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 α-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 $\text{b}$ 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-independent 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 $\text{b}$ kinase.     

The role of calcium ion as a mediator of the effects of angiotensin II, catecholamines, and vasopressin on the phosphorylation and activity of enzymes in isolated hepatocytes.

Angiotensin II, catecholamines, and vasopressin are thought to stimulate hepatic glycogenolysis and gluconeogenesis via a cyclic AMP-independent mechanism that requires calcium ion. The present study explores the possibility that angiotensin II and vasopressin control the activity of regulatory enzymes in carbohydrate metabolism through Ca2+-dependent changes in their state of phosphorylation. Intact hepatocytes labeled with [32P]PO43- were stimulated with angiotensin II, glucagon, or vasopressin and 30 to 33 phosphorylated proteins resolved from the cytoplasmic fraction of the cell by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. Treatment of the cells with angiotensin II or vasopressin increased the phosphorylation of 10 to 12 of these cytosolic proteins without causing measurable changes in cyclic AMP-dependent protein kinase activity. Glucagon stimulated the phosphorylation of the same set of 11 to 12 proteins through a marked increase in cyclic AMP-dependent protein kinase activity. The molecular weights of three of the protein bands whose phosphorylation was increased by these hormones correspond to the subunit molecular weights of phosphorylase (Mr = 93,000), glycogen synthase (Mr = 85,000), and pyruvate kinase (Mr = 61,000). Two of these phosphoprotein bands were positively identified as phosphorylase and pyruvate kinase by affinity chromatography and immunoprecipitation, respectively. Incubation of hepatocytes in a Ca2+-free medium completely abolished the effects of angiotensin II and vasopressin on protein phosphorylation but did not alter those of glucagon. Treatment of hepatocytes with angiotensin II, glucagon, or vasopressin stimulated phosphorylase activity by 250 to 260%, inhibited glycogen synthase activity by 50%, and inhibited pyruvate kinase activity by 30 to 35% (peptides) to 70% (glucagon). The effects of angiotensin II and vasopressin on the activity of all three enzymes were completely abolished if the cells were incubated in a Ca2+-free medium while those of glucagon were not altered. The results imply that angiotensin II, catecholamines, and vasopressin control hepatic carbohydrate metabolism through a Ca2+-requiring, cyclic AMP-independent pathway that leads to the phosphorylation of important regulatory enzymes.     

Role of extracellular calcium and calcium efflux in the activation of hepatic glycogenolysis by calcium-dependent hormones

The role of extracellular calcium in the glycogenolytic effects of calcium-dependent hormones was examined in a rat liver perfusion system. Decreasing the perfusate CaCl2 concentration resulted in a concentration-dependent inhibition of glucose output by maximal concentrations of vasopressin (20 nM) and angiotensin II (10 nM), but not of glucagon (1.4 nM), cyclic AMP (100 microM), dibutyryl cyclic AMP (10 microM) or phenylephrine (5 microM). However, the effect of phenylephrine was inhibited when livers were perfused with CaCl2-free perfusate containing 0.5 mM EGTA in a duration-dependent manner. These effects were exerted through the inhibition of the maximal response of each hormone, and were associated with a parallel decrease in phosphorylase activation but not with changes in tissue cyclic AMP concentrations. When livers were preloaded with 45Ca for 45 min and then washed for either 15 min or 45 min, these hormones elicited a rapid and transient 45Ca efflux regardless of the perfusate calcium concentration. The sequential perfusion of two hormones resulted in the loss of 45Ca efflux by the second hormone. These results suggest that the glycogenolytic effects of vasopressin and angiotensin II depend on the extracellular calcium and that of phenylephrine primarily on the cellular calcium. It was also demonstrated that these calcium-dependent hormones mobilize calcium from the same pools. However, the mobilization of cellular calcium does not necessarily correlate directly with the glycogenolytic actions of vasopressin and angiotensin II.     

Hormonal stimulation of cyclic AMP accumulation and glycogen phosphorylase activity in calcium-depleted hepatocytes from euthyroid and hypothyroid rats.

Activation of glycogen phosphorylase by hormones was examined in hepatocytes isolated from euthyroid and hypothyroid female rats and incubated by Ca2+-free buffer containing 1 mM-EGTA. Basal glycogen phosphorylase activity was decreased in Ca2+-free buffer. However, the activation of hepatocyte glycogen phosphorylase, in the absence of extracellular Ca2+, in response to adrenaline, glucagon or phenylephrine was slightly lower, whereas that by vasopressin was abolished. The activation of glycogen phosphorylase by phenylephrine, adrenaline or isoproterenol (isoprenaline) in hepatocytes from euthyroid rats incubated in the absence of Ca2+ was not accompanied by any detectable increase in total cyclic AMP. The log-dose/response curves for activation of phosphorylase by phenylephrine or low concentrations of adrenaline were the same in hepatocytes from hypothyroid as compared wit euthyroid rats, whereas the response to isoproterenol was greater in hepatocytes from hypothyroid rats. However, the increases in total cyclic AMP accumulation caused by adrenaline or isoproterenol were greater in hepatocytes from hypothyroid rats than in hepatocytes from euthyroid rats. The increases in cyclic AMP accumulation caused by adrenaline or isoproterenol in Ca2+-depleted hepatocytes from hypothyroid rats were blocked by propranolol, a beta-adrenergic antagonist. In contrast, propranolol was only partially effective asan inhibitor of the activation of glycogen phosphorylase by phenylephrine or adrenaline in hepatocytes from hypothyroid rats and ineffective on phosphorylase activation in cells from euthyroid rats. These data indicate that the alpha-adrenergic activation of glycogen phosphorylase is not affected by the absence of extracellular Ca2+, and the extent to which total cyclic AMP was increased by adrenergic amines did not correlate with glycogen phosphorylase activation.     

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.     

Synergistic stimulation of the Ca2+ influx in rat hepatocytes by glucagon and the Ca2+-linked hormones vasopressin and angiotensin II

Glucagon was added to isolated rat hepatocytes, either alone or together with vasopressin or angiotensin II, and the effects on the initial 45Ca2+ uptake rate were investigated. Addition of glucagon alone which increased cyclic AMP content of the cells slightly increased the initial 45Ca2+ uptake rate. When glucagon was added together with vasopressin or angiotensin II--both of which when added separately increase the initial 45Ca2+ uptake rate but did not affect the cellular content of cyclic AMP--the measured initial 45Ca2+ uptake rate was larger than the sum of that seen with each hormone alone. This indicates that glucagon and Ca2+-linked hormones synergistically enhanced the Ca2+ influx in rat hepatocytes. These effects of glucagon can be mimicked by dibutyryl cyclic AMP or forskolin, suggesting that cyclic AMP augments both the resting Ca2+ and the vasopressin- or angiotensin II-stimulated influx. Measurement of the initial 45Ca2+ uptake rate as a function of the extracellular Ca2+ concentration indicated that the increase in the Ca2+ influx resulting from single or combined glucagon and vasopressin administration occurred through a homogeneous population of Ca2+ gates. These hormones were found to raise both the apparent Km for external Ca2+ and the apparent Vmax of the Ca2+ influx. The maximal increase in these two parameters was observed when the two hormones were added together. This suggests that glucagon and vasopressin synergistically stimulate the same Ca2+ gating mechanism. The dose-response curves for the action of glucagon or vasopressin applied in the presence of increasing concentrations of vasopressin or glucagon, respectively, showed that each hormone increases the maximal response to the other without affecting its ED50. It is proposed that glucagon and the Ca2+-linked hormones control the cellular concentration of two intermediates which are both necessary to allow Ca2+ entry into the cells.     

Activation of protein kinase and glycogen phosphorylase in isolated rat liver cells by glucagon and catecholamines.

In liver cells isolated from fed female rats, glucagon (290nM) increased adenosine 3':5'-monophosphate (cyclic AMP) content and decreased cyclic AMP binding 30 s after addition of hormones. Both returned to control values after 10 min. Glucagon also stimulated cyclic AMP-independent protein kinase activity at 30 s and decreased protein kinase activity assayed in the presence of 2 muM cyclic AMP at 1 min. Glucagon increased the levels of glycogen phosphorylase a, but there was no change in total glycogen phosphorylase activity. Glucagon increased glycogen phosphorylase a at concentrations considerably less than those required to affect cyclic AMP and protein kinase. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine, potentiated the action of glucagon on all variables, but did not increase the maximuM activation of glycogen phosphorylase. Epinephrine (1muM) decreased cyclic AMP binding and increased glycogen phosphorylase a after a 1-min incubation with cells. Although 0.1 muM epinephrine stimulated phosphorylase a, a concentration of 10 muM was required to increase protein kinase activity. 1-Methyl-3-isobutyl xanthine (0.1 mM) potentiated the action of epinephrine on cyclic AMP and protein kinase. (-)-Propranolol (10muM) completely abolished the changes in cyclic AMP binding and protein kinase due to epinephrine (1muM) in the presence of 0.1mM 1-methyl-3-isobutyl xanthine, yet inhibited the increase in phosphorylase a by only 14 per cent. Phenylephrine (0.1muM) increased glycogen phosphorylase a, although concentrations as great as 10 muM failed to affect cyclic AMP binding or protein kinase in the absence of phosphodiesterase inhibitor. Isoproterenol (0.1muM) stimulated phosphorylase and decreased cyclic AMP binding, but only a concentration of 10muM increased protein kinase. 1-Methyl-3-isobutyl xanthine potentiated the action of isoproterenol on cyclic AMP binding and protein kinase, and propranolol reduced the augmentation of glucose release and glycogen phosphorylase activity due to isoproterenol. These data indicate that both alpha- and beta-adrenergic agents are capable of stimulating glycogenolysis and glycogen phosphorylase a in isolated rat liver cells. Low concentrations of glucagon and beta-adrenergic agonists stimulate glycogen phosphorylase without any detectable increase in cyclic AMP or protein kinase activity. The effects of alpha-adrenergic agents appear to be completely independent of changes in cyclic AMP protein kinase activity.     

Synergistic stimulation of Ca2+ uptake by glucagon and Ca2+-mobilizing hormones in the perfused rat liver. A role for mitochondria in long-term Ca2+ homoeostasis.

A perfused liver system incorporating a Ca2+-sensitive electrode was used to study the long-term effects of glucagon and cyclic AMP on the mobilization of Ca2+ induced by phenylephrine, vasopressin and angiotensin. At 1.3 mM extracellular Ca2+ the co-administration of glucagon (10 nM) or cyclic AMP (0.2 mM) and a Ca2+-mobilizing hormone led to a synergistic potentiation of Ca2+ uptake by the liver, to a degree which was dependent on the order of hormone administration. A maximum net amount of Ca2+ influx, corresponding to approx. 3800 nmol/g of liver (the maximum rate of influx was 400 nmol/min per g of liver), was induced when cyclic AMP or glucagon was administered about 4 min before vasopressin and angiotensin. These changes are over an order of magnitude greater than those induced by Ca2+-mobilizing hormones alone [Altin & Bygrave (1985) Biochem. J. 232, 911-917]. For a maximal response the influx of Ca2+ was transient and was essentially complete after about 20 min. Removal of the hormones was followed by a gradual efflux of Ca2+ from the liver over a period of 30-50 min; thereafter, a similar response could be obtained by a second administration of hormones. Dose-response measurements indicate that the potentiation of Ca2+ influx by glucagon occurs even at low (physiological) concentrations of the hormone. By comparison with phenylephrine, the stimulation of Ca2+ influx by vasopressin and angiotensin is more sensitive to low concentrations of glucagon and cyclic AMP, and can be correlated with a 20-50-fold increase in the calcium content of mitochondria. The reversible uptake of such large quantities of Ca2+ implicates the mitochondria in long-term cellular Ca2+ regulation.     

Effects of catecholamines and glucagon on glycogen metabolism in human polymorphonuclear leukocytes.

Addition of 10 micron of the alpha-adrenergic agonist phenylephrine to polymorphonuclear leukocytes suspended in glucose-free Krebs-Ringer bicarbonate buffer (pH 6.7) activated phosphorylase, inactivated glycogen synthase R maximally within 30 s, and resulted in glycogen breakdown. Phenylephrine increased 45Ca efflux relative to control of 45Ca prelabelled cells, but did not affect cyclic adenosine 3',5'-monophosphate (cAMP) concentration. The effects of phenylephrine were blocked by 20 micron phentolamine and were absent in cells incubated at pH 7.4. The same unexplained dependency of extracellular pH was observed with 2.5 nM--2.5 micron glucagon, which activated phosphorylase and inactivated synthase-R, but in addition caused a 30-s burst in cAMP formation. 25 nM glucagon also increased 45Ca efflux. The activation of phosphorylase by phenylephrine and possibly also by glucagon are thought mediated by an increased concentration of cytosolic Ca2+ activating phosphorylase kinase. The effects of 5 micron isoproterenol or 5 micron epinephrine were independent of extracellular pH 6.7 and 7.4 and resulted in a sustained increase in cAMP, an activation of phosphorylase and inactivation of synthase-R within 15 s, and in glycogenolysis. The effects of both compounds were blocked by 10 micron propranolol, whereas 10 micron phentolamine had no effect on the epinephrine action. The efflux of 45Ca was not affected by either isoproterenol or epinephrine. The beta-adrenergic activation of phosphorylase is consistent with the assumption of a covalent modification of phosphorylase kinase by the cAMP dependent protein kinase. Phosphorylation of synthase-R to synthase-D can thus occur independently of increase in cAMP, but the evidence is inconclusive with respect to the cAMP dependent protein kinase also being active in this phosphorylation.     

Regulation of platelet phosphorylase.

A sensitive fluorimetric enzyme assay was developed for study of activation of glycogen phosphorylase (EC 2.4.1.1) in intact platelets and in platelet extracts. Activity was calculated as AMP independent (activity in the absence of AMP), total (activity in the presence of 1 mM AMP), and AMP dependent (difference between AMP independent and total). The following observations were made with intact rat platelets. (1) Stimulation of platelets with thrombin caused a 7-fold increase in total activity, with increases in both AMP-dependent and AMP-independent activities. Maximum activation was obtained within 10 s after addition of thrombin. (2) The divalent cation ionophore A23187 caused a similar, though less pronounced, activation of phosphorylase. (3) Acceleration of glycogenolysis by inhibition of respiration with cyanide caused similar changes in phosphorylase activity but with the maximum effect observed only after 45 s. (4) Dibutyryl cyclic AMP had two effects; it partially activated phosphorylase and blocked further activation by thrombin, but not A23187. Similar effects were observed with human platelets, but low resting levels of phosphorylase activity could not be maintained so that changes were not as large as with rat platelets. Experiments with extracts of rat platelets gave the following results. (1) Phosphorylase activity in many extracts of non-stimulated platelets could be increased by incubation with Mg2+-ATP and Ca2+; ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) partially inhibited. (2) In some extracts there was essentially no activation by incubation with Mg2+-ATP and Ca2+, but addition of cyclic AMP GAVE PARTIAL ACTIVATIon while addition of rabbit muscle phosphorylase kinase gave full activation. (3) Incubation of extracts of thrombin-stimulated platelets caused conversion of AMP-dependent to AMP-indeptndent activity. It is concluded that platelet phosphorylase exists in an inactive and two active forms. Conversion of the inactive to the active forms and of the AMP-dependent to the AMP-independent form is catalyzed by a kinase(s) that requires Ca2+ for full activity and is activated through a cyclic AMP-mediated process. The major change following physiological stimulation is an increase in both active forms, with little change in their ratio.     

Studies on alpha-adrenergic activation of hepatic glucose output. Studies on role of calcium in alpha-adrenergic activation of phosphorylase.

The role of Ca2+ ions in alpha-adrenergic activation of hepatic phosphorylase was studied using isolated rat liver parenchymal cells. The activation of glucose release and phosphorylase by the alpha-adrenergic agonist phenylephrine was impaired in cells in which calcium was depleted by ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) treatment and restored by calcium addition, whereas the effects of a glycogenolytically equivalent concentration of glucagon on these processes were unaffected. EGTA treatment also reduced basal glucose release and phosphorylase alpha activity, but did not alter the level of cAMP or the protein kinase activity ratio (-cAMP/+cAMP) or impair viability as determined by trypan blue exclusion, ATP levels, or gluconeogenic rates. The effect of EGTA on basal phosphorylase and glucose output was also rapidly reversed by Ca2+, but not by other ions. Phenylephrine potentiated the ability of low concentrations of calcium to reactivate phosphorylase in EGTA-treated cells. The divalent cation inophore A23187 rapidly increased phosphorylase alpha and glucose output without altering the cAMP level, the protein kinase activity ratio, and the levels of ATP, ADP, or AMP, The effects of the ionophore were abolished in EGTA-treated cells and restored by calcium addition. Phenylephrine rapidly stimulated 45Ca uptake and exchange in hepatocytes, but did not affect the cell content of 45Ca at late time points. A glycogenolytically equivalent concentration of glucagon did not affect these processes, whereas higher concentrations were as effective as phenylephrine. The effect of phenylephrine on 45Ca uptake was blocked by the alpha-adrenergic antagonist phenoxybenzamine, was unaffected by the beta blocker propranolol, and was not mimicked by isoproterenol. The following conclusions are drawn: (a) alpha-adrenergic activation of phosphorylase and glucose release in hepatocytes is more dependent on calcium than is glucagon activation of these processes; (b) variations in liver cell calcium can regulate phosphorylase alpha levels and glycogenolysis; (c) calcium fluxes across the plasma membrane are stimulated more by phenylephrine than by a glycogenolytically equivalent concentration of glucagon. It is proposed that alpha-adrenergic agonists activate phosphorylase by increasing the cytosolic concentration of Ca2+ ions, thus stimulating phosphorylase kinase.     

Glucagon and the Ca2+-linked hormones angiotensin II, norepinephrine, and vasopressin stimulate the phosphorylation of distinct substrates in intact hepatocytes

Recent studies have demonstrated that angiotensin II, catecholamines, and vasopressin can stimulate the phosphorylation of hepatic cytosolic proteins via a Ca2+-linked cyclic AMP-independent mechanism. The present study used high resolution, two-dimensional gel electrophoresis to determine if the proteins phosphorylated in response to the Ca2+-linked hormones were distinct from those affected by glucagon acting via the cyclic AMP-dependent pathway. Intact hepatocytes labeled with [32P]PO4(3-) were stimulated with glucagon, angiotensin II, l-norepinephrine, and vasopressin and over 100 phosphorylated proteins resolved by two-dimensional electrophoresis and autoradiography. Six important enzymes known to be regulated through covalent modification were positively identified, including phosphorylase, phosphofructokinase, pyruvate kinase, fructose-6-phosphate, 2-kinase, phenylalanine hydroxylase, and fructose-1,6-bisphosphatase. Computer analysis of the autoradiograms from control and hormone-treated cells demonstrated that glucagon increased the phosphorylation state of 12 phosphoproteins and reduced the phosphorylation of one protein with a Mr = 21,000 and a pI = 5.9. The Ca2+-linked hormones stimulated the phosphorylation of 7 phosphoproteins and also reduced the phosphorylation state of the 21,000-dalton protein. Angiotensin II, l-norepinephrine, and vasopressin had equivalent effects on protein phosphorylation. There were six protein substrates uniquely affected by glucagon and one phosphoprotein uniquely stimulated by the Ca2+-linked hormones. Seven substrates were affected by stimulation of the cell with either glucagon or the Ca2+-linked hormones. These results demonstrate that, while there is overlap in the substrates affected by glucagon and the Ca2+-linked hormones, each pathway is able to affect the phosphorylation of unique substrates. This finding suggests that the two types of hormones may have some distinct effects on hepatic function.U     

On the role of intra-adrenal unesterified cholesterol in the steroidegenic effect of corticotropin

Addition of 10 μM of the α-adrenergic agonist phenylephrine to polymorphonuclear leukocytes suspended in glucose-free Krebs-Ringer bicarbonate buffer (pH 6.7) activated phosphorylase, inactivated glycogen synthase R maximally within 30 s, and resulted in glycogen breakdown. Phenylephrine increased ⁴⁵Ca efflux relative to control of ⁴⁵Ca prelabelled cells, but did not affect cyclic adenosine 3′,5′-monophosphate (cAMP) concentration. The effects of phenylephrine were blocked by 20 μM phentolamine and were absent in cells incubated at pH 7.4.The same unexplained dependency of extracellular pH was observed with 2.5 nM–2.5 μM glucagon, which activated phosphorylase and inactivated synthase-R, but in addition caused a 30-s burst in cAMP formation. 25 nM glucagon also increased ⁴⁵Ca efflux. The activation of phosphorylase by phenylephrine and possibly also by glucagon are thought mediated by an increased concentration of cytosolic Ca²⁺ activating phosphorylase kinase.The effects of 5 μM isoproterenol or 5 μM epinephrine were independent of extracellular pH 6.7 and 7.4 and resulted in a sustained increase in cAMP, an activation of phosphorylase and inactivation of synthase-R within 15 s, and in glycogenolysis. The effects of both compounds were blocked by 10 μM propranolol, whereas 10 μM phentolamine had no effect on the epinephrine action. The efflux of ⁴⁵Ca was not affected by either isoproterenol or epinephrine. The β-adrenergic activation of phosphorylase is consistent with the assumption of a covalent modification of phosphorylase kinase by the cAMP dependent protein kinase.Phosphorylation of synthase-R to synthase-D can thus occur independently of increase in cAMP, but the evidence is inconclusive with respect to the cAMP-dependent protein kinase also being active in this phosphorylation.     

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.     

Difference in the mechanism of action of alpha-adrenergic agonists and vasopressin or angiotensin II in stimulating hepatic glycogenolysis; a role of extracellular calcium concentration

The role of extracellular calcium in hormone-induced glycogenolysis was examined in a rat liver perfusion system by manipulating the perfusate calcium concentration and by using calcium antagonistic drugs. When the perfusate contained 1 mM CaCl2, 5 microM phenylephrine, 20 nM vasopressin, and 10 nM angiotensin II caused a persistent increase in glucose output and phosphorylase activity as well as a transient increase in 45Ca efflux from 45Ca preloaded liver. Verapamil hydrochloride (20-100 microM) inhibited the activation of glucose output by these hormones in a dose-dependent manner. This inhibitory effect was also associated with the inhibition of hormone-induced activation of phosphorylase and 45Ca efflux. In the absence of CaCl2 in the perfusate, the glycogenolytic effect of phenylephrine and its inhibition by verapamil were obtained equally as in the presence of CaCl2. However, the effects of vasopressin and angiotensin II were markedly attenuated and were not inhibited any further by verapamil. The substitution of diltiazem hydrochloride for verapamil produced essentially identical results. Cyclic AMP concentrations in the tissue did not change under any of these test conditions. The results indicate that the glycogenolytic effect of alpha-adrenergic agonists depends on intracellular calcium but those of vasopressin and angiotensin II on extracellular calcium, and support the concept that calcium antagonistic drugs inhibit the glycogenolytic effects of calcium-dependent hormones at least by inhibiting the mobilization of calcium ion from cellular pools.     

Modulation of Glycogen Phosphorylase Activity Affects 5‐Phosphoribosyl‐1‐Pyrophosphate Availability in Rat Hepatocyte Cultures

The effect of modulation of the rate of glycogenolysis on the availability of 5‐phosphoribosyl‐1‐ pyrophosphate (PRPP) was investigated in rat hepatocyte cultures. Dibutyryl cyclic AMP (dbcAMP), forskolin and glucagon, activating glycogen phosphorylase through activation of protein kinase A (PKA), were found to raise PRPP availability by 44%–56%. Arg‐vasopressin and phenylephrine, activating glycogen phosphorylase through the phosphoinositide cascade, did not affect PRPP availability. dbcAMP, but not phenylephrine, increased the degradation of pre labeled glycogen by 57%. Caffeine and CP‐91149, inhibitors of glycogen phosphorylase, decreased PRPP availability by 33% and 43%, respectively. The finding that induction of glycogenolysis enhances, and inhibition of glycogenolysis decelerates PRPP generation suggests that glycogenolysis is a major contributor to PRPP generation in liver tissue in the basal (postabsorptive) state.     

On the Role of Calcium Ions in the Regulation of Glycogenolysis in Mouse Brain Cortical Slices

Abstract: 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; α-1,4-glucan:orthophosphate glucosyltransferase) induced by noradrenaline and by depolarization. Activation of phosphorylase by 100 μM noradrenaline is mediated by β-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 m M K⁺ or 100 μ M 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 halfmaximal activation of phosphorylase occurs at about 12 m M 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.     

The role of calcium and cyclic adenosine 3',5'-monophosphate in the regulation of glycogen metabolism in phagocytozing human polymorphonuclear leukocytes.

Incubation of human polymorphonuclear leukocytes in a glucose-free Krebs-Ringer bicarbonate buffer for 2 h resulted in glycogen depletion, decreased phosphorylase activity and increased synthase-R activity. Addition of dialyzed latex particles to starved leukocytes revealed a very rapid ingestion rate (half-maximal ingestion within 30 s). This uptake is followed by glycogenolysis associated with an immediate two-fold increase in phosphorylase a activity and a synthase-R to -D conversion within 30 s. Furthermore, in rapid time-course experiments with phagocytozing cells we found that the concentration of cyclic AMP increased by 93% within 15 s and returned to baseline values at 1 min. In a medium without added calcium and with 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid, phagocytosis was blocked, cyclic AMP formation decreased by 50% and phosphorylase activation was abolished, but the conversion of synthase-R to -D was preserved. Addition of calcium ions to cells suspended in a calcium-free buffer without added latex results in phosphorylase activation and glycogenolysis, but not in cyclic AMP increase or synthase-R to -D conversion. Measurements of 45Ca efflux during phagocytosis suggest an initial increase in cytosolic calcium obtained by a release of membrane-bound 45Ca. Activation of phosphorylase during phagocytosis is thus presumably due to an increase in cytosol Ca2+ and subsequent activation of phosphorylase kinase, and is independent of the simultaneous increase in concentration of cyclic AMP. Phosphorylation of synthase R to the D form does not depend on the presence of Ca2+ in the extracellular phase.     

Purification and characterization of two cyclic AMP-independent casein/glycogen synthase kinases from rat liver cytosol

Two cyclic AMP-independent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) (casein kinase 1 and 2) have been purified from rat liver cytosol by a method involving chromatography on phosphocellulose and casein-Sepharose 4B. Both kinases were essentially free of endogeneous protein substrates and capable of phosphorylating casein, phosvitin and I-form glycogen synthase, but were inactive on histone IIA, protamine and phosphorylase b. They were neither stimulated by cyclic AMP, Ca2+ and calmodulin, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. The casein and glycogen synthase kinase activities of each enzyme decreased at the same rate when incubated at 50 degrees C. Casein kinase 1 and casein kinase 2 showed differences in molecular weight, sensitivity to KCl, Km for casein and phosvitin and Ka for Mg2+, whereas their Km values for ATP and I-form glycogen synthase were similar. The phosphorylation of glycogen synthase by these kinases correlated with a decrease in the +/- glucose 6-phosphate activity ratio (independence ratio). However, casein kinase 1 catalyzed the incorporation of about 3.6 mol of 32P/85000 dalton subunit, decreasing the independence ratio from 83 to about 15, whereas the phosphorylation achieved by casein kinase 2 was only about 1.9 mol of 32P/850000 dalton subunit, decreasing the independence ratio to about 23. The independence ratio decrease was prevented by the presence of casein but was unaffected by phosphorylase b. These data indicate that casein/glycogen synthase kinases 1 and 2 are different from cyclic AMP-dependent protein kinase and phosphorylase kinase.     

The role of protein kinase C in the inactivation of hepatic glycogen synthase by calcium-mobilizing agonists.

The regulation of glycogen synthase by Ca2+-mobilizing hormones was studied by using rat liver parenchymal cells in primary culture. Long-term exposure of hepatocytes to 4 beta-phorbol 12-myristate 13-acetate (TPA) resulted in a decrease in vasopressin or ATP inhibition of glycogen synthesis and glycogen synthase activity, without any change in the activation of glycogen phosphorylase. In contrast, treatment with TPA did not diminish the effects of glucagon, isoprenaline or A23187 on glycogen synthase or phosphorylase. TPA treatment for 18 h did not change specific [3H]vasopressin binding, but abolished protein kinase C activity in a concentration-dependent manner. The effects of TPA to decrease protein kinase C activity and to reverse the inactivation of glycogen synthase by vasopressin were well correlated and were mimicked by mezerein, but not by 4 alpha-phorbol. However, 1 microM-TPA totally inhibited protein kinase C activity, but reversed only 60% of the vasopressin effect on glycogen synthase. It is therefore concluded that Ca2+-mobilizing hormones inhibit glycogen synthase partly, but not wholly, through a mechanism involving protein kinase C.