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S Keppens

Publications and source records attributed to S Keppens.

At least 37 records · Page 2Linked to original sources
PubMed1988

Involvement of a plasma membrane phosphodiesterase in the negative control of cyclic AMP levels by vasopressin in rat hepatocytes.

Vasopressin has been shown previously to lower the glucagon-induced increase of cyclic AMP levels in isolated rat hepatocytes by way of an enhanced phosphodiesterase (EC 3.1.4.17) activity. Five phosphodiesterase inhibitors were tested for their ability to prevent vasopressin from lowering cyclic AMP levels in intact hepatocytes and for their inhibitory effect in vitro on soluble and particulate phosphodiesterase activities partially purified from hepatocytes. Three soluble activities have been separated by DEAE-cellulose chromatography: a phosphodiesterase hydrolyzing both cyclic AMP and cyclic GMP, a form stimulated by cyclic GMP and a cyclic AMP-specific activity. The absence of any statistically significant correlation between the in vivo (in intact cells) and the in vitro (on isolated phosphodiesterases) potencies of the inhibitors does not support a role for the cytosolic phosphodiesterases in mediating the vasopressin-induced decrease in cyclic AMP levels. No statistically significant correlation was observed between the inhibition of the vasopressin effect on cyclic AMP accumulation and the inhibition of phosphodiesterase activity either associated with the native plasma membranes or solubilized from these membranes with 0.4 M NaCl. In contrast, a statistically significant correlation was observed between the degree of inhibition of the vasopressin effect in the intact cells and the degree of inhibition of the intrinsic phosphodiesterase still associated with the plasma membranes after high-salt treatment. These data indicate that a phosphodiesterase activity integral to the plasma membrane is very likely involved in the negative control of cyclic AMP levels by vasopressin.

Animals↗
PubMed1988

Periportal and perivenous hepatocytes respond equally to glycogenolytic agonists.

We have used the technique of short-term infusion with digitonin to obtain hepatocytes originating either from the periportal or the perivenous zone of the liver acinus [(1985) Biochem. J. 229, 221-226]. Total glycogen phosphorylase content and sensitivity to cyclic AMP-dependent and calcium-mediated glycogenolytic agonists were very similar for both cell sub-populations and did not differ from the values obtained for control cells. We conclude therefore that there is an apparent absence of metabolic zonation as far as receptor-mediated glycogenolysis and glycogenolytic potency is concerned.

Adenosine Triphosphate↗
PubMed1988

Differences in liver glycogen-synthase phosphatase activity in rodents with spontaneous insulin-dependent and non-insulin-dependent diabetes.

The activation of glycogen synthase after addition of glucose to isolated hepatocytes became impaired in BB rats after the development of insulin-dependent diabetes. This defect was associated with a decreased hepatic synthase phosphatase activity. Both features correspond closely to previous observations on alloxan-diabetic rats. In contrast, in hyperinsulinaemic db/db mice with a similarly increased plasma glucose concentration (non-insulin-dependent diabetes), the synthase phosphatase activity was essentially normal. We conclude that the decreased hepatic synthase phosphatase activity in insulin-dependent diabetes in rodents is due to the lack of insulin, rather than to the increased intrahepatic glucose concentration.

Animals↗
PubMed1986

Characterization of the liver P2-purinoceptor involved in the activation of glycogen phosphorylase.

Evidence has been presented for the existence in rat liver of P2-purinoceptors which are involved in the control of glycogenolysis. Isolated rat hepatocytes and purified liver plasma membranes have been used to study the binding of the ATP analogue adenosine 5'-[alpha- [35S]thio]triphosphate (ATP alpha [35S]) to these postulated P2-purinoceptors. The nucleotide analogue behaves as a full agonist for the activation of glycogen phosphorylase in isolated hepatocytes, 0.3 microM being required for half-maximal activation. Specific binding of ATP alpha [35S] to hepatocytes and plasma membranes occurs within 1 min and is essentially reversible. The analysis of the dose-dependency at equilibrium indicates the presence of binding sites with Kd of 0.23 microM with hepatocytes and Kd of 0.11 microM with plasma membranes. The relative affinities of 10 nucleotide analogues were deduced from competition experiments for ATP alpha [35S] binding to hepatocytes, and these correlated highly with their biological activity (activation of glycogen phosphorylase in hepatocytes). For all the agonists, binding occurs in the same concentration range as the biological effect. These data clearly suggest that the detected binding sites correspond to the physiological P2-purinoceptors involved in the regulation of liver glycogenolysis. The rank order of potency of some ATP analogues suggests that liver possesses the P2Y-subclass of P2-purinoceptors.

Animals↗
PubMed1985

P2-purinergic control of liver glycogenolysis.

Purinergic agonists cause a dose-dependent activation of glycogen phosphorylase in isolated rat hepatocytes. Half-maximally effective concentrations are 5 X 10(-7)M for ATP, 2 X 10(-6)M for ADP, and about 5 X 10(-5) M for AMP and adenosine. This potency series indicates the presence of P2-purinergic receptors. The mode of action of ATP appears to be identical with that of the Ca2+-dependent glycogenolytic hormones angiotensin, vasopressin and alpha 1-adrenergic agonists. (1) They all require Ca2+ for phosphorylase activation; (2) they do not increase cyclic AMP levels; (3) they are susceptible to heterologous desensitization by vasopressin and phenylephrine; (4) they lower cyclic AMP concentrations in hepatocytes stimulated by glucagon, most probably mediated by an enhanced phosphodiesterase activity.

Adenine Nucleotides↗
PubMed1984

Vasopressin and angiotensin control the activity of liver phosphodiesterase.

Vasopressin and angiotensin are able to lower the glucagon-induced increase of cyclic AMP levels in isolated hepatocytes. Results presented are in favour of an enhanced phosphodiesterase activity to account for this cyclic AMP lowering effect. In particular, vasopressin prevents exogenous cyclic AMP from activating glycogen phosphorylase: in the presence of phosphodiesterase inhibitors, the hormone becomes unable to decrease glucagon-induced cyclic AMP levels. This anti-glucagon effect of vasopressin and angiotensin might be physiologically more important than their glycogenolytic effect; indeed, the latter is very transient in nature and, in addition, requires higher hormone concentrations [Bréant, Keppens & De Wulf (1981) Biochem. J. 200, 509-514] than those needed for the anti-glucagon effect, as reported here.

Angiotensin II↗
PubMed1982

The liver angiotensin receptor involved in the activation of glycogen phosphorylase.

Specific angiotensin binding to rat hepatocytes and purified liver plasma membranes was measured by using biologically active [(3)H]angiotensin (sp. radioactivity 14Ci/mmol). The kinetic parameters for angiotensin binding to hepatocytes are: K(+1) (association rate constant). 100mum(-1).min(-1); K(-1) (dissociation rate constant), 2min(-1); K(d) (dissociation constant). 30nm; maximal binding capacity, 0.42pmol/10(6) cells or 260000 sites/cell. Angiotensin binding to membranes is profoundly affected by GTP (0.1mm) and NaCl (100mm); these regulatory compounds greatly enhance both the rate of association and of dissociation and also the extent of dissociation. K(d) amounts to 10nm in the presence of GTP+NaCl and to 1.5nm in their absence; maximal binding capacity is 0.70pmol/mg of protein, both with or without GTP+NaCl. The relative affinities of 11 angiotensin structural analogues were deduced from competition experiments for [(3)H]angiotensin binding to hepatocytes and to membranes (in the latter case, GTP + NaCl were not included, in order to study the higher affinity state of the receptor). These are highly correlated with their biological activity (activation of glycogen phosphorylase in hepatocytes). Binding to membranes occurs in the same concentration range as the biological effect. On the other hand, the existence of numerous spare receptors is suggested by the observation that binding of the agonists to hepatocytes requires 25-fold higher concentrations than those needed for their biological activity. These data clearly suggest that the detected binding sites correspond to the physiological receptors involved in the glycogenolytic action of angiotensin on rat liver.

Angiotensin II↗
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↗
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↗
PubMed1979

The nature of the hepatic receptors involved in vasopressin-induced glycogenolysis.

We have found a close correlation between the known vasopressor potency of arginine vasopressin and fourteen structural analogs, and the ability of these peptides to activate glycogen phosphorylase in isolated rat hepatocytes; there was no relation with the known antidiuretic activity of the analogs. We have also found that the pA2 values characterizing the known antivasopressor capacity of five analogs against vasopressin were close to those obtained for their inhibition of the vasopressin-induced activation of hepatic glycogen phosphorylase. We propose therefore that the hepatic receptors responsible for the glycogenolytic activity of vasopressin share characteristics with and appear therefore related to those responsible for pressor activity in vivo.

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↗