Pituitary Adenylate Cyclase- Activating Polypeptide: A Potent

Activator of Human Intestinal Ion Transport"

M. FTJCHS, K. ADERMANN, H. R. RAAB? W. G. FORSSMANN, AND M. KUHN Lower Saxony Institute for Peptide Research

Feodor-Lynen Str. 31 bDepartment of Abdominal and Transplantation Surgery

Hannover Medical School 0-30625 Hannover, Germany

INTRODUCTION

Pituitary adenylate cyclase-activating peptide (PACAP) was originally isolated from ovine hypothalamic extracts, using its pronounced stimulation of adenylate cyclase in rat anterior pituitary cells as an isolation criterion.' The two molecular forms isolated, C-terminally amidated PACAP-( 1-27) and PACAP-( 1 -38), show high sequence homology to vasoactive intestinal polypeptide Although PACAP was originally identified in the brain, it is now known to have a wide variety of biological activities in other tissues. Morphological studies demonstrated that in several animal species and in humans, PACAP as well as its specific binding sites are widely distributed in the gastrointestinal tract. In human tissue, PACAP-like immunoreactivity was observed in nerve fibers in all layers of the gut wall, including the intramural ganglia, in colocalization with VIP.3 Abundant [Iz5I]-PACAP binding sites were found in both the mucosa and in the muscle layers of the inte~tine.~.~ The presence of PACAP as well as abundant binding sites in the human intestine suggests a role for this peptide as a neuromodulator in the regulation of intestinal functions. Indeed, PACAP causes potent relaxation of human intestinal muscle in vitro.6 It has also been shown that PACAP stimulates active ion transport in isolated rat intestinal mucosa and in the cultured human colon carcinoma cell line T84,',* suggesting that this neuropeptide may have a physiological role in the control not only of gastrointestinal motility but also of intestinal secretion. No information exists, however, on the effects of PACAP on electrolyte transport in the human intestine, and, therefore, this study

aThis work was supported by Deutsche Forschungsgerneinschaft (DFG Ku 1037/1-1). Address for correspondence: Michaela Kuhn, Niedersachsisches Institut fur Peptid-Forschung,

Feodor-Lynen-Str. 3 1. D-30625 Hannover, Germany. Temporary address (until September 1997): Howard Hughes Medical Institute, University of Texas, Southwestern Medical Center at Dallas, Dallas, TX 75235-9050. Tel: (49) (511) 5466 360/386; fax: (49) (511) 5466101/102.

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was designed to characterize its effects on the small and large bowel and to compare it with VIP and other intestinal peptides.

METHODS

Specimens

Tissues used in this study were obtained from 20 patients undergoing elective abdominal surgery at Hannover Medical School between July 1994 and September 1995. The study was conducted in accordance with the Declaration of Helsinki, and it was approved by the Human Ethics Committee of Hannover Medical School. The clinical conditions necessitating removal of the small (n = 5) or large intestine (n = 15) in these patients were carcinomas of the stomach, gut, liver, or pancreas. Tissues used for experimental studies were taken from macroscopically normal areas distant from the pathologic tissue. A separate series of experiments was conducted with colonic specimens obtained from four patients with chronic bowel inflammation because of Crohn's disease.

Ussing Chamber Studies

Segments for experimental use were cut from the surgically resected specimens immediately after resection, opened along the mesenteric border, and then transported in oxygenated ice-cold Krebs-Ringer bicarbonate solution to the laboratory. The Krebs-Ringer bicarbonate solution contained (mM) 140 Na', 124 C1-, 21 HC03-, 5.4 K , 2.4 HPOJ2-, 0.6 HzP04-, 1.2 Mg2+, 1.2 Ca2', and 10 glucose. Osmolarity was adjusted to 300 mosmoVL, with mannitol and pH adjusted to 7.4. The specimens were then prepared by dissecting the muscle layers, and sheets of the isolated mucosa were mounted in modified Ussing chambers with an exposed surface area of 1 Both sides of the mucosa were bathed with Krebs-Ringer bicarbonate solution and oxygenated by a gas-lift system with 95% O2 / 5% C02. Each chamber was connected to voltage clamp amplifiers (VCC 600 or VCCMC6, Physiological Instruments, San Diego, CA). Fluid resistance was determined before mounting and automatically adjusted during the experiment. Transepithelial potential differences were measured with Ringer-agar bridges connected to calomel half cells with reference to the mucosal solution. The tissues were voltage clamped using Ag/AgCl electrodes in 3 M KCl. The short-circuit current (Isc in PA) was considered positive for cation flow from the mucosal to the serosal side. Responses ( A h ) were determined as the difference between the basal Isc and the maximum change in Isc observed in response to substances tested.

Data Analysis

Results are presented as the mean maximal increases in Isc (AIsc) 2 SEM (n = number of tissues). Statistical differences between mean values were determined by analysis of variance followed by the Fisher protected least-significant-difference test

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for comparison of different means; p values of less than 0.05 were considered statistically significant.

RESULTS

Effects of PACAP-27 on Human Intestinal Mucosa

After an equilibrium period of 30 min, the effect of peptides on Isc was assessed using a cumulative concentration-response protocol. Addition of PACAP-27 to the serosal bath solution elicited a rapid (within 1 min), marked, and sustained increase in Isc. In both human jejunal and colonic mucosa, stimulation of Isc occurred at 0.1 nM PACAP-27 (threshold concentration), and a maximal Isc response was obtained at 1 pM. In the colon, the Isc responses were clearly proportional to the cumulative addition of increasing PACAP-27 concentrations; at each concentration the maximal Isc response was reached after 15-20 minutes (FIG. 1A). In the jejunum, the secretory responses to low concentrations (1-10 nM) of PACAP-27 were often rapid and transient and not proportional to the cumulative additions of the peptide; at higher concentrations (0.1 and 1 pM) of PACAP the Isc responses became smoother and prolonged (FIG. 1A). Pooled data show that the magnitude of the maximal Isc responses to PACAP-27 (1 pM) were the same in both intestinal segments (FIG. 1B). Addition of PACAP-27 to the luminal solution had no effect on Isc in either tissue, indicating the localization of specific receptors in the basolateral side of the epithelium in both human small and large bowel. The maximal Isc increases evoked by PACAP-27 were significantly inhibited by the subsequent addition to the serosal solution of 0.1 mM bumetanide, indicating that stimulation of chloride secretion is responsible for the stimulation of Isc.

Pretreatment of intestinal tissues with the neuronal blocker, tetrodotoxin (1.2 pM, serosal side), had no significant effect on the magnitude of the Isc responses to PACAP-27 (data not shown).

Comparison with Other Secretugogues

Recently, a common VIP-PACAP receptor has been characterized in human intestinal epitheli~m.~ To investigate the possibility that the secretory responses to PACAP-27 are mediated by this receptor, in individual experiments with colonic mucosa, the responses to both peptides were compared. VIP resembled PACAP-27 in the time course and the magnitude of the Isc responses. The concentration- response curves of both peptides were parallel, with similar potencies and efficacies for stimulation of colonic chloride secretion (FIG. 2). Half-maximal secretory responses were obtained at 10 nM for both peptides. To test whether PACAP and VIP have additive effects, 1 pM VIP was added after 20 min of exposure to 1 pM PACAP- 27. The maximal Isc response to PACAP was not further increased by subsequent exposure to VIP ( A h : 145 ? 14 pA/cm2 before addition of VIP vs. 148 2 12 pA/ cm2 after addition of VIP).

The similar effects of PACAP and VIP support the view that PACAP acts on the intestinal epithelium by way of the VIP receptor and in turn activates apical

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10 nM

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0.1 1LM 100 nM

J. 0.1nM lny;*- 4 1OnM 1wnM

jejunum 2 mln

J . J . colon J.

100 pA

a

200-

-0- colon + jejunum

'

150-

100-

O.1nM 1nM lOnM 1WnM O.ipM

[PACAP-271 b

FIGURE 1. Effects of cumulative concentrations of PACAP-27 (serosally) on short-circuit current across isolated human jejunal and colonic mucosa. A: Original traces of two representa- tive Ussing chamber experiments. In the colon, the Isc responses were clearly concentration- dependent, whereas in the jejunum, the responses to low concentrations of PACAP-27 were often transient and not proportional to the cumulative additions of the peptide. B: Pooled data of all experiments with human colon (n = 23) and jejunum (n = 10).

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a“ 5 0 -

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-.- VIP

-0- PACAP-27

--)- guanylin

v I I I I

FIGURE 2. Effects of PACAP-27, VIP, and guanylin on short-circuit current across isolated human colonic mucosa. *p < 0.05 compared with the effect of guanylin (n = 10- 11).

cystic fibrosis transmembrane conductance regulator (CFTR) C1- channels through an increase in intracellular cyclic AMP.5,8 It has been shown that the CFTR channels can also be activated by a cyclic GMP-dependent pathway.1° Guanylin is a recently discovered intestinal peptide that stimulates specifically a receptor guanylate cyclase (GC-C), located in the brush-border membrane of the enterocytes. Subsequent in- creases in epithelial cyclic GMP activate the CFTR C1- channels and thereby chloride s e c r e t i ~ n . ~ . ~ ~ As shown in FIGURE 2, the concentration-response curve of guanylin for stimulation of Isc across the human colon was slightly shifted to the right as compared to PACAP and VIP; the maximal Isc responses, however, were similar. In contrast to PACAP and VIP, guanylin stimulated Isc exclusively after addition to the apical side of epithelia; serosal guanylin failed to elicit a response at doses as high as 1 pM.

Effect of PACAP-27 on Ion Transport in Colonic Specimens from Patients with Chronic Bowel Injlummation

To elucidate whether an increased secretory response to endogenous PACAP participates in the severe diarrhea of patients with chronic bowel inflammation, the effects of the peptide were evaluated in colonic specimens obtained from four patients with Crohn’s disease. As illustrated in FIGURE 3, the concentration -response curve was not significantly different from that obtained with macroscopically “normal” mucosa obtained from patients necessitating surgical removal of intestine because of tumoral diseases.

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2 0 0

+ controls + Crohn’s disease

1 5 0

a-

5 a 1 0 0

a

3. 0 v)

v - 5 0

0 0.1 nM 1 nM l O n M 100nM 0.1 pM

[PACAP-271

FIGURE 3. Comparison of the effects of PACAP-27 on short-circuit current across colonic specimens from patients with Crohn’s disease (n = 5 , from 4 patients) and across macroscopically “normal” colonic niucosa from patients with tumoral diseases (n = 23, from 9 patients).

DISCUSSION

To our knowledge, this is the first study to show that PACAP stimulates active electrogenic electrolyte transport (and thereby Isc) across human jejunal and colonic mucosa. The inhibitory effect of bumetanide suggests that stimulation of active chloride secretion is responsible for the increase in Isc. The increases in Isc were concentration dependent, and the threshold concentration for responsiveness to PACAP-27 was as low as 0.1 nM, which might support a physiological role of the endogenous peptide in the regulation of human intestinal ion transport. In a recent study it has been shown that VIP and both forms of PACAP relax the longitudinal muscle of human sigmoid colon in vitro.6 The ICso values of the peptides for this relaxing effect were about two orders of magnitude higher, as compared to the secretory effects documented for PACAP-27 and VIP in the present study, suggesting that the endogenous peptides might be predominantly involved in the local modulation of epithelial ion transport. The secretory action of PACAP is insensitive to tetrodo- toxin, which blocks propagated neuronal action potentials, suggesting that this effect of the peptide is not mediated indirectly by the liberation of neural components but lies on a direct interaction with the epithelium itself. It might be assumed that PACAP- 27 binds specifically to receptors in the basolateral membrane of the enterocytes, inasmuch as stimulation of chloride secretion is obtained exclusively after addition of PACAP to the basolateral, but not apical, side of the mucosa.

Shivers et ul. distinguished two types of PACAP receptors: the type-I PACAP receptor shows high affinity for PACAP but little affinity for VIP; the type-I1 PACAPI

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VIP receptor (formely named VIP receptor) shows similar affinity for both peptides.” From the present data, evidence is provided that in the human intestinal mucosa the increases in chloride secretion in response to PACAP-27 are mediated by the type- I1 PACAPNIP receptor: (1) PACAP and VIP activate electrogenic ion transport/ chloride secretion with the same potency and efficacy; (2) after induction of the maximal secretory response to PACAP-27 (1 pM), addition of 1 pM VIP has no further effect. It has been demonstrated that VIP affects epithelial transport processes directly by way of specific receptors within the basolateral membrane,”.l* and it is likely that PACAP also activates these receptors. The stimulation of adenosine 3‘3‘- cyclic monophosphate (CAMP) production in target cells underlies many of the biological effects of these peptides and an involvement of CAMP in the secretory responses of the intestinal mucosa seems likely with regard to the high expression of the CAMP-activated (CFTR) chloride channels in the apical membrane of the enterocytes. Guanylin is a recently discovered intestinal peptide that activates the same (CFTR) type of apical chloride channels but through a different cellular pathway, namely an increase in epithelial cyclic GMP.Io The efficacy of guanylin for activation of intestinal C1- secretion, however, is slightly less than that of PACAP and VIP, suggesting a higher efficiency of the cyclic AMP pathway for activation of intestinal chloride secretion. - In conclusion, the present results show that PACAP and VIP are potent stimulators

of electrogenic ion transport in both human small and large intestinal mucosa. Because expression of both peptides has been demonstrated in the intestinal nervous system in humans,) their participation in the physiological regulation of intestinal ion transport might be assumed. Experiments performed with surgically resected tissues from patients with Crohn’s disease show that the secretory effect of PACAP-27 on the chronically inflamed intestinal mucosa is largely preserved. Thus, it is conceivable that in certain disease states, such as intestinal inflammation, the local release of PACAP and/or VIP from intestinal nerve endings might be enhanced, leading to enhanced chloride secretion and, thereby, contributing to the severe diarrhea observed in these patients.

SUMMARY

To investigate the effects of PACAP-27 on electrolyte transport across the isolated human intestinal mucosa, changes in short-circuit current (Isc) were measured in Ussing chamber experiments. Serosally added PACAP-27 increased Isc in a concentra- tion-dependent manner, eliciting a similar maximal effect in both the jejunal and the colonic mucosa. Bumetanide inhibited Isc responses, indicating stimulation of C1- secretion. The potency and efficacy of PACAP-27 were comparable to those of VIP, suggesting that both peptides activate intestinal secretion by way of a common receptor located in the basolateral membrane of the intestinal epithelium.

ACKNOWLEDGMENTS

The authors wish to thank Mrs. Edda Kock for her excellent technical assistance. We are indebted to Dr. J. Jahne and Dr. J. Klempnauer, Department of Abdominal

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and Transplantation Surgery, Hannover Medical School, who provided specimens for study.

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