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PubMed1992

A variant of Glanzmann's thrombasthenia which fails to express a GPIIb-IIIa related epitope that is recognized by a specific monoclonal antibody (C17).

Binding of different antibodies to the GPIIb-IIIa complex in resting (AP2, EDU3, C17) or activated platelets (PAC1) was studied by flow cytometry in a patient with a platelet defect involving GPIIb-IIIa related functions. The patient has a mild history of bleeding. Aggregation induced by ADP and collagen were absent but normal response was obtained with ristocetin. Platelets from the patient do not bind fibrinogen. Perfusion studies with flowing blood showed that patient's platelets have a marked impairment in the process of spreading and aggregate formation on vascular subendothelium. Electrophoretic studies in SDS-polyacrylamide gels demonstrated the presence of normal amounts and normal mobility of GPIIb-IIIa. Fibrinogen was present in the patient's platelets (68-74% of controls). The binding of AP2 and EDU3 to patient's resting platelets was normal as assessed by flow cytometry. In contrast, a decreased presence of the C17 antigen (10 fold lower than control platelets) was detected in resting platelets and a markedly reduced binding of PAC1 was found in thrombin activated platelets. These studies suggest that C17 recognizes an epitope of the GPIIb-IIIa in resting platelets that is implicated in the regulation of adhesive and cohesive properties of GPIIb-IIIa. Studies on this patient might be helpful for the understanding of GPIIb-IIIa functions.

Adenosine Diphosphate↗
PubMed1975

Heterogeneity of the lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from pseudomonas aeruginosa PAC1 and its phage-resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide-containing fractions which were separated on Sephadex G-75. The low-molecular-weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher-molecular-weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low-molecular-weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low-molecular-weight polysaccharide and contained mainly high-molecular-weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low-molecular-weight polysaccharide similar to that obtained from strain PAC1 and PAC1R. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.

Alanine↗
PubMed2000

In vitro evaluation of VIP/PACAP receptors in healthy and diseased human tissues. Clinical implications.

The evaluation of peptide receptors in man is relevant to identifying the physiological target tissues of a given peptide and to selecting diseases with a sufficient receptor overexpression for diagnostic or therapeutic intervention. VIP/PACAP receptors have been evaluated in normal and diseased human non-neuronal tissues by using in vitro receptor autoradiography with 125I-VIP or 125I-PACAP in tissue sections. As assessed by subtype-selective VIP analogs, VIP receptors of the VPAC1 subtype are found in a wide variety of tissues including liver, breast, kidney, prostate, ureter, bladder, pancreatic ducts, gastrointestinal mucosa, lung, thyroid, adipose, and lymphoid tissues. VPAC2 receptors are predominantly found in vessels and smooth muscles, whereas PAC1 receptors are present in the adrenal medulla. VIP/PACAP receptors are expressed in the majority of the most frequently occurring human tumors, including breast, prostate, pancreas, lung, colon, stomach, liver, and bladder carcinomas, as well as lymphomas and meningiomas, predominantly as VPAC1 receptors, as do their tissues of origin. Although leiomyomas predominantly express VPAC2 receptors, glial tumors, pituitary adenomas, neuroblastomas, paragangliomas, pheochromocytomas, and endometrial carcinomas preferentially express PAC1 receptors. The very wide distribution of VIP/PACAP receptors in the normal human body is indicative of the key role of these peptides in human physiology and pathophysiology. Moreover, the receptor expression in tumors is the molecular basis for clinical applications of VIP/PACAP such as in vivo scintigraphy and radiotherapy of tumors as well as VIP/PACAP analog treatment for tumor growth inhibition.

Autoradiography↗
PubMed1995

Identification of outer membrane proteins as target antigens of Pseudomonas aeruginosa Homma serotype M.

Pseudomonas aeruginosa is routinely serotyped in Japan by using the Homma scheme. The serotypes (O serotypes) are based on the chemical structure of the O-polysaccharide portion of the lipopolysaccharide (LPS). However, the nature of the Homma serotype M antigen has remained obscure because strains classified as serotype M usually have the rough phenotype. I characterized the target antigen of serotype M. The results of Western blotting (immunoblotting) showed that commercially available typing monoclonal antibody (MAb) against serotype M specifically bound to outer membrane protein (Opr) G and that typing rabbit antiserum specific for serotype M mainly contained antibodies against Oprs F and H2. These Oprs were distributed among all P. aeruginosa strains tested, including the serotype standard, serotype M and nontypeable strains, and a series of LPS-core-defective mutants derived from strain PAC1. However, the rough mutants derived from strain PAC1 agglutinated with the anti-serotype M antibodies, whereas the smooth strains did not. LPS preparations from serotype M strains possessed few or no polysaccharide chains. These strains had higher levels of binding activity with anti-serotype M MAb, as well as with anti-lipid A MAb, which specifically bound to the cell surface of the rough-natured gram-negative bacterial strains with high activity. The anti-serotype M antiserum also contained rough-LPS-specific antibodies, but the epitope was distributed among only a few strains. The results suggested that the Oprs acted as the serotype M antigen and that LPS did not. In conclusion, the rough strains agglutinated with anti-Opr antibodies and were distinguished as serotype M from the smooth strains of other serotypes, because the antibodies were accessible to the cell surface lacking O polysaccharides. I supposed that Homma serotype M is an index of the rough nature of P. aeruginosa strains rather than one of the O serotypes.

Animals↗
PubMed1989

Characterization of DNA sequence-common and sequence-specific proteins binding to cis-acting sites for cleavage of the terminal a sequence of the herpes simplex virus 1 genome.

The terminal 500-base-pair alpha sequence of the herpes simplex virus 1 genome contains signals for cleavage (Pac1 and Pac2) of unit-length DNA molecules from concatemers in unique stretches of sequences designated Ub and Uc, respectively, and a cis site for cleavage designated DR1. We report that nuclear extracts from infected cells contain factors which form two DNA-virus-specific protein complexes with components of the a sequence. Purification of the factors forming the V2 complex yielded a protein with an apparent molecular weight of 82,000 binding to DNA in a non-sequence-specific manner. Addition of Mg2+ to the purified protein-DNA probe mixture resulted in exonucleolytic degradation of the DNA. The protein was identified as the virus-specific DNase with monoclonal antibody specific for the viral enzyme. The purification of the proteins forming the V4 complex yielded two proteins with molecular weights of greater than 250,000 and 140,000 corresponding to infected cell protein 1 and to an as yet unidentified protein, respectively. These proteins formed two DNA sequence-common bands with a number of DNA probes and one sequence-specific band with probes containing both Pac2 and DR1 but not with probes containing either site alone or Pac1 and DR1. Since the DNA probe containing Pac2 and DR1 inserted into viral genome or into amplicons induced specific cleavage of the DR1 sequence whereas the nonreactive probes failed to induce the cleavage, the formation of this sequence-specific DNA-protein complex is significant and may reflect a DNA-protein interaction essential for cleavage. The possible role of the proteins identified in this study for the cleavage-packaging of viral DNA into capsids is presented.

DNA Probes↗
PubMed2005

Increased platelet count and leucocyte-platelet complex formation in acute symptomatic compared with asymptomatic severe carotid stenosis.

OBJECTIVE: The risk of stroke in patients with recently symptomatic carotid stenosis is considerably higher than in patients with asymptomatic stenosis. In the present study it was hypothesised that excessive platelet activation might partly contribute to this difference. METHODS: A full blood count was done and whole blood flow cytometry used to measure platelet surface expression of CD62P, CD63, and PAC1 binding and the percentage of leucocyte-platelet complexes in patients with acute (0-21 days, n = 19) and convalescent (79-365 days) symptomatic (n = 16) and asymptomatic (n = 16) severe (> or =70%) carotid stenosis. Most patients were treated with aspirin (37.5-300 mg daily) although alternative antithrombotic regimens were more commonly used in the symptomatic group. RESULTS: The mean platelet count was higher in patients with acute and convalescent symptomatic compared with asymptomatic carotid stenosis. There were no significant differences in the median percentage expression of CD62P and CD63, or PAC1 binding between the acute or convalescent symptomatic and asymptomatic patients. The median percentages of neutrophil-platelet (p = 0.004), monocyte-platelet (p = 0.046), and lymphocyte-platelet complexes (p = 0.02) were higher in acute symptomatic than in asymptomatic patients. In patients on aspirin monotherapy, the percentages of neutrophil-platelet and monocyte-platelet complexes (p = 0.03) were higher in acute symptomatic (n = 11) than asymptomatic patients (n = 14). In the convalescent phase, the median percentages of all leucocyte-platelet complexes in the symptomatic group dropped to levels similar to those found in the asymptomatic group. CONCLUSION: Increased platelet count and leucocyte-platelet complex formation may contribute to the early excess risk of stroke in patients with recently symptomatic carotid stenosis.

Acute Disease↗
PubMed2003

Regulation of atrial natriuretic peptide secretion by cholinergic and PACAP neurons of the gastric antrum.

Atrial natriuretic peptide (ANP) released from enterochromaffin cells helps regulate antral somatostatin secretion, but the mechanisms regulating ANP secretion are not known. We superfused rat antral segments with selective neural agonists/antagonists to identify the neural pathways regulating ANP secretion. The nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) stimulated ANP secretion; the effect was abolished by hexamethonium but doubled by atropine. Atropine's effect implied that DMPP activated concomitantly cholinergic neurons that inhibit and noncholinergic neurons that stimulate ANP secretion, the latter effect predominating. Methacholine inhibited ANP secretion. Neither bombesin nor vasoactive intestinal polypeptide stimulated ANP secretion, whereas pituitary adenylate cyclase-activating polypeptide (PACAP)-27, PACAP-38, and maxadilan [PACAP type 1 (PAC1) agonist] each stimulated ANP secretion. The PAC1 antagonist M65 1) abolished PACAP-27/38-stimulated ANP secretion; 2) inhibited basal ANP secretion by 28 +/- 5%, implying that endogenous PACAP stimulates ANP secretion; and 3) converted the ANP response to DMPP from 109 +/- 21% above to 40 +/- 5% below basal, unmasking the cholinergic component and indicating that DMPP activated PACAP neurons that stimulate ANP secretion. Combined atropine and M65 restored DMPP-stimulated ANP secretion to basal levels. ANP secretion in the antrum is thus regulated by intramural cholinergic and PACAP neurons; cholinergic neurons inhibit and PACAP neurons stimulate ANP secretion.

Animals↗
PubMed2004

Principal role of glycoprotein VI in alpha2beta1 and alphaIIbbeta3 activation during collagen-induced thrombus formation.

OBJECTIVE: High-shear perfusion of blood over collagen results in rapid platelet adhesion, aggregation, and procoagulant activity. We studied regulation of alpha2beta1 and alphaIIbbeta3 integrin activation during thrombus formation on collagen. METHODS AND RESULTS: Blockade of glycoprotein (GP) VI by 9O12 antibody or of P2Y purinergic receptors permitted platelet adhesion but reduced aggregate formation, fibrinogen binding, and activation of alpha2beta1 and alphaIIbbeta3, as detected with antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of these integrins. Combined blockade of GPVI and P2Y receptors and thromboxane formation abolished integrin activation but still allowed adhesion of morphologically unstimulated, nonprocoagulant platelets. Exogenous ADP partly restored the suppressive effect of GPVI blockade on integrin alpha2beta1 and alphaIIbbeta3 activation. Adhesion was fully inhibited only with simultaneous blocking of GPVI and alpha2beta1, indicating that the integrin can support platelet-collagen binding in the absence of its activation. Blockade or absence of GPIbalpha only moderately influenced integrin activation and adhesion unless GPVI was inhibited. CONCLUSIONS: GPVI- and autocrine-released ADP induce affinity changes of alpha2beta1 and alphaIIbbeta3 during thrombus formation on collagen under flow. These integrin changes are dispensable for adhesion but strengthen platelet-collagen interactions and thereby collagen-induced platelet activation. Integrin activation during thrombus formation on collagen was studied using fluorescent-labeled antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of alpha2beta1 and alphaIIbbeta3 integrin, respectively. Glycoprotein VI blockade by 9O12 antibody or P2Y ADP receptors reduced integrin activation along with aggregate formation and fibrinogen binding but not alpha2beta1-dependent adhesion.

Adenosine Diphosphate↗
PubMed1999

Low-density lipoprotein enhances platelet secretion via integrin-alphaIIbbeta3-mediated signaling.

LDL is known to increase the sensitivity of human platelets for agonists and to induce aggregation and secretion independently at high concentrations, but its mechanism of action is largely obscure. To clarify how LDL increases platelet sensitivity, cells were incubated in lipoprotein-poor plasma and treated with collagen at a concentration that induced approximately 20% secretion of 14C-serotonin. Preincubation with LDL (30 minutes at 37 degreesC) enhanced secretion in a dose-dependent manner to 60+/-14% at a concentration of 2 g LDL protein/L. Similar stimulation by LDL was seen when secretion was induced by the thrombin receptor-activating peptide. This enhancement was strongly reduced (1) in the presence of monoclonal antibody PAC1 against activated alphaIIbbeta3, a polyclonal antibody against alphaIIb, and in the presence of the fibrinogen peptides GRGDS and HHLGGAKQAGDV; (2) in alphaIIbbeta3-deficient platelets; and (3) after dissociation of alphaIIbbeta3. In contrast, binding of 125I-LDL to normal platelets in the presence of PAC1, anti-alphaIIb, GRGDS, and HHLGGAKQAGDV, and to alphaIIbbeta3-deficient platelets was normal. LDL increased the surface expression of fibrinogen in lipoprotein-poor plasma and fibrinogen-free medium, suggesting that extracellular and granular fibrinogen bind to alphaIIbbeta3 after platelet-LDL interaction. Platelets deficient in fibrinogen (<0.5% of normal) or von Willebrand Factor (<1% of normal) but containing normal amounts of other ligands for alphaIIbbeta3 preserved responsiveness to LDL, indicating that occupancy of alphaIIbbeta3 was not restricted to fibrinogen. Inhibition of protein kinase C (bisindolylmaleimide) diminished fibrinogen binding and sensitization by LDL; inhibition of tyrosine kinases (herbimycin A) left fibrinogen binding unchanged but diminished sensitization by LDL. We conclude that an increased concentration of LDL, such as observed in homozygous familial hypercholesterolemia, sensitizes platelets to stimulation by collagen and thrombin receptor-activating peptide via ligand-induced outside-in signaling through integrin-alphaIIbbeta3.

Blood Platelets↗
PubMed2003

Smooth muscle--specific expression of CYP4A1 induces endothelial sprouting in renal arterial microvessels.

Cytochrome P450 (CYP) 4A1 has been characterized as the most efficient arachidonic acid omega-hydroxylase catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of the renal and cerebral microcirculation and a mitogen for smooth muscle cells. We constructed adenoviruses expressing the CYP4A1 cDNA or LacZ under the control of the smooth muscle cell-specific promoter SM22alpha (Ad-SM22-4A1 and Ad-SM22-nLacZ, respectively). Beta-galactosidase expression was detected in Ad-SM22-nLacZ-transduced vascular smooth muscle A7r5 and PAC1 cells, but not in Ad-SM22-nLacZ-transduced 3T3 fibroblasts or vascular endothelial cells. Likewise, CYP4A1 mRNA and protein were detected in Ad-SM22-4A1-transduced A7r5 and PAC1 cells. Ad-SM22-4A1-transduced A7r5 cells metabolized lauric acid to 12-hydroxy-lauric acid at a rate 5 times greater than that of cells transduced with Ad-SM22-nLacZ (4.79+/-1.77 versus 0.97+/-0.57 nmol 12-hydroxy lauric acid/10(6) cells per h). Smooth muscle-specific LacZ expression was also detected in microdissected renal interlobar arteries transduced with Ad-SM22-nLacZ. Arteries transduced with Ad-SM22-4A1 produced higher levels of 20-HETE (4.04+/-0.29 and 13.43+/-2.84 ng/mg protein in Ad-SM22-nLacZ-transduced and Ad-SM22-4A1-transduced arteries, respectively) and demonstrated a marked angiogenic activity measured as the total length of sprouting neovessels (12.63+/-3.66 mm in Ad-SM22-4A1-transduced vessels versus 1.79+/-0.89 mm in Ad-SM22-nLacZ-transduced vessels). This angiogenic activity represented endothelial cell sprouting and was fully blocked by treatment with HET0016, a selective inhibitor of CYP4A-catalyzed reactions. The inhibitory effect of HET0016 was reversed by addition of a 20-HETE agonist. We conclude that Ad-SM22-4A1 drives a smooth muscle-specific functional expression of CYP4A1 and demonstrates increased angiogenesis, presumably via increased production of 20-HETE.

3T3 Cells↗
PubMed2006

Neuropeptide signaling and hydrocephalus: SCO with the flow.

Congenital hydrocephalus affects 0.1-0.3% of live births, with a high mortality rate (approximately 50%) in the absence of surgical intervention. Although the insertion of shunts alleviates the symptoms of the majority of congenital cases, the molecular basis of hydrocephalus and the mechanisms of cerebrospinal fluid (CSF) circulation remain largely unknown. Two important players are the subcommissural organ/Reissner's fiber (SCO/RF) complex and the ventricular ependymal (vel) cells that together facilitate the flow of the CSF through the narrow canals of the ventricular system. In this issue of the JCI, Lang et al. demonstrate that overexpression of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I (PAC1) receptor gene results in abnormal development of the SCO and vel cells, leading to congenital hydrocephalus (see the related article beginning on page 1924). The ligand for the PAC1 receptor is the neuropeptide PACAP, which uncovers what the authors believe to be a novel role for this signaling cascade in the regulation of CSF circulation.

Animals↗
PubMed2006

PACAP, VIP, and PHI: effects on AC-, PLC-, and PLD-driven signaling systems in the primary glial cell cultures.

Pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and peptide histidine-isoleucine (PHI) are members of a superfamily of structurally related peptides widely distributed in the body and displaying pleiotropic biological activities. All these peptides are known to act via common receptors-VPAC1 and VPAC2. In addition, the effects of PACAP are mediated through its specific receptor named PAC1. The main signal transduction pathway of the mentioned receptors is adenylyl cyclase (AC)-->cAMP system. PACAP and VIP may also signal through receptor-linked phospholipase C (PLC)-->IP3/DAG-->PKC and phospholipase D (PLD)-->phosphatidic acid (PA) pathways. In the present article, we have studied the effects of PACAP, VIP, and PHI (0.001-5000 nM) on the AC-, PLC-, and PLD-driven signaling pathways in rat primary glial cell (astrocytes) cultures. All tested peptides dose-dependently and strongly stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in this experimental model, displaying the following rank order of potency: PACAP >> VIP > or = PHI. Their effects on PLC-IP3/DAG were weaker, while only PACAP and VIP (0.1-5 microM) significantly stimulated PLD activity. The obtained results showed that rat cerebral cortex-derived astrocytes are responsive to PACAP, VIP and PHI/PHM and possess PAC1 and likely VPAC-type receptors linked to activation of AC-cAMP-, PLC-IP3/DAG-, and PLD-PA signaling systems.

Adenylyl Cyclases↗
PubMed2006

PACAP stimulates the release of interleukin-6 in cultured rat Müller cells.

We have investigated the in vivo effect of PACAP on rat Müller cells that are the predominant glial element in the retina. Müller cells were treated with PACAP38, either alone or in the presence of the PACAP-selective antagonist, PACAP6-38. Cellular proliferation was determined by measuring the incorporation of bromodeoxyuridine, while interleukin-6 (IL-6) levels in the culture medium were examined using a B9 cell bioassay. In cultured rat Müller cells, the expression of PACAP receptor (PAC1-R) was assessed with immunohistochemistry using a PAC1-R-specific antiserum. PACAP stimulated IL-6 production in Müller cells at a concentration as low as 10(-12) M, which was not sufficient to induce cell proliferation. This elevation of IL-6 production was significantly inhibited by PACAP6-38. These data suggest that Müller cells are one of the target cells for PACAP, stimulating the release of IL-6, and providing a mechanism whereby PACAP exerts a significant neuroprotective effect in the retina.

Animals↗
PubMed2006

Expression of PACAP receptor mRNAs by neuropeptide Y neurons in the rat arcuate nucleus.

Neuropeptide Y (NPY) and pituitary adenylate cyclase-activating polypeptide (PACAP) exert opposite actions in energy homeostasis: NPY is a potent orexigenic peptide whereas PACAP reduces food intake. PAC1-R and VPAC2-R mRNAs are actively expressed in the arcuate nucleus of the hypothalamus which contains a prominent population of NPY neurons. By using a double-labeling in situ hybridization technique, we now show that a significant proportion of NPY neurons express PAC1-R or VPAC2-R mRNA. This observation indicates that PACAP may regulate the activity of NPY neurons, suggesting that the inhibitory effect of PACAP on food intake may be mediated, at least in part, through modulation of NPY neurotransmission.

Animals↗
PubMed2006

Cyclic AMP formation in C6 glioma cells: effect of PACAP and VIP in early and late passages.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) exert their actions via common receptors, VPAC1 and VPAC2, which are equally sensitive to both peptides, while PACAP stimulates its specific PAC1-type receptors. Both peptides potently stimulate cAMP production in different biological systems. In the present article, we examined the effects of PACAP and VIP on cAMP formation in C6 rat glioma cells used between passages 12-28 (early) and 120-136 (late). In the presence of the PDE inhibitor IBMX (0.1 mM), PACAP (0.1 microM) and VIP (1 microM) strongly stimulated cAMP synthesis in C6 cells in early passages, but not in C6 cells in late passages. In contrast, forskolin (10 microM), a direct activator of adenylyl cyclase, and isoprenaline (10 microM), a beta-adrenergic receptor agonist, strongly stimulated cAMP production in both early and late C6 cell passages. Concentration-dependent studies carried out in early passages with PACAP-38, PACAP-27, mammalian and chicken VIPs, and PHI/PHM peptides (1-5 microM) revealed that both forms of PACAP produced strong cAMP accumulation, VIP peptides were less effective than PACAP, while the cAMP effects of PHI/PHM peptides were noticeable only at the highest doses tested. These results suggest that C6 glioma cells in early passages possess functional PAC1 and possibly VPAC-type receptors, but either the density of PACAP/VIP receptors progressively declines or the PACAP/VIP receptor-Gs protein coupling becomes less effective through culture passages.

Animals↗
PubMed2004

VPAC2-R mediates the lipolytic effects of pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide in primary rat adipocytes.

The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are structurally and functionally related. Their actions have been shown to be mediated by three different receptor subtypes: PAC1-R, which has exclusive affinity for PACAP, and VPAC1-R and VPAC2-R, which have equal affinity for PACAP and VIP. We recently showed that PACAP38 induces lipolysis in rat adipocytes, and in the present study we examined whether VIP has similar effects and which of the three receptors mediates this PACAP/VIP action. We showed by RT-PCR that all three receptor subtypes are present in rat adipocytes. We demonstrated that VIP (1-100 nm), like PACAP38, stimulates lipolysis in isolated adipocytes, as determined by glycerol release. By a pharmacological approach, using antagonists and agonists specific for the receptor subtypes, we elucidated the mechanisms by which PACAP38 and VIP mediate their lipolytic effects. We found that antagonists of PAC1-R [PACAP(6-38)] and VPAC1-R (PG97-269) did not affect lipolysis induced by 0.1-100 nm PACAP38 or VIP, and that a VPAC1-R agonist [K15, R16, L27VIP(1-7)GRF(8-27)] did not affect lipolysis at 1-1000 nm. However, two different VPAC2-R agonists [Hexa-VIP(1-28) and Ro25-1553] clearly mimicked the lipolytic effect of PACAP38 and VIP. In addition, the VPAC2-R antagonist PG99-465 (100 nm) caused right-shifted dose-response curves of PACAP38- and VIP-induced lipolysis. These results therefore provide evidence that all three PACAP/VIP receptor subtypes are expressed in primary rat adipocytes, but that the VPAC2-R subtype is responsible for mediating the lipolytic effects induced by PACAP38 and VIP.

Adipocytes↗
PubMed1999

Molecular and functional characterization of pituitary adenylate cyclase-activating polypeptide (PACAP-38)/vasoactive intestinal polypeptide receptors in pancreatic beta-cells and effects of PACAP-38 on components of the insulin secretory system.

It has been previously demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) regulates insulin secretion. PACAP exerts its biological action by binding to at least three different receptor subtypes coupled to different signal transduction mechanisms. The signaling pathways underlying the insulinotropic effect of PACAP involve mainly the activation of adenylate cyclase to form cAMP, which directly and indirectly, through increased intracellular Ca2+, stimulates insulin exocytosis. In the present study we have characterized the functional and molecular expression of PACAP/vasoactive intestinal polypeptide receptors isoforms and subtypes and its isoforms in a beta-cell line and in isolated rat pancreatic islets. Although insulinoma cells express the messenger RNA encoding PAC1 (-R and -hop variants), VPAC1 and VPAC2, binding experiments indicate the preponderance of PAC1 over VPAC 1-2 receptors. We have also shown that the main signaling pathway of PACAP in beta-cells is mediated by adenylate cyclase, whereas the inositol 1,4,5-trisphosphate pathway is almost inactive. Furthermore, we have demonstrated that PACAP exerts long-term effects on beta-cells, such as transcriptional regulation of the insulin gene and genes of the glucose-sensing system (GLUT1 and hexokinase 1).

Adenylyl Cyclases↗
PubMed1999

A cloned frog vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide receptor exhibits pharmacological and tissue distribution characteristics of both VPAC1 and VPAC2 receptors in mammals.

Three receptor subtypes for the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been identified in mammals: the PAC1 receptor (PAC1-R) which is selectively activated by PACAP, and two VPAC receptors (VPAC1-R and VPAC2-R), which are equally stimulated by PACAP and VIP. The structures of PACAP and VIP have been well conserved during evolution, but little is known about VIP/PACAP receptors in nonmammalian species. An amphibian VIP/PACAP receptor complementary DNA (cDNA) has been cloned and characterized from a frog (Rana ridibunda) pituitary cDNA library. The predicted protein contains seven putative transmembrane domains and exhibits the highest sequence identity (65%) with the human VPAC1-R. The cloned cDNA was transiently expressed in LLC-PK1 cells, and its pharmacological profile was determined in comparison with the human VPAC1-R. Both PACAP and VIP stimulated cAMP accumulation through the cloned receptor with an EC50 of about 30 nM. In contrast, secretin, at concentrations that stimulate the human VPAC1-R, had no effect on cAMP production. RT-PCR analysis revealed the widespread distribution of this frog VIP/PACAP receptor in peripheral tissues. In situ hybridization histochemistry using a complementary RNA probe showed that the receptor gene is highly expressed in several hypothalamic and thalamic nuclei and to a lesser extent in the pallium and striatum. In the pituitary, the highest messenger RNA levels were detected in the distal lobe. Taken together, these data show that the cloned frog receptor shares several common features with both the VPAC1-R and VPAC2-R of mammals; the frog receptor exhibits the highest sequence identity with mammalian VPAC1-R, but the lack of effect of secretin and the brain distribution of the receptor are reminiscent of the characteristics of the mammalian VPAC2-R. The sequence of the frog receptor should thus prove useful to decipher the structure-activity relationships of the VIP/PACAP receptor family.

Adenylyl Cyclases↗