Henri De Wulf

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.

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.

Characterization of purinoceptors present on human liver plasma membranes.

Using ATPα35S as radioligand, we have detected the presence of specific purinoceptors on human liver plasma membranes. They are characterized by a K d value of 0.19 μM and a B max of 24 pmol/mg membrane protein. These purinoceptors belong to the P2Y subclass as demonstrated by the high degree of similarity with rat liver purinoceptors, previously shown to be P2Y[(1986) Biochem. J. 240, 367–371] and known to be involved in the control of liver glycogenolysis.

Characterization of the biological effects of 2‐methylthio‐ATP on rat hepatocytes: clear‐cut differences with ATP

1 In several tissues, 2‐methylthio adenosine triphosphate (2MeSATP) is a very potent P2Y‐purine agonist. In rat hepatocytes, 2MeSATP half‐maximally activated glycogen phosphorylase at 20 nm and was therefore about 25 times more effective than ATP (Ka 0.5–0.8 μm). This strong glycogenolytic potency of 2MeSATP suggests on its own the presence of P2Y‐purinoceptors in liver. 2 Displacement of the radioligand ATPα[35S] from its receptor however occurred at much higher concentrations of 2MeSATP than was anticipated on the basis of its glycogenolytic potency. 3 The interaction of 2MeSATP with the receptor, characterized with ATPα[35S] as radioligand, cannot be considered as a pure competitive interaction. 4 2MeSATP did not share the ability of ATP to counteract the effect of glucagon on the adenosine 3′:5′‐cyclic monophosphate levels. 5 2MeSATP barely increased the levels of inositol trisphosphate (IP3). 6 The glycogenolytic effect of 2MeSATP was completely abolished by pretreatment of the hepatocytes with phorbol myristic acetate. 7 It is tentatively concluded that 2MeSATP and ATP are interacting with different P2 purinoceptors.

The α-adrenergic control of rabbit liver glycogenolysis

Abstract We have confirmed that the electrical stimulation of the splanchnic nerve in the rabbit causes glycogenolysis m a cyclic AMP-independent way as found by Shimazu and Amakawa [1]; glycogen phosphorylase (1,4-α- d -Glucan: orthophosphate α-glucosyltransferase, EC 2.4.1.1) was activated, but phosphorylase b kinase (ATP: phosphorylase b phosphotransferase, EC 2.7.1.38) was not. We could, however, not confirm the observation of a decrease in phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) activity. Pretreatment of the rabbits with the α-adrenergic blocking agent phentolamine prevented the splanchnic nerve stimulation from activating glycogen phosphorylase. The addition of norepinephrine (10 −7 M) to isolated rabbit hepatocytes activated glycogen phosphorylase without an activation of phosphorylase b kinase. At 10 −6 M, norepinephrine activated both enzymes. Phentolamine blocked the activation of glycogen phosphorylase by norepinephrine at 10 −7 M but not at 10 −6 M. Absence of Ca 2+ from the incubation medium prevented norepinephrine (10 −7 M) from activating glycogen phosphorylase. The ionophore A 23187 also caused an activation of phosphorylase (but not of phosphorylase b kinase) provided that Ca 2+ was present in the incubation medium. These data indicate that sympathetic nervous control of liver glycogenolysis is achieved, via α-adrenergic receptors, by an increased concentration of cytosolic Ca 2+ ions which stimulate rather than activate phosphorylase b kinase. The neurotransmitter involved is most probably norepinephrine.

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.

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.

Induction of hepatic glycogen synthesis by glucocorticoids is not mediated by insulin

Administration of 0.1 or 1 mg of prednisolone to fed mice caused a 5-fold activation of glycogen synthase in the liver after 3h, without significant changes in the circulating levels of glucose or insulin, or the hepatic concentration of cyclic AMP. Adrenalectomized fasted rats responded to cortisol (10 mg) with an increased glycaemia and a progressive activation of hepatic glycogen synthase after 2-4 h. but without an increase in the very low insulinaemia. These results are incompatible with the prevailing hypothesis that glucocorticoids provoke hepatic glycogen synthesis through an extra secretion of insulin. It is discussed that the acute effect of glucocorticoids is to inhibit rather than stimulate the release of insulin.