Vasoactive Intestinal Peptide Agonists Research Articles

Future pharmacological therapy in hypertension.

Hypertension (HTN) is a widespread and growing disease, with medication intolerance and side-effect present among many. To address these obstacles novel pharmacotherapy is an active area of drug development. This review seeks to explore future drug therapy for HTN in the preclinical and clinical arenas. The future of pharmacological therapy in HTN consists of revisiting old pathways to find new targets and exploring wholly new approaches to provide additional avenues of treatment. In this review, we discuss the current status of the most recent drug therapy in HTN. New developments in well trod areas include novel mineralocorticoid antagonists, aldosterone synthase inhibitors, aminopeptidase-A inhibitors, natriuretic peptide receptor agonists, or the counter-regulatory angiotensin converting enzyme 2/angiotensin (Ang) (1-7)/Mas receptor axis. Neprilysin inhibitors popularized for heart failure may also still hold HTN potential. Finally, we examine unique systems in development never before used in HTN such as Na/H exchange inhibitors, vasoactive intestinal peptide agonists, and dopamine beta hydroxylase inhibitors. A concise review of future directions of HTN pharmacotherapy.

Abstract 632: Vasomera, a Novel VPAC2-Selective Vasoactive Intestinal Peptide Agonist, Improves Arterial Elastance and Ventriculo-Arterial Coupling in Rats with Induced Diastolic Dysfunction via Renoprival Hypertension

Vasomera™ is a first-in-class stable long-acting vasoactive intestinal peptide (VIP) agonist, with preferential actions on the G-protein-coupled VPAC2-receptors; VIP mediates cardiopulmonary regulation and has been proposed as a therapeutic target for both hypertension and systolic dysfunction. Here, the acute effects of Vasomera in load-independent function and ventriculo-arterial coupling were in evaluated in a rats with chronic diastolic dysfunction, mimicking heart failure with preserved ejection fraction (HFpEF). Diastolic dysfunction, as demonstrated by altered E/A ratios via echocardiography, was induced by renoprival hypertension secondary to renal wrapping (RW). Conditioned rats (n = 5) were instrumented for the determination of left-ventricular (LV) hemodynamics as well as load-independent fnction and ventriculo-arterial coupling (via pressure-volume relationships); data were evaluated before/after a single-dose continuous IV infusion of Vasomera (7.5 μg/kg/min IV). Vasomera decreased the estimated arterial elastance (Ea: -20 ± 5%, P < 0.05) with negligible changes in heart rate (-4 ± 2%). Improved inotropy (Ees: +28 ± 8% and PRSW: +22 ± 3%, P < 0.05) was observed post-treatment, suggesting improved ventriculo-arterial coupling (Ea/Ees: -37 ± 5%, P < 0.05). Concomitantly, Vasomera reduced filling pressures (EDP: -30 ± 8%, P < 0.05), accelerated the time-constant of relaxation (tau: -24 ± 3%, P < 0.05) and improved compliance (EDPVR: -26 ± 5%, P < 0.05). Vasomera, a novel VPAC2 agonist, improved arterial elastance and ventriculo-arterial coupling, while improving indices of diastolic function (i.e., lusitropy) in animals with chronic renoprival hypertension mimicking HFpEF.

Vasomera™, a Novel VPAC2-Selective Vasoactive Intestinal Peptide Agonist, Improves Ventriculo-Arterial Coupling and Decreases Myocardial Demand in Sheep with Induced Ischemic Heart Failure

Vasomera™, a Novel VPAC2-Selective Vasoactive Intestinal Peptide Agonist, Improves Ventriculo-Arterial Coupling and Decreases Myocardial Demand in Sheep with Induced Ischemic Heart Failure

Effect of VIP on Intracellular [Ca2+], Extracellular Regulated Kinase 1/2, and Secretion in Cultured Rat Conjunctival Goblet Cells

To determine the intracellular signaling pathways that vasoactive intestinal peptide (VIP) uses to stimulate high molecular weight glycoconjugate secretion from cultured rat conjunctival goblet cells. Goblet cells from rat bulbar and forniceal conjunctiva were grown in organ culture. Presence and localization of VIP receptors (VPAC1 and 2) were determined by RT-PCR, immunofluorescence microscopy and Western blot analysis. Intracellular [Ca(2+)] ([Ca(2+)]i) was measured using fura-2. Extracellular signal-regulated kinase (ERK)-1/2 activity was determined by Western blot analysis. High molecular weight glycoconjugate secretion was measured with an enzyme-linked lectin assay on cultured goblet cells that were serum-starved for 2 hours before stimulation with VIP, VPAC1-, or VPAC2-specific agonists. Inhibitors were added 30 minutes prior to VIP. Activation of epidermal growth factor receptor (EGFR) was measured by immunoprecipitation using an antibody against pTyr followed by Western blot analysis with an antibody against EGFR. Both VIP receptors were present in rat conjunctiva and cultured goblet cells. VIP- and VPAC-specific agonists increased [Ca(2+)]i and secretion in a concentration-dependent manner. VIP also increased ERK1/2 activity, VIP-stimulated increase in [Ca(2+)]i. Secretion, but not ERK1/2 activity, was inhibited by the protein kinase A inhibitor, H89. VIP-stimulated secretion was inhibited by siRNA for ERK2 but not by siRNA for EGFR. VIP did not increase the phosphorylation of the EGFR. In conclusion, in cultured rat conjunctival goblet cells, VPAC1 and 2 receptors are functional. VIP stimulates a cAMP-dependent increase in [Ca(2+)]i and glycoconjugate secretion, but not ERK1/2 activation. VIP does not activate with EGFR.

Anti-hyperglycemic, antioxidant and anti-inflammatory effects of VIP and a VPAC1 agonist on streptozotocin-induced diabetic mice

Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide with potent anti-inflammatory properties, and its receptor, VPAC1, mediates most of the anti-inflammatory effects of VIP. Diabetes mellitus is characterized by increased oxidation and inflammation due to persistent hyperglycemia. This research was performed to investigate the effects of VIP and a VPAC1 agonist on streptozotocin (STZ)-induced type 1 diabetic mice. Intraperitoneal injection of VIP and VPAC1 agonist (50 nmol/kg/day in saline) over a 28-day period (1) decreased food intake, (2) increased body weight, (3) improved visceral index, (4) increased the fasting plasma insulin levels, (5) decreased the fasting plasma glucose, (6) improved the glucose tolerance, (7) decreased pancreas H 2O 2 and malondialdehyde (MDA) and (8) increased total antioxidant activity (T-AOC) in the liver, spleen and pancreas. The results of histopathological and immunohistochemical analysis showed that VIP and the VPAC1 agonist improved the structure and cellularity of islets and ameliorated the insulin-secreting activity of islets. Additionally, administration of VIP or the VPAC1 agonist not only significantly decreased the plasma TNFα and CRP and promoted IL-10 in diabetic mice but also blocked the increased NF-κB activity of pancreatic tissue in diabetic mice. Furthermore, the VPAC1 agonist displayed stronger effects than VIP. These results show that both VIP and VPAC1 agonist ameliorated STZ-induced diabetes and protected mice against oxidative stress and inflammation associated diabetes, with VPAC1 being the receptor most responsible for these positive effects in diabetic mice.

γ-Glutamyl 16-diaminopropane derivative of vasoactive intestinal peptide: a potent anti-oxidative agent for human epidermoid cancer cells

We previously demonstrated that the gamma-glutamyl 16 amine derivative of vasoactive intestinal peptide (VIP) acts as structural VIP agonist with affinity and potency higher than VIP. Herein, we have evaluated the effects of VIP and gamma-Gln16-diaminopropane derivative of VIP (VIP-DAP3) on the proliferation and protection from oxidative stress induced by hydrogen peroxide (H2O2) on epidermoid carcinoma cell lines. We have found that 10(-11) M VIP-DAP3 completely antagonized the inhibition induced by H2O2 on both cell proliferation and S-phase distribution while these effects were only partially antagonized by equimolar concentrations of VIP. Moreover, both oxidative stress and intracellular lipid oxidation induced by H2O2 were reduced by VIP and completely antagonized by VIP-DAP3. Thereafter, we have found that H2O2 increased p38 kinase activity and both HSP70 and HSP27 expression. VIP and VIP-DAP3 again antagonized these effects partially or totally, respectively. H2O2 reduced the activity of extracellular signal-regulated kinases Erk-1/2 and Akt, signalling proteins involved in proliferation/survival pathways. Again VIP restored the activity of both kinases while VIP-DAP3 caused indeed an increase of their activity as compared to untreated cells. These data suggest that VIP-DAP3 has a stronger anti-oxidative activity as compared to VIP likely based on its super-agonistic binding on the putative receptor.

Differential expression of vasoactive intestinal peptide and its functional receptors in human osteoarthritic and rheumatoid synovial fibroblasts

Vasoactive intestinal peptide (VIP) has shown potent antiinflammatory effects in murine arthritis and ex vivo in human rheumatoid arthritis (RA) synovial cells. To investigate the potential endogenous participation of this system in the pathogenesis of RA, we analyzed the expression and regulation of VIP and its functional receptors in human fibroblast-like synoviocytes (FLS) from patients with osteoarthritis (OA) and patients with RA. The expression of VIP was studied by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunofluorescence in cultured FLS, and by immunohistochemical analysis in synovial tissue. The expression and function of the potential VIP receptors in FLS were studied by RT-PCR, determination of intracellular cAMP production, cell membrane adenylate cyclase (AC) activity, and interleukin-6, CCL2, and CXCL8 production in response to VIP or specific agonists and antagonists. VIP expression was detected in human FLS at the messenger RNA and protein levels, and it was significantly decreased in RA FLS compared with OA FLS. VIP receptor type 1 (VPAC1) was the dominant AC-coupled receptor in OA FLS, in contrast with RA FLS, in which VPAC2 was dominant. Tumor necrosis factor alpha-treated OA FLS reproduced the VIP and VPAC receptor expression pattern of RA FLS. The antagonistic effects of VIP on FLS proinflammatory factor production were reproduced by VPAC1- and VPAC2-specific agonists in OA FLS and RA FLS, respectively. VIP expression is down-regulated in RA and in tumor necrosis factor alpha-treated FLS, suggesting that down-regulation of this endogenous antiinflammatory factor may contribute to the pathogenesis of RA. In RA FLS, VPAC2 mediates the antiinflammatory effects of VIP, suggesting that VPAC2 agonists may be an alternative to VIP as antiinflammatory agents.

Neuropeptides as autocrine growth factors in cancer cells.

Neuropeptides can function as autocrine growth factors in cancer cells. High levels of bombesin (BB) and neurotensin (NT)-like immunoreactivity are present in small cell lung cancer (SCLC), a neuroendocrine tumor. Vasoactive intestinal peptide (VIP) stimulates and somatostatin (SST) inhibits the release of BB-like peptides from SCLC cells. BB-like peptides bind to BB(2) receptors, which are present on the cell surface. BB-like peptides stimulate the mitogen activated protein kinase (MAPK) cascade leading to increased expression of nuclear oncogenes and growth factors in SCLC cells. Due to the high density of neuropeptide receptors present on the cell surface, SST analogs have been radiolabeled to image neuroendocrine tumors. VIP receptors are present in many epithelial cancers including breast, colon, non-small cell lung cancer (NSCLC), pancreatic and prostate cancers. Due to the high density of VIP receptors on lung cancer cells, radiolabeled VIP agonists may be used to image these tumors. VIP receptor antagonists, such as VIPhybrid, inhibit the growth of cancer cell lines in vitro and in vivo. VIPhybrid and SR48692, a NT receptor antagonist, potentiate the cytotoxicity of chemotherapeutic drugs. These results suggest that neuropeptide receptor antagonists may be useful in the treatment of cancer.

Transglutaminase-mediated polyamination of vasoactive intestinal peptide (VIP) Gln16 residue modulates VIP/PACAP receptor activity.

Previous data showing an increase of receptor binding activity of [R16]VIP, a vasoactive intestinal peptide (VIP) structural analogue containing arginine at the position 16 of its amino acid sequence, have pointed out the importance of a positive charge at this site. Here, the functional characterization of three VIP polyaminated adducts (VIPDap, VIPSpd, and VIPSpm), obtained by a transglutaminase-catalysed reaction between the VIP Gln16 residue and 1,3-diaminopropane (Dap), spermidine (Spd), or spermine (Spm), is reported. Appropriate binding assays and adenylate cyclase enzymatic determinations have shown that these VIP adducts act as structural VIP agonists, both in vitro and in vivo. In particular, their IC50 and EC50 values of human and rat VIP/pituitary adenylate cyclase activating peptide (PACAP)1 and VIP/PACAP2 receptors indicate that VIPDap is a VIP agonist, with an affinity and a potency higher than that of VIP, while VIPSpd and VIPSpm are also agonists but with affinities lower than that of VIP. These findings suggest that the difference in adduct agonist activity reflects the differences in the positive charge and carbon chain length of the polyamine covalently linked with the VIP Gln16 residue. In addition, the data obtained strongly suggest that the length of polyamine carbon chain could be critical for the interaction of the agonist with its receptor, even though possible hydrophobic interaction cannot be ruled out. In vivo experiments on murine J774 macrophage cell cultures have shown the ability of these compounds to stimulate the inducible nitric oxide synthase activity at the transcriptional level.

Anti-inflammatory properties of the type 1 and type 2 vasoactive intestinal peptide receptors: role in lethal endotoxic shock.

Vasoactive intestinal peptide (VIP) is a neuropeptide that can modulate several immune aspects. Previous reports showed that VIP attenuates the deleterious consequences of septic shock by inhibiting the production of pro-inflammatory agents and stimulating the production of anti-inflammatory cytokines in activated macrophages. In this study, by using selective VIP agonists, we investigated the differential involvement of the VIP receptors in the anti-inflammatory role of VIP. Both the type 1 VIP receptor (VPAC1) agonist, [K(15), R(16), L(27)] VIP 1-7-GRF 8-27, and the type 2 VIP receptor (VPAC2) agonist, Ro25-1553, protected mice from lethal endotoxemia by inhibiting the macrophage-derived pro-inflammatory mediators IL-6, TNF-alpha, IL-12 and NO, and by stimulating the production of the anti-inflammatory cytokine IL-10. In addition, both VIP and VPAC1 agonist, but not the VPAC2 agonist, reduced in vitro and in vivo the expression of the co-stimulatory B7. 1/B7.2 molecules, and the subsequent stimulatory activity for T helper cells in stimulated macrophages. The higher effectiveness of the VPAC1 agonist compared with the VPAC2 agonist suggests that VPAC1 is the major mediator of the anti-inflammatory action of VIP. Since VIP and the two agonists appear to affect multiple cytokines and inflammatory factors, they might provide a more efficient therapeutical alternative to the use of specific cytokine antibodies or antagonists.

Functional characterization of a receptor for vasoactive-intestinal-peptide-related peptides in cultured dermal melanophores from Xenopus laevis.

A receptor for vasoactive-intestinal-peptide (VIP)-related peptides was functionally characterized in a cell line derived from Xenopus melanophores using a recently described microtiter-plate-based bioassay. Activation of the melanophore VIP receptor by VIP or the peptides pituitary-adenylate-cyclase-activating polypeptide (PACAP 38), PACAP 27, and helodermin stimulated intracellular 3'-5' cyclic adenosine monophosphate (cAMP) accumulation and pigment dispersion in the cells. Helodermin, with an EC50 (concentration of peptide inducing half-maximal melanosome dispersion) of 46.5 pM, was the most potent activator of pigment dispersion, followed by PACAP 38 > VIP > PACAP 27. A similar order of potencies was observed for the peptides to induce cAMP accumulation. The responses to VIP agonists were selectively inhibited by the VIP antagonists PACAP-(6-27) and (N-Ac-Tyr(1)-D-Phe2)-growth-hormone-releasing factor[GRF](1-29)-NH2. Taken together, the results suggest that the melanophores express a VIP receptor that shares certain characteristics of, but also differs significantly from, other previously identified VIP receptors.

Similar involvement of VIP receptor type I and type II in lymphocyte chemotaxis

Effects of vasoactive intestinal peptide (VIP) on T cell migration are mediated by structurally distinct types I (VIPR1) and II (VIPR2) G protein-associated receptors. The two receptor types were proposed to transduce opposite effects on human T cells, since cytokine-induced chemotaxis of VIPR1-bearing HuT 78 human T cells, in contrast to T cells that express VIPR2, was inhibited by VIP. We studied chemotactic effects of VIP and related agonists with different affinities for VIP- and peptide histidine-isoleucine (PHI)-related receptors. All, VIP, secretin (SEC), a specific ligand for VIPR1, helodermin (HEL), an activator of helodermin-preferring VIPR2, as well as PHI, stimulated chemotaxis into micropore filters of both normal human peripheral blood T and B cells. Involvement of VIPRs was supported by inhibition of VIP-related agonist-induced migration of T and B cells with a VIPR antagonist. Peripheral blood lymphocyte (PBL) chemotaxis to VIP, SEC, HEL and PHI was reduced by inhibition of tyrosine kinase and pertussis or cholera toxin, whereas inhibition of protein kinase C only affected SEC-induced chemotaxis of PBL significantly. VIP-related agonists induced deactivation of migration at high concentrations. Findings in PBL suggest that VIPR1 activation can stimulate normal T and B cell chemotaxis. Different signaling mechanisms may be involved in mediating chemotactic activation of VIPRs and PHIRs, which may allow further exploration of receptor-dependent mechanisms and signaling pathways of VIP as mediator of PBL functions.

Vasoactive intestinal peptide enhancement of antigen-induced differentiation of a cultured line of mouse thymocytes.

The prominence of vasoactive intestinal peptide (VIP) in rodent thymic neurons suggested that this potent mediator of T cell functions may alter developmental responses of thymocytes to T cell receptor (TCR) -dependent stimulation. CD4+8+ DPK cells derived from a thymic lymphoma of a TCR transgenic mouse respond to pigeon cytochrome C (PCC) antigen in association with distinct I-E MHC II haplotypes on antigen-presenting cells (APCs) by differentiating into CD4+8- T cells. The specific recognition of VIP by two types of homologous G-protein-coupled receptors (VIPR1 and VIPR2) on DPK cells was attributable predominantly to VIPR1 before and to VIPR2 after exposure to APCs and PCC, as assessed by quantification of the respective mRNAs. PCC-evoked differentiation of DPK cells was enhanced significantly by 1 to 100 nM VIP after 3 to 4 days. The effects of VIP analogs with VIPR type selectivity implied that VIP enhancement of differentiation of DPK cells was mediated principally by VIPR2. Differential reduction in the expression of each type of VIPR by transfection of DPK cells with plasmids encoding the respective antisense mRNAs confirmed the central role of VIPR2 in VIP-enhanced conversion to CD4+8- T cells. The suppression of DPK cell differentiation by inhibitors of adenylyl cyclase and protein kinase A suggested a transductional role for VIP-elicited increases in [cAMP]i. That the changes in frequency of CD4+8+ and CD4+8- DPK cells reflected principally differentiation was supported by the lack of consistent differences between the two subsets in the effects of VIP and VIPR2 agonist on cell number, viability, apoptosis, and proliferation. VIP may be one endogenous mediator that explains the unique thymic microenvironment for topographically specific development of T cells.

Vasoactive intestinal peptide prevents excitotoxic cell death in the murine developing brain.

Excitotoxic damage may be a critical factor in the formation of brain lesions associated with cerebral palsy. When injected at birth, the glutamatergic analog ibotenate induces mouse brain lesions that strikingly mimic human microgyria. When ibotenate is injected at postnatal day 5, it produces transcortical necrosis and white matter cysts that mimic human perinatal hypoxic-like lesions. Vasoactive intestinal peptide (VIP) has potent growth-related actions and neuroprotective properties that influence mitosis and neuronal survival in culture. The goal of this study was to assess the protective role of VIP against excitotoxic lesions induced by ibotenate in developing mouse brain. VIP cotreatment reduced ibotenate-induced microgyric-like cortical lesions and white matter cysts by up to 77 and 85%, respectively. VIP protective effects were reproduced by a peptide derived from activity-dependent neurotrophic factor (ADNF), a trophic factor released by VIP-stimulated astrocytes, and by stearyl norleucine VIP, a specific VIP agonist that does not activate adenylate cyclase. Neither forskolin, an adenylate cyclase activator, nor pituitary adenylate cyclase-activating peptide, provided VIP-like protection. VIP and neurotrophic analogs, acting through a cAMP-independent mechanism and inducing ADNF release, could represent new avenues in the understanding and prevention of human cerebral palsy.

Characterization of binding sites for beta-adrenergic agonists and vasoactive intestinal peptide in the rat harderian gland.

Vasoactive intestinal peptide (VIP) receptors and beta-adrenergic receptors were investigated in rat Harderian gland membranes using 125I-VIP and 125I-cyanopindolol (125I-CYP), respectively, as ligands. The receptor bindings were rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The stoichiometric data suggested the presence of two classes of VIP receptors with Kd values of 0.36 and 65.37 nM and binding capacities of 323 and 39,537 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP as follows: VIP > helodermin > rGRF > PHI > > secretin. Glucagon, somatostatin, insulin, and pancreastatin were ineffective at concentrations up to 1 microM. However, the stoichiometric data suggest the presence of one class of binding sites for 125I-CYP. The Kd for the single site was 290 pM with a binding capacity of 32 pmol/L. The pharmacological characterization of 125I-CYP binding to membranes showed that only isoproterenol, a beta-adrenergic agonist, and norepinephrine, an alpha beta-adrenergic agonist, was as effective as propranolol in inhibiting 125I-CYP binding to Harderian gland membranes. However, alpha 1- and alpha 2-adrenergic agonists and blockers such as methoxamine, prazosin, clonidine, and yohimbine were shown to be ineffective. These results demonstrate the presence of specific VIP and beta-adrenergic receptors in the Harderian gland and suggest a role for VIP and beta-adrenergic agonists in the physiology of this gland.

Structural requirements for the binding of the pituitary adenylate‐cyclase‐activating peptide to receptors and adenylate‐cyclase activation in pancreatic and neuronal membranes

PACAP (pituitary adenylate-cyclase-activating peptide)-binding receptors were investigated in membranes from the rat pancreatic acinar cell line, AR 4-2J, the rat hippocampus and the human neuroblastoma cell line NB-OK, by 125I-PACAP(1-27) (amino acid residues 1-27 of N-terminal amidated PACAP) binding and adenylate cyclase activation. The relative binding of 125I-PACAP(1-27) to the receptor, and ability to activate adenylate cyclase were PACAP greater than or equal to PACAP(1-27) greater than PACAP(2-38) greater than PACAP(1-9)-VIP(10-28)(PACAP-VIP) greater than PACAP(2-27) greater than [Ser9,Tyr13]VIP greater than [Tyr13]VIP greater than or equal to [Ser9]VIP greater than or equal to VIP(1-23)-PACAP(24-27)(VIP-PACAP) greater than VIP (vasoactive intestinal peptide). The N-terminal moiety of PACAP(1-27) was more important than the three amino acids at the C-terminus for 125I-PACAP(1-27)-binding site recognition. For rat pancreatic 125I-VIP-binding sites tested with 125I-VIP, the order of binding affinity was PACAP = PACAP(1-27) greater than or equal to VIP = [Ser9]VIP = [Tyr13]VIP = [Ser9,Try13]VIP greater than or equal to PACAP-VIP greater than or equal to VIP-PACAP greater than PACAP(2-38) = PACAP(2-27). Pancreatic 125I-VIP-binding sites, when compared to 125I-PACAP(1-27)-binding sites, showed little specificity and only weak coupling, so that PACAP and VIP-PACAP acted only as partial VIP agonists on adenylate cyclase.

Solubilization and hydrodynamic characterization of guanine nucleotide-sensitive vasoactive intestinal peptide-receptor complexes from rat intestine

The purpose of this work was to solubilize vasoactive intestinal peptide (VIP) receptors from rat small intestinal plasma membranes and to analyze the nature and function of its molecular form(s) in a nondenaturing environment. Membranes were incubated with 3 nM 125I-VIP, washed, and treated with 1% Triton X-100. Chromatography on Sephadex G-50 showed that 60% of the extractable radioactivity was eluted with macromolecular components in the void volume. This radioactive material was dramatically reduced when 1 microM unlabeled VIP was present in the incubation medium or when membranes were pretreated with trypsin or dithiothreitol. Macromolecular components that had bound 125I-VIP were further chromatographed on Sephacryl S-300. Two peaks were observed: a major one (80%) and a minor one (20%) with Stokes radii of 5.2 and 3.1 nm, respectively. The labeling of both components was inhibited by unlabeled VIP or peptide with NH2-terminal histidine and COOH-terminal isoleucine amide (a VIP agonist). The presence of GTP (0.1 mM) in the incubation medium of membranes completely abolished the labeling of the 5.2-nm component but did not affect that of the 3.1-nm one. Moreover, GTP induced dissociation of 125I-VIP from the 5.2-nm component isolated by Sephacryl S-300 chromatography. This effect was time dependent and nucleotide specific. In contrast, GTP did not affect the stability of the 3.1-nm component. After cholera toxin catalyzed [32P]ADP-ribosylation of membranes, chromatography of solubilized material on Sephacryl S-300 showed that a peak of 32P radioactivity was coeluted with the 5.2-nm component.(ABSTRACT TRUNCATED AT 250 WORDS)

Ac-Tyr 1hGRF discriminates between VIP receptors from rat liver and intestinal epithelium

The vasoactive intestinal peptide (VIP) stimulates adenylate cyclase activity in rat liver and intestinal epithelium with low and high efficacy, respectively. The human growth hormone releasing factor (hGRF) derivative with acetylated N-terminus e.g. Ac-Tyr 1hGRF binds to VIP receptors in both tissues with a similar affinity. However, Ac-Tyr 1hGRF is a partial VIP agonist with high intrinsic activity in liver (50% that of VIP) whereas it behaves as a VIP antagonist in intestine. These results further argue for a possible heterogeneity of VIP receptor-coupled adenylate cyclase among tissues on a pharmacological basis.

Solubilization of rat lung vasoactive intestinal peptide receptors in the active state. Characterization of the binding properties and comparison with membrane-bound receptors.

We demonstrate here that rat lung membrane vasoactive intestinal peptide (VIP) receptors can be extracted in the active state using digitonin. Sepharose 4B gel filtration chromatography was utilized to demonstrate the formation of specific binding complexes between 125I-VIP and solubilized receptors. A rapid soluble receptor assay was established to separate 125I-VIP-receptor complexes from free 125I-VIP, which entailed differential precipitation of the 125I-VIP-receptor complex with polyethylene glycol and bovine gamma-globulin. Using this assay, several detergents were tested for their suitability to extract active VIP receptors, and most favorable results were obtained with digitonin, as judged by specific binding of 125I-VIP to the solubilized receptors. Time course studies indicated that the binding of 125I-VIP to digitonin extract was more rapid than to rat lung membranes. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the digitonin extract, as in the membrane. The values for the dissociation constants (Kd) were 200 pM for Class I and 8 nM for Class II receptors while the values for binding capacity (Bmax) were 200 and 2300 fmol/mg for Class I and II sites, respectively. Although the binding parameters of the two classes were similar to those in the membrane, the pharmacological properties were different, as evidenced by the inability of rat growth hormone releasing factor, a potent VIP agonist in the membrane, to displace specifically bound 125I-VIP from solubilized receptors. The ability to solubilize active VIP receptors represents an important step toward purification of the functional protein.

Solubilization of the liver vasoactive intestinal peptide receptor. Hydrodynamic characterization and evidence for an association with a functional GTP regulatory protein.

Vasoactive intestinal peptide (VIP) receptors were solubilized using the nondenaturing detergent Triton X-100 after occupancy of rat liver membrane-bound receptors with 125I-VIP. Gel filtration and ultracentrifugation on sucrose density gradients revealed the existence in the soluble macromolecular fraction of two labeled components: a major (80%) heavy component and a minor (20%) light one. The two components exhibit the following hydrodynamic parameters: Stokes radius, 5.8 nm: s20,w, 5.98 s; Mr, 150,000; frictional ratio, 1.52 for the major; and Stokes radius, 3.0 nm: s20,w, 3.98 s; Mr = 52,000; frictional ratio, 1.12 for the minor component. The labeling of these components was specific in that it dramatically decreased when unlabeled VIP was added together with 125I-VIP. The pharmacological specificity was also assessed by using 10 nM histidylisoleucineamide (a VIP agonist). Many lines of evidence indicate that the light component (Mr = 52,000) is the VIP-receptor complex while the heavy component (Mr = 150,000) is a ternary complex consisting of VIP, the receptor, and a guanine nucleotide regulatory protein, probably Ns. GTP is required to dissociate 125I-VIP from the heavy component whereas it is ineffective on the light component. This effect is nucleotide specific. After cholera toxin-induced [32P]ADP ribosylation of liver membranes, a high peak of 32P radioactivity containing the alpha subunit (Mr = 42,000) of the Ns protein is coeluted with the heavy component on Sephacryl S-300. By mild urea (2 M) treatment, the heavy component is converted into the light without significant dissociation of 125I-VIP. When a Triton extract of membranes prelabeled with 125I-VIP is treated with dithiobis(succinimidyl propionate) subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals a major band corresponding to Mr = 150,000. Alternatively, when prelabeled membranes are directly treated with the cross-linker, a major complex of Mr = 51,000 is observed. This may be related to different accessibility of the cross-linker to the site at which the receptor and the Ns protein interact in the two conditions. In conclusion, these data represent initial reports on the successful solubilization of functional VIP-receptor complexes and provide evidence for an interaction between liver VIP-receptor complexes and a GTP-binding protein.