Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent relaxant of the rat ileum

Peptides, Vol. 14, pp. 587-592, 1993 0196-9781/93 $6.00 + .00 Printed in the USA. Copyright © 1993 Pergamon Press Ltd.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Is a Potent

Relaxant of the Rat Ileum

STAVROS K A T S O U L I S , * t I A N D R E A S C L E M E N S , t H A R A L D S C H W O R E R , t W E R N E R C R E U T Z F E L D T t A N D W O L F G A N G E. S C H M I D T * t

*Laboratory of Molecular Gastroenterology, I. Department of Medicine, University of Kiel, Schittenhelmstr. 12, D-2300 Kiel I, Germany and

tDivision of Gastroenterology and Endocrinology, Department of Medicine, University of GOttingen, Robert-Koch-Str. 40, D-3400 Ggttingen, Germany

Received 19 October 1992

KATSOULIS, S., A. CLEMENS, H. SCHWORER, W. CREUTZFELDT AND W. E. SCHMIDT. Pituitao, adenylate ~\vclase activating polypeptide (PACAP) is a potent relaxant of the rat ileum. PEPTIDES 14(3) 587-592, 1993.--The effect and mode of action of pituitary adenylate cyclase activating polypeptide (PACAP) were studied in rat ileal strips. PACAP relaxed, concentration dependently, rat ileum and was 50 times more potent than the structurally related vasoactive intestinal polypeptide (VIP). The inhibitory action of PACAP was not modified by TTX, omega-conotoxin, adrenergic, or ganglionic blockade, antagonists of adrenoreceptors and muscarinic receptors, indicating a direct myogenic effect probably through specific PACAP receptors. The lack of cross-tachyphylaxis between PACAP and VIP suggests that both peptides act by activation of distinct receptors. Structure- function analysis revealed that the N-terminal region of the PACAP molecule is crucial for biological activity.

PACAP fragments PACAP receptor subtypes Nonadrenergic/noncholinergic neurotransmitter

Vasoactive intestinal polypeptide

PITUITARY adenylate cyclase activating polypeptide (PA- CAP) was originally isolated from ovine hypothalami on the basis of its ability to stimulate adenylate cyclase in rat anterior pituitary cells (20). PACAP exists in two bioactive molecular forms (21), PACAP(1-38) and PACAP(1-27), comprising 38 or 27 amino acids residues, respectively. Both peptides are C- terminally amidated, and PACAP(1-27) constitutes the N-terminal portion of PACAP(1-38) (21). cDNA cloning of PACAP precursors in sheep, rat, and man revealed that the predicted PACAP sequence is identical in all three species (15,25). PA- CAP is a novel member of the secretin/glucagon/VIP family of peptides, exhibiting closest structural homology to VIP (about 68%). Binding studies demonstrated the existence of at least two PACAP receptor subtypes. Specific binding sites for PACAP (PACAP- 1 receptors) not accepting VIP as high-affinity ligand have been identified in the adrenal medulla (43), hy- pothalamus (8), other brain regions (8,17,19,30,39), a neuroblastoma cell line (3), AR 4-2 J cell membranes (1), and testis (8). In contrast, splenocytes (38), liver (28), lung (17), pancreas

(18,31,32), duodenum (8), and other peripheral organs (8) express binding sites for PACAP that interact with VIP at similar affinity (VIP/PACAP-2 receptors).

The presence of PACAP in the autonomic nervous system and smooth muscle layers of the gastrointestinal tract of various species, including rat and man (37), indicates a possible role of this peptide in the regulation of gastrointestinal motility. Recently, we demonstrated that PACAP contracts guinea pig ileum in vitro via the release of acetylcholine (ACh) and a noncholinergic/nonadrenergic neurotransmitter (13). In the rat gastrointestinal tract (23), guinea pig taenia coli (34), and porcine small intestine (33), relaxant responses to PACAP have been reported. The present study investigates the effect and mode of action of PACAP in the isolated rat ileum in vitro. Results were compared to the action of the structurally related VIP. To identify which part of the PACAP molecule is re- sponsible for biological activity, several PACAP fragments and one chimeric molecule combining the N-terminal part of PA- CAP and C-terminal part of VIP were tested.

Requests for reprints should be addressed to Dr. Stavros Katsoulis, Laboratory of Molecular Gastroenterology, I. Department of Medicine, Christian-Albrechts University of Kiel, Schittenhelmstr. 12, D-2300 Kiel 1, Germany.

587

588 KATSOULIS ET AL.

METHOD

Wistar rats of either sex (150-200 g) were killed by a blow on the head and exsanguination via the carotid arteries. Two intact (whole wall) segments of 1.5-2.0 cm of the ileum, 5-10 cm proximal to the ileo-cecal junction, were quickly removed and placed vertically in longitudinal orientation in 3-ml ther- mostatically controlled (37°C) organ baths, containing contin- uously oxygenated (5% CO2 and 95% 02) Krebs solution of the following composition (raM): NaCI 118, CaC12 2.5, MgSO4 1.18, NaHCOs 25, KH2PO4 1.18, and glucose 5.55. Muscle strips were mounted under a resting tension of 1.0 g and equilibrated for 30-60 rain until spontaneous phasic contractions of stable am- plitude occurred. The organ bath solution was renewed in 15- 20-rain intervals. Experiments were begun when reproducible responses to ACh (100 nM) were obtained. Muscular activity was measured isometrically with Grass FT03 force-displacement transducers connected to a Grass multichannel polygraph (Model 79D).

In a first series of experiments, concentration-response curves for PACAP, PACAP fragments, and VIP were performed by adding increasing concentrations of peptides in a noncumulative manner at intervals of 20-30 rain. Under these conditions, no tachyphylaxis was observed. Only one concentration-response curve was carried out on each strip. At the end of each experiment, a maximally effective concentration of papaverin (50 uM) was added to the bathing medium and left in contact with the tissue until maximal relaxatory response occurred. Potencies (ECs0) were calculated by interpolation from the appropriate dose-response curve. Effectiveness of peptides refers to the maximal response produced by papaverin (50 uM).

In a second series of experiments, the relaxing responses to maximally effective concentrations of PACAP (120 nM) and VIP (1.2 ~zM) were established in the absence and presence of various drugs. The preparations were incubated with the drug to be tested for 20-30 rain before the second addition ofpeptide.

In order to investigate cross-tachyphylaxis between PACAP and VIP in a third series of experiments, the relaxing responses to maximally effective concentrations of PACAP (120 nM) and VIP ( 1.2 uM) were determined without and after desensitization to the relaxant responses of VIP or PACAP, respectively. De- sensitization to the relaxing response of PACAP or VIP was obtained by administering the maximally effective concentration of VIP ( 1.2 ~M) twice or PACAP ( 120 nM) thrice, respectively, at 15-rain intervals without washing out. The second or third application of peptide was almost without effect, indicating tachyphylaxis. Responsiveness to PACAP or VIP in preparations desensitized to either peptide was assessed within 2-4 min from the last addition of the respective peptide, without washing out.

All data are expressed as mean _+ SEM of n experiments. Bonferroni's modification of Student's t-test for unpaired data adapted for multiple comparisons as described by Wallenstein (4 I) was used for statistical evaluation of differences. A p-value of less than 0.05 was considered to indicate significant differences.

Drugs and Peptides

Vasoactive intestinal polypeptide (V1P), omega-conotoxin GVIA, apamin, hexamethonium chloride, acetylcholine chloride, atropine sulphate, arterenol hydrochloride, guanethidine, tetrodotoxin, 4-aminopyridine, tetraethylammonium, propranolol, and papaverin were used (all from Sigma Chemie, Munich, Germany). Phentolamine was a gift from Ciba Geigy Switzer- land; PACAP and its fragments were synthesized in our laboratory, as described previously (30). Synthetic peptides were

purified by reverse-phase HPLC. The quality of the synthetic peptides was analyzed by time-of-flight mass spectrometry.

RESULTS

Effects ~?[ Various Drugs on the Inhibitory Action q/'PACAP and VIP in Rat Ileal Strips

Pituitary adenylate cyclase activating polypeptide (5-120 nM) and VIP (0.15-1.2 #M) caused a concentration-dependent relaxation of rat ileal strips (Fig. 1). The relaxant effects of PACAP (120 nM) and VIP (1.2 #M) were not modified by atropine (1 ~M) or a combination of phentolamine (10 #~¢') and propranolol ( 10 uM), while those effects produced by their respective agonists ACh (100 nM) and noradrenalin (10 #M) were abolished. TTX (1 ~M), an inhibitor of nerve conduction, and omega-conotoxin (100 nM), which selectively blocks N-type voltage-sensitive calcium channels, also failed to inhibit the relaxant effect of both peptides. Preincubation of the tissues with guanethidine (5 #M) and hexamethonium (100 uM), which inhibited adrenergic or ganglionic neurotransmission, respectively, did not significantly alter the effectiveness of PACAP (120 nM) and VIP (1.2 uM). To characterize the signal-transduction pathways that may mediate the inhibitory effects of PACAP and VIP, we examined the effects of both peptides in the presence of the potassium channel blockers apamin, tetraethylammonium (TEA), and 4- aminopyridine (4-AP). Rat ileal strips exposed to the bee venom apamin ( 100 nM) or TEA (300 taM) showed unaffected relaxant responsiveness to PACAP (120 nM) and VIP (1.2 uM). The maximally relaxant action of PACAP (120 nM) was slightly, but

PACAP

30 nM 120nM 50pM

Papaverin

VIP

X

150 nM 300 nM

x x

600 nM 1.2 pM 50 pM

Papaverln

0.2g i 2 min

FIG. 1. Typical tracings illustrating the dose-dependent relaxant effect of PACAP and V1P on the rat ileum. At the end of each experiment a maximal relaxation was induced by papaverin (50 uM). Doses are indicated as final concentrations of substances in the organ bath. PACAP and VIP were applied at 20-rain intervals. X indicates washout.

PACAP: A P O T E N T RAT ILEUM R E L A X A N T 589

TABLE 1

EFFECTS OF VARIOUS D R U G S ON THE RELAXANT ACTION OF PACAP AND V1P ON RAT ILEUM

Relaxation in the Presence of Drug (%)

Drugs PACAP VIP ( M)* ( 120 nM) ( 1.2 uM )

None 108 +_ 10 109 + 9 TTX(10 6) 100+_ 6 116_+ l0 Omega-conotoxin (10 7) 105 _+ 9 109 _+ 9 Hexamethonium (10 4) 90 _+ 10 90 _+ 8 Guanethidine (5 × 10 6) 90 _+ 9 106 _+ 6 Atropine(10 6) 97_+ 6 88_+ 6 Phentolamine (10 s)

+ propranolol (10 -5) 105 _+ 10 100 _+ 10 Apamin (10 7) 81 _+ 10 96 _+ 10 TEA (3 × 10 4) 95_+ 10 91+ 4 4-AP(3× 10 4) 67+ 7t 101_+ 5

The results are mean _+ SEM of 5-12 individual deter- minations. The effects of PACAP (120 nM) or VIP (1.2 uM) in the absence of drug were regarded as 100%. The incubation time of drugs with the tissue was 15-20 min. TTX, tetrodotoxin; TEA, tetraethylammonium; 4-AP, 4- aminopyridine.

* Molar concentration. t Indicates a value for p < 0.05.

nevertheless significantly, reduced to 67% (p < 0.05) in the presence of 4-AP (300 uM). In contrast, the maximal relaxation induced by VIP ( 1.2 uM) was not influenced by 4-AP (300 uM). These results are summarized in Table 1.

To investigate whether PACAP and VIP elicit relaxation of rat ileal strips through activation of the same receptor sites, we used the phenomenon of desensitization. Muscle strips desensitized with VIP remained fully responsive to PACAP and vice versa (Fig. 2).

Concentrati(m-Response Curves (?/PAC4P. Its Fragments, and VIP

Concentration-response curves for relaxant effect of PACAP, various PACAP fragments, and VIP obtained in rat ileal strips are illustrated in Fig. 3. A more complete description of results, including potencies (ECs0), relative potencies, effectiveness, and relative effectiveness, is presented in Table 2.

Pituitary adenylate cyclase activating polypeptide (5-120 nM), [Cys34]PACAP(I-34) (12-216 nM), [Asn24,Ser25,Ile26, Leu27,Asn28]PACAP(1-28) [hybrid peptide comprising of the first 23 amino acid residues of PACAP, extended by the C-terminal part (position 24-28) of VIP] (4-192 nM), PACAP(I -27) (7-180 nM), PACAP(1-23) (60 nM-3.24 uM), and VIP (0.15- 1.2 ~M) relaxed rat i leum in a dose-dependent manner. The PACAP fragments (18-38), (3-25), and (3-19) were without any effect at concentrations up to 10 uM. The effectiveness of the PACAP fragments, expressed as percent of the maximally relaxant response induced by papaverin (50 ,M) , showed nearly the same order compared to PACAP, and was of the range of 79-90%. VIP (efficacy of 60 + 4%) was slightly, but not significantly, less effective as compared to PACAP (efficacy of 79 _+ 4%). On the basis of ECs0 values, PACAP and [Asn24,Ser25,11e26,LeuZT,AsnZS]PACAP(1-28) exhibited similar potency to induce relaxation of rat ileum. [Cys34]PACAP(I-34)

PACAP (120 nM) ~i PACAP (120 nM) + delenlltization with VIP (1,2 ~M)

PACAP (120 nM) + desensitization with PACAP (120 nM) x 2 o i

i VIP (1.2 .M)

i VIP + desensitization with VIP (1.2 ~M) VIP (12 ~M) + deserlsitization with PACAP (120 nM)

FIG. 2. Influence of desensitization with V1P or PACAP on the maximal relaxant response induced by PACAP (120 nM) (left part of the panel) or by VIP ( 1.2 ~M) (right part of the panel). Data are expressed as percent of the maximal response, obtained by PACAP (120 nM) or V1P (1.2 uM) in the absence of desensitization. Columns represent means of eight individual experiments from eight rats. Vertical bars indicate SEM *, p < 0.05 compared with the response without desensitization. The desensitization protocol used is described in detail in the Method section.

and PACAP(1-27) were moderately less potent compared to PACAP (relative potency of both peptides = 35%, p < 0.05), while PACAP(1-23) showed a marked decrease in potency (relative potency = 10%). The structurally related VIP was approx- imately 50 times less potent than PACAP.

DISCUSSION

Our results show that PACAP, as well as the structurally related peptide VIP, induces dose-dependent relaxant effects in isolated rat ileal strips. This action of PACAP and VIP is con- sistent with its action in other preparations of intestinal (23,33- 35), tracheal (2), and vascular (2,42) smooth muscle.

It is likely that the inhibitory action of PACAP on the rat ileum follows a direct effect of the neuropeptide on inhibitory PACAP receptors, located on the smooth muscle membrane. Several arguments substantiate this conclusion. The PACAP-

• PACAP V [Cysa4I-PACAP (1-34)

100 [] IAnni4,3erIS,IIe|e,LouIV.AonleI-P&CAP (1-28)

~,~ O PACAP (I-27) at411 80 •• vIpPACAP (I-23) d~'~ ''~I~ / ~

k 60 _ -.

.I~ 40

zo ,~ o

0 . 7 / /A ld I I I I I I I I [ h L l i l l l d t I I I 1111 [ I I I I IH I [

- 9 - 8 - '7 - 6 - 5

peptide log (M)

FIG. 3. Concentration-response curves for the relaxant action of PACAP several PACAP fragments, and VIP in the isolated rat ileum, Relaxant response is expressed as percentage of maximal relaxation induced by papaverin (50 ~M). Each point represents the mean _+ SEM of 8-12 experiments.


TABLE 2

A COMPARISON OF THE POTENCIES AND EFFICACIES OF PACAP, ITS FRAGMENTS. AND VIP ON THE RELAXATION OF RAT ILEUM

Maximal ECso Relaxation Relative Relative

Peptides (nM) (%) Potency Efficacy

PACAP 6 _+ 1 79 ± 4 100 100 [Cys34]PACAP(l-34) 17 _+ 4 85 ± 5 35 108 [Asn24,Ser25,11e26,Leu27,Asn28]PACAP(1-28) 9 _+ 2 90 _+ 4 67 114 PACAP(I-27) 17+ 1 89,+7 35 113 PACAP(I-23) 61 _+ 11 81 _+ 3 10 103 PACAP(3-25) 0 0 0 0 PACAP(3-19) 0 0 0 0 PACAP(18-38) 0 0 0 0 VIP 277 _+ 35 60 ,+ 4 2 76

Data are expressed as mean _+ SEM of 8-12 experiments. Maximal relaxation (%) refers to the maximal effect produced by each peptide compared with the maximal effect obtained by papaverin (50/~M). Potencies (ECs0) were calculated by linear regression analysis. Relative potency and relative efficacy are expressed as percent, compared with PACAP set as 100%.

induced relaxation was preserved in the presence of TTX, omega- conotoxin, hexamethonium, and guanethidine, which indicated that the relaxation was not of neuronal origin. The relaxation in response to PACAP was neither antagonized by adrenergic nor by muscarinic receptor blockade, suggesting that the occu- pation of their receptor sites is not involved in this response. The same conclusions can be drawn tbr the inhibitory effect of VIP on rat ileum, since pharmacological analysis revealed identical results as discussed for PACAP. However, PACAP was fig tyfold more potent and slightly (19%) more effective in eliciting relaxation of rat ileum, as compared to VIP. Other reports studying the effects of PACAP and VIP claimed either equal potency of both peptides (2,32,34) or enhanced potency of PA- CAP over VIP (1,13,20,23,42).

Since PACAP is exclusively present within the intrinsic neuronal system of the mammalian (rat) gut (37) and exogenous PACAP relaxes rat ileum, it can be hypothesized that PACAP may act as an inhibitory nonadrenergic/noncholinergic (NANC) neurotransmitter, like the structurally related VIP. Further studies are needed before definite conclusions can be drawn on the role of PACAP in the regulation of gut motility.

Although PACAP and VIP relax smooth muscle preparations of various species (2,10,11,23,33-35,42), both peptides induce neurogenic contraction in the guinea pig ileum through release of ACh and substance P ( 13,14). Similar differential effects have been reported for other gastrointestinal peptides: the gut-brain peptide neurotensin relaxes rabbit (12) and rat (16) ileum, but contracts guinea pig ileum (22) and rat fundus (27), thus indicating region- and species-specific actions.

The inhibitory action of PACAP and VIP on rat ileal strips was further analyzed in the presence of potassium channel blockers, since potassium channels are involved in the mecha- nisms of smooth muscle relaxation (6,7,29). Both apamin and TEA did not alter the inhibitory effect of PACAP and VIP. In contrast, 4-AP, which blocks various types of K + channels (29,40), reduced slightly, but significantly, only the inhibitory effect of PACAP, without influencing the response to VIP. A further characterization of the 4-AP-sensitive K + channel, which seems to be coupled to the PACAP receptor, is not possible from the present experiments. In contrast to our results obtained in the rat ileum, the relaxant effect of PACAP in the guinea pig

taenia coli was sensitive to apamin (34). Accordingly, different signal-transduction pathways may mediate the inhibitory effect of PACAP in different species and/or gut regions. Because of the lack of potent and specific PACAP and VIP receptor antagonists, we used the method of desensitization to examine possible interactions between PACAP and VIP at the receptor level. This approach has been successfully employed to characterize the receptor types involved in the contractile effect of PACAP and VIP in guinea pig ileum (13). Induction oftachyphylaxis to PA- CAP did not modify the effect of VIP. Similarly, the effect of PACAP preserved in rat ileal strips desensitized to VIP. Ac- cording to the classification of Shivers et al. (36) and Schmidt et al. (32), two types of PACAP receptors can be distinguished: the PACAP-1 (PACAP-preferring) receptor shows high affinity to PACAP, but does not recognize VIP as ligand (4,8,17,26,30,36), whereas VIP/PACAP-2 receptor binds PACAP and VIP equipotently (8,17,28,32,36). The latter has been previously designated "high-affinity VIP receptor" (32,36). A third receptor type that preferentially binds VIP compared to PACAP (a monospecific VIP receptor) has not yet been identified. How- ever, the results from the present study raise the possibility that such a monospecific VIP receptor might be involved in the inhibitory action of VIP in rat ileum. This speculation is supported by the following observations:

1. the absence ofcross-tachyphylaxis between PACAP and VIP, in particular the preservation of the relaxant effect of VIP after desensitization with PACAP, and

2. different sensitivity of VIP- and PACAP-induced relaxation in response to 4-AP.

Further studies, including binding experiments, are needed to reinforce this hypothesis. The existence of a monospecific VIP receptor, distinguishable from the VIP/PACAP-2 receptor, has also been postulated for the relaxant effect of VIP in the guinea pig taenia coli (34). Apamin, a selective inhibitor of calcium- activated potassium channels, abolished the relaxant effect of PACAP, whereas the relaxant effect of VIP remained unaffected (34). The effect of VIP remained unchanged in the combined presence of PACAP and apamin. On the basis of these results, Schw6rer et al. (34) suggested the existence of a VIP-preferring receptor that is not activated by PACAP.

PACAP: A P O T E N T RAT ILEUM R E L A X A N T 591

The results from our structure-activity experiments revealed the importance of the N-terminal part of PACAP for its biological activity. Removal of two or more amino acids from the N-ter- minus leads to a loss of bioactivity. This observation is in agree- ment with results obtained by structure-function experiments in ileal smooth muscle of guinea pig (13) and binding studies performed on plasma membranes from rat brain (30) and rat pancreatic acini (31,32). Interestingly, the omission of histidine in position 1 resulted in a compound with substantially reduced, but not abolished, binding activity (9). The C-terminally trun- cated derivatives [CysS4]PACAP(1-34), PACAP(1-27), PA- CAP(I-23) , and the chimeric PACAP-VIP compound [Asn 24, Ser25,11e26,Leu27,Asn28]PACAP(1-28) were able to elicit a similar maximal response compared to PACAP. The potency to induce relaxation of [Asn24,Ser~5,Ile26,Leu27,Asn~8]PACAP(1-28) was comparable to that of PACAP, while [Cys34]PACAP (1-34) and PACAP(I-27) showed slightly decreased potencies; PA- CAP(1-23) exhibited a markedly diminished potency in comparison to PACAP.

Thus, it appears that the C-terminal part of the PACAP molecule contributes mainly to the affinity of PACAP receptors.

The data derived from our structure-activity investigation con- firm previous reports concerning structural requirement of PA- CAP receptors in ileal smooth muscle of guinea pig (13), plasma membranes from rat brain (30), and rat pancreatic acini (31,32). Interestingly, similar structure-activity relationships have been reported for VIP on its ability to bind on guinea pig and human membrane homogenates of the lung and to relax tracheal smooth muscle of guinea pig (24).

ACKNOWLEDGEMENTS

The authors express their gratitude to G. Mundkowski and Dr. C. Morys-Wortmann for valuable help with peptide synthesis, and to Dr. B. Zimmermann and Dr. K. Eckart, Max-Planck Institute for Experi- mental Medicine, for support with mass spectrometry. Parts of this study belong to a M.D. thesis (A.C.); some results have been published in abstract form (5). This work was supported by the Deutsche Forschungs- gemeinschaft (Schm 805/4-1), the German-Israeli Foundation for Sci- entific Research and Development (GIF project 1-79-063.2/88), the Eli Lilly European Gastroenterology Award 1990 (W.E.S.), and the Dr. Norbert-Henning Award for Gastroenterology 1992 (W.E.S.).

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Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent relaxant of the rat ileum