Piotr Poloczek

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.

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.

Impairment of hormone-stimulated cardiac adenylate cyclase activity in the genetically obese (fa/fa) Zucker rat

The age-related development of the capacity of the cardiac adenylate cyclase system to be stimulated with secretin, vasoactive intestinal peptide (VIP), glucagon, the β-adrenergic agonist isoproterenol, Gpp(NH)p, and NaF was compared in obese (fa/fa) Zucker rats and their lean (FA/?) littermates. The obese (fa/fa) Zucker rats tested developed postweaning obesity associated with marked hypertriglyceridemia, mild hyperglycemia, and hyperinsulinism. At 4 weeks, there was already a 57% reduction in secretin-VIP-stimulated adenylate cyclase activity in fa/fa rats. At 12 weeks, the secretin-VIP-stimulation was reduced by 77%, and glucagon-and isoproterenol-stimulations by 16–21%. At 45 weeks, secretin-VIP-stimulation was reduced by 91%, glucagon- and isoproterenol stimulations by 34–42%, and Gpp(NH)p- and NaF-stimulations by 16–23%. The reductions of isoproterenol-, Gpp(NH)p-, and NaF-stimulations were totally or partially reversed in 30-week old fa/fa animals submitted for 5 weeks to severe food restriction that almost normalized the altered blood parameters. In sharp contrast, food restriction imposed a further decrease in secretin-VIP- and glucagon-stimulated adenylate cyclase activities. This pattern of impaired secretin-VIP-stimulated adenylate cyclase activity appeared limited to cardiac membranes in obese animals as the responses of liver, brain and anterior pituitary adenylate cyclase activities to secretin and/or VIP were unaltered. These results suggest that secretin-VIP receptors coupled to adenylate cyclase were rapidly and specifically altered in the heart of fa/fa Zucker rats.

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.

Binding in vitro of vasoactive intestinal peptide on isolated acini of rat parotid glands

Binding of 125I-labelled vasoactive intestinal peptide (VIP) to rat parotid acini was saturable, temperature-dependent and reversible, and reflected interaction with a single class of binding sites. Parotid glands possessed approx. 400 fmol binding sites per mg protein and binding of the tracer to these sites could be inhibited by VIP [concentration for half-maximal effect (KD), 24 nM], by the peptide histidine isoleucine (KD, 140 nM), by secretin (KD, 470 nM) and by the human pancreatic growth hormone-releasing factor (hpGRF; KD, 3200 nM). In the same acini preparation, 10 microM VIP also stimulated amylase release 4-fold and increased cyclic AMP 11-fold. Thus, VIP might be a neurotransmitter in the rat parotid gland.

Inhibitory effects of pirenzepine on muscarinic stimulation of rat pancreas

The binding properties and pharmacological effects of pirenzepine were compared to those of atropine in isolated pancreatic acini and pancreatic membranes of rats. In the first preparation, pirenzepine and atropine blocked [N-methyl-3H]scopolamine ([3H]NMS) binding, pirenzepine being 110 times less potent than atropine (KD for pirenzepine 0.38 microM and for atropine 3.5 microM). A similar difference in potency was observed with respect to carbamylcholine stimulation of amylase secretion (IC50 for pirenzepine 4.5 microM and for atropine 30 nM) and calcium efflux (IC50 for pirenzepine 2.8 microM and for atropine 4 nM). Correspondingly, in rat pancreatic membranes, the KD values for pirenzepine and atropine were 250 and 1.5 nM, respectively. These data are compatible with the hypothesis that the in vitro antimuscarinic effect of pirenzepine on the rat pancreas is linked to the occupancy of a single homogeneous class of receptors with a low affinity for the antagonist.

Characterization and regulation of atrial natriuretic peptide (ANP)-R1 receptors in the human neuroblastoma cell line NB-OK-1

We characterized in membranes from the human neuroblastoma cell line NB-OK-1, an ANP-R1 receptor (Mr 130 kDa) for the atrial natriuretic peptide (ANP). This receptor recognized biologically active forms of ANP with high affinity but showed no affinity for truncated ANP forms. It was functional in that binding correlated with guanylate cyclase activation (a 2-fold increase in Vmax) with the following rank order of potency: rat ANP-(99-126) greater than human ANP-(99-126) greater than human ANP-(102-126) greater than porcine BNP (brain natriuretic peptide). The enzyme required free Mn2+ in addition to the Mn-GTP substrate (Km of about 0.3 mM for both basal and ANP-stimulated activity). In the presence of dithiothreitol, the dose-response curve of guanylate cyclase activation was shifted rightward by a factor of 30. ANP-R1 receptors were upregulated through protein synthesis in cells exposed to 1 mM carbamylcholine or 1 mM dibutyryl cyclic AMP for 8-24 h (ANP was ineffective).

Phospholipase A2 activity of pancreatic secretory factor, a new secretagogue isolated from the venom of Heloderma suspectum

Pancreatic secretory factor (PSF), an efficient pancreatic secretagogue recently isolated from the venom of Heloderma suspectum is shown to exert phospholipase A2 activity towards phosphatidylcholine. This activity is strictly dependent on calcium (apparent K a 40 nM) and has an optimum pH around 9. At pH 7.4 and in the presence of calcium, PSF retains 40% of its phospholipase A2 activity. These results are compared to the calcium dependency of the secretory effect of PSF on rat pancreatic acini. Taken collectively, the present data on PSF suggest that a similar endogenous phospholipase A2 activity might be involved in the late steps of stimulus‐secretion coupling in the exocrine pancreas.