Regulation of Ion Transport in the Porcine Intestinal Tract by Enteric Neurotransmitters and Hormones

Comp. Biochem. Physiol. Vol. 118A, No. 2, 309–317, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00311-8

SECTION E: REGULATION OF INTESTINAL ION TRANSPORT

Regulation of Ion Transport in the Porcine Intestinal Tract by Enteric Neurotransmitters and Hormones David R. Brown and Scott M. O’Grady University of Minnesota, Departments of Veterinary PathoBiology and Animal Science, 1988 Fitch Avenue, St. Paul, MN 55108-6010, U.S.A.

ABSTRACT. In the present paper, the mechanisms underlying the neural and hormonal regulation of mucosal ion transport in the pig intestinal tract are reviewed. The active transport of NaCl by isolated sheets of porcine intestinal mucosa is modulated by cholinergic and non-cholinergic neurons of undetermined neurochemical identity that lie in the submucosa. The application of electrical field stimulation to mucosa-submucosa preparations from porcine jejunum, ileum, or colon produces rapid elevations in short-circuit current which are inhibited by tetrodotoxin or omega-conotoxin GVIA, blockers of neuronal Na1 and Ca21 channels, respectively. In porcine ileum, these elevations in current are mimicked in large part by cholinergic agonists and have been attributed to anion secretion. The majority of classical neurotransmitters and gut peptides that have been examined to date increase active transepithelial anion secretion through interactions with G protein-coupled receptors associated with submucosal neurons or situated on the basolateral membranes of epithelial cells. A small number of neuropeptides interact with neuronal receptors to augment NaCl absorption or decrease anion secretion. Noradrenergic control of intestinal transport differs in the porcine small and large intestines, and displays considerable inter-species variability in its cellular underpinnings. Transport regulation by bombesin-like peptides may be mediated by receptors distributed in both the apical and basolateral membrane domains of epithelial cells in porcine colon. The transport process affected by these peptides may be linked to epithelial growth and differentiation. The pig intestinal tract appears to be a useful biological model for resolving the cellular mechanisms by which gut neurotransmitters and hormones act in regulating transepithelial ion fluxes. Its general relevance to human intestinal function is discussed. comp biochem physiol 118A;2:309–317, 1997.  1997 Elsevier Science Inc. KEY WORDS. Pig, secretion, absorption, norepinephrine, bombesin

INTRODUCTION The intestinal mucosa encompasses a vast surface area that participates in the metabolism and absorption of nutrients, the maintenance of water and electrolyte homeostasis, and the continuous defense of the host organism from infection by a variety of potentially life-threatening microorganisms entering or residing in the intestinal lumen. The complex nervous system enclosed within the walls of the intestines modulates active and passive ion fluxes across the mucosal epithelium in response to these demanding conditions. Although most investigations of intestinal function, including those concerned with the regulation of epithelial ion transport, have utilized rodents as experimental models, the use Address reprint requests to: David R. Brown, University of Minnesota, Department of Veterinary PathoBiology, 1988 Fitch Avenue, St. Paul, MN 55108-6010. Tel. (612) 624-0713; Fax (612) 625-0204; E-mail: [email protected]. Received 30 May 1996; accepted 31 May 1996.

of pigs in gastrointestinal research has greatly increased during the past decade. A review of the scientific literature reveals that the pig has become a valuable model for studies of nutrition; intestinal drug delivery; intestinal development; gut ischemia-reperfusion injury; bowel allotransplantation; intestinal infections; food hypersensitivity; and mucosal vaccination. The porcine gastrointestinal tract provides large amounts of tissue for the successful extraction of proteins, peptides and nucleic acids, and for the application of biochemical and molecular techniques in studying cell-to-cell signalling processes. It has traditionally been employed as a rich source of many regulatory peptides, including secretin, vasoactive intestinal peptide, gastrin-releasing peptide (GRP), galanin, cholecystokinin and peptide YY (41,42,66). In the context of this review, it has become especially useful in resolving the cellular mechanisms by which gut neurotransmitters and hormones act in regulating transepithelial ion fluxes across functionally-distinct intestinal segments.

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NEUROHUMORAL REGULATION OF EPITHELIAL ION TRANSPORT: AN OVERVIEW Control of Ion Transport by Cholinergic and Non-Cholinergic Submucosal Neurons The enteric nervous system of the pig is similar to its human counterpart in mean density of neurons, neurochemical characteristics, and presence of two distinctive submucosal plexuses: an outer (Schabadasch) plexus and an inner (Meissner) plexus (68,69,70). The first definitive investigation on the control of mucosal transport function by enteric neurons was performed by Hubel, who applied electrical field stimulation (EFS) techniques to preparations of the rabbit ileal mucosa mounted in Ussing flux chambers (35). In these initial studies of rabbit, and later human, ileal preparations that normally absorbed Na1 and Cl2, brief episodes of EFS produced increases in short-circuit current (Isc). These effects were attributable to net Cl2 secretion and were directly proportional to stimulus frequency. Tetrodotoxin (TTX), a blocker of neuronal Na1 channels and therefore of axonal conduction in enteric nerves, decreased spontaneous Isc and abolished the actions of EFS in both preparations. Mucosal responses to EFS were also reduced, but not eliminated by atropine, implying an involvement of endogenous acetylcholine and its interactions with muscarinic cholinergic receptors in neurally-mediated Cl2 secretion (35,36). Since these studies were conducted, there have been many similar reports of EFS action in other mucosal preparations from small and large intestine, obtained mostly from small mammals (11,22). In preparations of small intestinal or colonic mucosa from pigs that include the inner submucosal plexus, the application of EFS produces frequency-dependent increases in Isc, which are abolished by contraluminal application of TTX or the neuronal Ca21 channel blocker omega-conotoxin GVIA (18,32,76). In the distal colon, mucosal responses to EFS are manifested early in development (3). The actions of EFS are mimicked by Veratrum alkaloids or scorpion venom, substances that activate neuronal Na1 channels and depolarize submucosal neurons (7,72). Preparations of porcine ileum respond to EFS with increases in net anion secretion. TTX rapidly decreases Isc in these preparations as it does in ilea from other species. The toxin effect is due to increased Cl2 absorption (31). Based on these findings, it seems that ongoing activity in submucosal neurons acts to limit Cl2 absorption in the ileum. In about one-third of ileal sheets examined, EFS produces large increases in net residual flux accompanied by enhanced net Cl2 absorption. In this subset of tissues, EFS may increase electrogenic Na 1 /HCO 32 secretion (31). The effects of EFS in porcine small intestine and colon are reduced by atropine and hexamethonium, blockers of muscarinic and nicotinic cholinergic receptors respectively, indicating that they are partially mediated by cholinergic neurons as they are in intestinal epithelia from other spe-

D. R. Brown and S. M. O’Grady

cies. Moreover, the contraluminal application of acetylcholine or other choline esters (e.g., bethanechol and carbachol) produces transient increases in Isc in a manner similar to that of EFS in sheets of porcine jejunal, ileal, and distal colonic mucosa (3,17,18,19,72). Low concentrations (,1 µM) of these agonists preferentially activate muscarinic cholinergic receptors; higher agonist concentrations (.10 µM) may additionally activate nicotinic cholinergic receptors in these preparations. There appear to be segmental differences along the porcine intestinal tract in the mechanisms by which cholinergic agonists modify active ion transport. For example, blockade of neurotransmission by TTX dramatically increases the Isc-elevating potency of carbachol (CCh) in porcine jejunum, but not in ileum (17,18). This finding suggests that cholinomimetic drugs may interact directly with muscarinic cholinergic receptors on enterocytes in both small intestinal segments, but their effects in the jejunum may be dampened by a secondary activation of inhibitory enteric neurons. The ionic bases of agonistinduced Isc elevations also appear to vary with the intestinal segment under examination. In mucosal sheets from pig jejunum, ileum, and distal colon, the Isc response to CCh is dependent upon extracellular Cl2 concentration and CCh produces net Cl2 secretion in all three intestinal segments. Studies of small intestinal mucosae indicate that the initial mucosal response to CCh in ileal mucosa is significantly reduced by the removal of extracellular HCO32 ions, but the response remains unchanged in jejunal mucosa (18,19). Thus, unlike the jejunum, the ileum may secrete HCO32 as well as Cl2 in response to CCh. This hypothesis is supported by apparent differences in CCh effects on net luminal alkalinization in jejunal and ileal mucosa (12,19). Enteric cholinergic neurons may, therefore, differentially regulate the magnitude and characteristics of anion secretion along the length of the porcine small intestine. Non-cholinergic transmitter systems that contribute the remainder of EFS action have not been clearly identified. EFS effects are apparently not mediated by endogenous substance P or VIP in ileum, or by bombesin-like peptides in distal colon (31,76). On the other hand, several different neurotransmitter systems may act to modulate activity in submucosal neurons that control electrolyte transport. In porcine ileum, opioid peptides, norepinephrine (NE) and galanin reduce EFS-induced Isc changes and neuropeptide Y (NPY) inhibits mucosal Isc responses to EFS in both ileum and distal colon (Table 1). The luminal application of l-glutamate or l-asparagine increases Isc and produces net Cl2 secretion in sheets of porcine small intestinal mucosa. The effects of these amino acids, which may be mediated through an excitatory amino acid receptor, are inhibited by serosal TTX or pretreatment with the sensory neurotoxin capsaicin; in addition, they may involve neurokinin and nitric oxide signalling circuits (57). This study and others conducted in non-porcine intes-

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TABLE 1. Enteric neurotransmitter and hormones found to alter ion transport in porcine intestinal epithelia

Substance

Target cell(s)

Receptor type

N, E E E N E E N, E N (phase I) E (phase II) N

Muscarinic (M 3?) Muscarinic (M 3?) Muscarinic N.D. N.D. GC-A BB2 N.D. BB2 δ/ µ-OR

↑ ↑ ↑ ↑ ↓ ↑ ↑ ↑ ↑ ↑

N E N E N N N N N

N.D. H1..H 2 5-HT3 /5-HT4 (?) 5-HT2 /5-HT2A (?) N.D. N.D. N.D. N.D. α 2-AR

↓ ↑ ↑ ↑ ↑ ↑ ↓ ↑ ↑

E N E N E

α 1-AR NK1 NK1 SSTR(-2 or -5) N.D.

↑ ↑ ↑ ↑ ↑

Region

Acetylcholine

Jejunum Ileum* D. colon Atrial natriuretic peptide/ P. colon brain natriuretic peptide P. colon D. colon Bombesin† Jejunum D. colon Enkephalins

Ileum

Galanin Histamine 5-Hydroxytryptamine

Ileum D. colon Jejunum

Neuromedin U Neuropeptide Y Neurotensin Norepinephrine

Ileum Ileum D. colon Ileum Ileum

Substance P

D. colon Jejunum

Somatostatin VIP

Ileum D. colon

Action Cl secretion Cl and HCO3 secretion Cl secretion Cl secretion NaCl absorption Cl, K secretion Na, HCO 3 (?) and Cl secretion anion secretion (?) Na, K secretion NaCl absorption; ↓ NaHCO 3 secretion (?); ↓ EFSI secretion EFSI Cl secretion Cl secretion; ↓ NaCl absorption anion secretion (?) Na (HCO 3?) secretion; ↑ Cl secretion Cl secretion (?) Cl absorption; ↓ EFSI Cl secretion EFSI Cl secretion Cl secretion Na (HCO 3?) secretion‡; ↑ Cl absorption§; ↓ EFSI secretion Cl and Na secretion; ↓ Na absorption NaHCO 3 secretion K secretion (?) Cl absorption, ↓ HCO3 secretion (?) Cl secretion

References (18) (17,19) (72) (2) (2) (71) (16) (74,75) (52) (9) (73) (28) (29,30) (6) (8) (76) (7) (32) (72) (12,51) (12,51) (10) (72)

*This segment is anatomically referred to as distal jejunum in the pig (59). †See text for explanation of bombesin action. ‡Cl-absorbing tissues. §Cl-secreting tissues. Abbreviations: AR, adrenergic receptor; BB, bombesin receptor; E, epithelial cell; EFSI, electrical field stimulation-induced; GC-A, type A guanylate cyclase receptor; N, enteric neurons; N.D., not determined; OR, opioid receptor; SSTR, somatostatin receptor; CGRP: calcitonin gene-related peptide; GRP, gastrin-releasing peptide; VIP, vasoactive intestinal peptide.

tinal models (22) provide evidence suggesting that sensory neurons that reside in the porcine intestinal mucosa might regulate aspects of transepithelial ion transport occurring in response to amino acids and possibly other luminal stimuli. In a related vein, sheets of jejunal mucosa-submucosa from food-deprived pigs manifest exaggerated Isc elevations in response to the contraluminal application of prosecretory transmitter substances, such as serotonin and histamine (14). Location and Characteristics of Receptors for Regulatory Molecules Many types of regulatory substances affect electrolyte transport function in the small intestine and colon; this work is summarized in recent reviews (11,46). Acetylcholine (ACh), several biogenic amine neurotransmitters, and a variety of brain-gut peptides have been shown to alter active ion transport in mucosa-submucosa preparations from the

porcine intestinal tract. Most of these substances stimulate net Na 1 or Cl2 secretion through direct interactions with enterocyte receptors or indirect actions on enteric neurons. By inhibiting neuronal activity, a smaller number of putative transmitters (such as enkephalins, somatostatin, NPY, and NE) possess antisecretory or proabsorptive activities (Table 1). With few exceptions (see below), receptors for regulatory neurotransmitters and hormones are located in the basolateral membrane of epithelial cells or are associated with non-epithelial cells present in the underlying submucosa. They have been characterized and localized mainly in preparations of porcine small intestine by radioligand binding techniques and quantitative receptor autoradiography. Newer methods used to characterize receptors in the porcine digestive tract include mRNA hybridization analysis using specific cDNA probes, which has been applied to α2adrenergic receptors isolated from ileal submucosa (33). Immunohistochemical techniques in intestinal tissue using antisera directed towards unique peptide sequences in opioid

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TABLE 2. Characteristics of transmitter binding sites relevant to ion transport in porcine intestinal mucosa

KD (pM)†

Ligand

Location

α 2A-Adrenergic receptor (ileal submucosa) Meissner plexus Bombesin receptor (jejunal mucosa-submucosa) 160 Mucosal villi and crypts 3/890 6 120*,‡ Mucosal villi and crypts; submucosal ganglia 15** Villi . crypts 9*** Ileal mucosa Neurokinin-1 receptor (jejunal mucosa-submucosa) 120 Crypts . villi; Schabadasch plexus . Meissner plexus Neuropeptide Y receptor (colon) Submucosal ganglia Neurotensin receptor (ileal mucosa-submucosa) 17/370 6 34‡ Meissner and Schabadasch plexus Opioid receptor (ileal submucosa) 0.4 µM Villi, crypts 24 Submucosa

References

[3 H]Yohimbine

390 6 30

(33)

[125 I]GRP [3 H]QNB

393 6 10 6

(60) (17)

38 6 77 6 [125 I]BH-SP

320 6

[125 I]BH-NPY

409§

[125 I]Neurotensin [3 H]DAMGO [3 H]Diprenorphine

47 6 4.3 6 144 6

(18) (33) (51) (78) (61) (53) Brown, unpublished data

†Mean equilibrium constants obtained from saturation analyses of binding sites. Values are expressed as mean KD 6 S.E. Note that all values are in pM, except for [ 3 H]DAMGO affinity which is expressed in µM. ‡Two populations of binding sites. *Full thickness proximal jejunum. **Full thickness ileum. ***Ileal mucosa. §Determined in myenteric ganglia. Abbreviations: BH, Bolton-Hunter; DAMGO, [d-Ala 2, N-Me-Phe 4, Gly 5-ol]enkephalin; GRP, gastrin-releasing peptide; NPY, neuropeptide Y; QNB, quinuclidinyl benzilate; SP, substance P.

receptors have also been employed in an effort to localize these receptors within the porcine enteric nervous system (47). The receptors characterized so far are members of the G protein-coupled receptor superfamily and have relatively high affinities (picomolar range) for their cognate ligands (Table 2). Although their association with specific GTPbinding proteins has not been conclusively determined in intestinal epithelial cells, these receptors may be coupled to either Gi/o (α2A-adrenergic, NPY, and opioid receptors) or Gq/11 proteins (M3-muscarinic cholinergic, bombesin, neurokinin-1, and neurotensin receptors) based on studies in other systems. It is quite possible that some receptors may ‘‘promiscuously’’ couple to multiple G proteins and therefore may be capable of influencing several second messenger pathways or ion channel types within intestinal target cells simultaneously. In addition to their localization in the mucosa and submucosa, many of these same receptors are associated with the underlying myenteric plexus or smooth muscle layers where they affect intestinal motor function. In the following two subsections, we will turn our attention to some major themes that have emerged in the course of our studies on ion transport regulation in the pig intestinal tract. These will be discussed in terms of (a) segmental differences in the cellular mechanisms by which NE regulates transport in the intestinal tract of pigs and other mammalian species and (b) the modulation of secretagogue action by bombesin-like peptides that interact with receptors

associated with both basolateral and apical membranes of ion-transporting epithelial cells. SPECIES- AND SEGMENT-RELATED DIFFERENCES IN THE ADRENERGIC REGULATION OF INTESTINAL ION TRANSPORT Studies of adrenergic regulation of the mammalian distal small intestine and colon have revealed distinct mechanisms of action for epinephrine and NE in these two segments of the digestive tract (Table 3). Early in vitro studies with rabbit ileum demonstrated that catecholamines increase net Na1, Cl2 and fluid absorption, and inhibit anion secretion after their contraluminal application to mucosal sheets (20,25,34,37). Tyramine, which stimulates the release of endogenous NE from adrenergic nerve terminals in the intestine, mimicked the effects of NE (67). The actions of NE were found to be mediated by α2-adrenoceptors, which are localized in epithelial cells and submucosal neurons (20,37). Subsequent studies with rat and guinea pig small intestine also showed that NE produced changes in Isc consistent with inhibition of anion secretion. These effects were partially inhibited by TTX (13,39). Thus, a portion of the transport-related actions of NE were mediated by submucosal neurons, a finding that is consistent with the hyperpolarizing effects of NE on neurons located in the

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TABLE 3. Comparison of adrenergic regulation of electrolyte transport between species and intestinal segments*

Species rat rabbit

guinea pig human

Intestinal segment

Action

Adrenoceptor

Location

References (13,23) (54) (25) Hyun et al., 1985; (63,79,80) (27); (62) (39) (56) (64) (49)

Jejunum D. colon Ileum P. colon

↓ ↑ ↑ ↑

anion secretion NaCl absorption NaCl absorption, ↓ anion secretion NaCl and Ca 21 absorption

α2 α and β α2 α2

E, N ? E, N ?

D. colon Jejunum D. colon D. colon HT-29 cells

↑ ↓ ↑ ? ↓

K 1 secretion anion secretion K 1 secretion

β1 α2 ? α2 α2

E N ? E? E

VIP-induced [cAMP] i accumulation

*See Table 1 for comparative information on adrenergic influences in porcine intestinal tract. ? —indicates unknown or not determined. Abbreviations: [cAMP] i intracellular cyclic AMP concentration; D, distal; E, epithelial cells; N, enteric neurons; P, proximal.

guinea pig ileal submucosa (44). Binding studies using the selective α2-adrenergic ligand [3H]RX-821002 revealed that α2-adrenoceptors are preferentially expressed in rat jejunal crypt cells and that a gradient of receptor expression exists along the villus-crypt axis (50). This localization is consistent with direct inhibitory modulation of secretagogue action on anion secretion in crypt cells. Binding studies using epithelial cell membranes from human small intestine indicate that human intestinal epithelial cells express α2A-adrenergic binding sites which are preferentially localized to the basolateral membrane of crypt cells (77). As in other species, contraluminal administration of NE decreases Isc in porcine ileum through interactions with α2adrenoceptors. The ionic fluxes underlying this effect in adult animals have been attributed to either stimulation of net Na1 secretion in tissues exhibiting basal Na1 and Cl2 absorption or net Cl2 absorption in tissues exhibiting basal Cl2 secretion (31). Mucosal responses to NE are abolished by TTX as they are in rabbit ileum (37). Moreover, NE inhibits elevations in Isc produced by EFS or the ganglionic stimulant dimethylphenylpiperazinium. These results suggest that NE actions are mediated by submucosal neurons. The tyramine-stimulated release of endogenous NE from submucosal nerve terminals mimics the inhibitory actions of exogenous NE on EFS-induced Isc elevations in porcine ileum. Radioligand binding analyses and molecular hybridization studies confirm that specific α2A-adrenergic binding sites are associated with a neuronally-enriched fraction from porcine ileal submucosa and probably represent the population of α2-adrenoceptors regulating electrolyte transport in this gut segment (33). In this respect, the porcine ileum differs from the human ileum in that α2A-adrenoceptors are expressed only on neurons in the former tissue. Studies in colonic epithelia indicate that adrenergic influences on electrolyte transport exhibit considerable interspecies variability. For example, epinephrine increased net Na 1 and Cl21 absorption and decreased Isc in rat distal co-

lon in a manner similar to rabbit ileum, except that its action were mediated by both α- and β-adrenoceptors (54). Epinephrine or NE promoted K1 secretion in distal colon from guinea pigs (56) or rabbits (27,62), an effect that was mediated exclusively by β-adrenoceptors and was resistant to TTX. In these species, catecholamines seem to modulate colonic K1 transport through interactions with epithelial βadrenoceptors. There is conflicting evidence regarding the role of αadrenoceptors on ion transport in the rabbit distal colon. Alpha2-adrenergic binding sites have been identified in rabbit colonocytes where they are expressed at highest density in crypt epithelial cells (58). However, in a study by Albin and Gutman (1), the α1-selective adrenergic agonist phenylephrine stimulated Na 1 absorption by a phentolaminesensitive mechanism. Smith and McCabe (62) reported that epinephrine and NE acted primarily through β1-adrenoceptors to stimulate active K 1 secretion and did not alter NaCl transport in rabbit colon. Epinephrine interacts with α2-adrenoceptors to decrease Isc in the human distal colon (63,64). The ionic bases of this effect have not been determined, although they do not include changes in net NaCl transport. Epinephrine treatment was associated with a significant residual flux that is consistent with an inhibition of HCO 3 secretion or stimulation of cation secretion in this preparation. Binding studies with selective α2-adrenergic radioligands indicate that the α2-adrenergic binding sites are present in HT-29 cells, a human colon adenocarcinoma cell line (49). Native human colonocytes, particularly those in proximal colon, express α2A-adrenergic binding sites at high densities (77). NE produces a large and transient increases in Isc in distal colon from both neonatal and adult pigs (3,72). This mucosal response to NE differs from those observed in colonic mucosae from other species. Analyses of transepithelial Na and Cl fluxes indicate that NE inhibits net Na1 absorption and stimulates net Cl2 secretion. Most of the decrease in

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net Na1 absorption is accounted for by an increase in the serosa-to-mucosa unidirectional Na 1 flux indicating that a significant portion of the effect of NE on Na1 transport is due to Na1 secretion. Unlike the porcine ileum, the effects of NE in the distal colon were neither affected by TTX nor the eicosanoid synthesis inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA). Furthermore, the transport-related actions of NE were inhibited by the α1-adrenoceptor blocker prazosin but remained unaltered after yohimbine or propranolol, blockers of α2- and β-adrenoceptors respectively. Thus, it appears that NE stimulates colonic Cl2 and Na1 secretion in the pig through interactions with non-neural, possibly epithelial, α1-adrenoceptors. PEPTIDERGIC REGULATION OF INTESTINAL ION TRANSPORT: AN EMERGING ROLE FOR BOMBESIN-RELATED PEPTIDES GRP is a 27 amino acid peptide whose C-terminal amino acid sequence is homologous to the amphibian dermal peptide bombesin (BB) and to the mammalian peptides neuromedin B (NMB) and neuromedin C (65). At the present time, four receptors mediating the effects of BB-related peptides have been cloned from amphibian or mammalian tissues and pharmacologically defined; they are distinguished in part by their different affinities for GRP and NMB (43). A NMB-preferring BB1 receptor (BB1-R) and a GRP-preferring BB2-R are presently the best-characterized mammalian BB-R types (5,65). Studies in canine jejunum indicate that BB inhibits NaCl and fluid absorption (4). In rat, chinchilla, and guinea pig small intestine, BB stimulates Cl2 secretion (21,38). In guinea pig ileum, TTX does not inhibit the secretory actions of BB suggesting that the transport related actions of the peptide were not dependent upon activation of submucosal neurons within the preparation. In porcine jejunum, nearly 40% of GRP-induced Cl2 secretion is mediated by non-cholinergic submucosal nerves (16). The effects of BB and GRP in guinea pig ileum and porcine jejunum appear to be mediated by BB2-R. The effects of GRP-related peptides on electrolyte transport in the large intestine have only been investigated in the porcine distal colon in vitro (74). They are quite different from those described in the porcine jejunum. In colon, contraluminal administration of GRP produces a biphasic Isc response involving an initial increase in current (designated phase 1) followed by a prolonged decrease in Isc (phase 2). The phase 1 response is eliminated after pretreatment with TTX or the cyclooxygenase inhibitor indomethacin suggesting that GRP receptors are localized to neurons and perhaps inflammatory cells located in the submucosa. The phase 2 response is unaffected by TTX and indomethacin and is due to Na1 and K1 secretion. As a major portion of this peptide effect is blocked by serosal addition of bumetanide or Cl2 replacement, secretion of these cations appears

D. R. Brown and S. M. O’Grady

to be mediated to a large extent by basolateral Na-K-2Cl cotransport. It is interesting to note that the direct actions of GRP in porcine distal colon are similar to the effects of β-adrenoceptor-induced K1 secretion in rabbit and guinea pig distal colon, which is also sensitive to bumetanide. This result lends additional supporting evidence for independent regulation of cation and anion secretion in these mammalian species. Through the use of BB-related peptides (including GRP and NMB) and selective BB antagonists, the colonic phase 2 response to GRP was found to be mediated by BB2-R. These data are consistent with the presence of BB2-R in the basolateral membranes of epithelial cells. GRP is also effective in increasing Isc after its luminal application to colonic mucosa sheets from the pig; it exhibits a pharmacological profile that is similar to its actions after contraluminal administration. Thus, the BB-related peptides can potentially interact with either basolateral or apical BB2-R receptors on colonic epithelial cells to affect cation transport. To our knowledge, BB 2-R represents one of the few neuropeptide receptors that have been shown to be expressed on the luminal surface of the colon. A potential physiological role for apical BB2-R is unknown, but it is interesting to note that bovine milk reportedly contains BB-like peptides that stimulate smooth muscle contractions in guinea pig colon and rat uterus (40). The colonic epithelium may be responsive to dietary components or to products of microbial digestion within the lumen that are capable of generating these peptides. Indeed, this form of post-translational peptide processing occurs with exorphins and several other peptide classes (15). An alternate possibility is that BB-related peptides produced and released by immune cells located in the lumen might interact with these receptors to mediate secretory host-defense processes in the colon. Another interesting aspect of GRP action in the porcine distal colon relates to its ability to produce net Cl2 secretion when co-administered with prostaglandin E2 [PGE2 ; (75)]. This is a particularly interesting finding because neither GRP nor PGE2 stimulate Cl2 secretion when administered separately. PGE2 reduces net Cl2 absorption in porcine distal colon by decreasing the mucosa-to-serosa unidirectional Cl2 flux, an action which is dependent upon extracellular HCO3 (73). In the presence of GRP, however, a large increase in the serosa-to-mucosa unidirectional Cl2 flux occurs with a corresponding increase in tissue conductance and bumetanide-sensitive Cl2 secretion (Fig. 1). We hypothesize that PGE2 decreases Cl2 absorption by activating an anion conductance in the apical membrane that results in Cl2 recycling. The fact that the effects of PGE2 on Cl2 absorption are HCO3-dependent suggests that activation of Cl recycling may be an important mechanism for stimulating electroneutral HCO 3 secretion. In addition to modulating the actions of PGE2, GRP can enhance the ability of VIP to stimulate Cl2 secretion in porcine colon as well. GRP may function in combination with PGE2, VIP and per-

Intestinal Ion Transport in Swine

FIG. 1. A cellular model showing the proposed actions of

gastrin-releasing peptide (GRP) and prostaglandin E2 (PGE2) on transepithelial electrolyte transport across the porcine distal colon epithelium. See text for details.

haps other secretagogues to produce or enhance Cl secretion. It may do so by activating the bumetanide-sensitive Na-K-2Cl cotransporter that serves as the principle loading step for Cl2 across the basolateral membrane of colonic epithelial cells (45). BB-related peptides exhibit potent mitogenic activity in several epithelial tissues. The observations that 30% of human colonic cancer biopsies and 40% of colonic cancer cell lines manifest specific GRP binding sites suggests that this peptide family may play a key role in colon tumorigenesis (26,55). The transport-related actions of these peptides, particularly in colon, play some role in their mitogenic activity. Growth factors such as EGF, FGF, PDGF, and TGFα have been shown to stimulate Na-K-2Cl cotransport activity, an effect important in cell proliferation (48). At this time the relationships between cell cycle regulation and control of electrolyte transport pathways in epithelia are not well understood. However, as a number of neurohormones that regulate electrolyte transport in intestinal epithelia also control growth and proliferation, interrelationships between these cellular processes may indeed exist. CONCLUSIONS Over the past decade, a significant amount of work has been done in characterizing the mechanisms and regulation of electrolyte transport across the porcine intestine. These findings reveal that there is often a marked segmental heterogeneity in the mechanisms by which a particular neurotransmitter modulates active ion fluxes. In some respects, the properties of porcine intestinal segments are very similar to their human homologs, but there are some important differences particularly with respect to regulatory mechanisms.

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