J. Vet. Med. A 37, 123-129 (1990) 0 1990 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931-184X
Departamento de Biomedicina, Fisiologia, Facultad de Veterinaria, Universidad de Zaragoza, Spain
Effect of VIP on Sugar Transport in Rabbit Small Intestine in vitro M. P. ARRUEBO, V. SORRIBAS, M. J. RODRIGUEZ-YOLDI, M. D. MURILLO and ANAISABEL ALCALDE Address of authors: Prof. Dr. A. I. ALCADE,Departamento de Biomedicina, Fisiologia, Facultad de Veterinaria, Miguel Servet 177, 50013 Zaragoza, Spain
With 3 tables (Received for publication March 6, 1989)
Summary The vasoactive intestinal peptide (VIP) has shown to be widely distributed in the gastrointestinal mucosa, submucosa and nerves, and the existence of VIP receptors on the basolateral membrane of enterocytes has been recently reported for many species. The interaction of VIP with its receptors seemed to increase cyclic AMP level, and this nucleotide has been shown to be responsible for the intestinal secretion produced by VIP. The present study confirms that VIP inhibits the intestinal absorption of D-galactose. This effect seems to be due to the inhibition of the Na+-independent basolateral intestinal sugar transport system. RMI 12330A, described as adenylate cyclase inhibitors, blocked the VIP action. These findings suggest that cyclic AMP might be responsible for the inhibition of Na+-independent transport of D-galactose across the basolateral membrane. Moreover, results obtained to determine the possible role of calcium in the action of VIP suggest that Ca*+play a part, directly or indirectly, in the inhibition of the D-galactose transport across the basolateral membrane produced by VIP. Key words: VIP, D-galactose, intestinal transport, in vitro
Introduction Vasoactive intestinal peptide (VIP) is a 28-amino acid molecule identified and isolated from porcine intestine by SAID and MUTT in 1970. It was found to be located and distributed in the central and peripheral nervous systems UOHANSSON and LUNDBERG, 1981; MCDONALD et al., 1982) and in several organs and tissues (WEIHE and REINECKE, 1981; DEYet al., 1981; BLOOM and POLAK,1983). It has been shown that V I P is an important mediator or modulator of many physiological functions (ROSSELINet al., 1982; SAID,1984). VIP is widely distributed in the gastrointestinal wall, including the mucosa and submucosa (FURNESS and COSTA,1980). In the intestine VIP is present mainly in the nerve element, and it also seems to be present in the D1 cell in the crypt of Lieberkuhn (POLAKet al., 1974; SUNDLER et al., 1979). Nevertheless, specific endocrine VIP cells do not seem to be present in mammalian gastrointestinal tract.
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The existence of VIP receptors on the basolateral membrane of the small intestinal cells has been reported for the rat, rabbit and guinea pig (AMIRANOFF et al., 1980; BINDER et al., 1980; PRIETO et al., 1981; DHARMSATHAPHORN et al., 1983). The interaction of VIP with these receptors induces an increase in adenylate cyclase activity and intracellular cyclic AMP levels. VIP after binding to its receptor is rapidly internalized and most of the receptors are recycled back to the cell surface (LUISet al., 1988). It is known that cyclic AMP interacts with both, intestinal secretion of water and electrolyte, and sugar intestinal and NAFTALIN, 1975; MORETOet al., 1984). Recent studies carried absorption (HOLMAN out in our laboratory seem to indicate that VIP inhibits sugar absorption across rabbit jejunum in wiwo (RODRIGUEZ-YOLDI et a]., 1988). Not only VIP, but several intestinal hormones have been shown to interfere with the intestinal transport of nutrients (SORRIBAS et al., 1987). Furthermore, theophylline, and exogenous intestinal secretagogue which increases cyclic AMP, has been described as an inhibitor of intestinal sugar transport (RANDLESand KIMMICH, 1978; ILUNDAIN et al., 1985) and this inhibition seems to be mediated by the increase in both cyclic AMP and intracellular free calcium. Since VIP appears to interfere with different intestinal transport processes, it was considered of interest to investigate the action of VIP on intestinal D-galactose absorption.
Material and Methods Animals, incubation solutions The study was carried out o n male New Zealand rabbits weighing 2.0-2.5 kg. The animals were maintained on a standard rabbit, diet with free access to water. After killing by a blow on the head, the proximal jejunum (5cm distal to the ligament of Treitz) was removed and rinsed free of intestinal contents with ice-cold Ringer’s solution. The tissue was then striped of its serosal and external muscle layers. The Ringer’s solution contained in mmol/l: 140 NaC1, 10 KHCO,, 0.4 KHzPO+2.4 K 2 H P 0 4 , 1.2 CaClz and 1.2 MgCl2, and was continuously bubbled with 95 % O2/ 5 YO COz. When Caz+-free conditions were required, Caz+ was omitted from the bathing solution and 0.5mmol/l EGTA was added to remove the interstitial calcium. Extracellular space determinations Pieces of jejunum were incubated in Ringer’s solution at 37°C containing 0.02pCi/ml “Clabelled polyethylene glycol (mol. wt 4,000, PEG 4,000, Amersham) for 20 min. The pieces of mucosa were then gently blotted o n filter paper and weighed, then extracted in 1ml 0.1 mol/l HNO3 overnight. Aliquots of the extracts and aliquots of the bathing solutions were then counted. Following extraction the tissues were dried at 80 “C for 24 h and then reweighed. Tissue water was calculated as the difference between wet and dry weights. VIP at five concentrations ( 1 , 2 , 3 , 4 , 6 x 10-*mol/l) was present in the bathing solution from the start of the incubation, and had no significant effect either on the extracellular space (ranging from 0.098 f 0.02 t o 0.061 ? 0.002 ml/g wet weight), tissue water fraction (ranging from 0.902 k 0.002 to 0.891 +0.002ml/g wet weight) o r on cell water fraction (ranging from 0.834 LO.003 to 0.827 ? 0.004ml/g wet weight). N o t only VIP, but omitting calcium from the incubation solution in combination with EGTA, and the presence of the other effectors tested (RMI 12330A, trifluoperazine, verapamil) were assayed t o exclude the possibility that these conditions might affect the cell water ratio. The results obtained showed that none of these conditions modified cell water fraction. Sugar uptake measurements Strips of jejunum, weighing about 100mg were incubated at 37°C in lOml Ringer’s solution containing 0.01 pCi/ml “C D-galactose for 20 min. At the end of the experiment the tissues were washed with two or three gentle shaking in ice-cold Ringer’s solution and blotted carefully on both sides t o remove excess moisture. The tissue was weighed wet and extracted by shaking for 15 h in 0.5 ml 0.1 mol/l HNO,. Samples were taken from the bathing solutions and from the extracts of the tissues for radioactivity counting. All the modifiers were added to the incubation solution at the beginning of the incubation period. The results are expressed as pmol D-galactose/ml cell water after correction with the extracellular space.
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Effect of VIP on Sugar Transport
Table 1 . Effect of vasoactive intestinal peptide (VIP) on the steady-state cell water D-galactose accumulation and mucosal to serosal D-galactose fluxes am-s) in rabbit jejunum. Concentration of D-galactose 5 x 10-'moVl in sugar uptake measurements and 10-'mol/l in Jm-s determinations. The values presented are the means f S. E. The figures in brackets indicate the numbers of independent determinations
(VIP) mol/l
p o l D-galactose/ml cell water
Jm-s pmol D-gal cm-zh-1
0
1.47 f 0.005 (45) 2.76 f 0.25' (22) 3.23 i0.13" (29)
0.55 -I 0.04 (23) 0.35 f 0.03" (20) 0.30 i 0.02" (22)
2 x 10-8 4 x 10-8
* p < 0.001 compared with control value (first row). In order to prove the tissue viability in conditions in which calcium was removed and EGTA was added to the incubation medium, a histological study was carried out. After incubation at 37°C in 1Oml Ringer's solution, tissues samples from both, control and the condition in which calcium was absent and EGTA was added, were fixed in 10 YO buffered formaldehyde and embedded in paraffin. 5 pm sections were stained with haematoxylin-Eosin and PAS (Periodic Acid Schiff). Another part of the samples was embedded in methacrylate and 1 pm sections were stained by methenamine silver. Table 2 A and B. Effect of RMI 12330A and trifluoperazine on the action of VIP on D-galactose absorption. Sugar concentrations in the bathing solutions were 5 x 1O-'mol/l in accumulation experiments, and 10-2mol/l in Jm-s determinations. VIP concentration: 4 x 10-8mol/l. RMI 12330A and trifluoperazine (TFP) concentration were 10-'mol/l. Part A: Experiments with RMI 12330A. Part B: Experiments with trifluoperazine. The values presented are the means f S.E. of the number of determinations (in brackets) Table 2 A pmol D-galactose/ ml cell water
Control
VIP
Control
1.27 f 0.06 (41)
2.47 f 0.10 (41)
1.39 f 0.08"NS (17)
RMI 12330A VIP 1.32 fO.OSeNS (45)
* p < 0.001 test compared with VIP value. NS Not significantly different from control value. Table 2 B
(TFP)mol/l 0
Control (1)
VIP 10-4
Control (2)
VIP
pmol D-galactose/ ml cell water
Jm-s pmol x cm-2 x h-1
1.11 f 0.06 (22) 2.51 f 0.17 (32)
0.41 f0.03 (31) 0.22 0.01 (37)
1.44 f O.lO*NS (22) 1.34 f0.05*NS (33)
0.48 f 0.03XNS (36) 0.44 f 0.02+NS (35)
*
* p < 0.001 compared with VIP accumulation value. p < 0.001 compared with VIP Jm-s value. NS: Not significantly different compared either accumulation or Jm-s control (1) values. +
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Table 3 A and B. Effect of verapamil or the absence of calcium on VIP D-galactose transport action. mol/l. VIP concentration: 4 x lO-*mol/l. Part A: Experiments D-galactose concentration 5 x relative to calcium. Part B: Experiments with verapamil. The values are the mean k S. E. The figure in brackets is the number of independent estimates of the mean Table 3 A
(Caz+)= 1.2 mmol/l VIP Control 1.26 f 0.04 (56) ::-
2.22 (44)
p o l D-galactose / ml cell water (Ca2+)= 0 mmol/l Control VIP
* 0.12
1.07 (44)
p < 0.001 compared with VIP value.
#
* 0.05"#
0.89 f 0.03:># (46)
p < 0.001 compared with control value.
Table 3 B
p o l D-galactose / ml cell water (verapamil) 10-6 mol/l Control VIP
Control
VIP
1.27 f 0.04 (56)
2.17 k 0.03 (44)
::.
0.94 f 0.03::.# (44)
p < 0.001 compared with VIP value.
Iy
1.11 f0.05'># (46)
p < 0.001 compared with control value.
This morphological study has shown that the epithelium and the basement membrane seemed not to be modified either in control conditions or in conditions in which the calcium was omitted and the EGTA was added. Nevertheless, both classes of samples showed slight swelling of lymphatic vessels and oedema located in the lamina propria. These changes seemed not to be a sign of damage, and they appear a little more frequently in the tissues incubated without calcium plus EGTA, but neither tissue was significantly different.
Transepithelialflux measurements The stripped mucosa was mounted as a flat sheet in Ussing-type chambers. The bathing solutions on the mucosal and serosal surfaces of the tissue were maintained at 37°C using a circulating water bath. Both solutions contained D-galactose at the same concentration. Mucosal to serosal sugar fluxes were measured by adding 0.04 pCi/ml '4C-labelled D-galactose on the mucosal side. Samples were removed from serosal side at 20 min intervals for 80 min, after a 30 min preincubation period. O n e sample only was taken for counting from the radioactively labelled side. Samples of the radioactive solution were counted using a liquid scintillation counter. Materials D-galactose, VIP and DL-verapamil were obtained from SIGMA (St Louis, MO). PEG was obtained from MERCK. Trifluoperazine was a gift from Smith, Kline and French Laboratories. RMI 12330A was a gift from Merrel Dow Pharmaceuticals I N C (Cincinnati).
Statistics
Results are expressed as means Student's t-test.
* S. E. Statistical significance was evaluated by the two-tailed Results
Effect of vasoactive intestinal peptide (VIP) on steady-state cell water D-galactose accumulation and mucosal to serosal D-galactose fluxes um-s) in rabbit jejunum As the results in Table 1 show, the C/M ratio (cell water free sugar concentration/ sugar concentration in the bathing solution) was significantly (p < 0.001) increased by both
Effect of VIP on Sugar Transport
127
2 and 4 x mol/l VIP (2.76 f 0.25 and 3.23 k 0.13 respectively) compared with the control values (no addition of VIP) (1.47k 0.005). O n the other hand (see Table I), under control conditions D-galactose mucosal to serosal flux am-s) was 0.55 f 0.04, and both 2 and 4 x 10-8mol/l VIP significantly diminished D-galactose Jm-s fluxes (0.35 k 0.03 and 0.30 k 0.02 respectively). Effect of RMI 12330A and trifluoperazine on the action of VIP on D-galactose absorption RMI 12330A and trifluoperazine have been shown to prevent intestinal secretion
induced by cholera toxin. RMI 12330A inhibits adenylate cyclase activity (SIEGEL and WIECH,1976; SMITHand FIELD,1980), and both (RMI 12330A and trifluoperazine) are Ca*+-calmodulin antagonists (ILUNDAIN and NAFTALIN, 1979). RMI 12330A added to the medium did not significantly modify tissue D-galactose accumulation compared with control conditions, but when RMI 12330A and VIP were present simultaneously in the bathing solution, the increase in sugar accumulation produced by VIP disappeared (see Table 2 A). As was the case with Rh4I 12330A, trifluoperazine significantly diminished the tissue sugar accumulaton produced by VIP with no action on control conditions. Moreover, trifluoperazine blocked the effect of VIP on Jm-s D-galactose flux (see Table 2B). Role of calcium on the VIP effect on D-galactose intestinal transport The observation that both RMI 12330A and trifluoperazine decreased the effect of VIP suggests that not only CAMP, but Caz+ might have a role in the inhibition of D-galactose intestinal absorption produced by VIP. In order to study this point two experimental conditions were used. The first consisted of removing calcium from the medium, and in the second, verapamil (a calcium channel blocker) was added to the medium. The results in Table 3 A show that when calcium was absent, VIP did not increase D-galactose intestinal accumulation, but the absence of calcium slightly modified control D-galactose accumulation. Experiments with verapamil were carried out to corroborate the effect shown by calcium. When both verapamil and VIP were added simultaneously to the medium, the increase in D-galactose tissue accumulation produced by VIP disappeared (see Table 3 B). As the absence of calcium did, verapamil diminished control tissue accumulation of D-galactose.
Discussion VIP has been known to be a strong stimulant of intestinal secretion for over a decade (SCHWARTZ et al., 1974), and research into the effects of VIP on mucosal function in vivo in humans, dogs and rats have indicated that his net water and electrolyte secretion is associated with an increase of cyclic AMP (BEUBLER, 1980). The results obtained in the present study show that VIP reduced D-galactose Jm-s flux and increased sugar tissue accumulation (see Table 1). These findings suggest that VIP inhibits the I>-galactose exit across the basolateral border and they agree with results recently obtained in our laboratory about the effect of VIP on sugar absorption in vivo. All the experiments were carried out adding VIP to both the mucosal and serosal border. Nevertheless, when VIP was only present in the serosal border the results were similar (data not shown). The existence of VIP basolateral intestinal receptors has been widely reported. The VIP-receptor complex might be one step in the inhibition of D-galactose intestinal absorption. It has been postulated that the binding of VIP to its receptor stimulates adenlyate cyclase activity and increases the level of cyclic AMP (SCHWARTZ et al., 1974). Theophylline and other secretagogues have been shown to diminish D-galactose absorption by the mediation of an increase in both cyclic AMP and free Ca*+. In order to explain the role of cyclic AMP and free Caz+on VIP effect, RMI 12330A described as adenylate cyclase inhibitoris and trifluoperazine, were assayed. The results
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ARRUEBO, SORRIBAS, RODRIGUEZ-YOLDI, MURILLOand ANAISABELALCALDE
obtained show that when RMI 12330A or trifluoperazine were present in the bathing solution with VIP, the VIP effect on D-galactose absorption disappeared, but neither, RMI 12330A nor trifluoperazine significantly modified control condition (see Table 2). These results seem to corroborate the role of the increase in cyclic AMP produced by VIP, in the inhibition of basolateral D-galactose transport. Since RMI 12330A and trifluoperazine produced adenylate cyclase inhibition, they have been shown to interact with Caz+ in intestinal transport processes. An increase in cytosolic Ca2+ is reported to contribute to intestinal sugar basolateral transport inhibition (ILUNDAIN et al., 1985). In order to study the role of CaZ+on VIP intestinal transport effects, both calcium-free medium and verapamil (a clacium-channel blocker) were assayed, and both blocked the increase in D-galactose tissue accumulation produced by VIP. These findings indicate that the VIP effect might be mediated by changes in cytosolic Ca2'. Nevertheless, both calcium absence and verapamil produced a reduction of D-galactose tissue accumulation in control condition. This fact is in accordance with the role of calcium in the control of sugar efflux across the serosal border, so a decrease in intracellular calcium (by exposure to Caz+-freebathing solutions or verapamil) appears to increase serosal sugar efflux (ILUNDAIN et al., 1985). It might be that VIP has a direct effect on enterocytes, as indicated for instance, by the presence of binding sites on the basolateral membrane. A possible explanation of VIP effects on sugar intestinal transport is that VIP binds to its basolateral receptor and as a result adenylate cyclase, located in the basolateral membrane, is stimulated. The level of cyclic AMP would be increased and it may be responsible for VIP inhibition of D-galactose basolateral efflux by acting on the Na+-independent sytem of transport. Cytosolic free Ca2+might contribute to this effect, but the precise role of Caz+ in this process awaits further clarification. Acknowledgements The authors wish to thank Merrel Dow Pharmaceuticals Inc. for supplying RMI 12330A and Smith, Kline and French Laboratories for the gift of trifluoperazine. The work was supported by a grant from D G A CN-1/86. We thank Prof. MARfN and Prof. LUJANfor helping in the histological determinations.
Zusamrnenfassung In vitro-Effekt von VIP auf den Zuckertransport des Kaninchen-Dunndarms Vasoaktives Intestinales Peptid (VIP) ist in der Mucosa, Submucosa und in Nervenfasern des Gastrointestinaltrakts weit verbreitet. Ferner ist iiber das Vorhandensein von VIP-Rezeptoren in der basolateralen Membran von Enterozyten bei vielen Species berichtet worden. Die Bindung von VIP an seinen Rezeptor scheint den CAMP-Spiegel in der Zelle zu erhohen und auf dieses Nucleotid ist erwiesenermasen die durch VIP verursachte intestinale Sekretion zuriickzufiihren. Die vorkiegende Studie bestatigt den hemmenden Effekt von VIP auf die intestinale Galactoseabsorption. Der Effekt scheint auf eine Hemmung des Na+-unabhangigen basolateralen intestinalen Zuckertransportsystems zuriickzugehen. RMI 12330A und trifluoperazin, Hemmer der Adenylcyclase, blockieren den Effekt von VIP. Diese Befunde sprechen dafiir, dag die Hemmung des D-Galactose-Transports durch die basolaterale Membran durch CAMP verursacht wird. Ferner scheinr nach weiteren Untersuchungen Caz+ direkt oder indirekt an der durch VIP hervorgerufenen Hemmung des D-Galactose-Transports durch die basolaterale Membran beteiligt zu sein.
References AMIRANOFF,B., M. LABURTHE,and G. ROSSELIN,1980: Characterization of specific binding sites for vasoactive intestinal peptide in rat intestinal epithelial cell membranes. Biochim. Biophys. Acta 27, 215-224. BEUBLER,E., 1980: Influence of vasoactive intestinal polypeptide on net water flux and cyclic adenosine 3'-5' monophosphate formation in the rat jejunum. Arch. Pharmacol. 313, 243-247.
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BINDER,H. J., G. F. LEMP,and J. D. GARDNER, 1980: Receptors for vasoactive intestinal peptide and secretin in small intestinal epithelial cells. Am. J. Physiol. 238, G190-G196. BLOOM,S. R., and J. M. POLAK,1983: Regulatory peptides and the skin. Clin. Exp. Dermatol. 8, 3-18. DHARMSATHAPHORN, K., V.HARNS,D.J. YAMASHIRO, R.J. HUGHES, H.J. BINDER, and E.M. WRIGHT,1983: Preferential binding of vasoactive intestinal polypeptide to basolateral membrane of rat and rabbit enterocytes. J. Clin. Invest. 71, 21-35. DEY,R. D., W. A. SHANNON, and S. I. SAID,1981: Localization of VIP immunoreactive nerves in airways and pulmonary vessels of dogs, cats and human subjects. Cell Tissue Res. 220,231 -238. FURNESS, J. B., and M. COSTA, 1980: Types of nerves in the enteric nervous system. Neuroscience 5, 1-20. HOLMAN, G.D., and R.J. NAFTALIN,1975: Accumulation of D-galactose at the serosal border of rabbit ileum. The effects of theophylline. J. Physiol. 249, 49-51. ILUNDAIN, A., A. I. ALCALDE,Y.BARCINA, and J. LARRALDE, 1985: Calcium dependence of sugar transport in rat small intestine. Biochim. Biophys. Acta 818, 67-72. ILUNDAIN, A., and R. J. NAFTALIN,1979: Role of Ca*+-dependentregulator protein in intestinal secretion. Nature 279, 446-448. JOHANSSON, O., and J. M. LUNDBERG, 1981: Ultrastructural localization of VIP-like immunoreactivity in large dense-core vesicles of cholinergic-type nerve terminals in cat exocrine glands. Neuroscience 6, 847-862. LUIS,J., A.M. MARTIN,A. EL BAITARI, J. MARVALDI, and J. PICHON,1988: The vasoactive intestinal peptide (VIP) receptor: recent data and hypothesis. Biochimie 70, 1311-1322. MCDONALD, J. K., J. G. PARNAVELAS, A. N. KARAMANLIDIA, and N. BRECHA,1982: The morphology and distribution of peptide-containing nervous in the adult and developing visual cortex of the rat. 11. Vasoactive intestinal polypeptide. J. Neurocytol. 11, 825-837. MORETO,M., J. M. PLANAS,C. DE GABRIEL,and E. J. SANTOS,1984: Involvement of cellular cyclic AMP in theophylline-induced sugar accumulation in chicken intestinal epithelial cells. Biochim. Biophys. Acta 771, 68-73. POLAK,J. M., A. G. E. PEARSE,J. C. GARAND,and S. R. BLOOM,1974: Cellular localization of a vasoactive intestinal polypeptide in the mammalian and avian gastrointestinal tract. Gut 15, 720-724. PRIETO,J. C., M. LABURTHE, D. H. HOA,and G . ROSSELIN,1981: Quantitative studies of vasoactive intestinal peptide (VIP) binding sites and VIP-induced adenosine 3’, 5’-monophosphate production in epithelial cells from duodenum, jejunum, ileum, cecum, colon and rectum in the rat. Acta Endocrinol. 96, 100- 106. RANDLES,J., and G. A. KIMMICH,1978: Effects of phloretin and theophylline on 3-0-methylglucose transport by intestinal epithelial cells. Am. J. Physiol. 234, 64-73. RODRIGUEZ-YOLDI, M. J., M. P. ARRUEBO, A. I. ALCALDE, and M. MURILLO,1988: Influence of VIP on D-galactose transport across rabbit jejunum in wiwo. Rev. Esp. Fisiol. 44, 127-130. ROSSELIN, G., M. M A L E ~J.I ,BESSON, and W. ROSTENE,1982: A new regulator. The VIP Mol. Cell. Endocrinol. 27,243. SAID, S. I., and V. MUIT, 1970: Polypeptide with broad biological activity isolation from small intestine. Science 169, 1217-1218. SAID,S. I., 1984: Vasoactive intestinal polypeptide (VIP): Current Status. Peptides 5, 143- 150. SCHWARTZ, CH.J., D.V. KIMBERG, H. E. SHEERIN,M. FIELD, and S. I. SAID, 1974: Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J. Clin. Invest. 54, 536-544. SIEGEL,B. W., and N. L. WIECH,1976: RMI 12330A: An inhibitor of cholera toxin induced intestinal hypersecretion which also inhibits adenylate cyclase activity. Gastroenterology 70, A79, 937. SMITH,P. L., and M. FIELD,1980: In vim0 antisecretory effects of trifluoperazine and other neuroleptics in rabbit and human small intestine. Gastroenterology 78, 1545-1554. SORRIBAS, V., P. ARRUEBO, D. MURILLO,and A. I. ALCALDE, 1987: Effect of somatostatin on Dgalactose transport across the small intestine of rats. Comp. Biochem. Physiol. 86 A, 63-66. SUNDLER, F., J. ALUMETS, J. FAHRENKRUG, R. HAKANSON, and 0.B. SCHAPFALITZKY DE MUCKADELL, 1979: Cellular localization and ontogeny of immunoreactive vasoactive intestinal polypeptide (VIP) in the chicken gut. CeU Tissue Res. 196, 193-201. WEIHE,E., and M. REINECKE, 1981: Peptidergic innervation of the mammalian sinus nodes: vasoactive intestinal polypeptide (VIP)-like irnmunoreactivity in salivary glands. Life Sci. 25, 273-280.
Departamento de Biomedicina, Fisiologia, Facultad de Veterinaria, Universidad de Zaragoza, Spain
Effect of VIP on Sugar Transport in Rabbit Small Intestine in vitro M. P. ARRUEBO, V. SORRIBAS, M. J. RODRIGUEZ-YOLDI, M. D. MURILLO and ANAISABEL ALCALDE Address of authors: Prof. Dr. A. I. ALCADE,Departamento de Biomedicina, Fisiologia, Facultad de Veterinaria, Miguel Servet 177, 50013 Zaragoza, Spain
With 3 tables (Received for publication March 6, 1989)
Summary The vasoactive intestinal peptide (VIP) has shown to be widely distributed in the gastrointestinal mucosa, submucosa and nerves, and the existence of VIP receptors on the basolateral membrane of enterocytes has been recently reported for many species. The interaction of VIP with its receptors seemed to increase cyclic AMP level, and this nucleotide has been shown to be responsible for the intestinal secretion produced by VIP. The present study confirms that VIP inhibits the intestinal absorption of D-galactose. This effect seems to be due to the inhibition of the Na+-independent basolateral intestinal sugar transport system. RMI 12330A, described as adenylate cyclase inhibitors, blocked the VIP action. These findings suggest that cyclic AMP might be responsible for the inhibition of Na+-independent transport of D-galactose across the basolateral membrane. Moreover, results obtained to determine the possible role of calcium in the action of VIP suggest that Ca*+play a part, directly or indirectly, in the inhibition of the D-galactose transport across the basolateral membrane produced by VIP. Key words: VIP, D-galactose, intestinal transport, in vitro
Introduction Vasoactive intestinal peptide (VIP) is a 28-amino acid molecule identified and isolated from porcine intestine by SAID and MUTT in 1970. It was found to be located and distributed in the central and peripheral nervous systems UOHANSSON and LUNDBERG, 1981; MCDONALD et al., 1982) and in several organs and tissues (WEIHE and REINECKE, 1981; DEYet al., 1981; BLOOM and POLAK,1983). It has been shown that V I P is an important mediator or modulator of many physiological functions (ROSSELINet al., 1982; SAID,1984). VIP is widely distributed in the gastrointestinal wall, including the mucosa and submucosa (FURNESS and COSTA,1980). In the intestine VIP is present mainly in the nerve element, and it also seems to be present in the D1 cell in the crypt of Lieberkuhn (POLAKet al., 1974; SUNDLER et al., 1979). Nevertheless, specific endocrine VIP cells do not seem to be present in mammalian gastrointestinal tract.
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0931 - 184X/90/3702-0123$02.50/0
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ARRUEBO, SORRIBAS,RODRIGUEZ-YOLDI, MURILLOand ANAISABELALCALDE
The existence of VIP receptors on the basolateral membrane of the small intestinal cells has been reported for the rat, rabbit and guinea pig (AMIRANOFF et al., 1980; BINDER et al., 1980; PRIETO et al., 1981; DHARMSATHAPHORN et al., 1983). The interaction of VIP with these receptors induces an increase in adenylate cyclase activity and intracellular cyclic AMP levels. VIP after binding to its receptor is rapidly internalized and most of the receptors are recycled back to the cell surface (LUISet al., 1988). It is known that cyclic AMP interacts with both, intestinal secretion of water and electrolyte, and sugar intestinal and NAFTALIN, 1975; MORETOet al., 1984). Recent studies carried absorption (HOLMAN out in our laboratory seem to indicate that VIP inhibits sugar absorption across rabbit jejunum in wiwo (RODRIGUEZ-YOLDI et a]., 1988). Not only VIP, but several intestinal hormones have been shown to interfere with the intestinal transport of nutrients (SORRIBAS et al., 1987). Furthermore, theophylline, and exogenous intestinal secretagogue which increases cyclic AMP, has been described as an inhibitor of intestinal sugar transport (RANDLESand KIMMICH, 1978; ILUNDAIN et al., 1985) and this inhibition seems to be mediated by the increase in both cyclic AMP and intracellular free calcium. Since VIP appears to interfere with different intestinal transport processes, it was considered of interest to investigate the action of VIP on intestinal D-galactose absorption.
Material and Methods Animals, incubation solutions The study was carried out o n male New Zealand rabbits weighing 2.0-2.5 kg. The animals were maintained on a standard rabbit, diet with free access to water. After killing by a blow on the head, the proximal jejunum (5cm distal to the ligament of Treitz) was removed and rinsed free of intestinal contents with ice-cold Ringer’s solution. The tissue was then striped of its serosal and external muscle layers. The Ringer’s solution contained in mmol/l: 140 NaC1, 10 KHCO,, 0.4 KHzPO+2.4 K 2 H P 0 4 , 1.2 CaClz and 1.2 MgCl2, and was continuously bubbled with 95 % O2/ 5 YO COz. When Caz+-free conditions were required, Caz+ was omitted from the bathing solution and 0.5mmol/l EGTA was added to remove the interstitial calcium. Extracellular space determinations Pieces of jejunum were incubated in Ringer’s solution at 37°C containing 0.02pCi/ml “Clabelled polyethylene glycol (mol. wt 4,000, PEG 4,000, Amersham) for 20 min. The pieces of mucosa were then gently blotted o n filter paper and weighed, then extracted in 1ml 0.1 mol/l HNO3 overnight. Aliquots of the extracts and aliquots of the bathing solutions were then counted. Following extraction the tissues were dried at 80 “C for 24 h and then reweighed. Tissue water was calculated as the difference between wet and dry weights. VIP at five concentrations ( 1 , 2 , 3 , 4 , 6 x 10-*mol/l) was present in the bathing solution from the start of the incubation, and had no significant effect either on the extracellular space (ranging from 0.098 f 0.02 t o 0.061 ? 0.002 ml/g wet weight), tissue water fraction (ranging from 0.902 k 0.002 to 0.891 +0.002ml/g wet weight) o r on cell water fraction (ranging from 0.834 LO.003 to 0.827 ? 0.004ml/g wet weight). N o t only VIP, but omitting calcium from the incubation solution in combination with EGTA, and the presence of the other effectors tested (RMI 12330A, trifluoperazine, verapamil) were assayed t o exclude the possibility that these conditions might affect the cell water ratio. The results obtained showed that none of these conditions modified cell water fraction. Sugar uptake measurements Strips of jejunum, weighing about 100mg were incubated at 37°C in lOml Ringer’s solution containing 0.01 pCi/ml “C D-galactose for 20 min. At the end of the experiment the tissues were washed with two or three gentle shaking in ice-cold Ringer’s solution and blotted carefully on both sides t o remove excess moisture. The tissue was weighed wet and extracted by shaking for 15 h in 0.5 ml 0.1 mol/l HNO,. Samples were taken from the bathing solutions and from the extracts of the tissues for radioactivity counting. All the modifiers were added to the incubation solution at the beginning of the incubation period. The results are expressed as pmol D-galactose/ml cell water after correction with the extracellular space.
125
Effect of VIP on Sugar Transport
Table 1 . Effect of vasoactive intestinal peptide (VIP) on the steady-state cell water D-galactose accumulation and mucosal to serosal D-galactose fluxes am-s) in rabbit jejunum. Concentration of D-galactose 5 x 10-'moVl in sugar uptake measurements and 10-'mol/l in Jm-s determinations. The values presented are the means f S. E. The figures in brackets indicate the numbers of independent determinations
(VIP) mol/l
p o l D-galactose/ml cell water
Jm-s pmol D-gal cm-zh-1
0
1.47 f 0.005 (45) 2.76 f 0.25' (22) 3.23 i0.13" (29)
0.55 -I 0.04 (23) 0.35 f 0.03" (20) 0.30 i 0.02" (22)
2 x 10-8 4 x 10-8
* p < 0.001 compared with control value (first row). In order to prove the tissue viability in conditions in which calcium was removed and EGTA was added to the incubation medium, a histological study was carried out. After incubation at 37°C in 1Oml Ringer's solution, tissues samples from both, control and the condition in which calcium was absent and EGTA was added, were fixed in 10 YO buffered formaldehyde and embedded in paraffin. 5 pm sections were stained with haematoxylin-Eosin and PAS (Periodic Acid Schiff). Another part of the samples was embedded in methacrylate and 1 pm sections were stained by methenamine silver. Table 2 A and B. Effect of RMI 12330A and trifluoperazine on the action of VIP on D-galactose absorption. Sugar concentrations in the bathing solutions were 5 x 1O-'mol/l in accumulation experiments, and 10-2mol/l in Jm-s determinations. VIP concentration: 4 x 10-8mol/l. RMI 12330A and trifluoperazine (TFP) concentration were 10-'mol/l. Part A: Experiments with RMI 12330A. Part B: Experiments with trifluoperazine. The values presented are the means f S.E. of the number of determinations (in brackets) Table 2 A pmol D-galactose/ ml cell water
Control
VIP
Control
1.27 f 0.06 (41)
2.47 f 0.10 (41)
1.39 f 0.08"NS (17)
RMI 12330A VIP 1.32 fO.OSeNS (45)
* p < 0.001 test compared with VIP value. NS Not significantly different from control value. Table 2 B
(TFP)mol/l 0
Control (1)
VIP 10-4
Control (2)
VIP
pmol D-galactose/ ml cell water
Jm-s pmol x cm-2 x h-1
1.11 f 0.06 (22) 2.51 f 0.17 (32)
0.41 f0.03 (31) 0.22 0.01 (37)
1.44 f O.lO*NS (22) 1.34 f0.05*NS (33)
0.48 f 0.03XNS (36) 0.44 f 0.02+NS (35)
*
* p < 0.001 compared with VIP accumulation value. p < 0.001 compared with VIP Jm-s value. NS: Not significantly different compared either accumulation or Jm-s control (1) values. +
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ARRUEBO, SORRIBAS, RODRIGUEZ-YOLDI, MURILLOand ANA ISABEL ALCALDE
Table 3 A and B. Effect of verapamil or the absence of calcium on VIP D-galactose transport action. mol/l. VIP concentration: 4 x lO-*mol/l. Part A: Experiments D-galactose concentration 5 x relative to calcium. Part B: Experiments with verapamil. The values are the mean k S. E. The figure in brackets is the number of independent estimates of the mean Table 3 A
(Caz+)= 1.2 mmol/l VIP Control 1.26 f 0.04 (56) ::-
2.22 (44)
p o l D-galactose / ml cell water (Ca2+)= 0 mmol/l Control VIP
* 0.12
1.07 (44)
p < 0.001 compared with VIP value.
#
* 0.05"#
0.89 f 0.03:># (46)
p < 0.001 compared with control value.
Table 3 B
p o l D-galactose / ml cell water (verapamil) 10-6 mol/l Control VIP
Control
VIP
1.27 f 0.04 (56)
2.17 k 0.03 (44)
::.
0.94 f 0.03::.# (44)
p < 0.001 compared with VIP value.
Iy
1.11 f0.05'># (46)
p < 0.001 compared with control value.
This morphological study has shown that the epithelium and the basement membrane seemed not to be modified either in control conditions or in conditions in which the calcium was omitted and the EGTA was added. Nevertheless, both classes of samples showed slight swelling of lymphatic vessels and oedema located in the lamina propria. These changes seemed not to be a sign of damage, and they appear a little more frequently in the tissues incubated without calcium plus EGTA, but neither tissue was significantly different.
Transepithelialflux measurements The stripped mucosa was mounted as a flat sheet in Ussing-type chambers. The bathing solutions on the mucosal and serosal surfaces of the tissue were maintained at 37°C using a circulating water bath. Both solutions contained D-galactose at the same concentration. Mucosal to serosal sugar fluxes were measured by adding 0.04 pCi/ml '4C-labelled D-galactose on the mucosal side. Samples were removed from serosal side at 20 min intervals for 80 min, after a 30 min preincubation period. O n e sample only was taken for counting from the radioactively labelled side. Samples of the radioactive solution were counted using a liquid scintillation counter. Materials D-galactose, VIP and DL-verapamil were obtained from SIGMA (St Louis, MO). PEG was obtained from MERCK. Trifluoperazine was a gift from Smith, Kline and French Laboratories. RMI 12330A was a gift from Merrel Dow Pharmaceuticals I N C (Cincinnati).
Statistics
Results are expressed as means Student's t-test.
* S. E. Statistical significance was evaluated by the two-tailed Results
Effect of vasoactive intestinal peptide (VIP) on steady-state cell water D-galactose accumulation and mucosal to serosal D-galactose fluxes um-s) in rabbit jejunum As the results in Table 1 show, the C/M ratio (cell water free sugar concentration/ sugar concentration in the bathing solution) was significantly (p < 0.001) increased by both
Effect of VIP on Sugar Transport
127
2 and 4 x mol/l VIP (2.76 f 0.25 and 3.23 k 0.13 respectively) compared with the control values (no addition of VIP) (1.47k 0.005). O n the other hand (see Table I), under control conditions D-galactose mucosal to serosal flux am-s) was 0.55 f 0.04, and both 2 and 4 x 10-8mol/l VIP significantly diminished D-galactose Jm-s fluxes (0.35 k 0.03 and 0.30 k 0.02 respectively). Effect of RMI 12330A and trifluoperazine on the action of VIP on D-galactose absorption RMI 12330A and trifluoperazine have been shown to prevent intestinal secretion
induced by cholera toxin. RMI 12330A inhibits adenylate cyclase activity (SIEGEL and WIECH,1976; SMITHand FIELD,1980), and both (RMI 12330A and trifluoperazine) are Ca*+-calmodulin antagonists (ILUNDAIN and NAFTALIN, 1979). RMI 12330A added to the medium did not significantly modify tissue D-galactose accumulation compared with control conditions, but when RMI 12330A and VIP were present simultaneously in the bathing solution, the increase in sugar accumulation produced by VIP disappeared (see Table 2 A). As was the case with Rh4I 12330A, trifluoperazine significantly diminished the tissue sugar accumulaton produced by VIP with no action on control conditions. Moreover, trifluoperazine blocked the effect of VIP on Jm-s D-galactose flux (see Table 2B). Role of calcium on the VIP effect on D-galactose intestinal transport The observation that both RMI 12330A and trifluoperazine decreased the effect of VIP suggests that not only CAMP, but Caz+ might have a role in the inhibition of D-galactose intestinal absorption produced by VIP. In order to study this point two experimental conditions were used. The first consisted of removing calcium from the medium, and in the second, verapamil (a calcium channel blocker) was added to the medium. The results in Table 3 A show that when calcium was absent, VIP did not increase D-galactose intestinal accumulation, but the absence of calcium slightly modified control D-galactose accumulation. Experiments with verapamil were carried out to corroborate the effect shown by calcium. When both verapamil and VIP were added simultaneously to the medium, the increase in D-galactose tissue accumulation produced by VIP disappeared (see Table 3 B). As the absence of calcium did, verapamil diminished control tissue accumulation of D-galactose.
Discussion VIP has been known to be a strong stimulant of intestinal secretion for over a decade (SCHWARTZ et al., 1974), and research into the effects of VIP on mucosal function in vivo in humans, dogs and rats have indicated that his net water and electrolyte secretion is associated with an increase of cyclic AMP (BEUBLER, 1980). The results obtained in the present study show that VIP reduced D-galactose Jm-s flux and increased sugar tissue accumulation (see Table 1). These findings suggest that VIP inhibits the I>-galactose exit across the basolateral border and they agree with results recently obtained in our laboratory about the effect of VIP on sugar absorption in vivo. All the experiments were carried out adding VIP to both the mucosal and serosal border. Nevertheless, when VIP was only present in the serosal border the results were similar (data not shown). The existence of VIP basolateral intestinal receptors has been widely reported. The VIP-receptor complex might be one step in the inhibition of D-galactose intestinal absorption. It has been postulated that the binding of VIP to its receptor stimulates adenlyate cyclase activity and increases the level of cyclic AMP (SCHWARTZ et al., 1974). Theophylline and other secretagogues have been shown to diminish D-galactose absorption by the mediation of an increase in both cyclic AMP and free Ca*+. In order to explain the role of cyclic AMP and free Caz+on VIP effect, RMI 12330A described as adenylate cyclase inhibitoris and trifluoperazine, were assayed. The results
128
ARRUEBO, SORRIBAS, RODRIGUEZ-YOLDI, MURILLOand ANAISABELALCALDE
obtained show that when RMI 12330A or trifluoperazine were present in the bathing solution with VIP, the VIP effect on D-galactose absorption disappeared, but neither, RMI 12330A nor trifluoperazine significantly modified control condition (see Table 2). These results seem to corroborate the role of the increase in cyclic AMP produced by VIP, in the inhibition of basolateral D-galactose transport. Since RMI 12330A and trifluoperazine produced adenylate cyclase inhibition, they have been shown to interact with Caz+ in intestinal transport processes. An increase in cytosolic Ca2+ is reported to contribute to intestinal sugar basolateral transport inhibition (ILUNDAIN et al., 1985). In order to study the role of CaZ+on VIP intestinal transport effects, both calcium-free medium and verapamil (a clacium-channel blocker) were assayed, and both blocked the increase in D-galactose tissue accumulation produced by VIP. These findings indicate that the VIP effect might be mediated by changes in cytosolic Ca2'. Nevertheless, both calcium absence and verapamil produced a reduction of D-galactose tissue accumulation in control condition. This fact is in accordance with the role of calcium in the control of sugar efflux across the serosal border, so a decrease in intracellular calcium (by exposure to Caz+-freebathing solutions or verapamil) appears to increase serosal sugar efflux (ILUNDAIN et al., 1985). It might be that VIP has a direct effect on enterocytes, as indicated for instance, by the presence of binding sites on the basolateral membrane. A possible explanation of VIP effects on sugar intestinal transport is that VIP binds to its basolateral receptor and as a result adenylate cyclase, located in the basolateral membrane, is stimulated. The level of cyclic AMP would be increased and it may be responsible for VIP inhibition of D-galactose basolateral efflux by acting on the Na+-independent sytem of transport. Cytosolic free Ca2+might contribute to this effect, but the precise role of Caz+ in this process awaits further clarification. Acknowledgements The authors wish to thank Merrel Dow Pharmaceuticals Inc. for supplying RMI 12330A and Smith, Kline and French Laboratories for the gift of trifluoperazine. The work was supported by a grant from D G A CN-1/86. We thank Prof. MARfN and Prof. LUJANfor helping in the histological determinations.
Zusamrnenfassung In vitro-Effekt von VIP auf den Zuckertransport des Kaninchen-Dunndarms Vasoaktives Intestinales Peptid (VIP) ist in der Mucosa, Submucosa und in Nervenfasern des Gastrointestinaltrakts weit verbreitet. Ferner ist iiber das Vorhandensein von VIP-Rezeptoren in der basolateralen Membran von Enterozyten bei vielen Species berichtet worden. Die Bindung von VIP an seinen Rezeptor scheint den CAMP-Spiegel in der Zelle zu erhohen und auf dieses Nucleotid ist erwiesenermasen die durch VIP verursachte intestinale Sekretion zuriickzufiihren. Die vorkiegende Studie bestatigt den hemmenden Effekt von VIP auf die intestinale Galactoseabsorption. Der Effekt scheint auf eine Hemmung des Na+-unabhangigen basolateralen intestinalen Zuckertransportsystems zuriickzugehen. RMI 12330A und trifluoperazin, Hemmer der Adenylcyclase, blockieren den Effekt von VIP. Diese Befunde sprechen dafiir, dag die Hemmung des D-Galactose-Transports durch die basolaterale Membran durch CAMP verursacht wird. Ferner scheinr nach weiteren Untersuchungen Caz+ direkt oder indirekt an der durch VIP hervorgerufenen Hemmung des D-Galactose-Transports durch die basolaterale Membran beteiligt zu sein.
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