexual maturation and estrous cycle

Glycoproteins of the chromaffin-granule matrix: use of lectin blotting to distinguish several separate classes

Cytochrome b-561 is identical with chromomembrin B. a major polypeptide of chromaffin granule membranes

Co-localization of parathyroid hormone and secretory protein-I in bovine parathyroid glands: a double immunocytochemical study at the electron microscopical level

Immunohistochemical and immunocytochemical localization of myosin, chromogranin A and dopamine-B-hydroxylase in nerve cells in culture and in adrenal glands

Peptide mapping studies of the chromogranins and of two chromathn granule proteoglycans

Evidence for the presence of a chromogranin-like protein in bovine splenic nerve granules

The release of protein from the stimulated adrenal medulla

Chromogranin A: posttranslational modifications in secretory granules

Gel electrophoresis of soluble and insoluble proteins of noradrenergic vesicles from ox splenic nerve: a comparison with proteins of adrenal chromaffin granules

Co-localization of secretogranins/chromogranins with thyrotropin and luteinizing hormone in secretory granules of cow anterior pituitary

Primary structure of bovine chromogranin B deduced from cDNA sequence

Labelling of chromaffin granules in adrenal medulla with '5S-sulfate

Enhanced rate of expression and biosynthesis of neuropeptide Y after kainic acid-induced seizures

The primary structure of bovine chromogranin A: a representative of a class of acidic secretory proteins common to a variety of peptidergic cells

The primary structure of human secretogranin I (chromogranin B): comparison with chromogranin A reveals homologous terminal domains and a large intervening variable region

Chromogranin B (secretogranin I), a putative precursor of two novel pituitary peptides through processing at paired basic residues

GAWK, a novel human pituitary polypeptide: isolation, immunocytochemical localization and complete amino acid sequence

PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues

Phosphorylation of parathyroid secretory protein I

The distribution of GAWK-like immunoreactivity in neuroendocrine cells of the human gut, pancreas, adrenal and pituitary glands and its co-localisation with chromogranin B

Identification of the mucopolysaccharides in catecholamine-containing subcellular particle fractions from various rat, cat and ox tissues

Secretion of a chromafhn granule protein, chromogranin, from the adrenal gland after splanchnic stimulation

Subcellular distribution of acetylcholinesterase forms in chromathn cells

Calcitonin gene-related peptide and chromogranin A: presence and intra-axonal transport in lumbar motor neurons in the rat, a comparison with synaptic vesicle antigens in immunohistochemical studies

New bovine adrenal medullary peptide and its precursor

Pancreastatin distribution and plasma levels in the pig

Synaptophysin and chromogranin A immunoreactivities in senile plaques of Alxheimer's disease

Immunoreactivity of hormonally-characterized human endocrine cells against 3 novel anti-human chromogranin B (Bll and B13) and chromogranin A (Al 1) monoclonal antibodies

Chromogranins A and B and secretogranin II in hormonally identified endocrine cells of the gut and the pancreas

Matrix free Ca*+ . m isolated chromatlin vesicles

An enzyme(s) that converts glutaminyl-peptides into pyroglutamyl-peptides: presence in pituitary, brain, adrenal medulla

Chromogranin-reactive endocrine cells in argyrophilic carcinomas ('carcinoids') and normal tissue of the breast

The secretion of catecholamines, chromogranin A and neuropeptide Y from the adrenal medulla of the cat via the adrenolumbar vein and thoracic duct: different anatomic routes based on size

Immunoreactivities for chromogranin A and chromogranin B, and Secretogranin II in the guinea-pig endocrine pancreas

Immunoreactivities for chromogranin A and B, and secretogranin II in the guinea pig entero-endocrine system: cellular distributions and intercellular heterogeneities

Topology of chromogranins in secretory granules of endocrine cells

Chromogranin A (CgA) in the gastro-entero-pancreatic (GEP) endocrine system. II. CgA in mammalian entero-endocrine cells

Peptidasic activities associated with acetylcholinesterase are due to contaminating enzymes

Identification of ked-related proteases in chromathn granules by partial amino acid sequence analysis

Regulation of phenylethanolamine N-methyltransferase synthesis and degradation

Selective localization of the parathyroid secretory protein-I/adrenal medulla chromogranin A protein family in a wide variety of endocrine cells of the rat

The biosynthesis, intracellular processing, and secretion of parathormone

Cosecretion of secretory protein-I and parathormone by dispersed bovine parathyroid cells

Isolation and partial characterization of secretory protein I from bovine parathyroid glands

Similarity of secretory protein I from parathyroid gland to chromogranin A from adrenal medulla

Phosphorylation and dephosphorylation of chromaffin cell proteins in response to stimulation

Plasma chromogranin A as a marker of sympathochromaflin activity in humans

Distribution and partial characterisation of immunoreactivity to the putative C-terminus of rat pancreastatin

Immunological studies on the distribution of chromogranin A and B in endocrine and nervous tissues

Dopamine b-hydroxylase and other glycoproteins from the soluble content and the membranes of adrenal chromafhn granules: isolation and carbohydrate analysis

Isolation and characterization of chromogranins A, B and C from bovine chromafBn granules and a rat pheochromocytoma

Sex-related differences in chromogranin A, chromogranin B and 'secretogranin'I1 gene-expression in rat pituitary

Dexamethasone induces an 'increased biosynthesis of chromogranin A in rat pituitary gland

Isolation and immunological characterization of a glycoprotein from adrenal chromatlm granules

A novel 1745-dalton pyroglutamyl peptide derived from chromogranin B is in the bovine adrenomedullary chromathn vesicle

Effects of systemic pancreastatin on memory retention

Nucleotide sequence and cellular distribution of rat chromogranin B (secretogranin I) mRNA in the neuroendocrine system

A macro-molecular complex of bovine adrenal chromogranin A, adrenaline and ATP formed in vitro

Bioactivity of human pancreastatin and its localization in pancreas

Bioactivity of synthetic human pancreastatin on exocrine pancreas

Bioactivity of synthetic C-terminal fragment of rat pancreastatin on endocrine pancreas

Inhibitory effect of pancreastatin on pancreatic exocrine secretion in the conscious rat

Acetylcholine regulates pancreastatin secretion from the human pancreastatin-producing cell line (QGP-IN). C/in

Elevated plasma levels of pancreastatin (PST) in patients with non-insulin-dependent diabetes mellitus (NIDDM)

Isolation and characterization of a tumor-derived human pancreastatinrelated protein

Protein-carboxyl methylation in adrenal medullary cells

Chromostatin, a 20-amino acid peptide derived from chromogranin A, inhibits chromaffin cell secretion

Regulation of chromogranin A and chromogranin B (secretogranin I) synthesis in bovine cultured chromaffin cells

Chromostatin receptors control calcium channel activity in adrenal chromaffin cells

Presence and coexistence of chromogranin A and multiple neuropeptides in Merkel cells of mammalian oral mucosa

Composition and biogenesis of complex carbohydrates of ox adrenal chromathn granules

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

Hepatic golgi fractions resolved into membrane and content subfractions

Chromogranin A in uremia: progressive retention of immunoreactive fragments

Tyrosine sulfation and the secretory pathway

Biogenesis of nemoendocrine secretory vesicles

Biosynthesis of betagranin in pancreatic beta-cells. Identification of a chromogranin A-like precursor and its parallel processing with proinsulin

Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20 kDa N-terminal fragment (betagranin) by the concerted action of a Ca'+-dependent endonentidase and carboxvnentidase H

&Granins: 21 kDa_ co-secreted peptides of the insulin granule closely related to adrenal medullary chromogen. bedn

The molecular cloning of the chromo~a~n A-like precursor of &granin and pancreastatin from the endocrine pancreas

Immunolocalization of betagranin: a chromogranin A-related protein of the pancreatic B-cell

Bovine chromogranin A: its sequence and the distribution of its messenger RNA in endocrine tissues

The sequence of porcine chromogranin A messenger RNA demonstrates chromogranin A can serve as the precursor for the biologically active hormone

The bovine c~omo~a~n A gene: structural basis for hormone regul&on and generation of biologically active peptides

Primary structure of rat chromogranin A and distribution of its mRNA

Secretion of GAWK, a presumed product of chromogranin B processing, by a human medullary thyroid carcinoma cell line: effect of phorbol ester, forskolin and calciumionophore

Different processing of chromogranin 5 into GAWK-immunoreactive fragments in the bovine adrenal medulla and pituitary gland

Effect of pancreas&in on pan&eat& endocrine and exocrine secretion

A comparison of the insulinotropic and the insulin-inhibitory actions of gut peptides on newborn and adult rat islet cells

Effects of pancreastatin and chromogranin A on insulin release stimulated by various insulinotropic agents

Chromogranin A/pancreastatin-related molecules are richly contained in enteric neurons but not in perivascular sympathetic neurons

Condensing vacuole conversion and zymogen granule discharge in pancreatic exocrlne cells: metabolic studies

Assi~ment of secretogranin I locus to mouse chromosome 2 by in situ hybridization and interspecific backcross analysis

Elevated plasma chromogranin-A concentrations in prostatic carcinoma

Chromogranins or chromogranin-like proteins are present in lamellar bodies and pulmonary surfactant of rat alveolar type II cells

Ultrastructural localization of svnaotonhvsin to the secretory granules of normal glucag& and insulin cells in' human islets of Langerhans

I~un~yt~~~i~l localization and development of multiple kinds of neuro~ptid~ and nemoendocrine proteins in the chick ultimobranchial gland

Distribution and ontogeny of chromogranin A and tyrosine hydroxylase in the carotid body and glomus cells located in the wall of the common carotid artery and its branches in the chicken

Reflex splanchnic nerve stimulation increases levels of nroenkenhalin A mRNA and nroenkenhalin A-related neutides in the rat adrenal medulla

Highly sensitive enzyme immunoassay for bovine chromogranin A: application for studies of regional distribution in bovine centra! nervous system

Local&&on of chromogranin A and chromogranin B, met-enkephalin-Arg4-Gly7

Leu8 and PGP9.5-Like immunorea~tivity in the developing and adult rat adrenal medulla and extra-adrenal chromaflin tissue

Glycoproteins and proteoglycans 01 the chromaffin granule matrix

Biosynthetic relationship between the major matrix proteins of adrenal chromaffin granules

Immunological characteri~tion of the endoproteases PC1 and PC2 in adrenal chromaffin granules and in the pituitary gland

A specific soluble protein from the catecholamine storage vesicles of bovine adrenai medulla

Molecular organization of the chromaffin vesicles of the adrenal medulla

Release of catecholamines and specific protein from adrenal glands

Osmotic pressure of solutions of ATP and catecholamines relating to storage in chromaffin granules

Purification and characterization of a novel thiol protease involved in processing the enkephalin precursor

Sulfation of porcine parathyroid secretory protein I

Chromogranin A in the olfactory system of the rat

Light and electron microscopical immunocytochemical localization of pancreastatin-like immunoreactivity in porcine tissues

Chromogranin A-processing proteinases in purified chromaffin granules: contaminants or endogenous enzymes?

~denti~~tion of two glycoproteins of chromaffin granules as the carboxypeptidase H

Insulin hypoglycemia increases the levels of neuropeptide Y and calcitonin gene-related peptide, but not of chromogranins A-and B in rat chromaffin granules

Presence of chromoaranin A. B and C in bovine endocrine and nervous 'tissues: a comparative immunohistochemical study

Synaptic pathology in Alzheimer's disease. Immunologi~l data for markers of synaptic and large dense core vesicles

Chromogranin in bronchopulmonary neuroendocrine cells. Immunocytochemical detection in human, monkey, and pig respiratory mucosa

Secretogranin I (chromogranin B) messenger RNA accumulation is honnonally regulated in GH3B6 rat pituitary tumor cells

The ostrich pituitary contains a major peptide homologous to mammalian chromogranin A(l-76)

Synthesis of chromogranins and dopamine b-hydroxylase by perfused bovine adrenal glands

Tyrosine-O-sulfated proteins of PC12 pheochromocytoma cells and their sulfation by a tyrosylprotein sulfotransferase

Calcium-binding proteins in the parathyroid gland

C~omogranin A as a marker of neuroendocrine histogenesis of tumours: an immunoelectron microscopic study with considerations about the influence of fixation and embedding media on immunolabelling

Chromogranin B-like immunoreactivity in the mouse submandibular salivary gland during postnatal development

Localization of chromoaranins, non-neuron-specific enolase. and different forms of somatostatins in the submandibular salivary glands of-mice. 1. Dent. Res. 69, t49441499

Adrenal medullary chromaffin granule peptides: two major ovine peptides and their precursor

Primary structure of mouse chromogranin-B deduced from cDNA sequence

Galanin and pancreastatin inhibit stimulated insulin secretion in the mouse: comparison of effects

Distribution of chromogranin A and secretogranin I (chromogranin B) in neuroendocrine cells and tumors

Chromogranin A and B messenger rlbonucleic acids in pituitary and other normal and neoplastic human endocrine tissues

Detection of chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes

Immunohistochemical detection of chromogranin and neuron-specific enolase in pancreatic endocrine neoplasms

Immunohistochemical localization of epinephrine, norepinephrine, catecholamine-synthesizing enzymes, and chromogranin in neuroendocrine cells and tumors

Immunohistochemical localization of chromogranin in human hypophyses and pituitary adenomas

Specific endocrine tissue marker defined by a monoclonal antibody

Immunohistochemical localization of chromogranin in normal and neoplastic endocrine tissues

Pancreastatin inhibits insulin release from Rin m 5F cells: reversion by pertussis toxin

Distribution of mRNAs for chromogranins A and B and secretogranin II in rat brain

Temporal lobe epilepsy of the rat: differential expression of mRNAs of chromogranin B, secretogranin II, sy&ptin/ synaptophysin and p65 in subfields of the hippocampus. Molec

Cellular and cell-free processing of parathyroid secretory proteins

Identification and cell-free translation of mRNA coding for a precursor of parathyroid secretory protein

Immunocytochemical localization of a novel pituitary protein (7B2) within the rat brain and hypophysis

Two-dimensional immunoblotting analysis and immunocytochemical localization of the secretory polypeptide 7B2 in adrenal medulla

Immunoelectron microscopic localization of dopamine fi-hydroxylase and chromogranin A in adrenomedullary chromaiZn cells

Human chromogranin A-like immunoreactivity in the Bergmann glia of the rat brain

Purification and sequence of a novel ovine adrenal medullary peptide and its precursor

Effects of porcine pancreastatin on postprandial pancreatic exocrine secretion and endocrine functions in the conscious rat

The importance of the C-terminal amide structure of rat pancreastatin to inhibit pancreatic exocrine secretion

Inhibitory effect of pancreastatin on pancreatic exocrine secretions. Pancreastatin inhibits central vagal nerve stimulation

The human chromogranin A gene: chromosome assignment and RFLP analysis

Early fetal acquisition of the chromaflin and neuronal immunophenotype by human adrenal medullary cells. An immunohistological study using monoclonal antibodies to chromo&anin A, tiaptophysin, tyrosine hydioxylase

Regulation of svnthesis and secretion of chromoaranin-A bv calcium and 1,25-dihydroxycholecalcif&ol in c&u&l bo&e parathyioid cells

Chromogranin A immunoreactive peptides are major components of neocortical and limbic plaques in Alzheimer's disease

Chromogranin A-like immunoreactive neurites are major constituents of senile plaques

The distribution of chromogranin A like immunoreactivity in the human hippocampus coincides with the pattern of resistance to epilepsy-induced neuronal damage

Monodendritic neurons: a cell type in the human cerebellar cortex identified by chromogranin A-like immunoreactivity

Chromogranin A-like immunoreactivity in the human brain: distribution in bulbar medulla and cerebral cortex

The coregulation of secretion and cytoplasmic ribonucleic acid of chromogranin A and calcitonin by phorbol ester in cells that produce both substances

The effects of forskolin and calcium ionophore A23187 on secretion and cytoplasmatic RNA levels of chromogranin-A and calcitonin

The gene for human .-. _ chromogramn A (CgA) is located on chromosome 14

isolation and characterization of bovine pancreastatin

Biochemical and functional properties of large and small dense-cored vesicles in sympathetic nerves of rat and ox vas deferens

Secretory proteins from adrenal medullary cells are carboxylmethylated in oivo and released under their methylated form by acetylcholine

Chromogranin: widespread immunoreactivity in polypeptide hormone producing tissues and in serum

Plasma chromogranin A. Initial studies in human hypertension

Radioimmunoassay of chromogranin A in plasma as a measure of exocytotic sympathoadrenal activity in normal subjects and patients with pheochromocytoma

Chromogranin A: immunohistology reveals its universal occurrence in normal polypeptide hormone producing endocrine glands

Human chromogranin A: detection bv immunohistochemistry in cells and diverse polypeptide hormone producing tumors

Chromogranin A, the major catecholamine storage vesicle soluble protein

Human chromogranin A. Purification and characterization from catecholamine storage vesicles of human pheochromocytoma

Rapid radioimmunoassay of circulating chromogranin A: in vitro stability, exploration of the neuroendocrine character of neoplasia, and assessment of the effects of organ failure

Effect of porcine pancreastatin on endocrine function of canine pancreas

Pancreastatin-like immunoreactivity and insulin are released in parallel from the perfused porcine pancreas

Molecular cloning of chromogranin A from rat pheochromocytoma cells

Homologous pancreastatin inhibits insulin secretion without affecting glucagon and somatostatin release in the perfused rat pancreas

Pancreastatin inhibits insulin secretion as induced by glucagon, vasoactive intestinal peptide, gastric inhibitory peptide, and 8-cholecystokinin in the perfused rat pancreas

Neurofilament and chromogranin expression in normal and neoplastic neuroendocrine cells of the human gastrointestinal tract and pancreas

Chromogranin A-like proteins in the secretory granules of a protozoan, Paramecium tetraurelia

Gut endocrine cell population in coeliac disease estimated by immunocytochemistry using a monoclonal antibody to chromogranin

Chromoeranin B (secretoeranin I). a secretorv nrotem of the regulated

is also present in a tightly membrane-assGated form in PC12 cells

The organisation of the mouse chromogranin B (secretogranin I) gene

Chromatographic and electrophoretic behaviour of chromogranin obtained from bovine and equine adrenal medufla

Neuroendocrine cells in intestinal lamina propria. Detection with antibodies to chromogranin A

Glucocorticoid-and nerve growth factor-induced changes in chromogranin A expression define two different neuronal phenotypes in PC12 cells

Localization of pancreastatin immunoreactivity in porcine endocrine cells

Caz-' binding to chromaffin vesicle matrix uroteins: effects of DH, MJ? , and ionic strength

Chromogranins, widespread in endocrine and nervous tissue, bind Ca2+

Phylogenetic aspects of pancreastatin-and chromogranin-like immunoreactive cells in the gastro-entero-pancreatic neuroendocrine system of vertebrates

Effects of pancreastatin on pancreatic hormone secretion in the dog

Phylogenetic distribution of peptides related to chromogranins A and B

Tortora 0. and Solcia E. (1986) Chromogranin A, B and C immunoreactivities of mammalian endocrine cells. Distribution, distinction from costored hormones/prohormones and relationship with the argyrophil component of secretory granules

Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway

An antibody against secretogranin I (chromogranin B) is packaged into secretory granules

Purification of a sulfated secretory protein from the adenohypophysis. Immunochemical evidence that similar macromolecules are present in other glands

Chromogranin A, B and C: immunohistochemical localization in ovine pituitary and the relationship with hormone-containing cells

expression 'of parathyrbid hormone genes

Mechanism of secretion from the adrenal medulla. I. A microquantitative immunologic assay for bovine adrenal catecholamine storage vesicle protein and its application to studies of the secretory process

Regional distribution of chromogranin B 420-493-like immunoreactivity in the pituitary gland and central nervous system of man, guinea-pig and rat

Release of chromathn granule glycoproteins and proteoglycans from potassium-stimulated PC12 pheochromocytoma cells

Regulation of chromogranin B/secretogranin I and secretogranin II storage in GH4Cl cells

An immunological study on chromogranin A and B in endocrine and nervous tissues

Isolation and primary structure of tumor-derived peptides related to human pancreastatin and chromogranin A

Pancreastatin: molecular and immunocytochemical characterization of a novel peptide in porcine and human tissues

Secretion from the adrenal medulla: biochemical evidence for exocytosis

Comparison of chromogranins A, B, and secretogranin II in human adrenal medulla and pheochromocytoma

Ontogenesis of chromogranin A and B and catecholamines in rat adrenal medulla

The effect of pancreastatin on endocrine and exocrine pancreas. &and

Sympathetic axons and nerve terminals: the protein composition of small and large dense-core and of a third type of vesicles

cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases

Chromogranin A can act as a reversible processing enzyme inhibitor. Evidence from the inhibition of the IRCM-serine protease 1 cleavage of pro-enkephalin and ACTH at pairs of basic amino acids

C-terminal peptide of the GIP precursor and pancreastatin fragments on insulin secretion from isolated rat islets

Isolation of human pancreastatin fragment containing the active sequence from a glucagonoma

Production of GAWK (chromogranin-B 420-493)-like immunoreactivity by endocrine tumors and its possible diagnostic value

Immunological identification and characterization of chromogranins coded by Poly(A) mRNA from bovine adrenal medulla and pituitary gland and human phaeochromocytoma

Relationship of multiple forms of chromogranin

Chromogranin, an integral membrane protein

Analysis of the carbon-13 and proton NMR spectra of bovine chromaffin granules

A putative processing enzyme for proenkephalin in bovine adrenal chromafIin granule membranes

An immunochemical and immunohistochemical study on pancreastatin-like immunoreactivity using synthetic peptides

transplantation is not reflected by chromogranin A or basic fibroblast growth factor in ventricular fluid. h'xpl Neural

Chromogranin A biosynthetic cell populations in bovine endocrine and neuronal tissues; detection by in situ hybridization histochemistry

Rat adrenal medulla: levels of chromogranins, enkephalins, dopamine ,!I-hydroxylase and of the amine transporter are changed by nervous activity and hypophysectomy

Calcitonin and chromogranin A localization in medullary carcinoma of the thyroid by immunoelectron microscopy

Effects of pancreastatin on insulin, glucagon and somatostatin secretion by the perfused rat pancreas

Biochemistry of the chromogranin A protein family

Effect of secretagogues on chromogranin A synthesis in bovine cultured chromaffin cells. Possible regulation by protein kinase C

Proteolytic processing of chromogranin A in cultured chromaffin cells

Secretion from chromaffin cells is controlled by chromogranin A-derived peptides

Long term inhibitory effects of pancreastatin and diazepam binding inhibitor on pancreatic /?-cell deoxyribonucleic acid replication, polyamine content, and insulin secretion

Acetylcholinesterases: zymogens of neuropeptide processing enzymes?

Non-cholinergic actions of acetylcholinesterases: proteases regulating cell growth and development?

Acetylcholinesterase hydrolyses chromogranin A to yield low molecular weight peptides

Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans

Biochemistry of adrenal chromaffin granules

Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla

A specific soluble protein from the catecholamine storage vesicles of bovine adrenal medulla

Chromogranin immunoreactivity in the central nervous system. Immunochemical characterization, distribution and relationship to catecholamine and enkephalin pathways

Gonadotropin-releasing hormone regulates gonadotropin B-subunit and chromogranin-B messenge; ribonucleic acids in cultured-chromogranin-A-positive pituitary adenomas

Co-localization of chromogranin A and B, secretogranin II and neuropeptide Y in chromaffin granules of rat adrenal medulla studied by electron microscopic immunocytochemistry

Chromogranins A and B are co-localized with atria1 natriuretic peptides in secretory granules of rat heart

Extraction, purification, and partial characterization of bovine parathyroid secretory protein

Antigen receptor-regulated exocytosis in cytotoxic T lymphocytes

Suppression of chromogranin-A release from neuroendocrine sources in man: pharmacological studies

Is physiologic sympathoadrenal catecholamine release exocytotic in humans? Circuhtion

Neuroendocrine sources of chromogranin A in normal man: clues from selective stimulation of endocrine glands

Isolation and characterization of a tumor-derived human protein related to chromogranin A and its in vitro conversion to human pancreastatin-48

Plasma pancreastatin-like immunoreactivity in various diseases

Pancreastatin-like immunoreactivity in urine

Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion

KexZ-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes

Co-localization and secretion of parathyrin of stannius corpuscles (i~uno~~ve parathyroid hormone) and of secretory glycoproteins including SP-I in the European eel (AnguiBu angiEa L.)

Sorting-of progeny coronavirus from condensed secretory proteins at the exit from the tram-Golai network of AtT20 cells

Maturation of dense core granules in wild type and mutant Tetrahymeua thermophila

11953) Rate of turnover of eninenhrine in the adrenal medulla

Bi~h~ist~ of the ~kephalins and ~k~halin~n~ining peptides

Ultrastructural identification of human secretin cells by the immunogold technique. Their costorage of chromogranin A and serotonin

Ultrastructural localization of chromogranin: a potential marker for the electron microscopical recognition of endocrine cell secretory granules

Calcium and catecholamine interaction with adrenal chromogranins

Serotonin-, somatostatin-and chromogranin A-containing cells of the urethro-prostatic complex in the sheep. An immunocytochemical and immunofluorescent study

Chromogranin-and somatostatin-containing neuroendocrine cells in the porcine uterus. An imrnun~t~h~ic~ study

Choline& nerve terminals in the rat diaphragm are chromogranin A immunoreactive

A oronosed role for chromoeranin A as a glucocortikoid-r&ponsive autocrine inhibitor of pro-opiomelanocbrtin'secrktidn

Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs

Heterogeneitv of c~omo~anin Aderived peptides in bovine g& pancreas and adrenal medulla

Differential a~um~ation of catecholamines, proenkephalin A-derived anh*chromogranin A-de&ed peptides 'in the medium after chronic nicotine stimulation of cultured bovine adrenal chromaffin cells

Resemine-induced nrocessina of chromoaranin A in cultured bovine adrenal cells

Adrenal chromafhn granules and secretory granules from thyroid parafollicular cells have several common antigens

Immunological studies on the occurrence and properties of chromogranin A and B and secretogranin II in endocrine tumors

A high ratio of chromogranin A to synaptin/synaptophysin is a common feature of brains in Alzheimer and Pick disease

Chromogranins in rat brain: ch~a~te~~tion, to~graphi~l ~s~bution and reg~ation of synthesis

Divergent changes of chromogranin A/secretogranin II levels in differentiating human neuroblastoma cells

Undegraded chromogranin A is present in serum and enters the endocytotic lysosomal pathway in kidney

Synaptophysin and chromolgranins/secretogranins-widespread constituents of distinct types of neuroendocrine vesicles and new tools in tumor diagnosis

Identification of gastroenteropancreatic neuroendocrine cells in normal and neopiastic human tissues with antibodies against s~aptopbysin, chromogranin A, secretogranin I (chromo~anin B), and secretogranin II

Detection of chromogranin in neuroendocrine cells with a monoclonal antibody

Chromogranin from normal human adrenal glands: purification by monoclonal antibody a8inity chromatography and partial N-terminal amino acid sequence