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Biomedical subjects

J Christophe

Publications and source records attributed to J Christophe.

At least 145 records · Page 8Linked to original sources
PubMed1985

Stimulation by cholera toxin of ADP-ribosylation of membrane proteins, adenylate cyclase and insulin release in pancreatic islets.

In rat pancreatic islet membranes exposed to [alpha-32P]NAD, cholera toxin stimulated the labelling of three peptides with Mr close to 22 000, 42 000 and 48 000, respectively. In the islets, the toxin-stimulated ADP-ribosylation of the heavy form of the Ns alpha-subunit predominated over that of the light form, in mirror image of the situation found in the exocrine pancreas. When intact islets were preincubated with cholera toxin, the adenylate cyclase activity of a subcellular particulate fraction was increased. The responsiveness of adenylate cyclase to GTP was also augmented, but that to NaF was decreased. In intact islets, the production of cyclic AMP and the glucose-stimulated release of insulin were also enhanced after pretreatment with cholera toxin. These findings reveal the presence in pancreatic islets of the guanyl nucleotide regulatory protein of adenylate cyclase, with an unusual predominance of the heavy form of the Ns alpha-subunit.

Adenosine Diphosphate Ribose↗
PubMed1985

Uncoupling between beta-adrenoceptors and adenylate cyclase in dog ischemic myocardium.

We evaluated the effects of ischemic injury on the myocardial adenylate cyclase system, 5 h after ligation of the left anterior descending coronary in 5 anesthetized dogs. Crude cardiac membrane preparations were isolated from control and ischemic areas of ventricular myocardium and tested for: 1. L-(125I)iodocyanopindolol binding, in the absence and presence of +/- -isoprenaline and GTP, and 2. adenylate cyclase activity. The density of beta-adrenoceptors increased by 35% in membranes from ischemic areas while the proportion of receptors in a high affinity state for +/- -isoprenaline decreased from 43% to 20%. Adenylate cyclase activities in the basal state and under stimulation with NaF, forskolin, Gpp(NH)p, +/- -isoprenaline and VIP were all markedly and similarly reduced, being only about 30% of comparable activities in membranes from control areas. The +/- -isoprenaline subsensitivity of cardiac adenylate cyclase can, thus, be attributed to a defective enzymatic system and not to a reduction in the number of beta-adrenoceptors implying that the internal components of the system were more sensitive to acute ischemia than the outward oriented hormone receptors. It is tempting to ascribe this uncoupling to a functional depletion in the guanine nucleotide-binding regulatory protein Ns that might reflect a loss of high energy phosphate stores including GTP.

Adenylyl Cyclases↗
PubMed1985

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.

Amylases↗
PubMed1985

Indirect evidence against a contribution of the guanine nucleotide-binding inhibitory component of adenylate cyclase to impaired lipolysis in the epididymal adipose tissue of congenitally obese (ob/ob) mice.

The mixed adrenergic agonist epinephrine, at a 10 microM concentration, stimulated cyclic AMP production and glycerol release in the epididymal adipose tissue of ob/ob male mice. These effects when tested, respectively, after 7 min in the presence and after 60 min in the absence of theophylline were, however, 7- and 5-fold lower than in lean controls. The alpha-adrenergic blocker phentolamine and adenosine deaminase (which destroys extracellular adenosine) did not restore a normal lipolytic response to epinephrine in the adipose tissue of ob/ob mice. These data provide indirect evidence against a hyperactive mechanism in the coupling of alpha-adrenergic receptors and adenosine receptors to Ni, the guanine nucleotide-binding inhibitory component of adenylate cyclase, as the cause of reduced lipolysis in the adipose tissue of ob/ob mice.

Adenosine Deaminase↗
PubMed1985

Interaction of growth hormone-releasing factor (GRF) and 14 GRF analogs with vasoactive intestinal peptide (VIP) receptors of rat pancreas. Discovery of (N-Ac-Tyr1,D-Phe2)-GRF(1-29)-NH2 as a VIP antagonist.

Adenylate cyclase stimulation by GH-releasing factor (GRF) and 14 GRF analogs (modified in the N-terminal part) was compared to the capacity of the same peptides to inhibit [125I]iodo-vasoactive intestinal peptide (VIP) binding in rat pancreatic plasma membranes. These peptides interfered with VIP receptors as they inhibited [125I]iodo-VIP binding, and probably acted through VIP-preferring receptors as one of these peptides [(N-Ac-Tyr1,D-Phe2)-GRF(1-29)-NH2] selectively inhibited both VIP- and GRF-stimulated adenylate cyclase activities. In general, alterations in positions 6 and 7 (but not in positions 1-4) markedly reduced the affinity of the resulting GRF analog [based on Kact (concentration exerting half-maximal stimulation) values]. The intrinsic activity exerted by GRF analogs on adenylate cyclase was reduced by acetylation of the free NH2 group and by the replacement of Asp3, Ala4, Phe6, and Thr7 by the corresponding D-isomer. The presence of pCl-Phe6 and Trp6 also depressed this parameter. Substitution in GRF (or its N-acetylated derivative) by D-Phe2, D-Arg2, and D-Ala4 again reduced the intrinsic activity, whereas substitution of the natural L-amino acid residue by D-Ala2 and Phe4 gave superagonists.

Acetylation↗
PubMed1985

Structural requirements for the activation of rat anterior pituitary adenylate cyclase by growth hormone-releasing factor (GRF): discovery of (N-Ac-Tyr1, D-Arg2)-GRF(1-29)-NH2 as a GRF antagonist on membranes.

The efficacy and potency of 14 GH-releasing factor (GRF) analogs, substituted in position 1 to 7, on adenylate cyclase activation in crude homogenates from rat anterior pituitary were related to those of human pancreatic GRF(1-29)-amide and vasoactive intestinal peptide. Among several D-amino acid substitutions, that in position 2 was the only one to yield a super-agonist [with a Kact (concentration required for half-maximal adenylate cyclase activation) 2 times lower than that of GRF(1-29)-NH2]. By contrast, D-isomer substitution in position 1 and 3 was without effect and D-isomer substitution in position 4, 6, or 7 decreased the affinity of the analog. The N-acetylated analog of GRF was as potent and active as the parent peptide, and the identity of the amino acid in position 2 of (N-Ac-Tyr1)-GRF(1-29)-NH2 proved to be determining for enzyme activation, with D-Phe2 and D-Trp2 derivatives acting as partial agonists and the (N-Ac-Tyr1,D-Arg2) analog being an efficient competitive antagonist of GRF(1-29)-NH2. With use of this antagonist, it was possible to demonstrate that GRF and vasoactive intestinal peptide receptors represent distinct entities in the rat anterior pituitary.

Adenylyl Cyclases↗
PubMed1985

Effects of forskolin on adenylate cyclase activity and amylase secretion in the rat exocrine pancreas.

Forskolin stimulated adenylate cyclase activity 55-fold in crude rat pancreatic plasma membranes. Dose-response curves were better fitted by a two-component model with apparent Ka for forskolin of 0.8 microM and 85 microM corresponding, respectively, to 15% and 85% of total activity. Gpp (NH)p alone or the combined presence of GTP plus a hormone (secretin, VIP or CCK-8) potentiated activation through the high affinity forskolin component. These results are in favour of a dual mode of action of forskolin: a high affinity component related to the stimulatory guanine nucleotide-binding regulatory subunit, and a low affinity component more closely related to the catalytic subunit of the enzyme. In dispersed rat pancreatic acini, forskolin increased cyclic AMP levels 26-fold and potentiated the increase induced by secretin, VIP, and CCK-8. It also stimulated the phosphorylation of three particulate proteins (Mr = 21K, 25K and 33K). In terms of secretion, it raised amylase secretion by 60%, a weak effect comparable to that exerted by VIP but much lower than that of secretin or CCK-8. Forskolin did, however, potentiate the secretory effect of CCK-8 (a hormone inducing a redistribution of cellular calcium) while being without influence on the secretory effects of secretin and VIP.

Adenylyl Cyclase Inhibitors↗
PubMed1985

[Histiocytofibroma and Dupuytren's palmar fibromatosis. Comparative autohistoradiographic study of their proliferative and biosynthetic activities].

The proliferative rate and synthesis of proteins was studied in histiocytofibromas and in nodules of Dupuytren's palmar fibromatosis. Incorporations of tritiated thymidine and tritiated proline were used and revealed by autohistoradiography. In the two diseases, proliferation and protein synthesis occurred predominantly in cells of the vascular walls while the other cells constitutive of the lesions appeared less active.

Autoradiography↗
PubMed1984

Primary structure of helodermin, a VIP-secretin-like peptide isolated from Gila monster venom.

The complete amino acid sequence of helodermin isolated from the venom of Gila monster was elucidated. The peptide was shown to be a basic pentatriacontapeptide amide: His-Ser-Asp-Ala-Ile-Phe-Thr-Gln-Gln-Tyr-Ser-Lys-Leu-Leu-Ala-Lys-Leu-Ala- Leu-Gln-Lys- Tyr-Leu-Ala-Ser-Ile-Leu-Gly-Ser-Arg-Thr-Ser-Pro-Pro-Pro-NH2. A high degree of sequence similarities to secretin/VIP/PHI/(PHM)/GRF from mammal and bird was observed over the entire N-terminal 1-27 sequence. In particular, the amino acid residues in positions 3, 6 and 7 were found to be common to 9 peptides of the family. Another interesting feature of the structure of helodermin was its C-terminal -Pro-Pro-Pro-NH2 sequence. Isolation of helodermin was the first demonstration of the existence of a secretin/VIP-related peptide in an animal that is neither mammal nor bird.

Amino Acid Sequence↗
PubMed1984

Cleaving of disulfide bridges and apparent molecular weight of human prolactin variants as revealed by immunoperoxidase electrophoresis.

Five highly purified preparations of human pituitary prolactin, widely used in radioimmunoassays, were analyzed by immunoperoxidase electrophoresis with rabbit antisera raised against ovine and rat prolactin. When unreduced, the prolactin content of these preparations was separated into one major prolactin-like immunoreactive band of Mr 23,000, and four fainter immunoreactive bands of Mr's 16,000, 22,000, 25,000, and 45,000. Increasing the 2-mercaptoethanol concentration modified the relative proportions and the Mr of immunoreactive bands so that, after extensive reduction, only two immunoreactive bands were separated, one major band of Mr 25,000 and one fainter band of Mr 17,000. A direct relationship existed between the load of prolactin submitted to electrophoresis in the 0.25 to 75 ng range and the optical density of the separated bands.

Animals↗
PubMed1984

Effect of Bordetella pertussis toxin on ADP-ribosylation of membrane proteins, adenylate cyclase activity and insulin release in rat pancreatic islets.

Exposure of rat pancreatic islet membranes to [alpha-32P]-NAD+ in the presence of Bordetella Pertussis toxin (islet-activating protein) reveals the ADP-ribosylation of a peptide with a Mr close to 41 kDa, which corresponds to the alpha-subunit of the guanine nucleotide regulatory protein Ni. Islets removed from rats pretreated with the Bordetella Pertussis toxin display a specific increase in adenylate cyclase responsiveness to GTP and are characterized by a resistance to the inhibitory action of alpha2-adrenergic agonists upon either adenylate cyclase activity or glucose-induced insulin release.

Adenosine Diphosphate Ribose↗
PubMed1984

Inhibitory effects of quinidine on rat heart muscarinic receptors.

Quinidine inhibited binding of the labelled agonist [3H]oxotremorine M [( 3H]Oxo-M) and the labelled antagonist [3H]N-methylscopolamine [( 3H]NMS) to rat heart muscarinic receptors. Kinetic studies demonstrated that quinidine decreased the association rates (I50: 4 and 7.5 microM) and dissociation rates (I50: 100 and 68 microM) of [3H]Oxo-M and [3H]NMS, with different potencies. These cooperative effects explained the low Hill coefficients and apparent selectivity of quinidine competition curves.

Animals↗
PubMed1984

One-step isocratic high-performance liquid chromatographic purification of radioiodinated and radioiodinated-photoactivable derivatives of cholecystokinin.

N-Hydroxysuccinimidyl-3-(4-hydroxy-3-[125I]iodophenyl)propionate (the Bolton-Hunter reagent) was conjugated with (Thr 34, NLeu 37) cholecystokinin (CCK) 31-39 in anhydrous dimethylformamide-pyridine in 20% yield. The radiolabelled peptide was purified from the reaction mixture in one step, by isocratic elution from a C18 high-performance liquid chromatographic (HPLC) column with 35% aqueous acetonitrile-0.13% heptafluorobutyric acid as eluent. The concentration of the radiolabelled peptide was estimated by UV monitoring. The acylating photoactivable radioiodinated reagent N-hydroxysuccinimidyl-N-(4-azido-2-nitrophenyl)-3-[125I]iodotyrosi ne was synthesized, purified on a C18 HPLC column by isocratic elution with 65% acetonitrile-1 mM hydrochloric acid, then conjugated with (Thr 34, NLeu 37) CCK31-39. The resulting photoactivable radioiodinated CCK analogue was purified by isocratic elution on a C18 HPLC column with 39% aqueous acetonitrile-0.1 M triethylamine phosphate (pH 3.5). The binding ability of both tracers and their non-radioactive analogues to CCK receptors was tested on rat pancreatic plasma membranes. As compared to a KD of 4.5 nM for unmodified (Thr 34, NLeu 37) CCK31-39, the KD of the radioiodinated Bolton-Hunter derivative was 3 nM, and that of the photoactivable radioiodinated derivative was 19 nM.

Animals↗
PubMed1984

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 Ka 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.

Amylases↗
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↗