Jacques Winand

The pituitary adenylate cyclase activating polypeptide (PACAP I) and VIP (PACAP II VIP1) receptors stimulate inositol phosphate synthesis in transfected CHO cells through interaction with different G proteins.

The PACAP receptor (PACAP I receptor, selective for PACAP) and the PACAP II VIP1 receptor (recognizing PACAP and VIP with the same high affinity) were stably expressed in Chinese Hamster Ovary (CHO) cells. Cell lines expressing different receptor densities, as measured by binding saturation curves, were selected. Inositol phosphate production was stimulated dose dependently in all the cell lines by PACAP and VIP, and the order of potency of the agonists was identical to that of high affinity receptor occupancy. The stimulatory effect of a saturating peptide concentration was proportional to the total receptor density. At similar receptor densities, however, the PACAP receptor mediated stimulation was higher than the VIP receptor-mediated stimulation. Pretreatment of the cells with pertussis toxin for 8 h had no effect on receptor densities, did not alter the PACAP stimulated inositol phosphate synthesis by the cells expressing the PACAP I receptor but markedly inhibited the response of the cells expressing the PACAP II VIP1 receptor. Thus, the present results indicate that the two G(s)-coupled PACAP I and PACAP II VIP1 receptors may stimulate IP production. The maximal stimulation depended on the number of receptor expressed; the PACAP I and PACAP II VIP1 receptors probably activated the phospholipase C through G proteins of the G(q), and of the G(i)/G(o) families, respectively.

Discovery of a potent atrial natriuretic peptide antagonist for ANPA receptors in the human neuroblastoma NB-OK-1 cell line

The effects of seven competitive atrial natriuretic peptide (ANP) receptor antagonists were compared on cultured human neuroblastoma NB-OK-1 cells expressing exclusively ANPA receptors, by evaluating their capacity to inhibit [125I]ANP binding and to suppress ANP-stimulated cyclic GMP elevation. In ANP analogues with a shortened Cys7-Cys18 bridge, Asp13 and a hydrophobic Tic residue at position 16 expressed antagonistic activity, while Ala16 provoked lower antagonistic potency and Phe16 induced receptor activation. The binding affinity of A71915 ([Arg6, Cha8]ANP-(6-15)-D-Tic-Arg-Cys-NH2), the most potent antagonist (with a pKi of 9.18 and a pA2 of 9.48) was only 22 times less lower than that of the agonist ANP-(1-28).

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide stimulate two signaling pathways in CHO cells stably transfected with the selective type I PACAP receptor

The properties of the pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor were studied on a clone of Chinese hamster ovary cells (CHO) stably transfected with the recombinant receptor. PACAP(1-27), PACAP(1-38) and VIP inhibited [125I-acetyl-His1]PACAP (1-27) binding, stimulated cyclic AMP and inositol phosphates production and induced [Ca2+]i increase with the same order of potency: PACAP(1-27) = PACAP(1-38) > VIP. The concentrations required for half maximal receptor occupancy, IP3- and [Ca2+]i increase were not different for both PACAPs (1 nM) and 100-fold higher than those required for cyclic AMP increase (0.010 nM). These data suggest that the occupancy of a portion of the total receptors available was sufficient for maximal cyclic AMP production but not for maximal IP3 production. It is concluded that the possibility of the type I PACAP receptor being coupled to a transduction pathway is not located at the level of the ligand but rather at the level of the G-proteins.

Different antagonist binding properties of rat pancreatic and cardiac muscarinic receptors

The antagonist binding properties of rat pancreatic and cardiac muscarinic receptors were compared. In both tissues pirenzepine (PZ) had a low affinity for muscarinic receptors labelled by (3H)N-methylscopolamine [3)NMS) (KD values of 140 and 280 nM, respectively, in pancreatic and cardiac homogenates). The binding properties of pancreatic and cardiac receptors were, however, markedly different. This was indicated by different affinities for dicyclomine, (11-([(2-[diethylamino)-methyl)-1-piperidinyl] acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4) benzodiazepin-6-on) (AFDX-116), 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP) and hexahydrosiladifenidol (HHSiD). Pancreatic and cardiac muscarinic receptors also showed different (3H)NMS association and dissociation rates. These results support the concept of M2 receptor heterogeneity and confirm that M2 receptor subtypes have different binding kinetic properties.

Contrasting effects of PACAP and carbachol on [Ca2+]i and inositol phosphates in human neuroblastoma NB-OK-1 cells

The effects of PACAPs on [Ca2+]i were compared to those of carbachol in human neuroblastoma NB-OK-1 cells. PACAP(1-27) and PACAP(1-38) increased [Ca2+]i in a biphasic manner: a transient rise and a secondary plateau. The transient phase reflected the mobilization of [Ca2+]i pool(s) via the inositol phosphate pathway. The modest sustained plateau required extracellular Ca2+. Carbachol also increased [Ca2+]i in a biphasic manner, but it mobilized intracellular Ca2+ pool(s) with a higher efficacy than PACAPs, then greatly increased Ca2+ entry, this being accompanied by a more marked and prolonged elevation of IP3 and IP4 than with PACAPs. It is likely that cAMP-mediated phosphorylations due to PACAPs facilitated desensitization at the PACAP receptor-phospholipase C level, so that there was less Ca2+ handling through PACAP receptors than with muscarinic M1 receptors.

Atrial natriuretic peptide binds to ANP-R1 receptors in neuroblastoma cells or is degraded extracellularly at the Ser-Phe bond

ANP-R1 receptors for atrial natriuretic peptide (ANP) showed the following rank order of affinity in intact human neuroblastoma cells NB-OK-1: human ANP-(99-126) approximately human ANP-(102-126) approximately rat ANP-(99-126) (K1 17-32 pM) > human ANP-(103-126) > porcine brain natriuretic peptide (BNP). Analogues truncated at the C-terminal extremity or devoid of a disulphide bridge, such as rat ANP-(103-123), rat C-ANP-(102-121), rat ANP-(111-126), rat ANP-(99-109) and rat [desCys105,Cys121]ANP-(104-126) and chicken C-type natriuretic peptide, were not recognized. The occupancy of these high affinity ANP-R1 receptors led to marked cyclic GMP accumulation in the presence of 3-isobutyl 1-methylxanthine. An ectoenzymic activity, partly shed in the incubation medium, provoked the stepwise release of Phe-Arg-[125I]Tyr, Arg-[125I]Tyr and [125I]Tyr from rat [125I]ANP-(99-126), at an optimal pH of 7.0. Its inhibition by 1,10-phenanthroline, EDTA and bacitracin but not by thiorphan suggests the contribution of at least one neutral metalloendopeptidase, distinct from EC 3.4.24.11, for which ANP showed high affinity.

Functional characterization of muscarinic receptors in rat parotid acini

The muscarinic agonist, carbamylcholine, stimulated amylase secretion in rat parotid acini 6-fold, the 86Rb efflux 5-fold, the 45Ca efflux 5-fold and the accumulation of inositol monophosphate, bisphosphate, trisphosphate and tetrakisphosphate 4-, 4-, 3- and 3-fold, respectively. The EC50 of carbamylcholine on these parameters were 0.4, 0.5, 1.3, 12, 12, 6 and 9 microM, suggesting spareness between phospholipase C activation and amylase secretion. These muscarinic responses were inhibited by four muscarinic antagonists with an order of potency on all parameters and on receptor occupancy (using N-[methyl-3H]scopolamine as a tracer): atropine greater than hexahydrosiladifenidol greater than pirenzepine greater than AF-DX 116. The pA2 of these antagonists on carbamylcholine-stimulated amylase secretion were 9.72 for atropine, 8.14 for hexahydrosiladifenidol, 7.16 for pirenzepine and 6.22 for AF-DX 116, indicating that the parotid muscarinic receptors were of an M2 subtype 83-fold more sensitive to hexahydrosiladifenidol than to AF-DX 116.

Effector mechanisms of peptides of the VIP family

The present review is focused on the exocrine pancreas and liver where the only known effector mechanism of VIP is the activation of adenylate cyclase in plasma membranes. A two-state model of activation-deactivation of the enzyme visualizes the participation of VIP receptors and Ns, the guanyl nucleotide stimulatory protein of adenylate cyclase. In the rat pancreas, VIP and GRF receptors are indistinguishable and disulfide bridges influence their functional integrity. The antagonism of VIP and somatostatin perhaps requires, at the adenylate cyclase level, the contribution of Ni, the guanyl nucleotide inhibitory protein. The potentiation of VIP by various stimulants acting on Ca2+ movements may rely on later events, e.g., on a concerted activation of protein kinases. When comparing quantitatively peptide binding to receptors with adenylate cyclase activation, cyclic AMP levels and amylase secretion, a tool is at hand to tailor synthetic agonists and antagonists of VIP, with appropriate changes in the N-terminal moiety of the peptide (a good agonist allows efficient coupling of receptors to the adenylate cyclase system). Apart from stimulus-secretion coupling, VIP may influence protein synthesis in the rat pancreas, through the phosphorylation of ribosomal protein S6, and may alter the activity of the endoplasmic reticulum via the phosphorylation of Mr = 21 kDa and Mr = 25 kDa proteins. In rat liver membranes, high affinity VIP receptors are specifically labelled with 125I-helodermin and are coupled to adenylate cyclase (at variance with low affinity VIP receptors). These receptors are highly responsive to divalent cations and to guanyl nucleotides.

Vasoactive intestinal peptide receptors in pancreas and liver: structure-function relationship

In purified rat pancreatic plasma membranes, (D-Phe4)PHI interacts as a selective VIP agonist for rat pancreatic VIP-preferring receptors, based on binding selectivity and adenylate cyclase activation, therefore allowing us to discriminate between the participation of VIP-preferring and secretin-preferring receptors in VIP stimulation. VIP-preferring receptors also bind GRF. They rely on disulfide bridges for their functional integrity. Their coupling with adenylate cyclase, based on the intrinsic activity of VIP analogues, is poor when compared to that of hepatic VIP receptors. In fresh rat liver plasma membranes, high-affinity VIP receptors are specifically labeled with [125I]helodermin and [125I]His1, D-Ala NLeu27)GRF and are well coupled to adenylate cyclase while low-affinity VIP receptors are not. The first subtype of VIP receptors is highly responsive to guanyl nucleotides and is easily altered by dithiothreitol. Only after freezing and thawing are low-affinity hepatic VIP receptors coupled to adenylate cyclase. Concerning the chemical characterization of VIP receptors, 66- and 35-kDa peptides are detected after specific [125I]VIP cross-linking with double agents in rat pancreatic membranes. In contrast, in intact pancreatic acini, the main source of radioactivity has a molecular mass of 130-180 kDa (with no contribution of intramolecular disulfide bridges), and an 80-kDa peptide is also detectable. The 66-kDa species in membranes can conceivably derive from the 80-kDa species observed in intact cells. Its molecular mass is higher than that of the 56-kDa [125I]VIP cross-linked protein previously observed in rat liver membranes. Besides, species differences between rat and guinea pig pancreas are also evident.

Adenylate cyclase activity in the epididymal adipose tissue from obese-hyperglycaemic mice.

SummaryThe adenylate cyclase activities in 1,500 × g to 25,000 × g sediments of adipose tissue from 3 month old C57BL 6J/ob and lean mice were compared. Three anomalies were observed in obese mice: 1) a defect in basal (−80%) and fluoridestimulated (−50%) activities suggesting a decreased number of catalytic units per mg protein; 2) an impairment in stimulation by isoproterenol (−85%) with no change in apparent affinity for the hormone; and, 3) no stimulatory effect of ethanol upon guanyl nucleotide-stimulated activity. The two latter effects may be due to a reduced number ofβ-adrenergic receptors per mg protein and/or to an alteration in the transducing process. On the other hand, guanyl nucleotides as well as prostaglandin PGE1 exerted similar effects in the preparations from both lean and obese adipose tissue.