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Biomedical subjects

J H Exton

Publications and source records attributed to J H Exton.

At least 181 records · Page 10Linked to original sources
PubMed1982

Purification of rat liver phosphorylase kinase.

A rapid method for the purification of rat liver phosphorylase kinase 30,000-fold over homogenate values is described. The method allows the isolation of a near homogeneous preparation of phosphorylase kinase initially associated with the glycogen pellet to be accomplished within 24 h. The enzyme has Mr (apparent) = 1.3 million by gel filtration and is composed of subunits similar in size to those of skeletal muscle phosphorylase kinase. The enzyme is phosphorylated by the cAMP-dependent protein kinase: phosphate is incorporated into two of the subunits (Mr = 140,000 and Mr = 116,000) and is closely paralleled by activation of the enzyme. The enzyme is partially inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and is stimulated by 10(-8)-10(-6) M Ca2+. The pH optimum of the nonactivated enzyme is 7.0. Activation by cAMP-dependent protein kinase does not appear to alter the Ca2+ sensitivity of the enzyme. However, it results in a large increase in activity at pH 7 through 8, but not at pH below 6.5. Purified rat liver phosphorylase kinase thus shows many similarities to purified skeletal muscle phosphorylase kinase, but differs in respect to its incomplete inhibition by ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid and to the effects of phosphorylation by cAMP-dependent protein kinase on its pH activity profile and Ca2+ sensitivity.

Animals↗
PubMed1982

Studies of the interaction between glucagon and alpha-adrenergic agonists in the control of hepatic glucose output.

alpha-Adrenergic stimulation of hepatocytes prevented, in a dose-dependent manner, the stimulation of [U-14C]lactate conversion to [14C]glucose by glucagon and exogenously added cAMP and Bt2cAMP. The inhibition was referable to an interaction with adrenergic receptors which resulted in a small decrease in hepatic cAMP levels. Low concentrations of epinephrine (10 nM) were able to inhibit phosphorylase activation and glucose output elicited by low doses of glucagon (5 X 10(-11) M to 2 X 10(-10) M). The ability of epinephrine (acting via alpha 1-adrenergic receptors), vasopressin, and angiotensin II to elicit calcium efflux was inhibited by glucagon, suggesting that intracellular redistributions of Ca2+ are importantly involved in the gluconeogenic process. It is proposed that vasopressin, angiotensin II, and catecholamines, acting primarily via alpha 1-adrenergic receptors, are responsible for inhibition of glucagon mediated stimulation of gluconeogenesis by altering subcellular calcium redistribution and decreasing cAMP levels.

Adrenergic beta-Agonists↗
PubMed1982

Regulation of adrenergic stimulation of hepatic adenylate cyclase by divalent cations.

Liver plasma membrane adenylate cyclase was stimulated paradoxically by an alpha 2-adrenergic mechanism under conditions of low metal ion and low GTP concentrations. In untreated membranes, epinephrine stimulation was GTP-dependent and was mediated by beta-adrenergic receptors since it was completely blocked by propranolol, but unaffected by dihydroergocryptine. Pre-treatment of membranes to remove or reduce divalent cations and guanine nucleotides changed epinephrine stimulation to a form that was mediated by alpha 2-receptors since it was completely blocked by dihydroergocryptine, phenoxybenzamine and yohimbine, but not by propranolol or prazosin. The pre-treatment did not alter enzyme activation by isoproterenol or glucagon, alpha 2-Adrenergic stimulation of adenylate cyclase in depleted membranes required the presence in the assay of 1-2 mM Mg2+ and small amounts of exogenous GTP (less than or equal to 50 nM). Increasing the Mg2+ or GTP concentration in the assay produced a progressive reversal of epinephrine-stimulated activity from an alpha 2-adrenergic form to a predominantly beta-adrenergic form. Readdition of Ca2+ or Mg2+, but not Mn2+, into depleted membranes by incubation in the presence of metal reestablished the pattern of enzyme sensitivity to epinephrine to that seen with untreated membranes i.e., it changed from alpha 2- to beta-receptor mediation. Alterations in membrane and assay content of metal ions and GTP did not result in the activation of the enzyme by vasopressin or angiotensin II. These findings demonstrate the ability of Ca2+, Mg2+ and GTP to control the coupling of beta- and alpha 2-adrenergic receptors with liver adenylate cyclase. It is hypothesized that the cations act by regulating the interaction of the receptors with adrenergic agonists and/or the guanine nucleotide binding protein(s) which is postulated to be involved in control of the enzyme.

Adenylyl Cyclases↗
PubMed1982

Modulation by adrenalectomy and fasting of insulin effects in perfused hindlimb muscle.

Perfused hindlimb muscle from fed adrenalectomized rats accumulated more 2-deoxyglucose at submaximal concentrations of insulin in comparison to muscle from fed normal rats. However, in the fasted state, insulin-stimulated 2-deoxyglucose uptake was largely inhibited by adrenalectomy. Basal 2-deoxyglucose uptake did not differ between fed and fasted normal or adrenalectomized rats. The changes in insulin effects caused by adrenalectomy were due to altered hexose transport as shown by measurements of 3-O-methylglucose uptake and of intracellular free and phosphorylated 2-deoxyglucose. Muscles of fasted normal and fed or fasted adrenalectomized rats showed higher basal glycogen synthase --glucose-6-P/+glucose-6-P activity ratios than those of fed normal rats probably because of decreased glycogen content. However, muscles from fed or fasted adrenalectomized rats did not show any alterations in insulin effects on the activity ratio and half-maximal activation constant (A0.5) for glucose-6-P of glycogen synthase. Because of the dissociation of the effects of insulin on hexose transport and glycogen synthase in muscle of fasted adrenalectomized rats, it is concluded that the impairment in insulin-stimulated hexose transport in these animals is due to a defect lying beyond the interaction of insulin with its receptor.

3-O-Methylglucose↗
PubMed1981

Rat liver phosphorylase kinase. Stimulation by heparin.

Rat liver phosphorylase kinase activity was increased up to 7-fold by low concentrations of heparin, but other glycosaminoglycans were relatively ineffective. A half-maximal effect was seen with 0.5 units of heparin/ml, corresponding to 150 nM. The stimulation was rapid, i.e. occurring within 15 s, and was prevented or reversed by addition of the heparin-binding protein antithrombin III. The action of heparin was also sharply reduced by prior activation of the enzyme by MgATP. In the absence of heparin, the time course of the phosphorylase kinase reaction was concave upward, whereas in the presence of heparin, a constant rate was seen. The stimulatory effect of heparin was inversely proportional to the concentration of phosphorylase b. The data suggest that heparin reversibly stimulates a low activity form of liver phosphorylase kinase to a degree similar to that found with activation by phosphorylation.

Animals↗
PubMed1981

Fructose 2,6-bisphosphate. Hormonal regulation and mechanism of its formation in liver.

Vasopressin, phenylephrine, and A23187 cause an accumulation of fructose 2,6-bisphosphate in hepatocytes from fed rats, but not in Ca2+-depleted hepatocytes from fed rats or in phosphorylase kinase-deficient hepatocytes from (gsd/gsd) rats. The effect of vasopressin and phenylephrine is not found in hepatocytes from overnight-starved rats. Thus, the accumulation of fructose 2,6-bisphosphate by these agents may depend on the stimulation of glycogenolysis and on the resulting accumulation of hexose 6-phosphate. In support of this hypothesis, conditions are described for the enzymatic synthesis of fructose 2,6-bisphosphate from fructose 6-phosphate and Mg-ATP in liver extracts. Half-maximal activity (0.8 nmol/min.g) is obtained with about 60 microM fructose 6-phosphate, and the activity can be separated fom phosphofructokinase by ammonium sulfate fractionation. Treatment of rats or isolated hepatocytes with glucagon results in a 4-5-fold decrease in the maximal activity of this enzyme.

Animals↗
PubMed1981

Alpha 1-Adrenergic stimulation of Ca2+ mobilization without phosphorylase activation in hepatocytes from phosphorylase b kinase-deficient gsd/gsd rats.

Phenylephrine, vasopressin and the bivalent cation ionophore A23187 mobilized Ca2+ normally, but failed to activate phosphorylase, in hepatocytes from gsd/gsd rats with a deficiency of liver phosphorylase b kinase. These data provide strong evidence that phosphorylase b kinase is the site of action of the Ca2+ mobilized intracellularly during alpha 1-adrenergic activation of phosphorylase in liver cells.

Animals↗
PubMed1981

Studies on the interactions between insulin and epinephrine in the control of skeletal muscle glycogen metabolism.

Possible inhibitory effects of insulin on epinephrine-induced changes in the enzymes of glycogen metabolism in skeletal muscle were tested using a perfused rat hindlimb preparation. Epinephrine and/or insulin were infused over a wide range of concentrations. Insulin at 6 X 10(-9) M increased the activity ratio (--Glc-6-P/+Glc-6-P) of glycogen synthase from a basal value of 0.09 +/- 0.01 to 0.13 +/- 0.01 and caused a 23% decrease in the Ka for Glc-6-P. In contrast, epinephrine at 10(-7) M decreased the activity ratio to 0.05 +/- 0.01 and increased the Ka for Glc-6-P 6.3-fold. Insulin was without effect on the concentration of cAMP or the activity ratio (-cAMP/+cAMP) of cAMP-dependent protein kinase and caused a small decrease in the activity ratio (-AMP/+AMP) of phosphorylase, whereas epinephrine caused large increases in all these parameters. Insulin at 6 X 10(-11) to 6 X 10(-8) M had no inhibitory effect on the actions of 10(-8) or 10(-7) M epinephrine on glycogen synthase, phosphorylase or cAMP-dependent protein kinase at 30 min or at earlier times. Insulin (6 X 10(-9) M) also did not alter th concentration of cAMP in the presence of 10(-8) or 10(-7) M epinephrine. These data are not consistent with the view that insulin activates glycogen synthase by producing an inhibitor of cAMP-dependent protein kinase. Nor do they support the hypothesis that insulin acts by decreasing the activity of an inhibitor of a multisubstrate phosphoprotein phosphatase.

Animals↗
PubMed1981

Insulin inhibition of alpha-adrenergic actions in liver.

The effects of insulin on alpha-agonist (phenylephrine)- and [Arg8]vasopressin-induced Ca2+ and glucose release and mitochondrial Ca2+ fluxes in isolated perfused rat livers were examined. Insulin (6 nM) inhibited the ability of phenylephrine (1 and 0.5 microM) to elicit Ca2+ and glucose release, whereas it was without effect on vasopressin (10 and 2.5 nM) actions. Correspondingly, insulin inhibited the action of phenylephrine to induce a stable increase in mitochondrial Ca2+ uptake, but it did not affect the alteration caused by vasopressin. Phenylephrine and vasopressin caused transient increases in hepatocyte respiration. Insulin inhibited the effect of phenylephrine on this parameter, but not that of vasopressin. Insulin added alone did not alter any of the above parameters. It is concluded from these data that insulin does not alter cellular Ca2+ fluxes and respiration themselves, but selectively inhibits alpha-adrenergic stimulation of these processes. It is proposed that insulin acts either to inhibit binding of alpha-agonists to their specific plasma-membrane receptors or to alter generation and/or degradation of the putative alpha-adrenergic 'second messenger'. If this latter possibility is the case, then the alpha-adrenergic 'second messenger' must be different from the 'second messenger' of vasopressin.

Animals↗