Role of PACAP in the Regulation of Gastrointestinal Motility

Role of PACAP in the Regulation of Gastrointestinal Motility

STAVROS KATSOULIS AND WOLFGANG E. SCHMIDT a

Laboratory of Molecular Gastroenterology Gastrointestinal Unit

I. Department of Medicine Christian-Albrechts- University of Kiel

Schittenhelrnstrasse I 2 0-24105 Kiel, Germany

INTRODUCTION

Almost six years have elapsed since Miyata, Arimura, and colleagues reported the isolation of a novel 38-residue peptide from ovine hypothalamus. It was named, Pituitary Adenylate Cyclase Activating Peptide (PACAP) because of its marked ability to stimulate adenylate cyclase in rat pituitary cell cultures.' Subsequently, a second molecular form of this peptide comprising the N-terminal 27 residues of PACAP-38 was isolated and named PACAP-27.* PACAP belongs to the secretinlglucagonNIP peptide family and exhibits 68% sequence homology with VIP. PACAP is widely dis- tributed in the digestive tract,3 PACAP-specific immunoreactivity has been identified in intestinal nerve fibers, submucosal, and myenteric ganglia of several species, including humans.M Coexistence with VIP was demonstrated in some regions and The occurrence of PACAP in the gastrointestinal tract and its structural homology to VIP indicate that PACAP may play a role in the regulation of gastrointestinal motility.

The present paper summarizes the activity of PACAP on the contractility of smooth muscle preparations from various parts of the guinea pig, rat, pig, and human gastrointestinal tract. In vivo studies aimed to determine the physiological function of PACAP on gastrointestinal motility have not yet been performed. Since both prejunctional (presynaptic, neurogenic) and postjunctional (postsynaptic, myogenic) effects influence smooth muscle contractility, pharmacological, biochemical, and electrophysiological experiments were performed to evaluate the sites and mecha- nisms of action of PACAP.

ACTION OF PACAP ON GUINEA PIG INTESTINAL SMOOTH MUSCLE

Guinea Pig Ileum

The effect of a great number of peptides in the guinea pig ileum often is opposite to the action of these peptides in other regions of the gastrointestinal tract or in the

'Address for correspondence: Wolfgang E. Schmidt, M.D., I. Dept. of Medicine, CAU &el, Schittenhelmstr. 12, D-24105 Kiel, Germany. Tel.: 49-431-597-1395 or -1272; fax: 49-431-597-1427 or -1302; e-mail: weschmidt@ lmedmi-kiel.de.

364

KATSOULIS & SCHMIDT: PACAP 365

150 r

A= PACAP B= A + naloxone C= A + naloxone + dynorphin D= A + guanethidine E= A + hexamethonlum F= A + spantide C = A + omega-conoloxin 11: A + atropine I = A + apantide + omega-conotoxin J = A + TTK li= A + soniatostntin I.= A + dynorphin \I= A + atropine + *pantide N = A + atropine + omega-conotoxin 01 A t atropine + SP desensitization

. . . . . . -

A B C D E f C H l J K L M N O

FIGURE 1. Pharmacological characterization of the myotrnpic effect of PACAP-38 on isolated guinea pig ileal muscle strips. Data are given in percent of the contractile effect of PACAP- 38 (60 nM) as means 2 SE (n = 8-9). * p c 0.05. (From Katsoulis et al.'" Reproduced by permission.)

ileum of other species. For example, VIP is a relaxant in all gastrointestinal preparations with the exception of the guinea pig where it causes a neurogenic contraction.' Similar to structurally related VIP, PACAP-38, and PACAP-27 induce neurogenic contraction of ileal strips in a dose-dependent manner." No significant differences in efficacy and potency could be observed between both molecular forms of PACAP-38 and PACAP-27. In contrast, VIP was significantly less potent and less efficient compared to PACAP. As shown in FIGURE 1, the effect of PACAP-38 and PACAP-27 was abolished by TTX and partially inhibited by atropine, omega- conotoxin, somatostatin, dynorphin, and the substance P antagonist spantide. The atropine-resistant component could be completely inhibited either by addition of spantide or omega-conotoxin (FIG. 1). Induction of tachyphylaxis to substance P in atropinized preparations prevented the appearance of the atropine-resistant component of PACAP-induced contraction. Hexamethonium and guanethidine failed to inhibit the effect of PACAP. These results indicate that PACAP contracts the guinea pig ileum via the release of both acetylcholine (ACh) and substance P from postganglionic cholinergic and peptidergic nerves."

Since VIP revealed an identical mode of action, we have investigated whether both PACAP and VIP act through a common receptor by using the technique of desensitization."' Ileal strips exposed to high concentrations of PACAP (1 80 nM) or VIP (1 FM) for 20-25 min became desensitized to PACAP or to VIP, as indicated by application of the respective peptide 20-30 min later, which was without any effect. Ileal preparations desensitized with VIP ( 1 pM) remain fully responsive to PACAP (1 80 nM), whereas strips desensitized by PACAP (1 80 nM) did not respond

366

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0 .

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-

-

-

-

PACAP PACAP + dcacnsiliration with VIP PACAP + descnsilixnlion w i t h PACAP

T

VII' + Jcscnailixnlion with V I P p4 VII' vll' + dcscnrllirnllon with I'ACAI'

* * I* CI - 0

FIGURE 2. Effect of desensitization with PACAP or VIP on PACAP- or VIP-induced contraction of isolated guinea pig ileal muscle strips. Left part: response to PACAP (180 nM) without and after desensitization with either PACAP or VIP; right part: response to VIP (1000 nM) without and after desensitization with either PACAP or VIP. * p < 0.05 compared to control. (From Katsoulis er al. '" Reproduced by permission.)

to VIP (1 FM) (FIG. 2). Cross-tachyphylaxis is observed between PACAP-38 and PACAP-27. These observations and the demonstration of significantly different poten- cies and efficacies for PACAP and VIP are likely explained by the existence of at least two receptor types in this tissue. Functional studies, binding experiments, and molecular cloning have established the existence of at least three distinct receptors for PACAP and VIP that form two pharmacologically different types:"-" the PACAP/ VIP- 1 (PVl) receptor (formerly designated PACAP type 1 or PACAP-preferring receptor), the PACAPNIP-2 (PV2) receptor (formerly designated PACAPNIP type 2 or VIP or VIP- 1 receptor), and the PACAPNIP-3 (PV3) receptor (formerly designated PACAPNIP type 2 or classic VIP or VIP2 receptor). The PV 1 receptor shows high affinity for PACAP but does not bind VIP (type 1 receptor), whereas the PV2 and PV3 receptors equipotently interact with PACAP and VIP at high affinity (type 2 receptor).

Measurement of [3H]ACh release from the LMMP preparation of the guinea pig preloaded with [3H]choline revealed two effects of PACAP and VIP:Io stimulation of the spontaneous [3H]ACh release (FIG. 3(a), 3(b)) and inhibition of the electrically evoked [3H]ACh release. PACAP-38, PACAP-27, and VIP increased concentration- dependently the outflow of tritium from longitudinal smooth muscle strips incubated with [3H]choline (FIG. 3(a), 3(b)). In agreement with their effects on contraction, VIP- induced ACh release is abolished in the presence of PACAP, while the effect of PACAP persisted in the presence of VIP (FIG. 3(a), 3(b)). These findings suggest the existence of PVl and PV2PV3 receptors. The PACAP- and VIP-induced ACh release was inhibited by perfusion with Caz+-free medium and TTX, thereby indicating that


5

0

A= PACAP B= A + hexamelhonium C= A + scopolamine D= A + VIP desensitization E= A t omission 01 calcium

T , , F= A + TTX

A D C D E F

a

A= VIP B= A + hexomelhonium C= A + scopolcmine D= A + PACAP desensilizolion E= A + TTX f= A + omission of calcium

A B C D E F

b

FIGURE 3. Pharmacological characterization of the effect of PACAP (a) or VIP (b) on spontaneous 3[H]ACh release in longitudinal smooth muscle strips. Columns represent means ? SE of 3-6 experiments. * p < 0.05. (From Katsoulis et al."' Reproduced by permission.)

368 ANNALS NEW YORK ACADEMY OF SCIENCES

the release of ACh is of neuronal origin. Hexamethonium and scopolamine did not affect ACh release induced by PACAP and VIP, thus indicating that nicotinic and muscarinic receptors are not involved in the action of both peptides. These results allow us to conclude that PACAP and VIP might act at the presynaptic site or at cell bodies of postganglionic cholinergic neurons in this tissue. Since TTX abolished the stimulation of ACh release, the site of action of both peptides is probably located in the somato-dendritic region of postganglionic cholinergic neurons, because part of the TTX-resistant evoked release of neurotransmitters is assumed to be exerted through direct depolarization of nerve terminals. Electrophysiological studies on myenteric neurons from the guinea pig small intestine have shown that both PACAP- 38 and PACAP-27 evoke excitatory responses in 96% of the afterhyperpolarizing (AH)/type 2 neurons.'* The responses consist of membrane depolarization in associa- tion with increased input resistance and suppression of hyperpolarizing afterpoten- tials." In addition to their effects on spontaneous ['HIACh release, PACAP and VIP inhibited the electrically evoked [3H]ACh release indicating the presence of inhibitory presynaptic receptors.'"

Guinea Pig Taenia coli

Guinea pig taenia muscle strips relax in response to PACAP-38 and VIP.'"'' Neither TTX nor hexamethonium significantly change the effect of PACAP and VIP, indicating a direct myogenic action. PACAP and VIP are nearly equipotent in inducing relaxation. However, the effect of PACAP and VIP differed markedly in their sensitiv- ity to apamin, a selective blocker of calcium-activated potassium channels. Apamin reduces or abolishes the relaxant effect of PACAP but not of VIP,'"'' as shown in FIGURE 4. Our findings with apamin were later confirmed by McConalogue ef ~ 1 . ~ ' Additionally, they found that the relaxation produced by PACAP is reduced by suramin, which is thought to act as an antagonist of the P2,-purin receptors. Investiga- tions in muscle strips and dispersed taenia coli muscle cells show that the antagonist VIP10-28 inhibits relaxation induced by VIP only.*' PACAP6-38 only inhibits relaxation mediated by PACAP-38 or PACAP-27, implying interaction of PACAP and VIP with distinct receptors. This hypothesis is further supported in dispersed smooth muscle cells by the following findings: ( I ) the response to PACAP is preserved when PACAP receptors are protected with PACAP-38 or PACAP-27, whereas the action of VIP is only preserved when VIP receptors were protected with VIP; (2) only the response to VIP is accompanied by an increase in CAMP but not cGMP, and is blocked by the CAMP- and cGMP-kinase inhibitors, H-89 and KT-5823." Controversial results exist concerning the influence of CAMP-levels by PACAP. In contrast to our results, McConalogue et aL2" found that PACAP produces a concentration-dependent increase in CAMP levels simultaneously with the induced relaxation.

Inhibitory neurotransmission in the guinea pig coli probably involve VIP,22.23 ATP,24 and an apamin-sensitive component. The presence of PACAP-like immunoreactivity, which is localized immunohistochemically in varicose nerve fibers within the taenia coli, the underlying myenteric plexus and circular muscle,20 makes it likely that PACAP participates in inhibitory neural transmission in taenia cob. Electrical field stimulation (0.25 -4 Hz) elicit frequency-dependent relaxation and PACAP re-

KATSOULIS & SCHMIDT: PACAP

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FIGURE 4. Effect of tetrodotoxin (TTX), hexamethonium (HX), and apamin on relaxation of guinea pig taenia caeci muscle strips induced by PACAP-27 or VIP. Relaxant effect is expressed in percent of the control (first application) response. Data represent means ? SEM, n = 4-5. *p < 0.001. (From Schworer et ~ 1 . ' ~ Reproduced by permission.)

lease (FIG. 5).21 The frequencies eliciting half-maximal PACAP release and relaxation are 0.7 and 0.9 Hz, respectively. PACAP release is abolished in the presence of TTX (1 FM) and omission of Ca" (FIG. 5). Preincubation of muscle strips for 60 min with a combination of specific monoclonal PACAP-38 and PACAP-27 antibodiesZ5 or with VIP antiserum partially inhibit relaxation at all frequencies of stimulation.21 A combination of PACAP and VIP antibodies as well as of VIP antibodies plus apamin elicits additive inhibition (FIG. 6(a)). Desensitization with PACAP or VIP inhibit neurally induced relaxation at all frequencies, simultaneous desensitization with PA- CAP and VIP provokes additive inhibition (FIG. 6(b)) PACAP and VIP receptor antagonists reveal similar results. Since apamin seems to selectively and dose-dependently interfere with PACAP-, but not VIP-induced signaling (FIG. 7), it provides additive inhibition to the blockade of the VIP effect either by antibodies, receptor antagonists, or after VIP desensitization.

ACTION OF PACAP ON RAT GASTROINTESTINAL SMOOTH MUSCLE

Rat Stomach

Only a short report exists about the effect of PACAP on rat fundic strips. Huang et a1.26 showed that PACAP relaxes fundic strips in a dose-dependent fashion. Com- pared to VIP, PACAP is equipotent. Analysis of the mode of action and the receptors involved has not been performed.


- 600 -

a P

p 4 0 0 - 0 *I n

n 200 -

0 -

0.25 0.5 1 2 4

FREQUENCY (Hz)

FIGURE 5. Frequency-dependent release of PACAP induced by electrical field stimulation of guinea pig taenia coli muscle strips. PACAP release expressed as percent increase above basal level was significant at all levels of stimulation (p < 0.01) and abolished by tetrodotoxin (TTX) and in the absence of Ca2+ (From Jin et aL2' Reproduced by permission.)

Rat Small Intestine

PACAP-38, PACAP-27, and VIP produce a concentration-dependent relaxation of rat ileal strips (FIG. 8).27 The inhibitory action of PACAP was not modified by T T X , omega-conotoxin, adrenergic, or ganglionic blockade, antagonists of adrenore- ceptors and muscarinic receptors indicating a direct myogenic effect. PACAP is fifty times more potent than VIP in eliciting relaxation. Similar results have been reported by others.28 Apamin and tetraethylammonium failed to inhibit the relaxant effect of both PACAP and VIP. 4-Aminopyridine, which blocks various types of K' channels, reduces only the inhibitory effect of PACAP but does not influence the response to VIP. Induction of tachyphylaxis to PACAP did not modify the effect of VIP. Similarly, the effect of PACAP is preserved in rat ileal strips desensitised to VIP. These findings suggest that PACAP and VIP might act by activation of distinct receptors to induce relaxation.

Rat Colon

Mungan et a1.28 reported relaxant effects of PACAP and VIP in this tissue. We have investigated the descending relaxation phasez9 in a preparation devised by Costa and FurnesP' consisting of an isolated colonic segment on which graded radial stretch can be applied to induce the ascending contraction and descending relaxation components of the peristaltic reflex. Stretch of the caudad end produces ascending contraction only, whereas stretch of the orad end produces descending relaxation only. Orad stretch of colonic segments elicits descending relaxation and PACAP

KATSOULIS & SCHMIDT: PACAP

loo 1 37 1

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loo 1

0-1

P

0.25 0.5 1 2 4

FREQUENCY (HZ)

a

T/ +Apamin

0.25 0.5 1 2 4

FREQUENCY (Hz)

b

FIGURE 6. Inhibition of neurally induced relaxation by (a) VIP- plus PACAP-antibodies compared to VIP-antibodies plus apamin, or by (b) combined PACAP- plus VIP desensitization compared to VIP-desensitization plus apamin. Relaxation induced by electrical field stimulation was measured before and after a 60-min incubation with antibodies (a) or a 30-min incubation with peptides for desensitization (b). Apamin was added 15 min before measurements were made. Data are means 2 SEM of 5-7 experiments. (From Jin e r d 2 ' Reproduced by permission.)

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40 - 2 s = 2 0 -

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loo 1

0'

FIGURE 7. Apamin selectively inhibits relaxation induced by PACAP-38 or PACAP-27 but not by VIP in guinea pig taenia coli muscle strips. All peptides were administered at 1 pM. Data are means 2 SEM of 4 experiments. (From Jin et a/.*' Reproduced by permission.)

0 PACAP 0 [Cp"]-PACAP (1-S4)

0 [A.n".8.r".ll.",L.u",A~n"]-PAC*P (1-28)

0 PACAP (1-27) v PACAP ( 1 - a ~ )

VIP

I3

I , , , ,,,,,I , , , , ,,,,, , , , , ,,,,, , , , ,urJ

-9 -a -7 -0 -6 peptide log (Y)

FIGURE 8. Concentration-response curves for the relaxant action of PACAP, PACAP analogues, and VIP in isolated rat ileal muscle strips. Relaxant effects are expressed as percent of the maximal relaxation induced by papaverin (50 pM). Data are means 2 SEM of 8- 12 experiments. (From Katsoulis et al.*' Reproduced by permission.)

release in proportion to the degree of stretch. Electrical field stimulation at 4 Hz caused a 12-fold increase in PACAP release. PACAP release provoked by orad stretch or electrical stimulation is inhibited by the nitric oxide synthase inhibitor, NG-nitro-L-arginin (L-NNA).~~ These results are very similar to that previously reported for VIP.3' Preincubation of the segments with specific monoclonal PACAP-38 and PACAP-27 antibodies or VIP antibody inhibit descending relaxation at all degrees


of stretch. Preincubation with both PACAP and VIP antibodies virtually abolish descending relaxation. A similar pattern is observed with the antagonists PACAP6- 38 and VIP10-28 alone and in combination. Studies in dispersed colonic muscle cells showed that PACAP- and VIP-induced relaxation are inhibited by L-NNA, PACAP6- 38, and V1P10-28.29 In contrast, apamin has no effect on peptide-induced relaxation. Selective receptor protection by using N-ethylmaleimide32 with each peptide preserved the response to all three peptides, implying interaction of PACAP and VIP with a common receptor coupled to nitric oxide generation in muscle cells. Our data in colonic segments and dispersed colonic muscle cells indicate that PACAP and VIP are the main determinants of descending relaxation and that their effects reflect interplay with nitric oxide in neurons and muscle cells.

ACTION OF PACAP ON PORCINE SMALL INTESTINAL SMOOTH MUSCLE

Porcine small intestinal longitudinal muscle strips exhibited spontaneous phasic contractions that were not affected by TTX (1 yM), scopolamine (300 nM), or hexamethonium (100 pM). PACAP concentration dependently (10 nM- 1 p,M) inhibits the phasic contraction^.^^ Complete inhibition is observed at 300 nM PACAP. The inhibitory effect of PACAP was dose dependently antagonized by apamin (3 nM- 300 nM) and was preserved in the presence of VIP (10 nM-1 yM), which by itself only inconsistently inhibits phasic contractions. The inhibitory effect of VIP is insensitive to apamin. Our data suggest that PACAP probably acts on apamin-sensitive PVl receptors not interacting with VIP to induce inhibition of phasic contractions.

ACTION OF PACAP ON HUMAN SIGMOID COLONIC SMOOTH MUSCLE

PACAP-38, PACAP-27, and VIP inhibit spontaneous phasic contractions of the longitudinal muscle strips of the human sigmoid colon (FIG. 9) and relax concentration dependently carbachol precontracted preparation^.^^ Interestingly, the hybrid peptide

Serz5, Ile26, Leuz7, Asn2*]-PACAP-28 (comprising the first 23 amino acid residues of PACAP, extended by the C-terminal part, position 24-28, of VIP) is fully active.34 VIP is significantly less potent (about 6-fold) and less effective (about 50%) compared to PACAP. The effects of PACAP and VIP remain unchanged by TTX, by inhibition of phosphodiesterase activity, and by inhibition of nitric oxide synthesis (FIG. 10). Apamin only reduces the effect of PACAP, whereas tetraethylammonium only inhibited the action of VIP (FIG. 10). Our results indicate that PACAP and VIP mediate their relaxant effects via activation of specific PACAP, PV1-like receptors, and distinct VIP, probably PV2 and/or PV3 receptors, coupled to different potassium channels.

STRUCTURE-ACTIVITY RELATIONSHIPS

Structure-function studies with PACAP analogues, PACAP fragments, and PA- CAPNIP hybrid peptides have been performed on muscle strips of guinea pig ileum


PACAPl-38

PACAP1-17

Jio DM ( 0 a

VIP

FIGURE 9. Recordings of the inhibitory effect of PACAP-38, PACAP-27, a PACAPNIP hybrid peptide, and VIP on spontaneous contractions of the longitudinal muscle of the human sigmoid colon. Each peptide was added at 1 p,M. Preparations were preloaded with 10 mN; contractions were recorded isometrically. (From Schworer et ~ 2 1 . ’ ~ Reproduced by permission.)

and rat ileum. PACAP and its analogues [ C ~ S ~ ~ I - P A C A P - ~ ~ , [Amz4, Se?’, Ile26, Leu17, AsnZ8]-PACAP-28 (hybrid peptide comprising the first 23 amino acid residues of PACAP, extended by the C-terminal part, position 24-28, of VIP), PACAP-27, [Cys2’]-PACAP-25, PACAP-23, PACAP- 19, and VIP contract guinea pig ileum in a dose-dependent manner. PACAP fragments 18 -38, 3 - 19, 3 -25 at concentrations up to 20 pM fail to produce any effect. The degree of potency expressed as the ECSn of the tested peptides show remarkable differences. On the basis of ECsn values, PACAP-27 is slightly less potent than PACAP-38, while [CysZ5]-PACAP-25, PACAP- 23, and PACAP-19 show a marked decrease in potency compared to PACAP. [CYS~~] - PACAP-34 and S e P , IleZ6, Leuz7, AsnzR]-PACAP-28 are the only fragments in this study, showing a moderately increased potency compared to PACAP-38. The stucturally related peptide VIP is five times less potent than PACAP-38. The order of potency to induce contraction is [ C ~ S ~ ~ I - P A C A P - ~ ~ = Ser25, Hez6, LeuI7, AsnZ8]-PACAP-28 > PACAP-38 > PACAP-27 > VIP > PACAP-23 = [CysZ5]-PACAP- 25 > PACAP-19. The peptides tested do not exhibit the same effectiveness, expressed as percent of the maximal effect obtained by ACh (100 pM). PACAP-I9


- " x

X

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f. Y -

c(

X

X c(

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A TEA 4-AP NNLA

FIGURE 10. Pharmacological characterization of the relaxant effect of PACAP, PACAPl- 23VIP24-28, or VIP. Potassium channel inhibitors apamin (A), tetraethylammonium (TEA), or 4-aminopyridine (4-AP), or the inhibitor of nitric oxide synthase, N-nitro-L-arginine (L- NNA), were added to the organ bath 15 min prior to the respective peptide. Results are expressed as percent of inhibition of spontaneous phasic contractions observed 5 min before the peptide was added. Data are means ? SEM of 4-6 experiments. + p < 0.01. (From Schworer et ~ 1 . ~ ~ Reproduced by permission.)

is as effective as PACAP-38, while the remaining PACAP analogues display an increased efficacy. VIP shows the least efficacy (50% compared to PACAP-38). The order of effectiveness is: PACAP-27 = [Am2', Ser2h, IleZ7, Leu2*, A ~ n ~ ~ l - p A c A P - 2 8 = PACAP-23 > [ C ~ S ~ ~ I - P A C A P - ~ ~ = [Cys"]-PACAP-25 > PACAP-38 = PACAP- I9 > VIP. From these results, it can be concluded that the C-terminal portion of PACAP contributes mainly to the affinity of PACAP to its receptors, whereas the N-terminal portion of PACAP is essential for full activation of PACAP receptors in this preparation. The data derived from our structure-activity study are in agreement with results obtained by binding studies with PACAP and PACAP analogues performed on plasma membranes from rat brain" and rat pancreatic tumor cells AR 4-2 J."."

Similar results are obtained in rat ileal strips,27 as shown in FIGURE 8. PACAP- 38 (5- 120 nM), [ C ~ S ~ ~ I - P A C A P - ~ ~ (12-216 nM), [ A d 4 , Ser25, IleZ6, Leu2', Asn2*]- PACAP-28 (4-192 nM), PACAP-27 (7-180 nM), PACAP-23 (60 nM-3.24 pM), and VIP (0.15-1.2 pM) relax rat ileum in a dose-dependent manner. The PACAP fragments 18-38, 3-25, and 3-19 are without any effect at concentrations up to 10 pM. The effectiveness of the PACAP fragments, expressed as percent of the maxi- mally relaxant response induced by papaverine (50 pM), reaches 79- 90% compared to PACAP-38. VIP (efficacy of 60% 2 4) is slightly but not significantly less effective as compared to PACAP-38 (efficacy of 79% 2 4). On the basis of EC,, values PACAP-38 and [ A d 4 , Ser2,, Ile", AsnZ8]-PACAP-28 exhibit similar potency to induce relaxation of rat ileum. [cy~ '~] -PAcAP-34 and PACAP-27 were moderately less potent (relative potency of both peptides = 35%) compared to PACAP-38, while PACAP-23 shows a marked decrease in potency (relative potency = 10%). VIP is approximately fifty times less potent than PACAP-38. The results obtained in rat ileum reinforce the assumption that the N-terminal region of the PACAP molecule is crucial for biological activity on gastrointestinal smooth muscle.


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REFERENCES

MIYATA, A,, A. ARIMURA, R. R. DAHL, N. MINAMINO, A. UEHARA, L. JIANG, M. D. CULLER & D. H. COY. 1989. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem. Biophys. Res. Commun.

MIYATA, A., L. JIANG, R. R. DAHL, C. KITADA, K. KUBO, M. Fumo, N. MINAMINO & A. ARIMURA. 1990. Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP 38). Biochem. Biophys. Res. Commun. 170: 643-648.

ARIMURA, A,, A. SOMOGYVARI-VIGH, A. MIYATA, K. MIZUNO, D. H. COY & C. KITADA. 1991. Tissue distribution of PACAP as determined by RIA: Highly abundant in the rat brain and testes. Endocrinology 129: 2787-2789.

SUNDLER, F., E. EKBLAD, A. ABSOOD, R. H~KANSON, K. KOVES & A. ARIMURA. 1992. Pituitary adenylate cyclase activating peptide: A novel vasoactive intestinal peptide- like neuropeptide in the gut. Neuroscience 46: 439-454.

PORTBURY, A. L., K. MCCONALOGUE, J. B. FURNESS & H. M. YOUNG. 1995. Distribution of pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity in neurons of the guinea-pig digestive tract and their projections in the ileum and colon. Cell Tissue Res. 279: 385-392.

FURNESS, J. B., H. M. YOUNG, S. POMPOLO, J. C. BORNSTEIN, W. A. A. KUNZE & K. MCCONALOGUE. 1995. Plurichemical transmission and chemical coding of neurons in the digestive tract. Gastroenterology 108: 554-563.

KATSOULIS, S., W. E. SCHMIDT, A. CLEMENS, H. SCHWORER & W. CREUTZFELDT. 1992. Vasoactive intestinal polypeptide induces neurogenic contraction of guinea pig ileum. Involvement of acetylcholine and substance P. Regul. Pept. 38: 155- 164.

GRIDER, J. R., M. B. CABLE, S. I. SAID & G. M. MAKHLOUF. 1985. Vasoactive intestinal peptide as a neural mediator of gastric relaxation. Am. J. Physiol. 248: G73-G78.

GRIDER, J. R. & G. M. MAKHLOUF. 1988. Vasoactive intestinal peptide. Transmitter of inhibitory motor neurons of the gut. Ann. N.Y. Acad. Sci. 527: 369-377.

KATSOULIS, S., A. CLEMENS, H. SCHWORER, W. CRELJTZFELDT & W. E. SCHMIDT. 1993. PACAP is a stimulator of neurogenic contraction in the guinea pig ileum. Am. J. Physiol. 265: G295-G302.

SCH-R, H., R. SCHWARZHOFF, W. CREUTZFELDT& W. E. SCHMIDT. 1991. Characterization of a guanosine-nucleotide-binding-protein coupled receptor for pituitary adenylate cyclase activating peptide on plasma membranes from rat brain. Eur. J. Biochem. 202:

SCHMIDT, W. E., J. SEEBECK, M. HOCKER, R. SCHWARZHOFF, H. SCHAFER, H. FORNEFELD, C. MORYS-WORTMANN, U. R. FOLSCH & W. CREUTZFELDT. 1992. PACAP and VIP stimulate enzyme secretion in rat pancreatic acini via interaction with VIPPACAP-2 receptors: Additive augmentation of CCK-/carbachol-induced enzyme release. Pancreas

SHIVERS, B. D., T. J. GORCS, P. E. GOTTSCHALL & A. ARIMURA. 1991. Two high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions. Endocrinology 128: 3055 -3065.

HASHIMOTO, H., T. ISHIHARA, R. SHIGEMOTO, K. MORI & S. NAGATA. 1993. Molecular cloning and tissue distribution of a receptor for pituitary adenylate cyclase-activating polypeptide. Neuron 11: 333-342.

INAKAKI, N., H. YOSHIDA, M. MIZUTA, N. MIZUNO, Y. FUJII, T. GONOI, J.-I. MIYAZAKI & S. SEINO. 1994. Cloning and functional characterization of a third pituitary adenylate cyclase-activating polypeptide receptor subtype expressed in insulin-secreting cells. Proc. Natl. Acad. Sci. U.S.A. 91: 2679-2683.

ISHIHARA, T., R. SHIGEMOTO, K. MORI, K. TAKAHASHI & S. NAGATA. 1992. Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide. Neuron 8: 3 1 1 -3 19.

164: 567-574.

951-958.

8: 476-487.


17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

SPENCLER, D., C. WAEBER, C. PANTALONI, F. HOLSBOER, J. BOCKAERT, P. H. SEEBURG & L. JOURNOT. 1993. Differential signal transduction by five splice variants of the PACAP receptor. Nature 365: 170- 175.

CHRISTOFI, F. L. & J. D. WOOD. 1993. Effects of PACAP on morphologically identified myenteric neurons in the guinea pig small bowel. Am. J. Physiol. 264: G414-G421.

SCHWORER, H., S. KATSOULIS, W. CREUTZFELDT & W. E. SCHMIDT. 1992. Pituitary adenylate cyclase activating peptide, a novel VIP-like gut-brain peptide, relaxes the guinea- pig taenia caeci via apamin-sensitive potassium channels. Naunyn-Schmiedeberg’s Arch. Pharmacol. 346: 5 1 1 -514.

MARLEY. 1995. Histochemical, pharmacological, biochemical and chromatographic evidence that pituitary adenylyl cyclase activating peptide is involved in inhibitory neurotransmission in the taenia of the guinea-pig caecum. J. Auton. Nerv. Syst. 50:

JIN, J.-G., S. KATSOULIS, W. E. SCHMIDT & J. R. GRIDER. 1994. Inhibitory transmission in taenia coli is mediated by distinct vasoactive intestinal peptide and apamin-sensitive pituitary adenylate cyclase activating peptide receptors. J. Pharmcol. Exp. Ther. 270: 433-439.

GRIDER, J. R., M. B. CABLE, K. N. BITAR, S. 1. SAID & G. M. MAKHLOUF. 1985. VIP relaxant neurotransmitter in taenia coli of the guinea pig. Gastroenterology 89: 36-42.

GRIDER, J. R. & J. R. RIVIER. 1990. VIP as transmitter of inhibitory motor neurons of the gut: Evidence from the use of selective VIP antagonists and VIP antiserum. J. Pharmacol. Exp. Ther. 253: 738-742.

BURNSTOCK, G., T. COCKS, L. KASAKOV & K. H. WONG. 1978. Direct evidence for ATP release from non-adrenergic, non-cholinergic (“purinergic”) nerves in the guinea pig taenia coli and bladder. Eur. J. Pharmacol. 19: 145-149.

SCHWARZHOFF, R., H. SCHW~RER, H. FORNEFELD, C. MORYS-WORTMANN, S. KATSOULIS, W. CREUTZFELDT, U. R. FOLSCH & W. E. SCHMIDT. 1995. Specific monoclonal antibodies neutralize the action of PACAP 1-27 or PACAP 1-38 on intestinal muscle strips. Regul. Pept. 55: 57-67.

HUANG. M., H. SHIRAHASE & 0. P. RORSTAD. 1993. Comparative study of vascular relaxation and receptor binding by PACAP and VIP. Peptides 14: 755-762.

KATSOULIS, S., A. CLEMENS, H. SCHWORER, W. CREUTZFELDT & W. E. SCHMIDT. 1993. Pituitary adenylate cyclase-activating polypeptide is a potent relaxant of the rat ileum. Peptides 14: 587-592.

MUNGAN, Z., A. ARIMURA, A. ERTAN, W. J. ROSSOWSKI & D. H. COY. 1992. Pituitary adenylate cyclase-activating polypeptide relaxes rat gastrointestinal smooth muscle. Scand. J. Gastroenterol. 27: 375-380.

GRIDER, J. R., S. KATSOULIS, W. E. SCHMIDT & J . 4 . JIN. 1994. Regulation of the descending relaxation phase of intestinal peristalsis by PACAP. J. Auton. New. Syst.

COSTA, M. & J. B. FURNESS. 1976. The peristaltic reflex: An analysis of the nerve pathways and their pharmacology. Naunyn-Schmiedeberg’s Arch. Pharmacol. 294: 47-60.

GRIDER, J. R. 1993. Interplay of VIP and nitric oxide in the regulation of the descending relaxation phase of peristalsis. Am. J. Physiol. 264: G334-G340.

GRIDER, J. R. & G. M. MAKHLOUF. 1991. Identification of opioid receptors on gastric muscle cells by selective receptor protection. Am. J. Physiol. 260: G103-Gl07.

SCHWORER, H., W. E. SCHMIDT, S. KATSOULIS & W. CREUTZFELDT. 1991. The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on porcine small intestinal smooth muscle (Abstr.). Naunyn-Schmiedeberg’s Arch. Pharmacol. 343: 103.

SCHWORER, H., A. CLEMENS, S. KATSOULIS, H. KOHLER, W. CREUTZFELDT & W. E. SCHMIDT. Pituitary adenylate cyclase activating peptide is a potent modulator of human colonic motility. Scand. J. Gastroenterol. 28: 625 -632.

MCCONALOGUE, K., J. B. FURNESS, M. A. VREMEC, J. J. HOLST, K. TORN0E & P. D.

31 1-322.

SO: 151-159.


35. BUSCAIL, L., P. GOURLET, P. DE NEEF, D. GOSSEN, A. ARIMURA, A. MIYATA, A. COY, D. H. ROBBRECHT & J. CHRISTOPHE. 1990. Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating peptide) in membranes from the rat pancreatic acinar cell line AR 4-2J. FEBS Lett. 262: 77-81.

DISCUSSION OF THE PAPER

GABRIEL MAKHLOUF (Medical College of Virginia, Richmond): Could I just add a brief comment to this? We have looked at dispersed muscle cells, and PACAP has absolutely no effect on cyclic AMP, whereas VIP stimulates cyclic AMP in those cells. It troubles me to think that you are saying that a pituitary adenylate cyclase activating peptide does not activate adenylate cyclase. This is a system in which it doesn’t, and this receptor is obviously different from the three types of receptors that have been described. So we may anticipate a separate receptor; now that you’re going to look at the literature, perhaps you would take that into consideration. Shouldn’t this receptor be called something else to distinguish it from the three known receptors?

WOLFGANG E. SCHMIDT (Universiby oj’ Kiel, Germany): Thank you for this comment that highlights an intriguing problem. The question whether PACAP is able to stimulate CAMP production in guinea pig taenia coli is still controversial. If there is indeed no stimulatory effect of PACAP on adenylate cyclase, this may imply the existence of a monospecific VIP receptor that does not accept PACAP as a ligand. Although this is not a fascinating hypothesis, there is no definite evidence for the existence of such a receptor. I agree that we should look out for it in molecular terms, but it may be premature to name it right now.

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Role of PACAP in the Regulation of Gastrointestinal Motility