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.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

Effect of adrenalectomy on cellular calcium metabolism and on the response to adrenergic stimulation of hepatocytes isolated from male and female rats

The effects of adrenalectomy on cell calcium metabolism and on the effects of epinephrine on cAMP, phosphorylase a activity, and calcium efflux were studied in hepatocytes isolated from adult male and female rats. Adrenalectomy increased the total calcium of hepatocytes, all exchangeable calcium pools, and all calcium fluxes between the cellular pools in both sexes. After adrenalectomy, basal cAMP was elevated, phosphorylase a + b was decreased, but basal phosphorylase a activity was not changed. In adrenalectomized males and at all concentrations of epinephrine studied (1·10^?8?1·10^?5M) stimulation of calcium efflux was decreased and cAMP accumulation was enhanced, while the resulting phosphorylase a activation was depressed. In hepatocytes from adrenalectomized females there was a similar increase in cAMP accumulation induced by epinephrine, and a decrease in the stimulation of calcium efflux; however, the depression in phosphorylase a activation was much less and was significant only at 1·10^?8 and 1·10^?5M epinephrine. In the male, while activation of phosphorylase a shifted from a pure α-adrenergic response mediated by calcium to one also involving a cAMP-mediated β-adrenergic response, the contribution of the attenuated calcium signal was still significant. Hepatocytes from female rats did not show a comparable α- to β-shift, since the relative contribution of calcium and cAMP to phosphorylase activation was similar in sham-operated and adrenalectomized animals.     

Angiotensin II inhibits hepatic cAMP accumulation induced by glucagon and epinephrine and their metabolic effects

Incubation of isolated hepatocytes containing normal Ca2+ levels with angiotensin II, vasopressin or A23187 caused significant inhibition of the cAMP response to glucagon. Angiotensin II also inhibited cAMP accumulation induced by either glucagon or epinephrine in Ca2+-depleted hepatocytes. When submaximal doses of hormone were employed such that cell cAMP was elevated only 3-4-fold (approximately 2 pmol cAMP/mg wet wt cells) inhibition by angiotensin II was correlated with a decrease in phosphorylase activation. The data demonstrate that inhibition of hepatic cAMP accumulation results in reduced metabolic responses to glucagon and epinephrine and do not support the contention that the hepatic actions of glucagon are independent of cAMP.     

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.     

Modulation of glucagon effects by changes in extracellular pH and calcium

We have examined the influence of extracellular pH and calcium concentration on the action of glucagon on isolated rat hepatocytes, perfused liver or plasma membrane preparations. Incubation of rat hepatocytes with 10 nM glucagon at pH 7.4 caused an immediate increase in cAMP concentrations (8-fold), and this rise was almost 50% lower at acidic extracellular pH (6.9). This effect of pH could not be explained by an alteration of the hormone binding to its receptor for glucagon concentrations higher than 1 nM. The effect of acidosis on cAMP production was still present with non-hormonal effectors, such as 10 microM Gpp[NH]p, 30 microM forskolin or 10 mM NaF. This suggests a direct action of acidosis on the regulatory component Ns and/or on the catalytic subunit of adenylate cyclase. Acidic pH also depressed mitochondrial processes responsive to glucagon (NAD(P)H fluorescence, glutamine breakdown). Whatever the experimental model, calcium appeared to be required for maximal stimulation of cAMP production by glucagon. On perfused rat liver, glycogenolysis was depressed in the absence of extracellular calcium in the perfusate. In isolated hepatocytes, the stimulation of phosphorylase alpha activity by glucagon was modulated by extracellular calcium concentrations lower than 0.2 mM. This suggests that, although glucagon action is chiefly cAMP-mediated, its effect on calcium mobilization (affecting various cellular process, including cAMP production itself) should also be taken into account. This work also confirmed the importance of calcium in the stimulation of mitochondrial metabolism of glutamine by glucagon.     

Induction of cytosolic aspartate aminotransferase by glucagon in primary cultured rat hepatocytes

The activity and the mRNA content of cytosolic aspartate aminotransferase (EC 2.6.1.1) were examined in cultured rat hepatocytes. Addition of glucagon (1 x 10(-7) M) in the presence of dexamethasone (1 x 10(-7) M) caused about 2-fold increase in the activity and mRNA content. Dibutyryl cAMP (1 x 10(-4) M) could replace glucagon for this effect. Maximal induction of cytosolic aspartate aminotransferase mRNA was observed 8 h after their additions. Insulin (1 x 10(-7) M) did not inhibit the enzyme induction by glucagon or dibutyryl cAMP. These results suggest that the cytosolic aspartate aminotransferase gene is regulated by cAMP, and not by insulin.     

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.     

Effects of adrenalectomy on hormone action on hepatic glucose metabolism. Impaired glucagon activation of glycogen phosphorylase in hepatocytes from adrenalectomized rats.

The effects of adrenalectomy on glucagon activation of liver glycogen phosphorylase and glycogenolysis were studied in isolated hepatocytes. Adrenalectomy resulted in reduced responsiveness of glycogenolysis and phosphorylase to glucagon activation. Stimulation of cAMP accumulation and cAMP-dependent protein kinase activity by glucagon was unaltered in cells from adrenalectomized rats. Adrenalectomy did not alter the proportion of type I and type II protein kinase isozymes in liver, whereas this was changed by fasting. Activation of phosphorylase kinase by glucagon was reduced in hepatocytes from adrenalectomized rats, although the half-maximal effective concentration of glucagon was unchanged. No difference in phosphorylase phosphatase activity between liver cells from control and adrenalectomized rats was detected. Glucagon-activated phosphorylase declined rapidly in hepatocytes from adrenalectomized rats, whereas the time course of cAMP increase in response to glucagon was normal. Addition of glucose (15 mM) rapidly inactivated glucagon-stimulated phosphorylase in both adrenalectomized and control rat hepatocytes. The inactivation by glucose was reversed by increasing glucagon concentration in cells from control rats, but was accelerated in cells from adrenalectomized rats. It is concluded that impaired activation of phosphorylase kinase contributes to the reduced glucagon stimulation of hepatic glycogenolysis in adrenalectomized rats. The possible role of changes in phosphorylase phosphatase is discussed.     

Insulin-like stimulation of cardiac fuel metabolism by physiological levels of glucagon: involvement of PI3K but not cAMP

At concentrations around 10(-9) M or higher, glucagon increases cardiac contractility by activating adenylate cyclase/cyclic adenosine monophosphate (AC/cAMP). However, blood levels in vivo, in rats or humans, rarely exceed 10(-10) M. We investigated whether physiological concentrations of glucagon, not sufficient to increase contractility or ventricular cAMP levels, can influence fuel metabolism in perfused working rat hearts. Two distinct glucagon dose-response curves emerged. One was an expected increase in left ventricular pressure (LVP) occurring between 10(-9.5) and 10(-8) M. The elevations in both LVP and ventricular cAMP levels produced by the maximal concentration (10(-8) M) were blocked by the AC inhibitor NKY80 (20 microM). The other curve, generated at much lower glucagon concentrations and overlapping normal blood levels (10(-11) to 10(-10) M), consisted of a dose-dependent and marked stimulation of glycolysis with no change in LVP. In addition to stimulating glycolysis, glucagon (10(-10) M) also increased glucose oxidation and suppressed palmitate oxidation, mimicking known effects of insulin, without altering ventricular cAMP levels. Elevations in glycolytic flux produced by either glucagon (10(-10) M) or insulin (4 x 10(-10) M) were abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (10 microM) but not significantly affected by NKY80. Glucagon also, like insulin, enhanced the phosphorylation of Akt/PKB, a downstream target of PI3K, and these effects were also abolished by LY-294002. The results are consistent with the hypothesis that physiological levels of glucagon produce insulin-like increases in cardiac glucose utilization in vivo through activation of PI3K and not AC/cAMP.     

Flux through glycine cleavage system in isolated hepatocytes: effects of glucagon, cAMP, and calcium

The hepatic glycine cleavage system (GCS) is the principal route for the metabolism of glycine in mammals. Flux through the GCS in isolated rat hepatocytes was stimulated by about 100% by glucagon (10(-7) M), forskolin (10(-4) M), and dibutyryl cAMP (10(-4) M). The stimulation of flux through the GCS by these agents was accompanied by marked elevation of cellular cAMP levels. A significant correlation was observed between increased cellular cAMP levels induced by glucagon and stimulation of flux through the GCS by glucagon. Exclusion of calcium from the incubation medium reduced the basal flux by 38%, but did not affect the degree of stimulation of flux through the GCS by glucagon. A single intraperitoneal injection of glucagon to rats prior to isolation of hepatocytes resulted in a 76% stimulation of flux through the GCS. These hepatocytes with stimulated flux through the GCS showed reduced sensitivity for further stimulation by glucagon. Half-maximal stimulation of flux through the GCS occurred at 3.8 +/- 1.1 and 8.5 +/- 1.4 nM glucagon in hepatocytes isolated from control and glucagon-injected rats, respectively. We conclude that cAMP is involved in the regulation of flux through the GCS by glucagon.     

Characterization of responses of isolated rat hepatocytes to ATP and ADP

In isolated rat hepatocytes, ATP and ADP (10(-6) M) rapidly mobilize intracellular Ca2+ and increase the concentration of free cytosolic Ca2+ ([Ca2+]i) within 1-2 s. The increase in [Ca2+]i is maximal (2.5- to 3-fold) by about 10 s and is dose-dependent, with ATP and ADP being half-maximally effective at 8 X 10(-7) and 3 X 10(-7) M, respectively. At submaximal concentrations, the rise in [Ca2+]i is transient due to hydrolysis of the agonist. The increase in [Ca2+]i in response to ATP or ADP can be potentiated by low concentrations of glucagon (10(-9) M). In addition, the [Ca2+]i rise can be antagonized in a time- and dose-dependent manner by the tumor promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. Adenosine, at concentrations as high as 10(-4) M, does not alter [Ca2+]i. AMP is ineffective at 10(-5) M, but at 10(-4) M it increases [Ca2+]i approximately 1.5-fold after a 30-s lag and at a slow rate. Conversely, high concentrations (10(-4) M) of adenosine and AMP increases cell cAMP about 2- to 3-fold. ATP and ADP, at concentrations (10(-6) M) which near-maximally increase [Ca2+]i, do not affect hepatocyte cAMP. ATP and ADP increase the cellular level of myoinositol 1,4,5-trisphosphate (IP3), the putative second messenger for Ca2+ mobilization. The increase in IP3 is dose-dependent and precedes or is coincident with the [Ca2+]i rise. There is an approximate 20% increase in IP3 with concentrations of ATP or ADP which near-maximally induce other physiological responses. It is concluded that submicromolar concentrations of ATP and ADP mobilize intracellular Ca2+ and activate phosphorylase in hepatocytes due to generation of IP3. These effects may involve P2-purinergic receptors. In contrast adenosine and AMP interact with P1 (A2)-purinergic receptors to increase cAMP.     

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.     

Effect of DG5128 on epinephrine and glucagon induced glucose output from the isolated perfused rat liver

The effect of a specific alpha 2-adrenergic antagonist 2-[2-(4,5-dihydro-1.H-imidazol-2-yl)-1-phenyl-ethyl] pyridine dihydrochloride sesquihydrate (DG5128), on the glucose output by epinephrine and/or glucagon was studied using the perfused rat liver. The administration of DG5128 alone did not affect the glucose output. However, DG5128 produced a significant inhibition of the increased glucose output when induced by 10(-6) M epinephrine alone or 10(-6) M epinephrine plus 1.4 x 10(-10) M glucagon. There were no significant changes of the glucose output by 1.4 x 10(-10) M or 7.0 x 10(-11) M glucagon alone. On the other hand, addition of 1 mU/ml insulin to the perfusate suppressed the 7.0 x 10(-11) M glucagon-induced glucose output, but failed to decrease the 1.4 x 10(-10) M glucagon effect. DG5128 suppressed further the glucagon (7.0 x 10(-11) M)-induced increase of glucose output in the presence of insulin. These results suggest that DG5128 produces a hypoglycemic effect partly through an inhibition of the increased hepatic glucose output elicited by epinephrine and glucagon.     

Sex difference in cellular calcium metabolism of rat hepatocytes and in α-adrenergic activation of glycogen phosphorylase

Three topics were the subject of these investigations: (i) the difference between males and females in the basal calcium metabolism of hepatocytes; (ii) the source of the calcium which triggers the phosphorylase a stimulation induced by epinephrine through alpha-adrenergic receptors; (iii) the time relation between the rise in phosphorylase activity and the increase in calcium efflux. We found that there was no difference between males and females in total or exchangeable cell calcium. However, there were significant differences in the mitochondrial calcium pool and fluxes measured by steady-state kinetic analyses: they were smaller and the rate constants of mitochondrial calcium influx and efflux were lower in males than in females. The ⁴⁵Ca content of isolated mitochondria and microsomes was also significantly lower in males than in females. In both males and females, epinephrine stimulated phosphorylase activity and calcium efflux even in the absence of extracellular calcium, indicating that the principal source of calcium which triggers the enzyme stimulation is intracellular. During the first 10 min following stimulation by 10^?6 M epinephrine, the total cell calcium, ⁴⁵Ca and the mitochondrial calcium were significantly depressed in male hepatocytes. After 10 min, these changes were reversed and the cell or mitochondrial calcium content was greater than in controls. In females, on the other hand, changes could only be detected if the cells were transferred to calcium-free media before the stimulation. In both males and females, there was a good temporal relationship between the stimulation of calcium efflux and the rise in phosphorylase a activity when hepatocytes were exposed to increasing concentrations of epinephrine: both rose at least 75% in less than 15 s. We conclude that there are important differences in cellular calcium metabolism between males and females. The rise in cytosolic calcium induced by alpha-adrenergic activation is principally due to a mobilization of calcium from an intracellular pool, probably the mitochondria.     

Lack of effect of somatostatin on the stimulation of hepatic glycogenolysis by epinephrine in isolated canine hepatocytes

The effects of somatostatin on epinephrine's ability to stimulate glucose output have been examined in hepatocytes isolated from dogs fasted overnight. Half-maximal stimulation of phosphorylase a activity and glucose output occurred at an epinephrine concentration of approx. 5 X 10(-9) M. Somatostatin at 10, 100 or 1000 ng/ml had no effect on the ability of a maximal (1 X 10(-7) M) and a submaximal (1 X 10(-8) M) dose of epinephrine to activate phosphorylase at 2 min, or to stimulate glucose output over 20 min. Since the doses of somatostatin used in the present study are up to 50-fold higher than the blood concentrations commonly found when somatostatin is used in vivo to inhibit pancreatic hormone secretion, it seems unlikely that use of somatostatin in this way would affect stimulation of hepatic glycogenolysis by epinephrine in vivo.     

Modulation of the alpha 1-adrenergic control of hepatocyte calcium redistribution by increases in cyclic AMP

The Ca2+ content of hepatocytes from juvenile male rats (80-110 g) or adult female rats (135-155 g) displayed a biphasic dose-response curve to epinephrine. Low concentrations (less than or equal to 10(-7) M) caused efflux of Ca2+ from the cells, while higher concentrations (10(-6) M and 10(-5) M) induced net Ca2+ uptake which correlated with a large beta 2-adrenergic-mediated increase in cAMP (Morgan, N. G., Blackmore, P. F., and Exton, J. H. (1983) J. Biol. Chem. 258, 5103-5109). Calcium accumulation could be induced in cells from older male rats (180-230 g) by combining a Ca2+-mobilizing hormone with either exogenous cAMP or glucagon (10(-8) M). Readdition of Ca2+ in the presence of glucagon to cells treated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid also resulted in enhanced Ca2+ accumulation compared with controls. Addition of vasopressin plus glucagon to the medium perfusing male rat livers also led to cell Ca2+ accumulation, as evidenced by uptake of Ca2+ from the perfusate. Incubation of hepatocytes with antimycin A, oligomycin, and carbonyl cyanide m-chlorophenylhydrazone prevented net Ca2+ accumulation suggesting that mitochondria play a role in the uptake response. This was confirmed by isolation of mitochondria from cells incubated under conditions which promote Ca2+ accumulation. Within 5 min of incubation, the Ca2+ content of these mitochondria was increased 2-fold relative to controls, an effect which was inhibited by oligomycin. These studies demonstrate that a rise in hepatic cAMP can reverse hormonally induced Ca2+ mobilization and point to a major role for the mitochondria in this effect.     

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.     

Stimulation of inositol trisphosphate formation in hepatocytes by vasopressin, adrenaline and angiotensin II and its relationship to changes in cytosolic free Ca2+.

At maximally effective concentrations, vasopressin (10(-7) M) increased myo-inositol trisphosphate (IP3) in isolated rat hepatocytes by 100% at 3 s and 150% at 6 s, while adrenaline (epinephrine) (10(-5) M) produced a 17% increase at 3 s and a 30% increase at 6 s. These increases were maintained for at least 10 min. Both agents increased cytosolic free Ca2+ [( Ca2+]i) maximally by 5 s. Increases in IP3 were also observed with angiotensin II and ATP, but not with glucagon or platelet-activating factor. The dose-responses of vasopressin and adrenaline on phosphorylase and [Ca2+]i showed a close correspondence, whereas IP3 accumulation was 20-30-fold less sensitive. However, significant (20%) increases in IP3 could be observed with 10(-9) M-vasopressin and 10(-7) M-adrenaline, which induce near-maximal phosphorylase activation. Vasopressin-induced accumulation of IP3 was potentiated by 10mM-Li+, after a lag of approx. 1 min. However the rise in [Ca2+]i and phosphorylase activation were not potentiated at any time examined. Similar data were obtained with adrenaline as agonist. Lowering the extracellular Ca2+ to 30 microM or 250 microM did not affect the initial rise in [Ca2+]i with vasopressin but resulted in a rapid decline in [Ca2+]i. Brief chelation of extracellular Ca2+ for times up to 4 min also did not impair the rate or magnitude of the increase in [Ca2+]i or phosphorylase a induced by vasopressin. The following conclusions are drawn from these studies. IP3 is increased in rat hepatocytes by vasopressin, adrenaline, angiotensin II and ATP. The temporal relationships of its accumulation to the increases in [Ca2+]i and phosphorylase a are consistent with it playing a second message role. Influx of extracellular Ca2+ is not required for the initial rise in [Ca2+]i induced by these agonists, but is required for the maintenance of the elevated [Ca2+]i.