Evidence for multiple rat VPAC1 receptor states with different affinities for agonists.

We compare the binding properties of [125I-VIP] and [125I]-Ro 25 1553 to VPAC1 receptors, expressed in stably transfected CHO cells. [125I]-VIP labelled two VPAC1 receptor states, while [125I]-Ro 25 1553 labelled selectively a limited number of high-affinity receptors. This high-affinity state probably corresponds to an agonist-receptor-Gs ternary complex as its properties (guanyl nucleotides, EC50 values and maximal effect) were affected by cholera toxin pre-treatment. Both high- and low-affinity receptors participated in the adenylate cyclase activation. This suggested that agonists activate not only low-affinity uncoupled receptors by facilitating the ternary complex formation, but also activated the high-affinity ternary complex by accelerating the GTP binding to emptied, receptor-bound G proteins.

Ontogenic development of the adenylyl cyclase enzyme and the alpha s, alpha i1 and alpha i2 G-protein regulatory subunits from rat prostate.

The expression of alpha s, alpha i1 and alpha i2 G-protein subunits measured by immunoblot increased in the rat prostate during sexual maturation, supporting their involvement in proliferation/differentiation. Northern blotting gave transcripts of 1.8 and 4 kb for alpha s, 1.4 and 4.5 kb (mainly) for alpha i1, and 2.4 kb for alpha i2 with levels suggesting a differential regulation (at transcription or post-transcription for alpha s, transcription for alpha i1, and translation for alpha i2). The stimulatory effects of forskolin, vasoactive intestinal peptide (VIP) and isoproterenol on adenylyl cyclase activity increased between 0.5-3 mo, remained constant up to 12 mo and decreased thereafter, conceivably following the expression of VIP and beta-adrenergic receptors. However, G-protein activation of adenylyl cyclase (by GTP and Gpp[NH]p) was maximal at 0.5 mo and then decreased as it occurred with toxin-catalyzed ADP-ribose incorporation to alpha subunits suggesting that other factors are also involved in the regulation of G-protein activity during rat prostatic development.

Analysis of vasoactive intestinal peptide receptors and the G protein regulation of adenylyl cyclase in seminal vesicle membranes from streptozotocin-diabetic rats.

The present report describes the status of the vasoactive intestinal peptide (VIP) receptor/effector system of signal transduction in seminal vesicle from streptozotocin (STZ)-treated rats. STZ-treatment modified the binding parameters of the high-affinity sites for VIP in seminal vesicle: 0.78 +/- 0.10 and 2.54 +/- 0.30 nM for the dissociation constant (Kd) in control and diabetic rats, respectively; 0.07 +/- 0.01 and 0.15 +/- 0.03 pmol VIP/mg protein for the maximum binding capacity (Bmax) in control and diabetic rats, respectively. It was associated with a reduced potency of VIP on the stimulation of adenylyl cyclase activity in the diabetic state (ED50 = 64.0 +/- 20.0 nM) as compared to control (ED50 = 9.5 +/- 4.3 nM). In contrast, the stimulatory effects of GTP, Gpp[NH]p and forskolin on the enzyme activity were not modified in diabetic rats. The levels of G-protein subunits in rat seminal vesicle were studied by immunoblot of alpha s and alpha i subunits: whereas alpha i-subunit levels did not vary, those corresponding to alpha s subunit decreased after STZ treatment. In diabetic rats, low concentrations of Gpp[NH]p failed to inhibit forskolin-stimulated adenylyl cyclase activity, suggesting the absence of functional Gi in this condition. In conclusion, present results show a decrease in the sensitivity of the VIP receptor/effector system in seminal vesicle membranes from STZ-treated rats suggesting a physiopathological role for VIP in the seminal neuropathy observed in diabetes.

Neuroendocrine differentiation of the LNCaP prostate cancer cell line maintains the expression and function of VIP and PACAP receptors.

The molecular mechanisms involved in differentiation of prostate cancer cells to a neuroendocrine (NE) cell phenotype are not well understood. Here we used the androgen-dependent human prostate cancer cell line LNCaP to perform a systematic and broad analysis of the expression, pharmacology, and functionality of vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP) receptors. Reverse transcription polymerase chain reaction experiments, together with pharmacological approaches with a set of specific agonists and antagonists, demonstrated the presence of the three VIP/PACAP receptor subtypes (PAC1, VPAC1, and VPAC2 with a major role for VPAC1, acting through adenylate cyclase (AC) stimulation. An essentially similar pattern was observed by NE differentiated cells (4 days after serum deprivation) in spite of the important morphological changes observed. However, the expression of the prostate-specific antigen (PSA) decreased in NE cells (and increased again by dihydrotestosterone, DHT, treatment). The present demonstration of the induction of NE transdifferentiation in LNCaP cells by increasing concentrations of VIP adds value to previous observations on the role of cAMP in this process, an interesting topic in the comprehension of the molecular changes that are involved in the progression of prostate cancer to androgen independence.

Vasoactive intestinal peptide in the immune system: potential therapeutic role in inflammatory and autoimmune diseases.

Vasoactive intestinal peptide (VIP), a neuropeptide that is produced by lymphoid as well as neural cells, exerts a wide spectrum of immunological functions, controlling the homeostasis of the immune system through different receptors expressed in various immunocompetent cells. In the last decade, VIP has been clearly identified as a potent anti-inflammatory factor, which acts by regulating the production of both anti- and pro-inflammatory mediators. In this sense, VIP has been described to prevent death by septic shock, an acute inflammatory disease with a high mortality. In addition, VIP regulates the expression of co-stimulatory molecules, this being an action that may be related to modulating the shift toward Th1 and Th2 differentiation. We have recently reported that VIP prevents the deleterious effects of an experimental model of rheumatoid arthritis, by downregulating both inflammatory and autoimmune components of the disease. Therefore, VIP has been proposed as a promising candidate alternative treatment for acute and chronic inflammatory and autoimmune diseases such as septic shock, arthritis, multiple sclerosis, Crohn disease, or autoimmune diabetes.

Receptors for pituitary adenylate cyclase-activating peptide in human liver.

The presence as well as the pharmacological, molecular, and functional properties of pituitary adenylate cyclase-activating peptide (PACAP) receptors have been analyzed in human liver membranes compared in parallel with vasoactive intestinal peptide (VIP) receptors. [125I]PACAP-27 bound to two classes of receptors with high [dissociation constant (Kd) = 0.47 nmol/L] and low (Kd = 8.0 nmol/L) affinities that represented about 34% and 66% of total binding sites, respectively. The pharmacological profile of [125I]VIP and [125I]PACAP-27 binding to membranes supported the coexistence with VIP receptors of specific receptors for PACAP with a mol wt equal to 67.7K. When [125I]PACAP-27 was used, the order of potency of various related peptides for competition of tracer binding was PACAP-27 greater than PACAP-38 greater than VIP. Both PACAP-27 and VIP stimulated adenylate cyclase activity with similar efficacy, although PACAP-27 showed a potency (half-maximal efficient concentration or EC50 = 0.5 nmol/L) greater than that of VIP (EC50 = 4.1 nmol/L). When the two peptides were present simultaneously in the incubation medium, no additive effect on the stimulation of adenylate cyclase activity was observed, which suggests a unique receptor coupled to this enzyme.

Characterization of a novel VPAC(1) selective agonist and identification of the receptor domains implicated in the carboxyl-terminal peptide recognition.

Vasoactive Intestinal Polypeptide (VIP) interacts with a high affinity to two subclasses of G protein coupled receptors named VPAC(1) and VPAC(2), and has a 3 - 10 fold preference for VPAC(1) over VPAC(2) receptors. Selective ligands for each receptor subclass were recently described. [R(16)]-PACAP (1 - 23) and [L(22)]-VIP are two selective VPAC(1) agonists. Chimaeric human VPAC(2)-VPAC(1) recombinant receptors expressed in CHO cells were used to identify the receptor domains implicated in these two selective ligands recognition. The VPAC(2) preference for [R(16)]-PACAP (1 - 27) over [R(16)]-PACAP (1 - 23) did not require the receptor's NH(2)-terminus domain but involved the whole transmembrane domain. In contrast, the selectivity of [L(22)]-VIP depended only on the presence of the NH(2) terminus and EC(2) domains of the VPAC(1) receptor. The present data support the idea that in the GPCR-B family of receptors the different selective ligands require different domains for their selectivity, and that the peptides carboxyl terminal sequence (amino acids 24 - 27) folds back on the transmembrane receptor domain, close to the peptides, aminoterminus.

Proline residue 280 in the second extracellular loop (EC2) of the VPAC2 receptor is essential for the receptor structure.

Inspection of the amino acid sequence of the human VPAC1 and the VPAC2 receptors after alignment of the conserved residues indicates that the second extracellular loop (EC2) is one amino acid shorter in the VPAC1 receptor due to the lack of a proline residue in position 294. We hypothesized that this could be of importance for receptor structure and/or for ligand recognition. Insertion by directed mutagenesis of a proline in that position (294 VPAC1) had little consequence on the binding of several agonists but reduced the affinity for the VPAC1 antagonist. Coupling of the 294 VPAC1 receptor to adenylate cyclase was improved, as demonstrated by an increased affinity for VIP and other agonists, and by a shift of the VPAC1 antagonist to partial agonist behavior. Deletion of the proline 280 (DeltaPro280 VPAC2) in the VPAC2 receptor markedly reduced the apparent affinity for all the agonists tested. Replacement of the proline by a glycine residue had a smaller effect on the ligands affinities. The proline residue in the VPAC2 receptor EC2 is thus essential for the receptor structure, and the EC2 domain is involved in ligand recognition and receptor functionality.

Expression of vasoactive intestinal peptide (VIP) receptors in human uterus.

We show the existence of functional vasoactive intestinal peptide (VIP) receptors in normal human female genital tract (endometrium, myometrium, ovary and Fallopian tube) as well as in leiomyoma (a frequent uterine pathology). The correlation between VIP binding and stimulation of adenylyl cyclase activity for all studied tissues was linear (r = 0.86) suggesting the expression of VIP receptors throughout the human female genital tract. Immunodetection of VIP receptor subtypes gave different molecular weights for VPAC(1) (47 kDa primarily) and VPAC(2) (65 kDa), which may be due to different glycosylation extents. In conclusion, this study demonstrates the expression of both subtypes of VIP receptors and their functionality in human female genital tract, suggesting that this neuropeptide could play an important physiological and pathophysiological role at this level.

Stimulation of the adenylyl cyclase activity in human endometrial membranes by VIP and related peptides.

Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylyl cyclase activity in human endometrial membranes. The effect was dependent on the time and temperature of incubation as well as on the concentration of endometrial membrane proteins in the medium. In the presence of 1 microM GTP, half-maximal stimulation of adenylyl cyclase activity was observed at 25.0 +/- 7.0 nM VIP, whereas the maximal activity (at 1 microM VIP) corresponded to an increase of about 140% with respect to basal values (7.5 +/- 0.6 pmol cyclic AMP/min/mg of protein). However, the maximal stimulation of adenylyl cyclase activity was obtained with helodermin (1 microM) that increased the activity by 170% over the basal. The relative potency of VIP-related peptides upon the adenylyl cyclase activity was: helodermin (ED50 = 1.8 +/- 1.4 nM) > VIP (ED50 = 25.0 +/- 7.0 nM) > PHI (ED50 = 725.0 +/- 127.2 nM). Secretin had a faint effect upon the adenylyl cyclase activity and glucagon was completely inefficient at this level. The presence of alpha s and alpha i subunits of G proteins in human endometrium was detected by immunoblot. Preliminary results showed the presence of two classes of 125I-VIP receptors in human endometrial membranes with the following stoichoimetric parameters: high affinity receptor (Kd = 2.0 nM, binding capacity 0.1 pmol VIP/mg protein) and low affinity receptor (Kd = 0.43 microM, binding capacity 13.1 pmol VIP/mg protein). The present results together with the known presence of VIP in human uterus and the actions of this neuropeptide in the adjacent myometrial tissue support the idea that VIP and related peptides may have a role in human endometrium.

Vasoactive intestinal polypeptide receptor VPAC(1) subtype is predominant in rat prostate membranes.

BACKGROUND: The 28-amino-acid neuropeptide vasoactive intestinal peptide (VIP) might play an important role in the physiology of the prostate, since it stimulates glandular secretion, inhibits muscle contraction, stimulates proliferation of epithelial cells, and increases the secretion of prostate-specific antigen (PSA). This neuropeptide may act through interaction with two types of high-affinity receptors, named VPAC(1) and VPAC(2) receptors. Recently, selective agonists and antagonists for each receptor subtype were synthesized. We used them to identify the VIP receptor subclass expressed in rat prostatic tissue. METHODS: We tested the capacity of selective labeled and unlabeled agonists and antagonists of VPAC(1) and VPAC(2) receptors to bind to rat prostatic membranes and to stimulate or prevent the stimulation of adenylate cyclase activity. RESULTS: The following selective peptides were used: VPAC(1) agonist ([K(15), R(16), L(27)] VIP (1-7)/GRF (8-27)); VPAC(1) antagonist (PG 97-269); and VPAC(2) agonist (RO 25-1553). The IC(50) values of [(125)I]-VIP binding inhibition for the different peptides in rat prostatic membranes were: VIP (1.7 nM) < VPAC(1) agonist (20 nM) < VPAC(1) antagonist (40 nM) < VPAC(2) agonist (329 nM). The EC(50) values of adenylate cyclase stimulation were similar to the IC(50) values for each peptide, and the Ki values for the VPAC(1) antagonist, inhibiting the adenylate cyclase activity stimulated by VIP and the VPAC(1) agonist, were 22 and 35 nM, respectively. Comparison of binding of [(125)I]-VIP and of [(125)I]-RO 25-1553 indicates the presence of 80% of VPAC(1) and 20% VPAC(2) receptors. CONCLUSIONS: In rat prostate membranes, VPAC(1) receptors are largely predominant. Binding studies were compatible with a ratio of 80/20 of VPAC(1)/VPAC(2) receptors, whereas functionally only VPAC(1) receptors were detected.

Effects of chronic ethanol ingestion on the vasoactive intestinal peptide receptor-effector system from rat seminal vesicle membranes.

We studied the modifications of the vasoactive intestinal peptide (VIP) receptor/effector system from the rat seminal vesicle after chronic ethanol ingestion. Ethanol treatment resulted in a decreased height of the secretory epithelium of seminal vesicle as well as in a weight loss of this gland. These morphological changes were accompanied by an increase of immunoreactive vasoactive intestinal peptide (VIP) levels and a decrease of the stimulatory effect of VIP adenylate cyclase activity in the seminal vesicle. The loss of sensitivity of the enzyme to VIP was conceivably related to a decrease in the affinity of VIP receptors rather than to a decrease in their number. The changes in the affinity of the VIP receptors were accompanied with a lower sensitivity of VIP binding to GTP, which suggest an uncoupling between the receptor and the transductor molecules. However, chronic exposure to ethanol did not modify either the levels of G-protein subunits (alpha(s) and alpha(i1/2)) or the GTPase activity from seminal vesicle membranes. Moreover, ethanol feeding did not affect adenylate cyclase activity stimulated by forskolin or by Gpp(NH)p. Thus, ethanol-induced changes in the sensitivity of adenylate cyclase to VIP appear to be attributed to an alteration in the VIP-receptor/G-protein interphase rather than in the G-protein/adenylate cyclase connection.

Somatostatin inhibits VIP- and forskolin-stimulated cyclic AMP accumulation in enterocytes from rat jejunum.

This study demonstrates the dual regulation by somatostatin of vasoactive intestinal peptide (VIP)-stimulated and forskolin-stimulated cyclic AMP accumulation by isolated rat intestinal epithelial cells. Somatostatin non-competitively inhibited (IC50 = 1 microM) the stimulatory effect of VIP on cyclic AMP accumulation, suggesting that the two neuropeptides act through separate receptors. The cyclic AMP accumulation produced by forskolin (a diterpene that stimulates directly the catalytic subunit of adenylate cyclase) was also inhibited by somatostatin in a dose-dependent manner. However, somatostatin did not modify the stimulatory effect of VIP on adenylate cyclase activity in a membrane preparation from the same cells, making it difficult to explain the mechanism of somatostatin action at this level. The data presented here suggest that somatostatin may play a physiological role in the regulation of nutrient absorption and the release of gut hormones or exocrine secretions by intestinal epithelial cells through the modulation of cyclic AMP production.

Vasoactive intestinal peptide (VIP) stimulates rat prostatic epithelial cell proliferation.

BACKGROUND: Androgens play a major role in supporting normal growth and functional maintenance in the prostate. However, this gland contains an array of neuroendocrine peptides that can play a regulatory role in its physiopathology. Among these peptides, one of the best studied is vasoactive intestinal peptide (VIP), which is abundant in autonomic nerves surrounding both human and rat prostatic acini. This neuropeptide may act through interaction with two types of high-affinity receptors, named VPAC(1) and VPAC(2) receptors. Another regulatory peptide, the pituitary adenylate cyclase-activating peptide (PACAP), interacts with these receptors with the same affinity as VIP, but binds with higher affinity to PAC(1) receptors. Human prostate tumors and rat prostate show a major presence of VPAC(1) receptors, whereas various findings suggest a role for VIP in prostatic development. Here we studied the effects of VIP on the proliferation of rat prostatic epithelial cells in culture. METHODS: We studied the [(3)H]-thymidine uptake by rat prostatic epithelial cells in culture, characterized previously by using biomarkers such as cytokeratin and vimentin. In these cells we tested the effect of VIP and PACAP-27 on two different signaling pathways, the cyclic AMP (cAMP) and the inositol phosphate (IPs). RESULTS: The rat prostatic cells in culture were cytokeratin (5,6,8) and vimentin positive, indicating that the culture was predominantly epithelial. The proliferation curves showed that the cells followed different states of growth: a quiescent, an exponential proliferative, and a steady state. Cyclic AMP production, but not inositol phosphate production, was increased in the presence of VIP and PACAP-27, which suggests the expression of VPAC(1) and/or VPAC(2) receptors primarily. VIP significantly increased prostatic cell proliferation in a bimodal manner, as shown for dibutyryl cyclic AMP (dbcAMP), which suggests that the effect of VIP upon prostatic proliferation is cAMP-dependent. CONCLUSIONS: Here, we demonstrate that VIP increased [(3)H]thymidine uptake by rat prostatic epithelial cells in culture, conceivably by the activation of the adenylate cyclase.

G-protein regulation of adenylate cyclase activity in rat prostatic membranes after chronic ethanol ingestion.

BACKGROUND: The possibility that long-term ethanol ingestion might alter either vasoactive intestinal peptide (VIP) content, VIP binding to membrane receptors, G-protein levels or adenylate cyclase activity in rat prostate was tested, as ethanol produces serious alterations in the hypothalamic-pituitary-gonadal axis and several modifications on different elements on signal transduction pathways in other systems. METHODS: Prostatic membranes from control and ethanol-treated (for 4 weeks) rats were used to study adenylate cyclase stimulation as well as for the immunodetection of stimulatory (alpha(s)) and inhibitory (alpha(i)1-2) G-protein subunits. Studies on VIP binding and cross-linking to receptors were performed using [125I]VIP. Prostatic VIP content was estimated by radioimmunoassay. GTPase activity was quantified by measuring the amount of 32Pi released from [gamma-32P]GTP. RESULTS: Chronic ethanol ingestion resulted in an increased presence of VIP in the rat prostate without any change on the VIP receptor/effector system in this gland. By contrast, the basal adenylate cyclase activity as well as the dose-dependent stimulation of this enzyme by either the nonhydrolyzable GTP analogue Gpp(NH)p or the beta-adrenergic agonist isoproterenol were enhanced in prostatic membranes after ethanol intake. Moreover, an increase in the content of G-protein subunits (alpha(S) and alpha(i)1-2) was observed without any change in GTPase activity in this condition. These modifications were accompanied by a significant decrease in rat prostate weight and, consequently, the height of the secretory epithelium in this gland. CONCLUSIONS: Considering the role of VIP in the mechanisms of secretion and cell proliferation in the prostate, the observed increases in the prostatic content of VIP and G-protein subunits make conceivable that VIP and cAMP signal transduction could be involved in the atrophy of the rat prostate and in the alterations in the composition of seminal fluid that appear in the alcoholic syndrome.

Ontogeny of vasoactive intestinal peptide receptors in rat ventral prostate.

1. The stimulatory effect of VIP on rat prostatic adenylyl cyclase changes during postnatal development. It peaks at 2 months (peripubertal period), remains in a plateau between 3 and 12 months (adult period), and decreases at 24 months (old period). 2. The stimulation of rat prostatic adenylyl cyclase by the beta-adrenergic agonist isoproterenol follows a pattern rather similar to that of VIP. 3. The values of VIP binding capacity correlate well with those observed for adenylyl cyclase between 1 and 12 months, whereas there appears to exist a great number of uncoupled VIP receptors at 0.5 and 24 months. 4. The apparent molecular mass (51 kDa) of the rat prostatic VIP-receptor complex remains unaltered during ontogenic development.

Vasoactive intestinal polypeptide VPAC1 and VPAC2 receptor chimeras identify domains responsible for the specificity of ligand binding and activation.

In order to identify the receptor domains responsible for the VPAC1 selectivity of the VIP1 agonist, [Lys15, Arg16, Leu27] VIP (1-7)/GRF (8-27) and VIP1 antagonist, Ac His1 [D-Phe2, Lys15, Arg16, Leu27] VIP (3-7)/GRF (8-27), we evaluated their binding and functional properties on chimeric VPAC1/VPAC2 receptors. Our results suggest that the N-terminal extracellular domain is responsible for the selectivity of the VIP1 antagonist. Selective recognition of the VIP1 agonist was supported by a larger receptor area: in addition to the N-terminal domain, the first extracellular loop, as well as additional determinants in the distal part of the VPAC1 receptor were involved. Furthermore, these additional domains were critical for an efficient receptor activation, as replacement of EC1 in VPAC1 by its counter part in the VPAC2 receptor markedly reduced the maximal response.

Different vasoactive intestinal polypeptide receptor domains are involved in the selective recognition of two VPAC(2)-selective ligands.

A vasoactive intestinal polypeptide (VIP) analog, acylated on the amino-terminal histidine by hexanoic acid (C(6)-VIP), behaved as a VPAC(2) preferring agonist in binding and functional studies on human VIP receptors, and radioiodinated C(6)-VIP was a suitable ligand for binding studies on wild-type and chimeric receptors. We evaluated the properties of C(6)-VIP, its analog AcHis(1)-VIP, and the VPAC(2)-selective agonist Ro 25-1553 on the wild-type VPAC(1) and VPAC(2) receptors and on the chimeric receptors exchanging the different domains between both receptors. VIP had a normal affinity and efficacy on the chimeras starting with the amino-terminal VPAC(2) receptor sequence. The binding and functional profile of these chimeric receptors suggested that the high affinity of Ro 25-1553 for VPAC(2) receptors is supported by the amino-terminal extracellular domain, whereas the ability to prefer C(6)-VIP over VIP is supported by the VPAC(2) fifth transmembrane (TM5)-EC(3) receptor domain. These results further support the hypothesis that the central and carboxyl-terminal regions of the peptide (modified in RO 25-1553) recognize the extracellular amino-terminal region domain, whereas the amino-terminal VIP amino acids bind to the TM receptor core. VIP had a reduced affinity and efficacy on the N-VPAC(1)/VPAC(2) and on the N-->EC(2)-VPAC(1)/VPAC(2) chimeric receptors. C(6)-VIP behaved as a high-affinity agonist on these constructions. The antagonists [AcHis(1),D-Phe(2),Lys(15),Arg(16), Leu(27)]VIP(3-7)/GRF(8-27) and VIP(5-27) had comparable affinities for the wild-type receptors and for the two latter chimeras, supporting the hypothesis that these chimeras were properly folded but unable to reach the high-agonist-affinity, active receptor conformation in response to VIP binding.