Functional receptors for vasoactive intestinal peptide in cultured vascular smooth muscle cells from rat aorta

Specific binding sites for vasoactive intestinal peptide (VIP), a potent vasodilatory polypeptide, and its effect on formation of intracellular cyclic AMP levels were studied in cultured vascular smooth muscle cells (VSMC) from rat aorta. Specific binding of ¹²⁵I-labeled-VIP to cultured VSMCs was time- and temperature-dependent. Scatchard analysis of binding studies suggested the presence of two classes of high and low affinity binding sites for VIP; the apparent K_d and the number of maximal binding capacity were ∼8×10⁻⁹ M and 60,000 sites/cell (high-affinity sites) and ∼4×10⁻⁸ M and 140,000 sites/cells (low-affinity sites), respectively. Unlabeled VIP competitively inhibited the binding of ¹²⁵I-labeled-VIP to its binding sites, whereas neither peptides structurally related to VIP, nor other vasoactive substances affected the binding. VIP stimulated formation of intracellular cyclic AMP in cultured VSMCs in a dose-dependent manner; the stimulatory effect of VIP on cyclic AMP formation was not blocked by propranolol and was additive with isoproterenol. The present study first demonstrates the presence of specific receptors for VIP in VSMCs functionally coupled to adenylate cyclase system. It is suggested that VIP exerts its vasodilatory effect through its specific receptors distinct from β-adrenergic receptors.     

Conformational and dynamic considerations in the design of peptide hormone analogs

Efforts to understand the chemical-physical basis for peptide hormone and neurotransmitter action requires integration of conformational parameters and biological properties. Since most peptide hormones are conformationally flexible, the question arises as to which of the manifold of conformations is of biological significance. In molecular terms, it is necessary to carefully distinguish chemical-physical features important to binding (the binding message) from those involved in transduction (the biological activity message). One approach to this involves the design, synthesis, and conformational analysis of semirigid hormone analogs. The distinction between binding and transduction can best be examined by evaluation of full biological profiles of partial agonists, antagonists, and analogs with prolonged biological activity. Using this multidisciplinary approach, we have prepared several semirigid [Pen¹]-oxytocin antagonist analogs and evaluated their conformational properties and biological activities. Specific conformational features can be related to inhibitory activities in several cases. On the basis of structure–activity relationships and conformational considerations, we have designed a series of conformationally restricted cyclic and acyclic analogs of the linear peptide α-melanotropin. Some of these peptides have exceptionally prolonged in vivo activity (weeks), and others exhibit superagonist potency (10,000 times the native hormone). We have evidence that potency and prolonged activity have different structural and conformational requirements. It is suggested that potency is primarily a function of receptor recognition (the binding message), whereas prolonged activity is related to transduction (the biological activity message).     

Cyclic enkephalin analogs containing a cystine bridge

Two conformationally constrained enkephalin analogs were synthesized by substitution of cysteines in positions 2 and 5 and oxidative disulfide bond formation. In the guinea pig ileum assay the obtained cyclic analogs, [D-Cys²-L-Cys⁵]enkephalinamide and [D-Cys²-D-Cys⁵]enkephalinamide, showed potency ratios of 37.9 ± 0.8 and 73.3 ± 0.9, respectively, relative to [Met⁵]enkephalin. The extremely high potency of the analogs was shown to be a consequence of the conformational restrictions introduced by cyclization. Rat brain membrane binding studies with [³H]naloxone and [³H](D-Ala², D-Leu⁵)enkephalin as radiolabels revealed a moderate preference of both analogs for μ-receptors over δ-receptors. Furthermore, the cystine-containing analogs were shown to be highly resistant to enzymatic degradation.     

Enkephalin analogs modified in the aromatic ring of the N-terminal tyrosine residue

Two analogs of Leu⁵-enkephalin, (1-O-methyltyrosine,5-leucine)-enkephalin and [1-(3′-amino)-tyrosine,5-leucine]-enkephalin, were synthesized by classical methods. Both analogs show high biological potency after injection into the lateral brain ventricle of the rat. In both cases substitution of the Tyr residue of enkephalin leads to a pronounced prolongation of analgesic action, as compared with the unsubstituted peptide.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 1. Molecular characterization of the binding site

Using mono[125I]iodinated vasoactive intestinal peptide (125I-VIP), a very high number of specific binding sites for VIP were identified at the surface of the human melanoma cell line IGR39. The Scatchard analysis of competitive displacement experiments between native VIP and 125I-VIP was consistent with the existence of two classes of VIP-binding sites. IGR39 cells possess 0.54 x 10(6) high-affinity sites with a dissociation constant (Kd) of 0.66 nM and 1.3 x 10(6) sites of moderate affinity with a Kd of 4.7 nM. Pharmacological studies indicated that the order of potency in inhibiting 125I-VIP binding of the VIP/secretin family peptides was VIP much greater than peptide histidine methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin. Glucagon has no effect on the binding of the labelled peptide. By means of photoaffinity labelling a polypeptide of Mr 63,000 was characterized. The labelling of this species was completely abolished by native VIP. The order of potency of VIP-related peptides in inhibiting 125I-VIP cross-linking to its receptor was the same as in the competition experiments. The glycoprotein nature of the VIP-binding sites of IGR39 cells has been investigated by affinity chromatography on wheat-germ-agglutinin-Sepharose.     

Short peptide constructs mimic agonist sites of AT1R and BK receptors

Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT₁R and BKRB₁ or BKRB₂ G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S–S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac¹-AngII and Toac⁰-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac³-AngII and Toac³-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT₁R and BKRB cyclic constructs based on the structure of the CXCR₄, an α-chemokine GPCR-type receptor.     

Binding of vasoactive intestinal peptide and its stimulation of adenylate cyclase through two classes of receptors in rat liver membranes effects of 12 secretin analogues and 2 secretin fragments

1. Vasoactive intestinal peptide (VIP) receptors were identified in crude rat hepatic membranes by ¹²⁵I-labelled VIP binding and by the ability of VIP to stimulate adenylate cyclase activity. The specificity of these receptors was evaluated by the capacity of secretin, synthetic secretin analogues, and secretin fragments to inhibit ¹²⁵I-labelled VIP binding and to stimulate adenylate cyclase. 2. The results were compatible with the existence of two classes of VIP binding sites that could be distinguised according to their affinity for VIP and their specificity. High-affinity sites were more specific for VIP as secretin was 175 times less potent than VIP for recognition of these sites while being only 33 times less potent than VIP for recognition of low-affinity sites. 3. Secretin analogues, monosubstituted in position 2, 3, 4, or 6 were less potent than secretin for adenylate cyclase stimulation as well as for the recognition of the two classes of receptors. [Val⁵]Secretin was more potent than secretin and appeared definitely more VIP-like than secretin; [Ala⁴, Val⁵]secretin were equipotent to secretin. 4. The fragment secretin (7–27) was unable to recognize VIP receptors and to stimulate adenylate cyclase. The substituted fragment [Gln[⁹,Asn¹⁵]secretin (5–27) recognized these receptors with weak potency but could not activate the enzyme.     

Synthesis of cyclic α-MSH peptides

Summary Cyclic lactam analogs of α-melanocyte stimulating hormone (α-MSH) have been shown to be potent agonists in the frog skin bioassay [Al-Obeidi, F. et al., J. Med. Chem., 32 (1989) 2555], demonstrating melanocortin-1 (MC1) receptor activity. We synthesized cyclic α-MSH(1–13) and α-MSH(4–10) lactam analogs. The peptides were synthesized using Fmoc chemistry. We improved the cyclization procedure: side chains of Asp⁵ and Lys¹⁰ from the deprotected peptide were coupled in DMF to form a cyclic lactam, using an excess of PyBOP reagent and DIEA as a base. The cyclization reaction was completed within 1 h and was almost quantitative. We also synthesized an α-MSH analog cyclized via a disulphide bridge. The peptides were tested for their selectivity for the rat MC4 receptor. Cyclization and substitutions at position 7 dramatically influenced the selectivity for the rMC4 receptor.     

Respirable powder formulation of a shortened vasoactive intestinal peptide analog for treatment of airway inflammatory diseases

The aim of present study was to develop a respirable powder (RP) of a shortened vasoactive intestinal peptide (VIP) analog for inhalation. VIP and C‐terminally truncated VIP analogs were synthesized with a solid‐phase method. A structure‐activity relationship (SAR) study was carried out in terms with binding and relaxant activities of the peptides. Prepared RP formulation of a shortened VIP analog was physicochemically characterized by morphological, in vitro aerodynamic, and pharmacological assessments. The SAR study demonstrated that the N‐terminal 23 amino acid residues were required for biological activity of VIP. Upon chemical modification of VIP(1–23), [R^{15, 20, 21}, L¹⁷]‐VIP(1–23) was newly developed, which had higher binding activity in rat lung and smooth muscle relaxant effect in mouse stomach than VIP(1–23). The [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)‐based RP, [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP, exhibited fine in vitro inhalation performance. Airway inflammation evoked by sensitization of antigen in rats was attenuated by pre‐treatment with the [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP at a dose of 50 μg‐[R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/rat as evidenced by a 70% reduction of recruited inflammatory cells in bronchoalveolar lavage fluid. On the basis of these results, [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP might be a promising agent for treatment of airway inflammatory diseases.     

Synthesis and activity of partial retro-inverso modified atrial natriuretic factor analogs

The synthesis and pharmacological activity of partial retro-inverso modified rat atrial natriuretic factor (rANF) analogs is described. The route to these compounds utilized a combination of solution and solid-phase methods. The analogs prepared all contain a reversed amide bond (psi[NHCO]) at the Ser 25 to Phe26 linkage. This bond has been suggested to play a key role in the metabolic inactivation of ANF. The analogs are of comparable potency to the endogenous peptide rANF1-28 in binding to cultured rat vascular smooth muscle cells, in relaxing serotonin contracted rabbit aortic rings, and as natriuretic/diuretic agents in anesthetized rats. None of the peptides has an extended duration of action in vivo.     

Identifying structural determinants of potency for analogs of apelin-13: Integration of C-terminal truncation with structure–activity

Apelin peptides function as endogenous ligands of the APJ receptor and have been implicated in a number of important biological processes. While several apelinergic peptides have been reported, apelin-13 (Glu-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe) remains the most commonly studied and reported ligand of APJ. This study examines the effect of C-terminal peptide truncations and comprehensive structure–activity relationship (SAR) for a series of analogs based on apelin-13 in an attempt to develop more potent and stable analogs. C-terminal truncation studies identified apelin-13 (N-acetyl 2–11) amide (9) as a potent agonist (EC₅₀ = 4.4 nM). Comprehensive SAR studies also determined that Arg-2, Leu-5, Lys-8, Met-11, were key positions for determining agonist potency, whereas the hydrophobic volume of Lys-8 was a specific determinate of activity. Plasma stability studies on the truncated 10-mer peptide 28 (EC₅₀ = 33 nM) indicated the primary sites of cleavage occurred between Nle-3 and Leu-4 and also between Ala-5 and Ala-6. These new ligands represent the shortest known apelin peptides with good functional potency.     

Vasoactive Intestinal Polypeptide-Related Peptides Modulate Tyrosine Hydroxylase Gene Expression in PC12 Cells Through Multiple Adenylate Cyclase-Coupled Receptors

Abstract: We investigated the receptor mechanisms by which vasoactive intestinal polypeptide (VIP) and related peptides exert their effects on tyrosine hydroxylase (TH) gene expression. VIP, secretin, and peptide histidine isoleucine (PHI) each produced increases in TH gene expression, as measured by increases in TH mRNA levels and TH activity. The concentrations at which the effects of these peptides were maximal differed for TH activity and TH mRNA. Moreover, maximal increases in TH activity were 130-140% of control, whereas maximal increases in TH mRNA were 250% of control. The concentration dependence of the increases in TH mRNA in response to the three peptides was analyzed by fitting the data to nonlinear regression models that assume either one or two components to the response. The data for secretin fit best to a model that assumes a single component to the increase in TH mRNA levels. The data derived for PHI and VIP fit best to models that assumed two components to the TH mRNA response. These data suggested that there may be more than one receptor or signal transduction mechanism involved in the response to the various peptides. We examined whether the peptides exerted their effects through common or multiple second messenger systems. The ability of maximally active concentrations of these peptides to stimulate increases in TH mRNA was not additive, indicating that the peptides work through a common receptor or signal transduction pathway. Each peptide stimulated increases in protein kinase A (PKA) activity. Secretin and VIP were ineffective in increasing TH mRNA levels in a PKA-deficient mutant PC12 cell line (A 126-1B2). Moreover, the adenylate cyclase antagonist 2′,5′-dideoxyadenosine prevented the increase in TH mRNA produced by each peptide. Thus, each peptide requires an intact cyclic AMP second messenger pathway to produce changes in TH gene expression, suggesting that the complex pattern of response to VIP and PHI revealed by concentration-response analysis was due to the actions of these peptides at multiple receptors. To evaluate this possibility, we examined the effect of several peptide receptor antagonists on the increase in TH gene expression elicited by VIP, PHI, and secretin. The secretin antagonist secretin (5–27) (20 μM) had no significant effect on VIP or PHI stimulation of TH gene expression, but reduced the effect of secretin. The VIP antagonist VIP (10–28) (20 μM) reduced the effect of VIP on increasing TH mRNA, but had no significant effect on the response of TH mRNA to secretin or PHI. Interestingly, the VIP antagonist [Ac-Tyr¹,D-Phe²]-growth hormone releasing factor [GRF(1–29)] amide (20 μM) potentiated the effect of VIP on elevating TH mRNA levels, but had no effect on secretin-stimulated TH mRNA induction. To determine whether this response was mediated through the cyclic AMP pathway, we examined the effects of the VIP antagonist [Ac-Tyr¹,D-Phe²]-GRF(1–29) amide on VIP stimulation of PKA activity. Although the antagonist had no effect alone, it enhanced stimulation of PKA activity by VIP. Taken together, these findings indicate that VIP and secretin stimulate TH mRNA through different adenylate cyclase-linked receptors and that a second VIP receptor may modulate TH induction by inhibiting VIP stimulation of PKA.     

Neurohypophyseal hormones and behavior: effects of intracerebroventricularly injected hormone analogs in mice.

Neurohypophyseal hormones evoke spontaneous behavioral changes in mice. This study compares the potency of four naturally occuring neurohypophyseal hormones and of ten analogs with amino acid residue replacements selected in such a manner as to cover each residue position of the hormones with the exception of the cystine residue. Peptides were administered intraventricularly and the sum of foraging, scratching and squeaking, recorded at 30 second intervals during a 30 min session, was measured as a function of peptide dose. The most potent group of peptides is represented by the neurohypophyseal hormones as well as the five analogs [Hly⁸] vasopressin, [Δ³-Pro⁷]AVP, [Thi³]LVP, [Abu⁴]AVP and [Abu⁴]LVP. [Leu⁴]LVP showed significant activity but was far less potent than the natural hormones. None of the remaining analogs enhanced activity with an increase in peptide dose. This group included both peptides with C-terminal modifications and those in which the tyrosine (position 2) or the asparagine residue (position 5) of the hormones were substituted by alanine. The neurohypophyseal hormone-induced behavioral results of this study reveal a structure-function relationship, which is in its most important conclusions, identical to the conformation-activity model proposed for endocrine activities of neurohypophyseal peptides.     

Evidence for a new subclass of calcitonin/ calcitonin gene-related peptide binding site in rat brain

Binding sites for calcitonin and calcitonin gene-related peptide are widely distributed in the central nervous system. In this study, binding of [¹²⁵I]-alpha-rat calcitonin gene-related peptide and [¹²⁵I]-salmon calcitonin in adjacent sections of rat brain revealed clearly distinct patterns of binding in most regions although in some restricted areas such as parts of the ventral striatum, including the nucleus accumbens, there was some overlap in the patterns of binding. In the primary olfactory cortex, which bound only calcitonin gene-related peptide, salmon calcitonin was very weak in inhibiting the binding of calcitonin gene-related peptide. In the nucleus accumbens, high affinity binding of calcitonin and calcitonin gene-related peptide at their homologous receptors was observed, with affinity constants for calcitonin and calcitonin gene-related peptide of 1.4 × 10⁹ M⁻¹ and 1.2 × 10⁹ M⁻¹ respectively. Cross competition studies in this nucleus demonstrated that salmon calcitonin was able to compete for [¹²⁵I]-rat calcitonin gene-related peptide labelled sites with high affinity, with an affinity constant of 0.8 × 10⁹ M⁻¹. However, rat calcitonin gene-related peptide was less potent in inhibiting the binding of [¹²⁵I]-salmon calcitonin labelled sites with only 28% inhibition at 10⁻⁶M. Further characterization of the calcitonin sensitive calcitonin gene-related peptide labelled sites demonstrated that a range of calcitonin analogs inhibited the binding of [¹²⁵I]-rat calcitonin gene-related peptide with the same order of potency as the analogs competed for [¹²⁵I]-salmon calcitonin labelled sites. Digital substraction mapping revealed calcitonin-sensitive calcitonin gene-related peptide binding sites over parts of the ventral striatum, including mid-caudal nucleus accumbens and fundus striati; over the lateral border of the lateral bed nucleus of the stria terminalis; part of the central amygdaloid nucleus; the organum vasculosum of the lamina terminalis and area postrema and over the wings of the dorsal raphe.These results demonstrate the existence of a new subtype of calcitonin/calcitonin gene-related peptide binding site, which has high affinity for the two otherwise biochemically distinct peptides.     

Interaction of vasoactive intestinal peptide with HeLa cells: activation of cyclic AMP-dependent protein kinase and lack of effect on DNA synthesis

Vasoactive intestinal peptide (VIP) stimulated protein kinase activity in HeLa cells. Maximal activation by the peptide required the simultaneous presence of a phosphodiesterase inhibitor. The response was dose-dependent in the 0.3–10 nM range, half-maximal stimulation being observed at 1 nM VIP. This value agrees with the concentration of VIP required for half-maximal stimulation of cyclic AMP production as well as with the K_d of the high affinity binding sites for VIP in the same cell system (15). Secretin also stimulated protein kinase activity but with a 300-times lower potency than VIP. When DNA synthesis in Hela cells was studied, no effect of VIP could be seen in a 0.1–100 nM range of peptide concentration.     

Synthesis and evaluation of novel lipidated neuromedin U analogs with increased stability and effects on food intake

Neuromedin U (NMU) is a 25 amino acid peptide expressed and secreted in the brain and gastrointestinal tract. Data have shown that peripheral administration of human NMU decreases food intake and body weight and improves glucose tolerance in mice, suggesting that NMU receptors constitute a possible anti‐diabetic and anti‐obesity drug target. However, the clinical use of native NMU is hampered by a poor pharmacokinetic profile. In the current study, we report in vitro and in vivo data from a series of novel lipidated NMU analogs. In vitro plasma stability studies of native NMU were performed to investigate the proteolytic stability and cleavage sites using LC–MS. Native NMU was found to be rapidly cleaved at the C‐terminus between Arg²⁴ and Asn²⁵, followed by cleavage between Arg¹⁶ and Gly¹⁷. Lipidated NMU analogs were generated using solid‐phase peptide synthesis, and in vitro potency was investigated using a human embryonic kidney 293‐based inositol phosphate accumulation assay. All lipidated analogs had preserved in vitro activity on both NMU receptors with potency improving as the lipidation site was moved away from the receptor‐interacting C‐terminal octapeptide segment. In vivo efficacy was assessed in lean mice as reduction in food intake after acute subcutaneous administration of 1, 0.3, 0.1, and 0.03 µmol/kg. These lipidated NMU analogs prolonged the anorectic effect of NMU in a dose‐dependent manner. This was likely an effect of improved pharmacokinetic properties because of improved vitro plasma stability. Accordingly, the data demonstrate that lipidated NMU analogs may represent drug candidates for the treatment of obesity. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational constraints in angiotensin IV to probe the role of Tyr2, Pro5 and Phe6

The aromatic amino acids Tyr and Phe in angiotensin IV (Ang IV) were conformationally constrained by the use of β‐Me substituted analogs, or cyclic constrained analogs. None of these modifications was allowed for Tyr¹, while only e‐β‐MePhe⁶ substitution resulted in an AngIV analog with high IRAP potency and selectivity versus AP‐N or the AT₁ receptor. This indicates an important role of the orientation of the Phe⁶ for inducing selectivity. Pro⁵ replacement with 2‐aminocyclopentanecarboxylic acid maintained IRAP potency and abolished AT₁ affinity. These results confirm the importance of conformational constrained amino acids to generate selectivity in bioactive peptides. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Examination of the active secondary structure of the peptide 101.10, an allosteric modulator of the interleukin‐1 receptor,by positional scanning using β‐amino γ‐lactams

The relationship between the conformation and biological activity of the peptide allosteric modulator of the interleukin‐1 receptor 101.10 (D ‐Arg‐D ‐Tyr‐D ‐Thr‐D ‐Val‐D ‐Glu‐D ‐Leu‐D ‐Ala‐NH₂) has been studied using (R)‐ and (S)‐Bgl residues. Twelve Bgl peptides were synthesized using (R)‐ and (S)‐cyclic sulfamidate reagents derived from L ‐ and D ‐aspartic acid in an optimized Fmoc‐compatible protocol for efficient lactam installment onto the supported peptide resin. Examination of these (R)‐ and (S)‐Bgl 101.10 analogs for their potential to inhibit IL‐1β‐induced thymocyte cell proliferation using a novel fluorescence assay revealed that certain analogs exhibited retained and improved potency relative to the parent peptide 101.10. In light of previous reports that Bgl residues may stabilize type II′β‐turn‐like conformations in peptides, CD spectroscopy was performed on selected compounds to identify secondary structure necessary for peptide biological activity. Results indicate that the presence of a fold about the central residues of the parent peptide may be important for activity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and characterization of the colistin peptide polymyxin E1 and related antimicrobial peptides.

Two strategies were developed to synthesize the acylated cyclic peptides know as polymyxins. Synthesis of polymyxin E1 and several analogs enabled us to evaluate the minimum inhibitory concentration of individual compounds against Gram-negative bacteria. In this study we also report the first identification of two component peptides in the complex polymyxin fermentation product colistin, a Thr2Ser isoform and an acyl group isomer. Both of these peptides, as well as a known component peptide, Leu7Ile, were similar to polymyxin E1 in potency, suggesting that conservative mutations in the colistin family are functionally inconsequential. In contrast, the acyclic analogs of all of these peptides were inactive, indicating that the characteristic lariat structure of the polymyxins is necessary for antimicrobial activity.     

Cyclic AMP-stimulating effect of vasoactive intestinal peptide in isolated epithelial cells of rat ventral prostate

Vasoactive intestinal peptide (VIP) has been shown to increase cyclic AMP content in isolated epithelial cells of rat ventral prostate. The stimulatory effect of VIP was dependent on time and temperature and was potentiated by a phosphodiesterase inhibitor. At 15°C, the response occurred in the 1·10⁻¹⁰−10⁻⁷ M range of VIP concentrations. Half-maximal stimulation of cellular cyclic AMP was obtained at 1.4 nM and maximal stimulation (3-fold basal level) at about 100 nM VIP. Chicken VIP and porcine secretin were agonists of porcine VIP but exhibited a 2-times higher and a 170-times lower potency, respectively. A high concentration (1·10⁻⁶ M) of glucagon, somatostatin, neurotensin, substance P, Met-enkephalin or Leu-enkephalin did not modify cAMP levels. The finding of a VIP-stimulated cAMP system in rat prostatic epithelial cells together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, as well as the presence of VIP-containing neurones innervating the male genitourinary tract, strongly suggest that VIP may be involved in prostatic growth regulation and function.