key: cord-0889539-5lmrthmb
authors: Homaidan, Fadia R.; Torres, Alfonso; Donowitz, Mark; Sharp, Geoffrey W.G.
title: Electrolyte transport in piglets infected with transmissible gastroenteritis virus Stimulation by verapamil and clonidine
date: 1991-10-31
journal: Gastroenterology
DOI: 10.1016/0016-5085(91)90713-u
sha: 4f51fbf167ca0d3eef1bff57a9df606733591db3
doc_id: 889539
cord_uid: 5lmrthmb

Abstract The effects of clonidine, an α 2-adrenergic agonist, and verapamil, a Ca2+ channel blocker, on Na2+ and Cl− absorption were studied in stripped jejunal mucosa from control and transmissible-gastroenteritis-virus-infected piglets. All infected piglets developed severe diarrhea 18–24 hours after oral inoculation. Jejunum from infected animals, as compared with control jejunum, had decreased mucosal-to-serosal, serosal-to-mucosal, and net Na+ and Cl− fluxes. Clonidine and verapamil caused a decrease in short-circuit current and stimulation of Na+ and Cl− absorption in control jejunum. In infected piglets, although the jejunum exhibited severe villus atrophy, both drugs stimulated Na+ and Cl− absorption and the magnitude of Na+ and Cl− absorption was similar in control and transmissible-gastroenteritis-infected jejunum. In contrast, d-glucose stimulated Na+ absorption, and the decrease in shortcircuit current caused by verapamil and clonidine, were decreased in transmissible-gastroenteritis-infected jejunum. Such pharmacological stimulation of Na+ and Cl− absorption might be useful in the management and treatment of certain viral diarrheal diseases.

piglets. All infected piglets developed severe diarrhea 18-24 hours after oral inoculation. Jejunum from infected animals, as compared with control jejunum, had decreased mucosal-toserosal, serosal-to-mucosal, and net Na' and Cl fluxes. Clonidine and verapamil caused a decrease in short-circuit current and stimulation of Na' and Cl-absorption in control jejunum. In infected piglets, although the jejunum exhibited severe villus atrophy, both drugs stimulated Na' and Cl-ahsorption and the magnitude of Na' and Cl-absorption was similar in control and transmissihle-gastroenteritis-infected jejunum. In contrast, n-glucose stimulated Na' absorption, and the decrease in shortcircuit current caused by verapamil and clonidine, were decreased in transmissihle-gastroenteritisinfected jejunum. Such pharmacological stimulation of Na' and Cl-absorption might he useful in the management and treatment of certain viral diar-rhea1 diseases.

cute viral gastroenteritis is a common disease of A animals and humans that can produce severe morbidity and mortality in the very young (1). In general, viral enteric infections are self-limiting. Enteric viruses appear to selectively affect the mature enterocytes lining the upper portion of the intestinal villi. Viral replication in these mature enterocytes leads to premature sloughing of infected enterocytes with resulting villus atrophy (2,3), malabsorption, and disordered electrolyte transport due to the lack of a functional epithelium involved in digestion and absorption of nutrients. There may also be an increase in secretory function caused by an increase in the number of immature enterocytes and crypt epithelium hyperplasia (24). The mortality associated with viral gastroenteritis is for the most part caused by severe water and electrolyte loss, rather than by any irreversible intestinal tissue damage.

Management of viral diarrheas typically consists of dietary adjustments and oral rehydration therapy. If the nutritional and electrolyte requirements are stabilized and secondary bacterial infections prevented, the infected animal can overcome the critical phase of the infection, allowing time for the intestinal mucosa to recover normal function.

Electrolyte transport in mammalian small intestine is regulated by many factors including the enteric nervous system, inflammatory cells, and hormones and agents that act directly or indirectly on the epithelial transporting cells. Some of the control may be exerted via changes in the intracellular Ca*+ concentration ([Ca"],). Increasing [Caz+li by the use of Ca'+ ionophores such as A23187 causes a decrease in Na' and Cl-absorption and stimulates Cl secretion (5). Conversely, decreasing [Ca'+], by the use of calcium channel blockers increases Na' and Cl absorption (6) . Verapamil, a calcium channel blocker, and clonidine, an a,-adrenergic agonist, have been found to increase active Na' and Cl absorption in rabbit ileum (7) (8) (9) (10) (11) .

The mechanism of action of verapamil is thought to be by inhibition of calcium entry through the plasma membrane, thus decreasing [Ca"'],. The action of clonidine may be, in part, through decreas- ing intracellular levels of calcium, but it may also be due to an as yet unidentified action that mimicks the effects of decreased .

Neonatal piglets are highly susceptible to a variety of viral and bacterial enteric infections that result in severe diarrhea. In addition, neonatal, conventional, specific pathogen-free, and gnotobiotic piglets have been used as animal models for the study of human viral enteritis that also affects children (12) (13) (14) (15) . In this study, we describe the effects of verapamil and clonidine on electrolyte transport in the small intestine of normal piglets, and of piglets with severe villus atrophy from transmissible gastroenteritis (TGE) caused by a coronavirus infection.

Conventional crossbred healthy 2-5-day-old piglets were obtained from a commercial herd. Piglets were removed from the sow, kept in isolation for 24-48 hours before any use, and fed a commercial diet (SPF Lac, New York, NY) supplemented with gentamicin, 0.5 mg/mL (TechAmerica Group, Inc., Elwood, KS). A total of 11 control uninfected and 12 TGE-virus-infected piglets were used in these studies.

A virulent strain of porcine TGE coronavirus, obtained from the University of Nebraska (Lincoln, NE) (161, was used as a 1:lO bacteria-free suspension of fecal material collected from an experimentally infected gnotobiotic piglet. Piglets were orally infected with 1.0 mL of the viral suspension and were killed 48 hours later at the peak of severe diarrhea and villus atrophy that occurred in all test animals. Control piglets were kept uninfected (in a pathogenfree facility) for 48 hours before the experiments. Segments of jejunum, adjacent to all of the segments used in the electrolyte transport studies, were fixed with 10% buffered formalin and processed for histological evaluation by conventional procedures (17).

Control uninfected piglets remained healthy during their isolation period until death. All TGEvirus-infected piglets developed severe diarrhea within 18-24 hours after oral inoculation. 

Active jejunal electrolyte transport was measured by the Ussing chamber-voltage clamp technique according to the method described by Donowitz and Asarkof (5) for use in rabbits. Piglets were killed with an overdose of sodium pentobarbital. Jejunal intestinal mucosa was removed and For comparision of Isc and transport in control stripped from its underlying muscle, mounted between two and infected tissues, the data in Tables 1 and 2 controls). However, infected jejunum had signifiafter gassing with 95% O,-5% CO,. Glucose (10 mmol/L) cantly different Na' mucosal-to-serosal fluxes (6.9 & and mannitol (10 mmol/L) were added to the serosal and 0.9 pEq * cm-' . h-' in infected tissues vs. 17.6 f 2.4 mucosal bathing fluids, respectively, at the time of mounting the tissue. Transmural potential difference (PD), shortcircuit current (1s~) and conductance (G) were measured. An automatic voltage clamp, corrected for fluid resistance between the PD sensing bridges, provided continuous shortcircuiting of the tissue. The major transport measurement reported in this paper is the Isc, which is equivalent to the electrical sum of all electrogenic ion transport processes occurring simultaneously. Unidirectional fluxes of "Na and %l were also measured 20-100 minutes after the addition of isotopes on tissues matched to differ in conductance by not more than 25%. In these experiments two 20 minute flux periods were measured before the addition of any drug, then verapamii or clonidine were added to the serosal side and three 20 minute flux periods were determined. Fluxes from untreated tissue were also measured for time controls. The effects of the two drugs were evaluated in both normal (n = 5) and virus-infected piglets (n = 4 for studying the effects of verapamil and n = 5 for clonidine). Nine of the 10 animals studied were paired with respect to the two drug treatments. In the ion flux experiments, a negative sign (-) indicates net secretion and a positive sign (+) indicates net absorption.

Statistical analyses were performed with Student's t test for paired and unpaired data. All results are expressed as means 2 SE.

["Na]NaCl and [36C1]HC1 were obtained from Amersham, Arlington Heights, IL. Verapamil and clonidine were purchased from Sigma Chemical Co., St. Louis, MO. . h-' in control tissues; P < 0.01); Na'

cme2 . h-' in infected tissues vs. 3.2 2 0.3 FEq . cm-' . serosal-to-mucosal fluxes (8.5 + 0.7 pEq . cm-* . h-' h-' in controls; P < 0.001); Clan mucosal-to-serosal in infected tissues vs. 14.5 k 2.1 pEq . cm-' . h-' in fluxes (3.2 f 0.8 FEq . cm-* . h -' in infected tissues controls; P < 0.05); net Na' fluxes (-1.6 + 0.7 p,Eq. vs. 10.0 + 1.5 FEq. cm-' . h-' in controls; P < 0.01); Table 1 J, flux; ms, mucosal to serosal; sm, serosal to mucosal.

and net Cl-fluxes (-0.8 +-1.0 PEq . cm-' . h-' in infected tissues vs. 2.9 + 0.9 FEq. cm-' . h-' in controls; P < 0.05). Cl-serosal-to-mucosal fluxes in infected piglets were not significantly different than those in controls (3.9 k 1.1 FEq * cm-' -h-' in infected vs. 7.1 k 1.5 FEq * cm-' * h-' in control piglets). Figure 2 and Table 1 and Figure 3 and Table 2 show the effects of 10 Fmol/L verapamil and 3 kmol clonidine, respectively, on Isc and Na+ and Zl-fluxes in control and infected jejunum. Verapamil and clonidine both decreased Isc in control and virus-infected piglets. The decrease in Isc (measured 20 minutes after drug addition) caused by verapamil (AIsc = -25.8 k 2.0 ~A/cm'; starting Isc = 23.6 + 4.6 PA/cm'; n = 7; Figure 2 ) or clonidine (AIsc = -19.8 + 1.5 PA/cm'; starting Isc = 18.5 k 4.4 PA/cm*; n = 8; Figure 3 ) in infected tissue, was significantly smaller (P < 0.02 in both cases) than the effect of either drug on Isc in control tissue. In control tissue the decrease in Isc caused by verapamil was 51.5 f 3.0 PA/cm' (n = 9) (starting Isc = 47.3 + 6.9 @/cm'), and the decrease in Isc caused by clonidine was 46.2 -+ 2.8 ~A/cm'; (n = 9; starting Isc = 44.1 + 6.5 FA/cm'). The reduced response to verapamil or clonidine in the infected piglet might be explained by the reduced surface area of the tissue exposed to the drugs ( Figure  1 ) caused by the loss of mature enterocytes. Potential difference values were significantly decreased in control jejunum from -2.6 + 0.4 mV (n = 10) to -0.1 2 0.2 mV (n = 5; P I 0.01) after the addition of verapamil, and to -0.5 k 0.1 mV (n = 5; P I 0.01) after the addition of clonidine. In infected jejunum also, PD values were decreased from -1.9 + 0.5 mV (n = 9) to 0.3 f 0.5 mV (n = 4; P I 0.05) after the addition of verapamil, and to 0.3 + 0.5 mV (n = 5; P I 0.05) after the addition of clonidine. The magnitudes of the changes in PD were similar in control and infected jejunum. Conductance was not significantly changed in either control or virus-infected jejunum by the addition of either drug. In normal tissue, G values increased from -22.9 + 2.7 mS * cm-' (n = 10) to -27.2 k 6.3 mS . cm-' (n = 5) after the addition of verapamil, and to -29.0 -+ 6.6 mS . cm-' (n = 5) after the addition of clonidine. In infected tissue, G values increased from -12.7 * 1.7 mS . cm-* (n = 9) to -10. 3 ? 4.0 mS * cm-' (n = 4) after the addition of verapamil, and to -12.2 + 5.2 mS * cm-' (n = 5) after the addition of clonidine.

The decrease in Isc, caused by verapamil and clonidine, correlated with increased Na' and Cl absorption (Tables 1 and 2) . Verapamil caused significant increases in net Na' and Cl-absorption in control jejunum (Table 1) . This increase in absorption is caused by increases in the mucosal-to-serosal Nat and Cl-fluxes. In virus-infected jejunum, verapamil caused a significant increase in Na' absorption but only a slight, nonsignificant increase in net Cl-absorption; these changes were also caused by increases in the mucosal-to-serosal Na' and Cll fluxes. The magnitude of the increases in net Na' and Cl-fluxes and mucosal-to-serosal fluxes were similar in control and infected tissues.

Clonidine also significantly increased net Na' and Cll absorption ( Table 2 ). The increase in Na' and Clabsorption caused by clonidine was mainly because of an increase in ion movement from the mucosal-to-serosal surface with some increase in the serosal-tomucosal ion movement for Na' only. In virus-infected jejunum, the effect of clonidine was also to increase net Na+ and Cl-absorption by increasing the mucosalto-serosal fluxes. The magnitude of the clonidine induced increase in mucosal-to-serosal fluxes of Na' and Cl-was greater in the control than infected tissues but there was no significant difference in the increase in net Na+ and Cl fluxes in infected compared with control jejunum.

In separate studies, the maximum increase in Isc was determined in response to 10 mmol/L glucose added to the mucosal surface. Glucose-stimulated Isc (assumed to be Na' transport) was significantly decreased in infected tissue as compared to controls; AIsc caused by the addition of mucosal glucose in infected jejunum was 13.6 + 5.1 t.&crn' (n = 11) vs. 72.5 2 10.2 @/cm' in control tissue (n = 12; P < 0.005).

The search for antisecretory drugs for the therapeutic management of bacterial diarrheas mediated by enterotoxins, particularly cholera, has gained attention during recent years (18). This has not been the case to the same extent with viral diarrhea1 diseases in which electrolyte and water disturbances are not thought to be associated with enterotoxins, but rather with the loss of mature absorptive enterocytes and their replacement by immature cryptlike cells (2-4). Approaches for the potential treatment of neonatal intestinal viral infections have followed immunological and nonimmunological lines, the latter including the use of protease inhibitors or glycoproteins blockers (19). The TGE virus is known to infect the mature enterocytes lining the intestinal villi of jejunum and ileum of young piglets. After infected enterocytes are shed, they are replaced by cells migrating from the crypts at an accelerated rate (2, 3, 20, 21) . Davidson et al (22) have suggested that the defective epithelial function after human rotavirus infection is primarily caused by retarded differentiation of the uninfected migrating enterocytes. These enterocytes, isolated from atrophied villi during acute diarrhea, had an enzymatic profile typical of crypt cells, being rich in thymidine kinase and low in sucrase activities. The current study examines several aspects of the Na' absorptive processes in the jejunum of TGE-infected animals. Mucosal-to-serosal Na' and Cl fluxes (Tables 1 and 2), serosal-to-mucosal Na' and Cl fluxes and net Na' and Cl fluxes as well as glucosedependent Na' absorption were all decreased in jejunum from TGE-virus-infected animals. It was found previously (7-ll), that verapamil and clonidine increase Na' and Cl absorption in rabbit ileum. In the present study, verapamil and clonidine not only stimulated Na' and Cl-absorption in the jejunum of normal piglets, but also in the jejunum of TGE-virus-infected piglets. Surprisingly, the magnitudes of the verapamil-and clonidine-induced increases in Na' and Cl-absorption were similar in normal and virus-infected jejunum. These data are notable because it has been thought that the luminal membrane NaCl cotransport system may be absent in piglet jejunum after infection with TGE virus (23). The evidence for this was the lack of 5'-cyclic adenosine monophosphate (CAMP)-mediated or furosemidemediated antiabsorptive effects. One possible explanation for the discrepancy may lie in the fact that our results demonstrate stimulation of absorption, and such stimulation may be necessary before antiabsorptive effects can occur. Alternatively, the piglets used in the two studies differed considerably in age (4-7 days vs. 17 days).

Glucose-stimulated Na+ transport in the small intestine has been shown to be defective in TGE-infected animals (24) because of the absence of the highaffinity n-glucose carriers (25). This defect in Na-Dglucose transport was confirmed in the current study in which n-glucose-stimulated Na' absorption was markedly reduced in TGE-infected tissue when compared with normal tissue. Oral rehydration therapy is effectively used in the treatment of viral diarrhea1 diseases in children, further confirming that the defect in glucose-dependent Na' transport is only partial (26,27). Thus, these different Na' absorptive processes, which are present in small intestinal Na' absorbing epithelial cells, were affected differently in TGE-infected jejunum. n-glucose carriers have been shown, with antibodies raised to the purified Na-Dglucose carrier, to be present predominantly in the villus tips, whereas the location at which neutral NaCl absorption takes place is unknown. These results are consistent with neutral NaCl absorption being present throughout the villi, whereas the Na-Dglucose transporter is found predominantly in the villus tips, the site of the most severe histological damage in TGE diarrhea.

Histological observations in this study, showing severe villus atrophy and replacement of the intestinal mucosa by flat cuboidal immature intestinal cells present in the affected jejunal sections, support the conclusion that verapamil and clonidine induce Na' and Cl-absorption either in some remaining mature villus enterocytes not killed by the virus, in the immature villus cells, in crypt cells that are capable of absorbing Na' and Cl-, or in all three of these cell types. Which of these possibilities is correct awaits identification of the location of the neutral NaCl absorptive process. Earlier studies in somewhat older piglets have shown that at the height of diarrhea, the GASTROENTEROLOGY Vol. 101, No. 4 epithelium consists predominantly of immature cells that have migrated to the villi in a relatively undifferentiated state (21,23,24). As mentioned earlier, these cells were not able to generate CAMP-mediated or furosemide-mediated antiabsorptive effects, but only serve as the site for CAMP-mediated anion secretion, suggesting the absence of neutral NaCl cotransporters in these cells. The mucosa of the infected tissue was shown to be composed of cells closely resembling those occurring in normal crypts, in that they have similar enzyme activities (23). In addition to the effects on Na' absorption, another transport process was significantly affected in TGE-infected jejunum. The process is that represented by the verapamil-and clonidine-induced decrease in Isc. Initially, it was thought that this process represented inhibition of bicarbonate secretion; however, it has recently become obvious that a decrease in Isc in this setting can represent several transport processes involved in hydrogen ion transport (28). These current studies do not shed light on the nature of this transport process but do suggest that whatever that process, it is probably localized to the same cells which are responsible for D-glucose-stimulated Na' absorption and are likely to be the jejunal villus tip cells.

The fact that under the conditions of the study, verapamil and clonidine caused an increase in Na' and Cl absorption in jejunal segments of TGE-virusinfected piglets, opens the possibility of using drugs to pharmacologically stimulate NaCl absorption, especially those which lower intracellular Ca'+, for the management of diarrhea1 diseases. Clinical trials to evaluate the effectiveness of these and similar drugs in piglets might be valuable to veterinary medicine. It might be also important to test the efficacy and safety of these drugs in humans, because the piglet disease bears many similarities to human rotavirus enteritis

Diagnostic procedures for viral, rickettsial, and chlamydial infections

Jejunal epithelium in transmissible gastroenteritis of swine. An electron microscopic and histochemical study

Epithelial cell dynamics in transmissible gastroenteritis of neonatal pigs

Calcium dependence of basal electrolyte transport in rabbit ileum

Ca'+ in the control of active intestinal Na and Cl References

Dopamine stimulation of active Na and Cl absorption in rabbit ileum. Interaction with o,-adrenergic and specific dopamine receptors

Cat+ channel blockers stimulate ileal and colonic water absorption in vivo

Clonidine and lidamidine (WHR-1142) stimulate sodium and chloride absorption in the rabbit intestine

Ca'+ channel blockers interact with u,-adrenergic receptors in rabbit ileum

Enterocyte cu,-adrenergic receptors: yohimbine and p-aminoclonidine binding relative to ion transport

Transmissible gastroenteritis in piglets: a model of infantile viral diarrhea

Effects of environmental and dietary factors on human rotavirus infection in gnotobiotic piglets

Diarrhea caused in gnotobiotic piglets by the reovirus-like agent of human infantile gastroenteritis

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