Characterization of binding sites for VIP-related peptides and activation of adenylate cyclase in developing pancreas VALERIE LE MEUTH, NICOLE FARJAUDON, WAFA BAWAB, ERIC CHASTRE, GABRIEL ROSSELIN, PAUL GUILLOTEAU, AND CHRISTIAN GESPACH Institut National de la Sank et de la Recherche Mkdicale, U55, Hpital Saint-Antoine, 75571 Paris Cedex 12; and Laboratoire du Jeune Ruminant, Institut National de la Recherche Agronomique 35042 Rennes, France

LE MEUTH, VALERIE, NICOLE FARJAUDON, WAFA BAWAB, ERIC CHASTRE, GABRIEL ROSSELIN, PAUL GUILLOTEAU, AND CHRISTIAN GESPACH. Characterization of binding sites for VIPrelated peptides and activation of adenylate cyclase in developing pancreas. Am. J. Physiol. 260 (Gastrointest. Liver Physiol. 23): G265-G274, 1991.-HPLC-purified 1251-labeled vasoactive intestinal peptide (VIP) bound in a specific, saturable, and reversible manner to pancreatic plasma membranes isolated from newborn calves, from milk-fed calves at 28 and 119 days, and from weaned calves at 119 days. A series of VIP analogues, including pituitary adenylate cyclase-activating polypeptide (PACAP), displaced 1251-VIP binding and activated adenylate cyclase in the same order of relative potency: PACAP> helodermin > VIP, PACAP> PHM (human peptide with NH2-terminal histidine and COOH-terminal methionine amide). At maximally effective concentrations, these five peptides produced the same two- to threefold increase of adenylate cyclase activity in pancreatic membranes from newborn and %-day-old calves, and fourfold in ruminant or preruminant animals at 119 days. The activation constant for PACAPranged from 0.1 to 0.34 nM throughout the postnatal development. Helospectin I and II were three times less potent than VIP in inhibiting 1251-VIP binding. At concentrations up to 0.1 PM, secretin, rat and human growth hormone-releasing factors, glucagon, oxyntomodulin, the truncated form of glucagon-like peptide-l lacking the 6 NH2-terminal amino acid sequence (TGLP-1), GLP-2, gastric inhibitory peptide, gastrin, CCK, and insulin had no effect on binding. Scatchard plots from 28and 119-day-old calves were compatible with the presence of two classes of 1251-VIP binding sites: one with a high affinity for VIP and a low binding capacity (& = 0.11-0.4 nM, B,,, = 66-174 fmol/mg protein) and the other with a low affinity and high binding capacity. At birth, only one class of binding sites was observed (Kd = 0.4 nM, B,,, = 858 fmol/mg protein). The covalently cross-linked PACAP-preferring 1251-VIP binding site is a glycoprotein of 55 kDa with higher sensitivity to PACAP vs. helodermin and VIP. Our results suggest that calf pancreatic functions might be regulated at an early stage of postnatal development by PACAP receptors linked to CAMP generation.

development of calf pancreas; PACAP-preferring sites; adenylate cyclase activation; pharmacological molecular identification; pancreatic enzymes

VIP binding properties;

and functional maturation of the alimentary canal is associated with the cellular proliferation, differentiation, and organogenesis that occur during fetal and postnatal life (6). Exocrine and endocrine secretions are regulated by various hormones, neurome-

THE MORPHOLOGICAL

0193~1857/91

$1.50 Copyright

diators, paracrine agents, and growth factors after activation of the membrane receptors coupled to transduction systems. This coupling leads to the elaboration and amplification of intracellular messengers (15). In mammals, the digestive tract adapts to milk intake at birth and subsequently to weaning and an adult-type diet. We previously observed prominent differences in the functional activity and molecular components of the histaminergic and peptidergic receptors in the gastrointestinal mucosa during fetal and postnatal development in humans and rats (7, 14, 15). In rat gastric epithelia, these differences were found to be temporally associated during development with the milk diet, weaning, or precocious weaning. In the rat intestine, marked changes in the activity of the receptors for vasoactive intestinal peptide (VIP) receptor activity were observed during ontogenic development (7). These changes are reflected by a gradual reduction in the generation of adenosine 3’,5’-cyclic monophosphate (CAMP) in intestinal epithelial cells, coupled with a decrease in the affinity of the VIP receptor. All these events may be due to modification of the molecular architecture of the intestinal VIP receptors, which were identified as autoradiographic bands of 65 kDa in 19-day-old rat fetuses, and as 76-kDa bands in adult rats. VIP stimulates pancreatic secretion of amylase, water, and bicarbonate (40), and VIPergic fibers are present in the pancreas (25). Stimulation by VIP is mimicked by secretin and other structurally related peptides such as helodermin (10, 35). Previous studies demonstrated that rat and guinea pig pancreatic acinar cells possesstwo classesof receptor activated by VIP, the porcine peptide with NH2-terminal histidine and COOH-terminal isoleutine (PHI), and by secretin (3, 8, 13, 24, 30, 36, 44, 45). One class has been designated as “VIP-preferring” receptors because of their high affinity for VIP and lower affinity for PHI and secretin. The other class consists of “secretin-preferring” receptors, which have a high affinity for secretin and a lower affinity for VIP and PHI. Occupation of these receptor sites is associated with increases in cellular CAMP and amylase secretion. Raufman et al. (35) have shown that like VIP, PHI, and secretin, Gila monster lizard venom interacts both with the VIP-preferring receptors and secretin-preferring receptors. This venom, which contains the secretin-related peptides helodermin and helospectin (34, 43), was

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found to enhance amylase secretion and CAMP levels in guinea pig and rat pancreatic acini (10, 35). On the other hand, the presence of highly specific secretin receptors has been recently demonstrated in human pancreatic membranes (38). Robberecht et al. (38) suggested that the human pancreas also possesses another set of “helodermin-preferring” receptors activated by VIP and PHI but not by secretin. In agreement with this, VIP is a weak stimulator of adenylate cyclase, bicarbonate, and amylase secretion in humans (28, 38). Taken together, these reports reveal the complexity of the expression of membrane receptors activated by the secretin family of peptides in the mammalian pancreas (3, 8, 13, 19, 23, 24, 29,30,33,36,41,44,45). Moreover, the novel neuropeptide PACAP was recently isolated from ovine hypothalamus, showing a 68% amino acid sequence homology with porcine VIP in the NH2-terminal region (31). This peptide with 38 residues stimulates adenylate cyclase in rat anterior pituitary cells and so was designated the pituitary adenylate cyclase-activating polypeptide. PACAP contained a Gly-Lys-Arg sequence at positions 28-30, suggesting the presence of a shorter amidated peptide with 27 residues, PACAP(20, 31). So far, characterization of VIP pancreatic receptors has been confined to identifying their binding sites and determining their pharmacological, functional, and molecular properties, and no information was available about the ontogenic development of the membrane receptors activated by VIP and its related peptides in the pancreas. The objective of the present study was therefore to investigate the binding of l’“I-labeled VIP in calf pancreatic membranes prepared at the following stages of postnatal development: 1) at birth, 2) in milk-fed calves (preruminants) at 28 and 119 days, and 3) in weaned calves at 119 days (ruminants). For this purpose, we first purified monoiodinated 12’I-VIP using high-performance liquid chromatography (HPLC) and studied its binding to calf pancreatic membranes. Second, the pharmacological specificity of the VIPergic sites was evaluated by comparing the ability of VIP and its related peptides to inhibit membrane binding to VIP. Third, the molecular components of the proteins binding 12”1-VIP were identified by covalent cross-linking of the label to its receptors in calf pancreatic membranes using the bifunctional reagent dithiobis-succinimidyl propionate (DSP) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Last, adenylate cyclase activation induced by VIP was compared with that induced by its naturally occurring analogues. Part of this work has already been published as an abstract (1). METHODS

Animals. Newborn Holstein-Friesian male calves were provided by the Station de Recherches sur la Vache Laitiere (Institut National de la Recherche Agronomique, Saint-Gilles, France). The animals were divided into four groups: those killed at birth and at 28 days of postnatal life and preruminants and ruminants killed at 119 days. The calves killed at birth were not given any food. Those in the three other groups were fed two colostrum meals (25 g/kg) during the first 2 days of life

AND

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and then a milk substitute with a base of spray-dried skim milk and tallow containing 25% crude protein, 22% fat, 43% lactose, 3% starch, and 7% minerals. Because the calves in the second and third groups were exclusively milk-fed, they were maintained at the preruminant stage until they were killed at 28 or 119 days. The amount of dry matter in their feed was progressively increased from 370 to 2,680 g/day between days 7 and 119. From day 29, the ruminant calves in the fourth group were given, ad libitum, water, hay, and a commercial concentrate comprising 22% crude proteins, 2% fat, 14% cellulose and nitrogen-free extract, 52% starch, and 10% minerals. As the amount of milk substitute was gradually reduced to nothing between days 29 and 56, group 4 calves were functional ruminants from days 57 to 119. Preparation of pancreatic plasma membranes. After the calves were killed, the pancreas was rapidly excised, dissected from the adipose tissue, weighed, and cut into 3- to 5-mm fragments. The tissue was then homogenized at 4°C with a polytron in 10 mM tris( hydroxymethyl)aminomethane (Tris) . HCl buffer (20% wt/vol) containing 1 mM EDTA, 30 mM NaCl, and 5 PM phenylmethylsulfonyl fluoride (PMSF), pH 7.5, using three bursts of 10 s each. The resulting homogenate was filtered through nylon gauze and centrifuged for 10 min at 600 g and 4°C in a Sorvall RC-2B apparatus (Newtown, CT). Plasma membrane-enriched particles were obtained from this supernatant centrifugation for 30 min at 25,000 g and 4°C. Membranes were stored frozen at -80°C until use. Association and dissociation of 1251-VIP in calf pancreatic plasma membranes. Porcine VIP was labeled with 1251at the tyrosine residues in positions 10 and 22 by the chloramine-T method and purified by HPLC, as previously described (7). After separation, experiments were conducted on the peak containing 1251-VIP monoiodinated at tyrosine 10. Conditions of apparent equilibrium for 12”1-VIP binding were obtained by incubating pancreatic plasma membranes (30 pg protein/tube) at 30°C for 60 min in 250 ~1 of 25 mM Tris HCl buffer containing 1% bovine serum albumin (BSA), 1 mg/ml bacitracin (TBB buffer, pH = 7.5), and 50 pM 12”1-VIP alone or combined with either native VIP or its related peptides. Membrane-bound 12”1-VIP was separated at 4°C from free 12”1-VIP by centrifugation of duplicate ZOO-p1 aliquots at 25,000 g for 30 min through 1.25 ml of 25 mM Tris . HCl buffer containing 1% BSA. The resulting pellets were washed with 1 ml of Krebs-Ringer phosphate buffer containing 10% sucrose. The bottom of the microfuge tube was then cut off and counted in a Packard autogamma counter. Specific binding was calculated as the difference between the amount of 12”1-VIP bound in the absence of unlabeled VIP (total binding) and in the presence of 0.1 FM unlabeled VIP (nonspecific binding). After measurement of binding for 60 min at 30°C (i.e., the association step), the dissociation of 1251-VIP from plasma membranes was induced at 37°C in TBB buffer (spontaneous dissociation) or after the addition of VIP or helodermin, either alone or combined with 0.1 mM GTP (induced dissociation). The time zero to study the dissociation of ‘““I-VIP was determined in membranes diluted and centrifuged at 4°C after the association step. l

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Degradation of 1251-VIP and its binding sites. To evaluate the degradation of the ligand and its binding sites during the association step, pancreatic plasma membranes and 1251-VIP were preincubated at 30°C for 120, 90, 60, 30, 15, or 0 min in the binding assay buffer alone or in buffer containing the following protease inhibitors: PMSF (40 pg/ml), soybean trypsin inhibitor (STI) at 0.24 pg/ml, leupeptin (0.24 pg/ml), and pepstatin (4 pg/ ml). To assessreceptor degradation, preincubated membranes were then compared with fresh membranes in their capacity to bind 1251-VIP for 60 min at 30°C. The degradation of 12”1-VIP was measured at 30°C after the association step and under the conditions described above. The free ‘““I-VIP was separated and tested for its binding activity using fresh pancreatic plasma membranes. Covalent affinity labeling of 1251-VIP binding sites. About 100 pg membrane proteins were incubated for 60 min at 30°C in 1.25 ml TBB buffer containing 0.4 nM 12”1-VIP alone or combined with various concentrations of VIP and its related peptides. Membrane-bound 1251VIP was collected after centrifugation for 30 min at 25,000 g and 4°C. The pellets were washed with 1.5 ml Krebs-Ringer phosphate buffer and resuspended in 1 ml of 60 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES buffer, pH 7.5) by serial passages through a 26-gauge needle mounted on a l-ml syringe. The covalent labeling of the VIP binding sites by 1251VIP was initiated by adding 20 ~1 of 50 mM DSP in dimethyl sulfoxide, yielding a final concentration of 1 mM DSP (5). After 20 min incubation at 4°C the reaction was stopped by adding 500 ,ul of ice-cold 60 mM HEPES buffer (pH 7.5) containing 60 mM ammonium acetate as a reagent quench. Membranes were collected after centrifugation for 20 min at 25,000 g and 4°C and were solubilized for 30 min at 60°C in 60 mM Tris HCl buffer containing 10% (vol/vol) glycerol, 0.001% bromophenol blue, and 3% SDS. Nonspecific 12”1-VIP binding, evaluated in the presence of 0.1 PM VIP, did not exceed 30% of total binding. The solubilized membrane samples underwent electrophoresis on a 12% polyacrylamide slab gel. The gels were then fixed, stained with Coomassie Brilliant Blue R 250, destained, and dried. Kodak films (XAR) were exposed to the gels for 5-15 days using an intensifying screen. The apparent molecular mass of the radioactive bands was evaluated using a series of marker proteins of known molecular mass (BioRad, Paris, France). The radioactivity incorporated into the corresponding bands was measured by evaluating the relative absorbance, which was analyzed with a Biocom program (Paris, France). Detergent solubilization and lectin affinity chromatography. Affinity ‘251-VIP-labeled membranes were incubated for 2 h at 4°C in buffer containing 50 mM HEPES, 0.16 M NaCl, 1 mM MgC12, 1 mM CaC12,150 PM PMSF, 5 pg/ml leupeptin, and 1% Nonidet P-40 (NP-40) (pH 7.5). Insoluble material was removed by centrifugation at 4°C and 25,000 g for 20 min. Before use, the wheat germ agglutinin (WGA)-sepharose column was thoroughly washed with 100 ml of the same buffer containing 0.1% NP-40. The soluble fraction was applied to the column and eluted with buffer until the radioactivitv

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G267

returned to baseline levels. Buffer containing 0.5 M of N-acetylglucosamine as the complementary sugar was then added, and the column flow continued until the radioactivity returned to baseline. Fractions of 0.4 ml were collected and monitored for radioactivity in a gamma-counter. Assay of adenylate cyclase in plasma membranes. The standard incubation medium (final volume, 250 ~1) contained in 20 mM Tris HCl buffer (pH 7.5) 1 mM ATP, 5 mM MgC12, the ATP-regenerating system (10 mM creatine phosphate and 1 mg/ml creatine kinase), 0.4 mM 3-isobutyl-1-methylxanthine, 1 mM ethylene glycolbis( ,&aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 400 pg/ml bacitracin, 0.8% BSA (wt/vol), 20 PM GTP, and test substances. The reaction was initiated by the addition of membrane-bound adenylate cyclase, i.e., 20-40 pg of membrane protein. The mixture was incubated at 30°C for 15 min. CAMP was determined by radioimmunoassay (7). Data processing and statistical analysis. Data are derived from representative experiments, each of which was repeated at least three times with duplicate samples at each point. Results are expressed as means t SE. Where appropriate, statistical analysis was carried out by analysis of variance and Student’s t test. Differences between paired values were considered significant at P < 0.05. The specific binding data were plotted according to Scatchard, and in addition were analyzed by the LIGAND program of Munson and Rodbard (see Ref. 7). All calculations were performed on an HP-85 microcomputer (Hewlett-Packard) and an IBM-PC microcomputer. Peptides and chemicals. Highly purified natural porcine VIP and gastric inhibitory peptide (GIP) were purchased from Prof. V. Mutt (GHI Laboratory, Stockholm, Sweden). Synthetic ovine hypothalamic PACAPand its derivative PACAP-27, comprising the 27 NH2-terminal amidated residues, were synthesized by solid-phase techniques, as previously described (29). Porcine secretin was donated by Prof. E. Wunsch (Max Planck Institute fur Biochemie, Martinsried, FRG). PHM (human peptide with NH2-terminal histidine and COOH-terminal methionine amide), PHV (peptide with NH2-terminal histidine and COOH-terminal valine), rat PHI, helospectin I and II, human oxyntomodulin, glucagon-like peptide- (GLP-2), and TGLP-1 (the truncated form of GLP-1 lacking the six NH2-terminal amino acid sequence) were purchased from Peninsula Laboratories (St. Helens, UK). Human pancreatic growth hormonereleasing factor (hpGRF) and rat hypothalamic growth hormone-releasing factor (rhGRF) were given by Drs. J. Rivier and W. Vale (Salk Institute, San Diego, CA). Helodermin was purchased from Novabiochem and porcine pancreatic glucagon from the Novo Research Insti[Dtute (Bagsvaered, Denmark). [Acetyl-His’]VIP, Ala4]VIP, [D-Phe”]VIP and [D-Phe4]VIP were generously donated by Prof. P. Robberecht (Departement de Biochimie et de Nutrition, Universite Libre de Bruxelles, Brussels, Belgium) and Dr. D. H. Coy (Peptide Research Laboratories, New Orleans, LA), respectively. Human gastrin and the somatostatin analogue Sandostatin were from Beckman and Sandoz, respectivelv. Bacitracin,

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HEPES, EDTA, STI, pepstatin, leupeptin, NP-40, PMSF, N-acetylglucosamine, human growth hormone, cholecystokinin (CCK), and porcine insulin were purchased from Sigma (St. Louis, MO). DSP, acrylamide, N-N ’ -methylenebisacrylamide and protein molecular weight standards for SDS gel electrophoresis were obtained from Pierce Chemical (Rockford, IL), LKB (Bromma, Sweden), and Bio-Rad (Richmond, VA), respectively. 1251-labeled Na was from the Radiochemical Centre (Amersham, Buckinghamshire, UK).

nificantly to 61% in the presence of GTP plus 0.1 FM helodermin, suggesting that the 12”1-VIP binding sites are functionally coupled to a GTP binding protein of the signal-transduction systems (2). Under our experimental conditions, no degradation of the ‘““I-VIP binding sites was observed when they were incubated for 60 min at 30°C with either bacitracin added alone or combined with the other protease inhibitors (STI, leupeptin, pepstatin, and PMSF). At 28 days after birth, we observed 30% degradation of 1251-VIP after 90 min of incubation of pancreatic plasma membranes at 30°C. These properties were also observed at the other RESULTS stages considered: at birth, and in ruminant and preruAssociation and dissociation of 1251-VIP in calf pan- minant animals at 119 days of postnatal life. creatic plasma membranes. In membranes prepared from Pharmacological properties of 1251VIP binding sites in 28-day-old calves, the binding of 1251-VIP was saturable, calf pancreatic membranes. The pharmacological specificmaximal after 60 min incubation at 30°C, and stable ity of the 1251-VIP binding sites was determined using until 120 min (Fig. 1, left). Nonspecific binding averaged several natural and synthetic peptides structurally re26% of total binding. Specific binding constituted 14.5% lated to VIP. Figure 2 shows that 12”1-VIP binding is of the total added radioactivity. Thirty percent of the preferentially inhibited by PACAP(IC50 = 0.16 nM) ‘“‘I-VIP initially bound during the association step after and helodermin (IC50 = 0.23 nM) compared with VIP 60 min incubation at 30°C dissociated spontaneously UC = 0.63 nM). At 28 days after birth, the order of from the pancreatic membranes when they were postinpotrncy for competitive inhibition of this binding was cubated at 37°C for 120 min in the standard binding PACAP> helodermin > VIP, [acetyl-His’]VIP > buffer (Fig. 1, right). This dissociation accelerated and PACAP> PHM > [D-Ala4]VIP, [D-Phe2]VIP > [Dreached 50% 120 min after the addition of 0.1 PM VIP Phe4]VIP (Fig. 2). At 0.1 PM, rat PHI and PHV inhibited to the initial ‘251-VIP-receptor complex. The increase of 12”1-VIP binding by 80 and 90%, respectively (data not shown). Because the effect of helodermin on this binding the dissociation rate by VIP alone was not statistically different. The addition of 0.1 mM GTP accelerated the was three times more potent than that of VIP, we invesdissociation rate much more, since at 30 min postincutigated the action of two other natural peptides extracted bation, 42% of the 1251-VIP initially bound dissociated from lizard venoms: helospectin I and helospectin II. with 0.1 nM VIP, and 54% with 0.1 PM VIP (Fig. 1, Both were three times less effective than VIP: helodermin > VIP > helospectin I and II (data not shown). At right). At 30 min, GTP alone induced 37% dissociation of initial 1251-VIP binding, and this proportion rose sig- 0.1 mM, the guanyl nucleotide analogue GppNHp reduced 12”1-VIP binding by 20%. 3 When we tested other peptides structurally related to looTotal VIP, including secretin, rhGRF, hpGRF, TGLP-1, GLP2, pancreatic glucagon, human oxyntomodulin, and GIP, 7-

I 0

I 15

1

I 60

I

I 1 120

Time, min

and dissociation of l”“I-VIP in calf pancreatic membranes isolated at 28 days of postnatal development. Left: time course of lz51-VIP binding in pancreatic plasma membranes incubated at 30°C in the presence of ‘““I-VIP, either alone (A, total binding) or combined with 0.1 ,uM native VIP (0, nonspecific binding). Specific ““I-VIP binding (0) was calculated as difference between total and nonspecific radioactivity bound to membranes. Data are expressed as fmol/mg protein and are means t SE of 3 experiments. Right: time from calf pancreatic memcourse of the dissociation of bound ““I-VIP branes isolated at 28 days of postnatal development. After association ‘““I-VIP was separated by centrifugation at 4°C step, membrane-bound and then incubated at 37°C in 250 ~1 of binding assay buffer alone (0, spontaneous dissociation) or in buffer containing an excess of 0.1 PM VIP, either alone (0) or combined with 0.1 mM GTP (A). Results were compared with those observed in the presence of 0.1 mM GTP, alone (CI) or combined with 0.1 PM helodermin (A), or with 0.1 nM (0) or 0.1 PM VIP (A). Data are expressed as percentage of l’L”I-VIP specifically bound to pancreatic membranes at end of association step (%initial binding) and are means t_ SE of 4-7 experiments. FIG.

1. Association

-J-f\' 0 12

I

I

I

I

I

11

10

9

8

7

Peptides,

-Log[M]

I

FIG. 2. Effect of PACAP, helodermin, VIP, and their structurally related peptides on 1251-VIP binding in calf pancreatic membranes isolated at 28 days of postnatal development. Pancreatic membranes were incubated at 30°C for 60 min in the presence of 50 pM 12”1-VIP alone or combined with the different concentrations of peptides. n, PACAP-38; A, helodermin; l , VIP; V, [Ac-His’]VIP; +, PACAP-27; 0, PHM; v, [D-Ala4]VIP; q , [D-Phe2]VIP; o, [D-Phe4]VIP. Data are expressed as %maximal specific 1251-VIP binding and are means k SE of 4-11 experiments.

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we found they were ineffective at concentrations up to 0.1 PM. At the same concentrations, human growth hormone, gastrin, CCK, porcine insulin, and Sandostatin did not compete for ‘251-VIP binding. The pharmacological properties of the ““I-VIP binding sites in calf pancreatic membranes did not change at any of the stages of postnatal development examined, i.e., at birth, and at 119 days in ruminant and preruminant animals (data not shown). Scatchard membranes.

analysis of “‘I-

VIP binding in calf pancreatic

Table 1, indicating the Scatchard parameters of “‘I-VIP binding in calf pancreatic membranes prepared at birth, at 28 days, and at 119 days in ruminants and preruminants, shows that there was no significant variation in the Kd values of the high-affinity binding sites at any of these stages (Kd = 0.11-0.4 nM VIP). Only one class of binding sites was identified at birth (Kd = 0.4 nM). Two classes were observed at 28 and 119 days: one with a high affinity for lZ51-VIP (O.ll0.27 nM VIP) and a low binding capacity, and the other with a low affinity and a high binding capacity. At 28 days, Scatchard analysis revealed that helodermin interacted with 20- and 2-fold higher affinity than VIP on the high- and low-affinity ‘251-VIP binding sites, respectively. Therefore, helodermin was not more potent than VIP because it reduces the tracer degradation less than VIP. These binding data are consistent with our observations that helodermin and PACAPwere 4 and 20 times more potent, respectively, than VIP on adenylate cyclase activation in membranes prepared from 28-dayold calf pancreas (see below). We observed significant decreases in the 12”1-VIP binding capacity at 28 and 119 days compared with the neonatal period. This binding capacity was significantly greater at 119 days in the ruminants than at 119 and 28 days in the preruminants. Molecular identification of proteins binding 1251-VIP in calf pancreatic plasma membranes. The molecular com-

ponents of the proteins binding lz51-VIP in pancreatic plasma membranes were analyzed by SDS-PAGE after cross-linking with the heterobifunctional reagent DSP (Figs. 3 and 4). The molecular mass of the 12”1-VIP (3,300 Da) was subtracted from the apparent molecular mass of the proteins binding 12”1-VIP, determined according to their electrophoretic mobility. We observed that in membranes from 28-day-old calves one major autoradiographic band was resolved with an apparent molecular mass of 55 kDa, and two minor bands of 76 and 33 kDa. About 68% of the radioactivity was incorporated into the 1. Scatchard parameters of 1251-VIP binding sites in the pancreas during the postnatal development of the calf TABLE

Age,

days 0 28 119 (preruminant) 119 (ruminant)

Scatchard

Parameters

B “IRX 1,

Ki1,

KS 1, IlM

fmol/mg

0.4+0.1 0.11+0.05 0.14+0.1 0.27fO.l

858*260* 81f22*f 66?38*1174k25.t

nM

l.bO.4 2.9k1.2 2.6k-1

B “lax 2,

n

fmol/mg

445+90 1,806+908 739t139

5 5 4 4

Values are means f SE; n, no. of calves (pancreases). * Significantly different at P < 0.05 compared with ruminants at day 119. t Significantly different at P < 0.05 compared with animals at birth.

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ABCDE

FGH

Mr ,1O-3

-

76

-

55

-

33

FIG. 3. Autoradiographic profile and pharmacological specificity of proteins binding ““I-VIP in calf pancreatic membranes isolated at 28 days of postnatal development. Membranes were incubated for 90 min at 30°C in the presence of 0.4 nM ““I-VIP alone (lane A) or combined with the following natural peptides: 0.1 nM VIP (lane B) or 0.1 FM VIP (lane C), 0.1 nM helodermin (lane D) or 0.1 KM helodermin (lane E), 0.1 PM PHM (lane F), 0.1 PM hpGRF (lane C), and 0.1 PM secretin (lane H). Letters correspond to the following protein markers of known M,: a, phosphorylase B (92,500); b, bovine serum albumin (66,200); c, ovalbumin (45,000); cl, carbonic anhydrase (31,000). Similar results were obtained in another experiment.

55-kDa band, and 27 and 5% into the 76- and 33-kDa bands, respectively. The radioactivity incorporated into the 55-kDa protein was reduced by 70% in the presence of an excess of PACAP-38, helodermin, or VIP (Fig. 4). However, under the same conditions, these peptides only reduced the radioactivity incorporated into the 76-kDa band by 35%, suggesting that the 55-kDa protein was specific for binding and functional interaction with the VIP-like peptides. At 1 mM, GTP inhibited lz51-VIP cross-linking to the 55-kDa band by 30% (data not shown). In agreement with the results of our pharmacological study, covalent labeling of the 55-kDa protein was preferentially inhibited by helodermin (Fig. 3, lanes D and E) vs. VIP (lanes B and C). PHM was a much less effective inhibitor than helodermin or VIP at 0.1 PM (lane F), and GRF and secretin had no effect (lanes G and H). Densitometric analysis of the autoradiogram patterns showed dose-dependent inhibition of lz51-VIP cross-linking to the 55-kDa band (Fig. 4). Half-maximal inhibition of this cross-linking occurred at 0.1 nM PACAP-38 and 1 nM VIP, which is in good agreement with the relative potencies of the two neuropeptides on 12’1VIP binding: PACAP> VIP (Fig. 2). At 0.1 nM, helodermin was a more efficient inhibitor than VIP on the labeling of the 55-kDa band. Labeling of the ““I-VIP binding proteins in pancreatic membranes was comparable at all the postnatal stages considered (Fig. 5). The nonspecific 76-kDa band was more intensively labeled at 119 days in ruminants than at the other stages. Glycoprotein nature of the l”I- VIP binding sites in calf pancreatic membranes. To determine the glycoprotein

nature of the 12”1-VIP binding sites in pancreatic membranes, we tested the ability of the proteins binding 1251VIP to be retained on immobilized WGA. Accordingly, the affinity-labeled 1251-VIP binding sites were solubi-

Downloaded from www.physiology.org/journal/ajpgi at Washington Univ (128.252.067.066) on February 13, 2019.

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Peptides ,

-Lo&q

4. Dose-dependent inhibition by PACAP-38, helodermin, and VIP of the radioactivity incorporated in the 55-kDa protein binding ““I-VIP in calf pancreatic membranes isolated at 28 days of postnatal development. Binding was performed in the presence of 0.4 nM m’IVIP alone or combined with different concentrations of PACAP-38, VIP (lo-“’ to 10m7M), and helodermin (lo-” and 10e7 M). After crosslinking, the 55-kDa-labeled proteins were identified by electrophoresis and autoradiography (bottom). Absorbance of the corresponding bands was then determined. For each peptide, results are expressed as relative absorbance of the 55-kDa autoradiographic band vs. control absorbance measured in the presence of ‘251-VIP alone (100%). Similar results were obtained in 2 other experiments. FIG.

lized with NP-40 and applied to the WGA-Sepharose column. We observed that when the equilibration buffer containing 0.5 M N-acetylglucosamine was run through the column, -26% of the total radioactivity applied was retained and eluted (Fig. 6). This revealed the glycoproteinic nature of the ‘251-VIP binding sites in calf pancreatic membranes. Adenylute cyclase activation by VIP and its related peptides. The functional coupling of the ‘251-VIP binding

sites to the adenylate cyclase system was also investigated in calf pancreatic membranes (Table 2). At the different developmental stages considered, basal enzyme activity ranged from 0.06 to 0.15 nmol. min-’ .mg protein-‘. Sodium fluoride stimulated adenylate cyclase activity E-fold at birth and 4- to 8-fold in membranes isolated at 28 or 119 days. Similar evolution was observed for the stimulation induced by forskolin: 22-fold at birth and 6- to 13-fold in 28 or 119 day pancreases. In contrast, the increases in adenylate cyclase activity and CAMP levels induced by the maximally effective concentrations of VIP and PACAP(0.1 PM) were smaller in mem-

33

FIG. 5. Molecular components of ‘251-VIP binding sites in calf pancreatic membranes during postnatal development. Binding was performed in the presence of 0.4 nM iz51-VIP, either alone (lanes A, C, E, and G) or combined with 0.1 /IM VIP (lanes B, D, F, and H) on membranes prepared at birth (lanes A and B), at 28 days (lanes C and D), and at 119 days from ruminants (lanes E and F) and preruminants (lanes G and H). Two other experiments gave similar results.

branes from newborn calves (2-fold increase) than from postnatal animals [3- to 4-fold increase (Table 2 and Fig. 7)]. At the different stages considered, the four VIP analogues increased adenylate cyclase activity in the following order of potency (K,): PACAP> helodermin > PACAPand VIP. Thus PACAPwas 7-20 times more potent than VIP and 4-9 times more potent than helodermin (Table 2). At 0.1 and 1 PM, pancreatic glucagon, oxyntomodulin, TGLP-1, and GIP did not increase basal adenylate cyclase activity. In membranes from 28-day-old animals, rat PHI, PHV, and PHM were -10 times less potent than VIP (Fig. 8). At maximally effective concentrations, paired combinations of 30 nM PACAP-38, helodermin, and 0.1 PM VIP did not produce any additional increase in adenylate cyclase activity (data not shown). DISCUSSION

Our data document the existence of a new subtype of ‘251-VIP binding site in calf pancreatic membranes with unexpected high affinity for PACAPand helodermin. We extended this observation to show the functional coupling of these binding sites with adenylate cyclase, and we demonstrated that PACAP is a very potent and efficient neuropeptide in activating this membranebound enzyme. Binding of 1251-VIP was characterized by several biochemical properties specific to membrane receptors. In particular, it was a fast saturable process, which was temperature dependent and reversible. Binding of 1251VIP was optimal at a physiological or slightly basic pH of 7.5-8 (data not shown). Both binding and dissociation of ‘251-VIP were GTP dependent, suggesting that a G protein is activated in the signal transduction system stimulated by VIP and its analogues. Accordingly, in the calf pancreatic membranes, the 1251-VIP binding sites

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ACTION

OF

PACAP,

HELODERMIN,

0.5M

AND

VIP

ON

CALF

NAcGlc

FIG. 6. Affinity chromatography of proteins binding 1’51-VIP on WGA-sepharose in calf pancreatic membranes isolated at 28 days of postnatal development. WGA-bound material was eluted with 0.5 M N-acetylglucosamine (NAcGlc) in WGA buffer containing 0.1% Nonidet P40. Results are expressed in counts/min x lo-". Similar results were obtained in another experiment.

v

0

5

IO

15

20

25

Fraction

30

G271

PANCREAS

35

40

45

50

55

number

2. Adenylate cyclase activity in developing calf pancreas TABLE

Day Day

0

Day

0.1 Preruminant

Adenylate Basal NaF (lo-’ M) Forskolin (lo-" M) VIP (lo-’ M) PACAP(lo-‘M)

cyclase activity,

0.15t0.02*

0.96t0.3

0.78t0.09

1.36t0.43

2.04t0.38

0.12t0.02

0.42tO.l”

0.13t0.02

0.48kO.08"

min-’

Ruminant

- mg protein-’

0.07t0.01 0.55t0.07 0.78t0.24

0.10t0.02 0.43kO.06 0.59t0.09

0.25?0.04* 0.30t0.04"

0.38&0.04* 0.44t0.07*

.$ Oso5 Basal

5

k

-c Adenylate

PACAPHelodermin VIP

nmol-

0.06t0.02

119

28

cyclase activation,

0.27kO.18

K, values,

i

nM

1.57kO.34"r

0.1t0.02 0.47k0.14-t

0.34t0.04 2.82t1.12

0.23t0.07 0.87t0.27

5.55&0.97$§

2.12+0.48$§

6.58+1.56$

1.53&0.28*$

Values are means & SE of 4-7 experiments performed in duplicate. * Different at P < 0.01-0.05 from the values measured at birth. Other values measured during development were not statistically different from each other. At each stage of postnatal development, the K, values were statistically different and P < 0.01-0.05 for PACAPand helodermin (t), PACAPand VIP ($), and helodermin and VIP (§).

were functionally coupled to CAMP generation at the different stagesconsidered, with similar potency on binding UC50 = 0.6 nM VIP) and adenylate cyclase in 28day-old calves (K, = 2.1 nM VIP). This potency compared well with that previously observed in rat and guinea pig pancreas (I& = 0.13-0.4 nM VIP) (41). In calf pancreas, ?-VIP binding was inhibited by [D-Phe”]VIP and [D-Phe4]VIP at concentrations lOO- to ZOO-fold higher than VIP. An increase in hydrophobicity in position 2 or 4 does not favor interaction with or activation of VIPergic receptors. The [D-Ala4]VIP isomer was 70 times less potent than VIP, which confirms our earlier findings (16) that the NH2-terminal portion of VIP is essential for its intrinsic biological activity. Under the present experimental conditions, ‘251-VIP degradation was low (-30%), and the VIP binding sites were not degraded. It was previously shown (8) in membrane and cellular preparations from the digestive epithelia that the degradation of 1251-VIP is temperature sensitive. Here, in the presence of bacitracin, addition of the pro-

28 Days

0 5

-41’ 011

E

’

10

’

9

’

8

’

7

L -41 0-H

6

10

9

8

7

6

$ g

119 Days, 0

t

q,L

.I

011

L 10

I, 9

1 I 8

7

6

Pept ide

ruminants 0

44 011

10

9

8

7

6

-Lo&]

FIG. 7. Adenylate cyclase activation by VIP and its related peptides in calf pancreatic membranes during postnatal development. Adenylate cyclase activity was tested after addition of various concentrations of PACAP(H), helodermin (n), VIP (o), and PACAP(+). Results are means t SE for 4 experiments at each developmental stage.

tease inhibitors PMSF, soybean trypsin inhibitor, leupeptin, and pepstatin had no effect on the degradation of 1251-VIP or on its binding sites. VIP might be degraded by specialized enzymes such as enkephalinase, a membrane-bound endopeptidase that is present in the epithelial cells of various tissues and is capable of cleaving VIP (17). We showed here that [N-acetyl His’]VIP has a similar potency to that of VIP. The N-acetylation pro-

Downloaded from www.physiology.org/journal/ajpgi at Washington Univ (128.252.067.066) on February 13, 2019.

G272

ACTION

.-c aI 5 ki

OF

PACAP,

HELODERMIN,

-

$0.4f 5 E .-+s 0.2 _ .-> cl Q

t?4 0 ,”>

‘Basal 0

c 0 11

10 9 8 7 Pept ides, - Log [M ]

6

FIG. 8. Adenylate cyclase activity in calf pancreatic membranes isolated at 28 days of postnatal development. Effects of PACAP(u) and VIP (0) were compared with those produced by 3 of their related peptides: rat PHI (0), PHM (o), or PHV (A). Data are means for 2 experiments performed in duplicate. The following peptides, tested at 0.i PM, had-no effect on basal adenylate cyclase activity: pancreatic glucagon, human oxyntomodulin, TGLP-1, and GIP.

tects VIP from destruction by N-aminopeptidase (9). This enzyme, which attacks the NH2-terminal extremity of various gastrointestinal peptides, does not appear to break down VIP in pancreatic calf membranes. As VIP inhibits the binding of 1251-VIP at concentrations as low as 20 pM, it might act as a neurotransmitter and hormone in the calf pancreas. In this connection, the VIP concentration in the peripheral blood of the calf after a meal ranged from 10 to 40 pM (22). In the calf pancreas, the VIPergic binding sites differed from those already described and were characterized by 1) a higher affinity for PACAP and helodermin than for VIP, 2) a reduced affinity for [D-Ala4]VIP, and 3) the absence of interaction with secretin and rat hypothalamic and human pancreatic GRFs. The VIP analogues we tested displayed the same order of potency as regards their effects on 1251-VIP binding (PACAP> helodermin > VIP, PACAP> PHM) and on adenylate cyclase (PACAP> helodermin > VIP, PACAP> PHM, PHV, PHI). Because paired combinations of PACAP-38, helodermin, and VIP did not produce additive stimulation, these peptides might activate the same pool of receptor-transducer system, since they also produced similar extent of adenylate cyclase activation. The Scatchard representation of 12”1-VIP binding to pancreatic membranes from 28day-old calves and 119day-old ruminants and preruminants is curvilinear. Thus two classes of binding sites were identified in calves at these stages of development and only one type of binding site with high affinity for VIP at birth. The postnatal development of the pancreas is therefore associated with the appearance of binding sites with low affinity and -with a decrease in the density of the high-affinity sites. After weaning, the capacity of the high-affinity binding sites increased, and coordinated increases in adenylate cyclase activation by PACAP-38, helodermin, and VIP were observed at 28 and 119 days after birth, suggesting that these ontogenetic changes are probably concomitant

AND

VIP

ON

CALF

PANCREAS

with the relative density of the high- and low-affinity sites. These changes in receptor affinity and capacity may therefore reflect the functional activation of the pancreas by the three peptides, depending on the physiological and nutritional status during the postnatal development. For example, it is well known that the specific interaction of VIP with its binding sites is associated with selective disappearance of the high-affinity binding sites, while the affinity of the residual sites is not modified (15). On the other hand, the genes encoding the pancreatic enzymes amylase, lipase, and trypsin showed increased expression during the 0- to 28-day period, and this was accompanied by a remarkable augmentation of the amylase specific activity after birth (26). Thus the maturation of the VIPergic binding sites may take place in concert with the capacity of the developing calf pancreas to elaborate, store, and secrete specific digestive enzymes. Cross-linked preparations of HPLC-purified 1251-VIP and plasma membranes from the developing calf pancreas migrated on SDS-PAGE as a major band of 55 kDa and two minor bands of 76 and 33 kDa. The major affinity-labeled band of 55 kDa exhibited a specific pattern of inhibition by the VIP analogues, since the labeling was preferentially inhibited by PACAPvs. helodermin and VIP. Incorporation of the radioactive affinity label into the 55-kDa band was not inhibited by an excess of human pancreatic GRF or secretin. This pharmacological specificity (PACAP> helodermin > VIP > PHM) resembled the apparent affinities of the 1251-VIP binding sites and adenylate cyclase for the VIP analogues. We therefore suspect that in calf pancreatic membranes, this 55-kDa protein constitutes the high-affinity binding sites involved in the PACAP-induced adenylate cyclase activation. In contrast, 1251-VIP incorporation into the minor affinity-labeled band of 76 kDa was slightly inhibited by an excess of PACAPor VIP, suggesting that this membrane component may constitute the low-affinity/high-capacity binding sites. This membrane protein pattern was similar at all the stages of development investigated, except in 119-day-old ruminants, for which the 76-kDa protein displayed higher incorporation and partial inhibition with an excess of VIP. In calf pancreatic membranes, the PACAP-preferring binding sites had electrophoretic mobilities compatible with the membrane receptors of the VIP family of peptides, such as secretin, VIP, glucagon, somatostatin, GRF, GIP, and TGLP-1, previously identified in other tissues and animal species (15, 18). Thus in rat and guinea pig pancreas (29, 4l), the corresponding 1251-VIP binding sites were 45- to 63-kDa (major polypeptide) and 30-, 77-, and 80-kDa proteins (minor polypeptide). Chastre et al. (5) and Nguyen et al. (32) obtained two bands with similar molecular masses of 60 and 90 and 53 and 77 kDa in human and rat liver, respectively. However, the action of PACAPand helodermin was not investigated in these models. On the basis of these observations and other recent findings, it seems justifiable to designate the 12”I-VIP binding sites in calf pancreatic membranes as “PACAP-preferring” binding sites. We demonstrated the presence of a glycosylated portion in these sites, as observed previously for many membrane

Downloaded from www.physiology.org/journal/ajpgi at Washington Univ (128.252.067.066) on February 13, 2019.

ACTION

OF

PACAP,

HELODERMIN,

receptors coupled to G proteins (11, 12, 15, 32). These findings allow us to speculate that the variations in the molecular masses of the VIP binding sites indicated above might result from the existence of different glycosylation levels for the membrane receptors of the VIP family of peptides in different tissues (11). The 55 and 33-kDa proteins may also originate from the proteolytic cleavage and deglycosylation of the 76-kDa protein. This cleavage may be induced by the binding of the ligand, because it causes a conformational change in the receptor and favors the action of proteases (27). In summary, the present results suggest that both PACAP and helodermin may play a physiological role in pancreatic secretion at an early stage in postnatal development. The calf pancreas constitutes an excellent model for exploring this hypothesis at the level of the hormonal and neuroparacrine regulations of the digestive enzymes and hydroelectrolytic fluids in the mammalian digestive tract. Accordingly, the fetal exocrine pancreas containing differentiated acinar cells and zymogen granules responds to established stimulants of the adult calf pancreas, including CCK (42). Preliminary studies showed that PACAP stimulated amylase release from the rat pancreas and that PACAPappears to be present in neuronal fibers of the rat gut (A. Arimura, personal communication). In a recent report, Buscail et al. (4) characterized highly selective receptors for PACAPin membranes from the rat pancreatic acinar cell line AR42J, and found that the rat pancreatic PACAP receptor was a 65kDa binding protein (4). Helodermin, which has been identified in humans and animals, including calves, also stimulates water, salt, and amylase secretions in the pancreas (21, 35, 37, 39). This peptide is present at high concentrations in the thyroid and has been detected in the pancreas and salivary glands (21,37). Taken together, our data support the concept that in the developing calf pancreas, HPLC-purified 12”1-VIP interacts with functional PACAP receptors regulating CAMP generation. In future studies, it will be of interest to examine the interaction of ‘251-PACAP with calf pancreatic membranes so as to identify the cell populations bearing functional PACAP receptors and to obtain a complete picture of the biological effects regulated by the PACAP pathway in the exocrine pancreas. We are grateful to Dr. Patrick Robberecht (Universite Libre de Bruxelles, Brussels, Belgium) and Dr. Akira Arimura (Tulane University, Belle Chasse, LA) for providing PACAPand -27 and for permission to cite unpublished data. We also thank Yves Issoulie for photographic reproductions. We gratefully acknowledge La Region de Bretagne for the support to this work and fellowship to V. Le Meuth. Address for reprint requests: C. Gespach, INSERM, U55, Hopital, Saint-Antoine, 75571 Paris Cedex 12, France. Received

4 December

1989; accepted

in final

form

24 September

1990.

REFERENCES 1. BAWAB, W., N. FARJAUDON, V. LE MEUTH, E. CHASTRE, G. ROSSELIN, P. GUILLOTEAU, AND C. GESPACH. Helodermin-preferring lZ51-VIP binding sites during the postnatal development of the pancreas (Abstract). Regul. Peptides 26: 141, 1989. 2. BIRNBAUMER, L., J. ABRAMOWITZ, AND A. M. BROWN. Receptoreffector coupling by G proteins. Biochim. Biophys. Acta 1031: 163224,199O.

AND

VIP

ON

CALF

PANCREAS

G273

3. BISSONNETTE, B. M., M. J. COLLEN, H. ADACHI, R. T. JENSEN, AND J. D. GARDNER. Receptors for vasoactive intestinal peptide and secretin on rat pancreatic acini. Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9): G710-G717, 1984. A. CAUVIN, P. DE NEEF, D. GOSSEN, A. 4. BUSCAIL, L., P. GOURLET, ARIMURA, A. MIYATA, D. H. COY, P. ROBBERECHT, AND J. CHRISTOPHE. Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating peptide) in membranes from the rat pancreatic acinar cell line AR 4-25. FEBS Lett. 262: 77-81, 1990. 5. CHASTRE, E., W. BAWAB, C. FAURE, S. EMAMI, F. MULLER, A. BOUE, AND C. GESPACH. Vasoactive intestinal peptide and its receptors in fetuses with cystic fibrosis. Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G561-G569, 1989. 6. CHASTRE, E., S. EMAMI, AND C. GESPACH. Expression of membrane receptors and (proto) oncogenes during the ontogenic development and neoplastic transformation of the intestinal mucosa. Life. Sci. 44: 1721-1742, 1989. 7. CHASTRE, E., S. EMAMI, G. ROSSELIN, AND C. GESPACH. Ontogenie development of vasoactive intestinal peptide receptors in rat intestinal cells and liver. EndocrinoZogy 121: 2211-2221, 198% 8. CHRISTOPHE, J. P., T. P. CONLON, AND J. D. GARDNER. Interaction of porcine vasoactive intestinal peptide with dispersed pancreatic acinar cells from the guinea pig. Binding of radioiodinated peptide. J. Biol. Chem. 251: 4629-4634, 1976. 9. CHRISTOPHE, J., M. SVOBODA, M. WAELBROECK, J. WINAND, AND P. ROBBERECHT. Vasoactive intestinal peptide receptors in pancreas and liver. Structure-function relationship. Ann. NY Acad. Sci. 527: 238-256,1988. 10. DEHAYE, J. P., J. WINAND, C. DAMIEN, F. GOMEZ, P. POLOCZEK, P. ROBBERECHT, A. VANDERMEERS, M. C. VANDERMEERS-PIRET, M. STIEVENART, AND J. CHRISTOPHE. Receptors involved in helodermin action on rat pancreatic acini. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G602-G610, 1986. 11. DICKINSON, K. E. J., M. SCHACHTER, C. M. M. MILES, D. H. COY, AND P. S. SEVER. Characterization of vasoactive intestinal peptide (VIP) receptors in mammalian lung. Peptides 7: 791-800, 1986. 12. EL BATTARI, A., J. LUIS, J. M. MARTIN, J. FANTINI, J. M. MULLER, J. MARVALDI, AND J. PICHON. The vasoactive intestinal peptide receptor on intact human colonic adenocarcinoma cells (HT29D4). Evidence for its glycoprotein nature. Biochem. J. 242: 185191,1987. 13. GARDNER, J. D., T. P. CONLON, M. L. FINK, AND M. BODANSZKY. Interaction of peptides related to secretin with hormone receptors on pancreatic acinar cells. Gastroenterology 71: 965-970, 1976. 14. GESPACH, C., Y. CHEREL, AND G. ROSSELIN. Development of sensitivity to CAMP-inducing hormones in the rat stomach. Am. J. Physiol. 247 (Gastrointest. Liver Physiol. 10): G231-G239, 1984. 15. GESPACH, C., S. EMAMI, AND E. CHASTRE. Membrane receptors in the gastrointestinal tract. A Review. Biosci. Reports 8: 199-232, 1988. A.-M. LHIAUBET, A. FOURNIER, S. ST. 16. GESPACH, C., S. EMAMI, PIERRE, G. ROSSELIN, AND W. ROSTENE. Structure-activity relationship of vasoactive intestinal peptide fragments in the human gastric cancer cell line. IRCS Med. Sci. 12: 724-725, 1984. 17 GOETZL, E. J., S. P. SREEDHARAN, C. W. TURCK, R. BRIDENBAUGH, AND B. MALFROY. Preferential cleavage of aminoand carboxyl-terminal oligopeptides from vasoactive intestinal polypeptide by human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11). Biochem. Biophys. Res. Commun. 158: 850854, 1989. 18. GOKE, R., T. COLE, AND J. M. CONLON. Characterization of the receptor for glucagon-like peptide-l (7-36) amide on plasma membranes from rat insulinoma-derived cells by covalent cross-linking. J. Mol. Endocrinol. 2: 93-98, 1989. 19. GOSSEN, D., P. POLOCZEK, M. SVOBODA, AND J. CHRISTOPHE. Molecular architecture of secretin receptors: the specific covalent labeling of a 51-kDa peptide after cross-linking of [‘251]iodo-secretin to intact rat pancreatic acini. FEBS Lett. 243: 205-208, 1989. 20. GOTTSCHALL, P. E., I. TATSUNO, A. MIYATA, AND A. ARIMURA. Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase activating polypeptide. Endocrinology 127: 272-277, 1990. T., P. PERSSON, R. HAKANSON, A. ABSOOD, G. 21. GRUNDITZ, BOTTCHER, C. RERUP, AND F. SUNDLER. Helodermin-like peptides in thvroid C cells: stimulation of thvroid hormone secretion and ” Y

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G274

22.

23.

24.

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26.

27.

28.

29.

30.

31.

32.

33.

ACTION

OF

PACAP,

HELODERMIN,

suppression of calcium incorporation into bone. Proc. Natl. Acad. Sci. USA 86: 1357-1361, 1989. GUILLOTEAU, P., I. LE HUEROU, J. A. CHAYVIALLE, R. TOULLEC, C. BERNARD, A. MOUATS, AND J. H. BURTON. Changes with age and at weaning in the plasma and duodenal tissue concentrations of vasoactive intestinal peptide (VIP), gastric inhibitory polypeptide (GIP), and secretin in the calf (Abstract). Regul. Peptides 26: 161, 1989. JENSEN, R. T., C. G. CHARLTON, H. ADACHI, S. W. JONES, T. L. O’DONOHUE, AND J. D. GARDNER. Use of ‘“‘I-secretin to identify and characterize high-affinity secretin receptors on pancreatic acini. Am. J. Physiol. 245 (Gastrointest. Liver Physiol. 8): G186G195,1983. JENSEN, R. T., K. TATEMOTO, V. MUTT, G. F. LEMP, AND J. D. GARDNER. Actions of a newly isolated intestinal peptide PHI on pancreatic acini. Am. J. Physiol. 241 (Gastrointest. Liver Physiol. 4): G498-G502,1981. LARSSON, L.-I., J. FAHRENKRUG, 0. SCHAFFALITSKY DE MUCKADELL, F. SUNDLER, R. HAKANSON, AND J. F. REHFELD. Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc. Natl. Acad. Sci. USA 73: 3197-3200, 1976. LE HUEROU, I., C. WICKER, P. GUILLOTEAU, R. TOULLEC, AND A. PUIGSERVER. Specific regulation of the gene expression of some pancreatic enzymes during postnatal development and weaning in the calf. Biochim. Biophys. Acta 1048: 257-264, 1990. LUIS, J., J. M. MARTIN, A. EL BATTARI, J. MARVALDI, AND J. PICHON. The vasoactive intestinal peptide (VIP) receptor: recent data and hypothesis. Biochimie 70: 1311-1322, 1988. MAKHLOUF, G. M. Role of VIP in the function of the gut. In: Vasoactive Intestinal Peptide, edited by S. I. Said. New York: Raven, 1982, p. 425-446. MCARTHUR, K. E., C. L. WOOD, M. S. O’DORISIO, Z.-C. ZHOU, J. D. GARDNER, AND R. T. JENSEN. Characterization of receptors for VIP on pancreatic acinar cell plasma membranes using covalent cross-linking. Am. J. Physiol. 252 (Gastrointest. Liver Physiol. 15): G404-G412,1987. MILUTINOVIC, S., I. SCHULZ, AND G. ROSSELIN. The interaction of secretin with pancreatic membranes. Biochim. Biophys. Acta 436: 113-127, 1976. MIYATA, A., A. ARIMURA, R. R. DAHL, N. MINAMINO, A. UEHARA, L. JIANG, M. D. CULLER, AND D. H. COY. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem. Biophys. Res. Commun. 164: 567574, 1989. NGUYEN, T. D., J. A. WILLIAMS, AND G. M. GRAY. Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein. Biochemistry 25: 361-368, 1986. OKUMURA, K., S. IWAKAWA, K. I. INUI, AND R. HORI. Specific secretin binding sites in rat pancreas. Biochem. Pharmacol. 32:

AND

VIP

ON

CALF

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