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Biomedical subjects

P Robberecht

Publications and source records attributed to P Robberecht.

At least 181 records · Page 10Linked to original sources
PubMed1984

Importance of disulfide bonds in receptors for vasoactive intestinal peptide and secretin in rat pancreatic plasma membranes.

Vasoactive intestinal peptide (VIP), secretin, and C-terminal octapeptide of cholecystokinin (CCK-8) receptors were identified in rat pancreatic plasma membranes by the ability of these peptides to stimulate adenylate cyclase activity. The membrane preparation procedure was conducted through a series of steps including discontinuous sucrose density gradient fractionation. 5 mM beta-mercaptoethanol was added stepwise. Membrane preparations obtained stepwise were preincubated for 10 min at 25 degrees C in the presence of various concentrations of beta-mercaptoethanol or dithiothreitol before assaying adenylate cyclase. The use of the reducing agents exerted no effect on p[NH]ppG-, NaF-, and CCK-8- stimulated activities. By contrast, stimulation of adenylate cyclase by low VIP concentrations was specifically altered when beta-mercaptoethanol was used during tissue homogeneization at 5 degrees C. In addition, both VIP and secretin responses were highly sensitive towards a preincubation of 10 min at 25 degrees C in the presence of dithiothreitol. These results were likely to reflect alterations at the receptor level. 125I-VIP binding was, indeed, reduced after dithiothreitol preincubation, low concentrations of the thiol reagent decreasing the apparent number of high-affinity VIP receptors and higher dithiothreitol concentrations reducing the affinity of VIP receptors.

Adenylyl Cyclases↗
PubMed1984

Specific labelling by [125I]helodermin of high-affinity VIP receptors in rat liver membranes.

Helodermin, a newly isolated peptide from Gila Monster venom, is structurally related to VIP and secretin. When used as radioligand, [125I]helodermin bound rapidly and reversibly to crude rat liver membranes, the dissociation being accelerated by GTP. Competition binding curves of [125I]helodermin and [125I]VIP with unlabelled peptides showed the following order of decreasing affinity: VIP greater than helodermin greater than secretin greater than hpGRF(1-29)-NH2. The shape of binding curves and of concurrent adenylate cyclase activation is compatible with the specific labelling, by [125I]helodermin, of a class of high-affinity VIP receptors that is capable to stimulate adenylate cyclase.

Adenylyl Cyclases↗
PubMed1984

Effects of verapamil on the binding properties of rat heart muscarinic receptors: evidence for an allosteric site.

The calcium channel antagonist verapamil is known to inhibit competitively antagonist binding to rat heart muscarinic receptors. The present data suggest that this drug recognized two binding sites on the muscarinic receptors: 1) an allosteric site modulating the tracer dissociation rates and 2) the muscarinic drug binding site. The affinity of verapamil for the allosteric site and the efficacy of its effect on muscarinic ligand dissociation rates depended on the ligand studied.

Allosteric Site↗
PubMed1984

Multiple effects of short-chain alcohols on binding to rat heart muscarinic receptors.

Short-chain alcohols inhibited the equilibrium binding of agonists and antagonists to rat heart muscarinic receptors. Methanol, ethanol, propan-2-ol and propan-1-ol, when used at low concentrations, behaved as pseudo-competitive antagonists. Their rank order of potency paralleled their relative partition coefficients, suggesting that this inhibition was simply due to the interaction of the alcohols with a hydrophobic part of the receptor or with membrane lipids. The four alcohols increased the dissociation rate constant of [3H]oxotremorine M from the high-affinity agonist receptors and decreased the stability of this receptor state. These effects might reflect increased membrane fluidity and/or decreased hydrophobic interactions (see below). By contrast, the effects of alcohols on the association and dissociation rates of N-[3H]- methscopolamine (an antagonist) were not correlated to their relative octanol/water partition coefficient (a measure of their affinity for biophases ). Alcohols, at the relatively high concentrations necessary for increased membrane 'fluidity', are known to affect the relative stability of various protein conformations. We believe that the effects of alcohols on antagonist binding to rat heart muscarinic receptors reflected changes in the activation energy of association and dissociation reactions, the inhibition of equilibrium binding being mainly due to decreased 'hydrophobic interactions'.

1-Propanol↗
PubMed1984

Purification of a novel pancreatic secretory factor (PSF) and a novel peptide with VIP- and secretin-like properties (helodermin) from Gila monster venom.

A combination of three HPLC procedures applied to the venom of Gila monster (Heloderma suspectum) has led to the purification to homogeneity of two bioactive components: (i) a 17.5 kDa protein, isolated on the basis of its potent secretory effect on dispersed rat pancreatic acini, was accordingly designated PSF (pancreatic secretory factor); (ii) a 5.9-kDa peptide, designated helodermin, was purified on the basis of its ability to stimulate adenylate cyclase in rat pancreatic membranes. PSF was unable to activate adenylate cyclase and, conversely, helodermin was devoid of secretory action.

Adenylyl Cyclases↗
PubMed1984

Evidence that helodermin, a newly extracted peptide from Gila monster venom, is a member of the secretin/VIP/PHI family of peptides with an original pattern of biological properties.

Helodermin, a newly isolated peptide from the venom of Gila monster (Heloderma suspectum) was shown to stimulate the adenylate cyclase activity of rat pancreatic membranes as efficiently as secretin and VIP. It also increased cyclic AMP levels and inhibited [125I]VIP binding in rat pancreatic acini. Finally, helodermin activated adenylate cyclase in membranes from rat heart, rat brain, and human heart, showing properties analogous yet distinct from those of secretin, VIP and PHI.

Adenylyl Cyclases↗
PubMed1984

Pancreatic secretory factor (PSF), a protein from Gila monster venom stimulating enzyme secretion from rat pancreatic acini.

Pancreatic secretory factor (PSF), a 17.5-kDa protein purified from the venom of Gila monster (Heloderma suspectum), stimulated amylase secretion from dispersed rat pancreatic acini more efficiently than CCK-8, bombesin, carbachol and secretin, and without increasing 45Ca2+ efflux and cyclic AMP levels. The secretory action was dependent on the presence of extracellular calcium and was additive to the secretion induced by agents acting via cyclic AMP or via Ca2+ efflux.

Amylases↗
PubMed1984

A comparison between muscarinic receptor occupancy, adenylate cyclase inhibition, and inotropic response in human heart.

Binding to muscarinic receptors was compared with adenylate cyclase inhibition in membranes derived from human heart auricles, and with inhibition of the contraction of auricular muscle fibers. In the absence of GTP, agonists recognized two classes of receptors both of which bound antagonists with the same affinity. In the presence of GTP, both classes of receptors for agonists were converted into a single low affinity state. Carbachol and oxotremorine inhibited adenylate cyclase activity by 43%, pilocarpine being less efficient (-28%). The 3 agonists exerted similar inhibitory effects on the inotropic response, in 7 out of 9 preparations of electrically- and norepinephrine-stimulated fibers. Dose-effect curves suggested that spareness (or an amplification mechanism) was implicated in the occupancy of low affinity binding sites by carbachol and oxotremorine (but not by the partial agonist pilocarpine) and the resulting inhibition of both adenylate cyclase activity and contractile force.

Adenylyl Cyclase Inhibitors↗
PubMed1984

Secretin-induced changes in rate, contractility and adenylate cyclase activity in rat heart atria.

Secretin stimulated adenylate cyclase activity in crude membrane preparations from right and left rat atria, when tested in the presence of the potentiating activator forskolin. Its maximal effect on adenylate cyclase activity was greater in the right atrium than in the left atrium but the peptide efficiency was lower, in both atria, than that of D,L-isoproterenol. Secretin stimulated the rate of contraction of the spontaneously beating right atrium, but less efficiently than D,L-isoproterenol. Although this positive chronotropic action of secretin was inhibited by D,L-propranolol, it was probably not mediated by the release of endogenous catecholamines as: a) the inhibitory effects of D- and L-propranolol were similar and b) the efficiency of secretin on the in vitro beating of right atrium was the same in control and reserpinized rats. Secretin stimulated the force of contraction of spontaneously beating right atrium, even in the presence of propranolol, i.e. when the chronotropic effect of secretin was abolished. The hormone exerted also a positive inotropic effect on the electrically stimulated rat atrium. This effect was blocked neither by tetrodotoxin nor by propranolol. When comparing the dose-effect curves of secretin and D,L-isoproterenol on adenylate cyclase activation on the one hand, and on the stimulation of rate and contractility on the other hand, it is tempting to suggest that cyclic AMP might be involved in the modulation by secretin of the mechanical properties of rat atria.

Adenylyl Cyclases↗
PubMed1984

The in vitro chronotropic and inotropic effects of vasoactive intestinal peptide (VIP) on the atria and ventricular papillary muscle from Cynomolgus monkey heart.

The in vitro chronotropic and inotropic effects of vasoactive intestinal peptide (VIP) and of isoproterenol, two agents known to stimulate cardiac adenylate cyclase were compared on the heart from Cynomolgus monkey using the spontaneously beating right atrium, the electrically stimulated left atrium, and the electrically-stimulated ventricular papillary muscle. VIP increased concentration-dependently the rate of beating of the right atrium as well as the contractility of both atria but its efficiency was lower than that of D,L-isoproterenol. VIP also stimulated concentration-dependently, and this time as efficiently as D,L-isoproterenol, the contractility of papillary muscle. These VIP effects were unaltered by the neuronal blocker tetrodotoxin. In addition, the moderate inhibition exerted by the beta-adrenergic blocker D,L-propranolol on VIP effects argued against the implication of beta-adrenergic receptors in VIP effects. These results indicate that VIP exerts a direct stimulatory influence on the rate and contractility of Cynomolgus monkey heart.

Animals↗
PubMed1983

Effect of pH on binding of agonists and antagonists to rat heart muscarinic receptors.

The pH-dependence of antagonist and agonist binding to rat heart muscarinic receptors was investigated at 25 degrees C, in the absence and in the presence of GTP. The small inhibitory effect observed at the lowest pH investigated (pH 6.0) on [N-methyl-3H]methscopolamine chloride and [methyl-3H]oxotremorine-M acetate binding indicated that one or more amino acid residues of the receptor had to be deprotonated for optimal binding affinity. The low pK value of these residues (between 5 and 6) prevented their identification. The binding of scopolamine (pK 7.6) was favoured by a positive charge in the titratable amine, but binding with a lower affinity remained possible charge in the titratable amine, but binding with a lower affinity remained possible without this charge. GTP did not affect antagonist binding at any pH, but converted more than 90% of agonist binding sites into a low affinity conformation. In the absence of GTP, we observed a time- and pH-dependent conversion of the super-high- and high-affinity receptors to a low-affinity GTP-insensitive state. This conversion was markedly accelerated at high pH (above pH 8.0). In the presence of GTP, a positive charge on the titratable amine of pilocarpine (pK 7.05) and oxotremorine (pK 8.60) was required for binding. These results support the view that antagonist (e.g. methscopolamine) binding to receptors was largely facilitated by hydrophobic interactions, whereas agonist binding to low-affinity sites was mainly driven by ionic interactions.

Animals↗
PubMed1983

The adenylate cyclase activity in heart membranes from normotensive and spontaneously hypertensive rats, after chemical sympathectomy, suggests the presence of presynaptic secretin receptors.

Normotensive (WKY) and spontaneously hypertensive (SHR) male adult rats were sacrificed 2 and 3 weeks after 6-hydroxydopamine treatment. Untreated WKY and SHR rats served as controls. In rat heart membranes from WKY rats, 6-hydroxydopamine treatment increased guanosine 5'-O-(2-3-imido)-triphosphate (Gpp(NH)p)-, NaF-, D,L-isoproterenol- and glucagon-stimulated adenylate cyclase activities by 18-38% while secretin stimulation was unaffected. In heart membranes from SHR rats, Gpp(NH)p, NaF, D,L-isoproterenol, or glucagon stimulation of the enzyme was similarly increased by 14-38% whilst the low secretin responsiveness which is characteristic of these animals decreased even further (by 24-47%). These results are consistent with: (1) an up regulation of postsynaptic beta-adrenergic receptors coupled to adenylate cyclase after degeneration of adrenergic nerves, and (2) a differential response of secretin receptors coupled to adenylate cyclase in the two strains of rats: there was no change in WKY rats and a decreased response in SHR rats. The possible presence and contribution of presynaptic secretin cardiac receptors is considered.

Adenylyl Cyclases↗
PubMed1983

Increased number of high-affinity muscarinic receptors in rat heart after phenylhydrazine treatment.

The effect of phenylhydrazine-induced heart hypertrophy on muscarinic cholinergic binding sites was studied in heart membranes by determining total (with [3H]NMS) and high-affinity (with [3H]Oxo-M) binding sites and estimating low-affinity binding sites by difference. Phenylhydrazine induced an increased concentration of high-affinity binding sites without affecting the concentration of low-affinity binding sites. The KD values for the ligands were unaffected.

Animals↗
PubMed1983

Effects of full and partial beta-adrenergic agonists and antagonists on human lung adenylate cyclase.

The beta-adrenergic stimulation of adenylate cyclase in membranes from human lung was compared to that of adenylate cyclase in membranes with a majority of beta 2-adrenergic receptors (from rat lung) and in membranes with a homogeneous population of beta 2-adrenergic receptors (from rat erythrocytes and reticulocytes). In terms of adenylate cyclase stimulation, three full agonists (isoproterenol, epinephrine and norepinephrine), four partial agonists (procaterol, salbutamol, fenoterol and zinterol), and four antagonists (propranolol, metoprolol, atenolol and practolol) were tested. The potency (Kact or Ki) of the eleven beta-adrenergic agents, and the Hill coefficient (of 1) for the four antagonists tested indicated that the activation of human lung adenylate cyclase occurred through receptors of the beta 2-subtype only. Partial beta-adrenergic agonists were efficiently discriminated by the human lung preparation, as shown by distinct intrinsic activities. The mediocre efficacy and the relatively low potency of all beta-adrenergic agonists on adenylate cyclase suggested a relatively low density of beta 2-adrenergic receptors, as compared to the enzyme density.

Adenylyl Cyclase Inhibitors↗
PubMed1983

Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols.

The diterpene forskolin stimulated rat cardiac adenylate cyclase activity at least 20-fold and potentiated the effect of NaF. The stimulatory effect of forskolin was reduced in the presence of Gpp(NH)p. Ethanol markedly reduced the stimulation of adenylate cyclase by forskolin while potentiating NaF and Gpp(NH)p stimulation. The inhibitory effect of ethanol on forskolin stimulation appeared to be of a mixed type with both a competitive and a non-competitive component. Three other short-chain linear alcohols (methanol, propanol, butanol) also inhibited forskolin-stimulation, this effect being proportional to the number of carbon atoms.

Adenylyl Cyclases↗
PubMed1983

Topographical distribution of the secretin- and VIP-stimulated adenylate cyclase system in the heart of five animal species.

Adenylate cyclase stimulation by secretin and VIP was compared to the effect of glucagon, D,L-isoproterenol, Gpp[[NH]p, and NaF in atria and ventricles from rat, guinea pig, rabbit, dog and Cynomolgus monkey. In rat ventricular membranes, secretin was a better stimulant than VIP and was as active as D,L-isoproterenol. In rat auricular membranes both peptides were inactive. In guinea pig and rabbit heart membranes (ventricular and auricular) VIP and secretin were inactive. In dog and monkey atria, VIP stimulation of adenylate cyclase was comparable to that of D,L-isoproterenol, secretin being inactive. In dog ventricles, VIP was less efficient than D,L-isoproterenol, secretin being inactive. In monkey ventricles, by contrast, VIP was slightly more efficient than D,L-isoproterenol, secretin having a small effect only in left ventricles. The present results established a clear difference between animal species with respect to the efficacy of the peptides of the secretin/VIP family: the presence of "secretin-preferring" receptors in rat heart contrasted with the presence of "VIP-preferring" receptors in dog and monkey heart. Our results in dog and monkey hearts suggest that VIP might be a candidate for a physiological control of heart function.

Adenylyl Cyclases↗