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H De Wulf

Publications and source records attributed to H De Wulf.

At least 37 records · Page 2Linked to original sources
PubMed1982

Characteristics of the desensitization and resensitization of the cyclic AMP-independent glycogenolytic response in rat liver cells.

Vasopressin and alpha-adrenergic agonists were shown previously [Bréant, Keppens & De Wulf (1981) Biochem. J. 200, 509-514] to induce a heterologous, dose-dependent and receptor-mediated desensitization of the cyclic AMP-independent glycogenolytic response in isolated hepatocytes. The desensitized state of the hepatocytes can be preserved as long as the agonist is bound to its receptor. Conversely, washing the cells with a hormone-free buffer or displacement of the agonist from its receptor by a specific antagonist restores the responsiveness. The desensitization and its reversibility (i.e. resensitization) are obtained within minutes. The desensitization can be clearly elicited at temperatures as low as 5 degrees C, whereas the glycogenolytic response and the enhancement of the 45Ca flux are only obtained above 15 degrees C; the resensitization requires even higher temperatures. A tentative model is proposed to account for the observed effects.

Angiotensin II↗
PubMed1982

Pattern of protein phosphorylation in rat hepatocytes stimulated by glucagon or by the Ca2+-linked hormones.

We have adapted the high-resolution electrophoretic technique of O'Farrell to analyze phosphorylated proteins from rat hepatocytes. Total proteins were extracted from rat hepatocytes which had been incubated in the presence of [32P]phosphate and with two types of stimuli: glucagon on the one hand and the Ca2+-linked hormones on the other hand. About 200 phosphorylated polypeptides have been separated. Glucagon modifies the incorporation of [32P]phosphate in at least 17 polypeptides and dibutyryladenosine 3',5'-monophosphate mimics this hormonal effect, implying a common mechanism of action. Phenylephrine (in the presence of the beta-antagonist propranolol), vasopressin and angiotensin all modify the incorporation of [32P]phosphate in about 13 polypeptides; since the Ca2+ ionophore A23 187 reproduces the effect of these agents it may be concluded that Ca2+ mediates their effect. Not all the substrates affected by the two types of hormones are identical. Both types of stimuli increase the phosphorylation of a same set of seven proteins and decrease the phosphorylation of a same set of three proteins but seven proteins have their phosphorylation uniquely enhanced by glucagon whereas three other specific proteins get more phosphorylated by the Ca2+ -linked hormones. The clear differences between the patterns of protein phosphorylation observed in the presence of glucagon and dibutyryladenosine 3',5'-monophosphate on the one hand and by the Ca2+-linked hormones on the other hand strongly suggest different mechanisms of action for these two types of stimuli.

Angiotensin II↗
PubMed1982

On the role of calcium ions in the regulation of glycogenolysis in mouse brain cortical slices.

Using mouse brain cortical slices, we investigated the relative roles of cyclic AMP and of calcium ions as the intracellular messengers for the activation of glycogen phosphorylase (EC 2.4.1.1; alpha-1,4-glucan:orthophosphate glucosyltransferase) induced by noradrenaline and by depolarization. Activation of phosphorylase by 100 microM noradrenaline is mediated by beta-adrenergic receptors and does not require the copresence of adenosine. The role of the concomitant small increase in cyclic AMP is questioned. Short-term treatment with EGTA or LaCl3 abolishes the noradrenaline activation of phosphorylase, pointing to a critical role of extracellular calcium. Depolarization by 25 mM K+ or 100 microM veratridine produces a rapid and large (fourfold) activation of phosphorylase. Only veratridine increases the cyclic AMP levels; exogenous adenosine deaminase essentially blocks this cyclic AMP accumulation but not the phosphorylase activation. A half-maximal activation of phosphorylase occurs at about 12 mM K+. Addition of EGTA or LaCl3 reduces the effect of both depolarizations to a slight and transient activation of phosphorylase. These results indicate that activation of glycogen phosphorylase by K+ or veratridine occurs by a cyclic AMP-independent and calcium-dependent mechanism. The calcium dependency of brain phosphorylase kinase renders this kinase the prime target enzyme for regulation of glycogenolysis by calcium ions.

Animals↗
PubMed1981

Heterologous desensitization of the cyclic AMP-independent glycogenolytic response in rat liver cells.

Vasopressin and alpha-adrenergic agonists are known to be potent cyclic AMP-independent Ca2+-dependent activators of liver glycogen phosphorylase. When hepatocytes are pre-incubated with increasing concentrations of vasopressin or of the alpha-agonist phenylephrine, they become progressively unresponsive to a second addition of the respective agonist. The relative abilities of six vasopressin analogues and of five alpha-agonists to activate glycogen phosphorylase and to cause subsequent desensitization are highly correlated, indicating that the same vasopressin and alpha-adrenergic receptors are involved in both responses. About 5-times-higher peptide concentrations are needed to desensitize the cells than to activate their glycogen phosphorylase, whereas the concentrations of alpha-agonists required for the desensitization are only twice those needed for the activation of phosphorylase. The desensitization is not mediated by a perturbation in the agonist-receptor interaction. It is clearly heterologous, i.e. it is not agonist-specific, and must therefore involve a mechanism common to both series of agonists. The evidence for a role of Ca2+ movements or phosphatidylinositol turnover is briefly discussed.

Adrenergic alpha-Agonists↗
PubMed1980

(3H)-vasopressin binding to isolated rat hepatocytes and liver membranes: regulation by GTP and relation to glycogen phosphorylase activation.

Specific vasopressin binding to rat hepatocytes and rat liver membranes was measured using biologically active (3H)-Tyr2-Lys8-vasopressin (8.5 Ci/mM). In both systems, vasopressin binding was found to be time-dependent, reversible, and saturable. The kinetic parameters for vasopressin binding were: apparent dissociation constants (Kd): 4.9 nM and 15 nM; maximal binding capacities: 0.83 pmoles/mg protein and 2.10(5) sites/Cell for purified membranes and intact cells respectively. The relative affinities of 19 vasopressin structural analogues were deduced from competition experiments and compared to the previously determined glycogenolytic (or antiglycogenolytic) potencies of these analogues. For both agonists and antagonists, a highly significant correlation was demonstrated between pKd and pKa (or pKi) values, suggesting that the detected binding sites are the physiological receptors involved in the glycogenolytic action of vasopressin on the rat liver. The affinity of antagonists for binding to these receptors is the same for both membranes and cells. In contrast, agonists which bind to vasopressin receptor sites have a higher affinity for purified membranes than for intact cells (Kd membranes/Kd cells = 8 +/- 1). GTP (0.1mM) reduced the affinity of agonists but not of antagonists for binding to membranes and abolished the differences between Kd values for binding to hepatocytes and membranes.

Animals↗
PubMed1977

Inactivation and reactivation of liver phosphorylase b kinase.

When crude rat liver preparations were incubated at 30degrees C, a gradual loss of phosphorylase kinase (ATP:phosphorylase b phosphotransferase, EC 2.7.1.38) activity was observed. This inactivation was Mg2+ dependent and was partially inhibited by sodium fluoride. Addition of Mg2+ ATP to the liver preparations, at any time throughout the incubation, caused a reactivation of the phosphorylase kinase and this was accelerated by micromolar concentrations of cyclic AMP. The reactivation process could be completely abolished by the addition of a heat stable protein kinase inhibitor, implicating cyclic AMP dependent protein kinase in the activation reaction. Both the low and the high activity forms of the enzyme required micromolar quantities of Ca2+ for full activity (KA = 0.6 micronM). The two forms exhibit quite different pH dependencies and at the physiological pH of liver (pH 7.4) their activities differed by a factor of 5-10. Conversion of the lower activity form into the higher seems to affect only the V - Km for muscle phosphorylase b (EC 2.4.1.1) was about 1 mg/ml for both enzyme forms.

Adenosine Triphosphate↗
PubMed1977

On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase.

We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an alpha-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase b kinase (EC 2.7.1.38). (b) The absence of Ca2+ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca2+ was present in the incubation medium. (d) Glucagon, cyclic AMP and three cyclic AMP-dependent hormones caused an enhanced uptake of 45Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate 45Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced 45Ca uptake and the phosphorylase activation. We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca2+ is most probably phosphorylase b kinase.

Angiotensin II↗