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.     

Modulation by thyroid status of cyclic AMP-dependent and Ca2+-dependent mechanisms of hormone action in rat liver cells. Possible involvement of two different transduction mechanisms in alpha 1-adrenergic action

The actions of hormones which are associated to cAMP-dependent and calcium-dependent mechanisms of signal transduction were studied in hepatocytes obtained from rats with different thyroid states. In cells from euthyroid and hyperthyroid rats, the metabolic actions of epinephrine were mediated mainly through alpha 1-adrenoceptors; beta-adrenoceptors seem to be functionally unimportant. In contrast, both alpha 1- and beta-adrenoceptors mediate the actions of epinephrine in hepatocytes from hypothyroid animals. Phosphatidylinositol labeling was strongly stimulated by epinephrine, vasopressin and angiotensin II in cells from eu-, hyper- or hypothyroid rats. However, metabolic responsiveness to vasopressin and angiotensin II was markedly impaired in the hypothyroid state. The glycogenolytic response to the calcium ionophore A-23187 was also impaired, suggesting that hepatocytes from hypothyroid rats are less sensitive to calcium signalling. The persistence of alpha 1-adrenergic responsiveness in the hypothyroid state suggests that the mechanism of signal transduction for alpha 1-adrenergic amines is not identical to that of the vasopressor peptides. alpha 1-Adrenergic stimulation of cyclic AMP accumulation was not detected in cells from hypothyroid rats. These data suggest that factors besides calcium and besides cAMP are probably involved in alpha 1-adrenergic actions. Metabolic responses to glucagon and to the cAMP analogue dibutyryl cAMP were not markedly changed during hypothyroidism, although cAMP accumulation produced by glucagon and beta-adrenergic agonists was enhanced. In hyperthyroidism, cell responsiveness to epinephrine, vasopressin, angiotensin II and glucagon was decreased, but sensitivity to cAMP was not markedly altered. The factors involved in this hyposensitivity to hormones during hyperthyroidism are unclear.     

Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate

The effects of submaximal doses of AlF4- to mobilize hepatocyte Ca2+ were potentiated by glucagon (0.1-1 nM) and 8-p-chlorophenylthio-cAMP. A similar potentiation by glucagon of submaximal doses of vasopressin, angiotensin II, and alpha 1-adrenergic agonists has been previously shown (Morgan, N. G., Charest, R., Blackmore, P. F., and Exton, J. H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4208-4212). When hepatocytes were pretreated with the protein kinase C activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), the effects of AlF4- to mobilize Ca2+, increase myo-inositol 1,4,5-trisphosphate (IP3), and activate phosphorylase were attenuated. Treatment of hepatocytes with PMA likewise inhibits the ability of vasopressin, angiotensin II, and alpha 1-adrenergic agonists to increase IP3 and mobilize Ca2+ (Lynch, C. J., Charest, R., Bocckino, S. B., Exton, J. H., and Blackmore, P. F. (1985) J. Biol. Chem. 260, 2844-2851). In contrast, the ability of AlF4- or angiotensin II to lower cAMP or inhibit glucagon-mediated increases in cAMP was unaffected by PMA. The ability of AlF4- to lower cAMP was attenuated in hepatocytes from animals treated with islet-activating protein, whereas Ca2+ mobilization was not modified. These results suggest that the lowering of cAMP induced by AlF4- and angiotensin II was mediated by the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase, whereas Ca2+ mobilization was not. Addition of glucagon, forskolin, or 8CPT-cAMP to hepatocytes raised IP3 and mobilized Ca2+. Both effects were blocked by PMA pretreatment, whereas cAMP and phosphorylase a levels were only minimally affected by PMA. The mobilization of Ca2+ induced by cAMP in hepatocytes incubated in low Ca2+ media was not additive with that induced by maximally effective doses of vasopressin, angiotensin II, or alpha 1-adrenergic agonists, indicating that the Ca2+ pool(s) affected by agents which increase cAMP is the same as that affected by Ca2+-mobilizing hormones which do not increase cAMP. These findings support the proposal that AlF4- mimics the effects of the Ca2+-mobilizing hormones in hepatocytes by activating a guanine nucleotide-binding regulatory protein (Np) which couples the hormone receptors to a phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphodiesterase. They also suggest that Np, PIP2 phosphodiesterase, or a factor involved in their interaction is activated following phosphorylation by cAMP-dependent protein kinase and inhibited after phosphorylation by protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Agonist-induced down-regulation of the angiotensin II receptor in primary cultures of rat hepatocytes

The ability of angiotensin II to down-regulate its receptor was tested on rat hepatocytes in primary culture for 4 h. Angiotensin II treatment decreased [3H]angiotensin II specific binding in a concentration- and time-dependent manner. The effect was maximum with 1 microM angiotensin II and after 2 h. There was a decrease in the maximum number of binding sites (56% of control) with no significant effect on the apparent dissociation constant. The down-regulation was blocked by the angiotensin II antagonist [Val4,Ile7]angiotensin III and was not induced by other hormones (e.g. vasopressin, norepinephrine, or glucagon) or by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate or A23187 ionophore. The decrease in angiotensin II receptors resulted in correlated decreases in the potency of angiotensin II to activate phosphorylase or lower glucagon-induced cAMP accumulation. However, high concentrations of the agonist were still able to elicit maximal responses in both parameters. Down-regulation of the receptor was not dependent upon active Gi, since it was still observed after ADP-ribosylation and inactivation of Gi by pertussis toxin. The above results indicate that the down-regulation of the hepatic angiotensin II receptor induced by its agonist is homologous and does not involve Gi, Ca2+, or protein kinase C. The correlation of receptor loss with decreases in the potency of angiotensin to activate phosphorylase and inhibit glucagon-induced cAMP accumulation is consistent with the idea that a single receptor population regulates two different messengers, i.e. calcium and cAMP.     

Characterization of the angiotensin II receptor in primary cultures of rat hepatocytes. Evidence that a single population is coupled to two different responses

The angiotensin II receptor of cultured rat hepatocytes was characterized using [3H]angiotensin II as radioligand. Binding at 23 degrees C was rapid (t1/2 = 0.65 min) with equilibrium being reached in 10-12 min. At this time, binding was completely reversible after 20 min (t1/2 = 3.5 min), indicating negligible internalization of the ligand. Analysis of the saturation binding curve showed one population of binding sites with an apparent KD of 8.6 nM and a Bmax of 35 fmol/mg of protein. The time courses of association and dissociation were also consistent with one class of binding sites with an apparent kinetically derived KD of 7.7 nM. The order of potency of different agonists and antagonists to increase cytosolic Ca2+ or phosphorylase a or inhibit the effects of angiotensin II on these parameters was the same as for their mimicry or reversal of angiotensin II inhibition of glucagon-induced cAMP accumulation, and was well correlated with their order of potency to inhibit angiotensin II specific binding. Treatment of cultured hepatocytes with dithiothreitol caused a time- and concentration-dependent inhibition of angiotensin II binding and corresponding alterations of angiotensin II effects on phosphorylase and cAMP. It also inhibited the actions of other hormones on phosphorylase. These results indicate that hepatocytes contain a homogeneous population of angiotensin II receptors that are coupled to two different biological effects apparently mediated by different G-proteins.     

Vasopressin and angiotensin II stimulate ureogenesis through increased mitochondrial citrulline production.

Vasopressin, angiotensin II, glucagon and epinephrine (through a cAMP-independent, alpha₁adrenergic mechanism), stimulate ureogenesis in isolated rat hepatocytes. Mitochondria, isolated from hepatocytes which were previously treated with these hormones, displayed an enhanced rate of citrulline synthesis in the presence of NH₄Cl as the nitrogen source. When mitochondria were incubated with glutamine as the nitrogen source, only those mitochondria isolated from hepatocytes previously treated with epinephrine or glucagon displayed an enhanced capacity to synthesize citrulline.When cells were incubated in the absence of extracellular calcium, the effects of vasopressin and angiotensin II on urea synthesis were abolished, whereas those of epinephrine and glucagon were only diminished. Mitochondria isolated from cells incubated under these conditions, showed that the effect of all these hormones on citrulline synthesis could still be observed. However, the effects of glucagon and epinephrine plus propranolol were larger than those of angiotensin II or vasopressin.Phosphatidylinositol labeling was significantly increased by epinephrine, vasopressin and angiotensin II both in the absence or presence of calcium. Cyclic AMP levels were significantly increased by glucagon or epinephrine but not by vasopressin or angiotensin II. The effect of epinephrine on cyclic AMP levels was blocked by propranolol both in the absence or presence of calcium.     

Studies on the mechanism of inhibition of hepatic cAMP accumulation by vasopressin

Vasopressin elicited a dose-dependent inhibition of glucagon-induced cAMP accumulation in isolated hepatocytes. This response was not diminished by incubation of cells with the calmodulin antagonists trifluoperazine or chlorpromazine and was only slightly reduced in Ca2+-depleted hepatocytes. Half-maximal inhibition of cAMP accumulation occurred at 8 X 10(-11) M vasopressin, a dose which does not increase cytosolic Ca2+ in hepatocytes. Direct activation of adenylate cyclase by forskolin was significantly inhibited by vasopressin in Ca2+-depleted cells. It is concluded that inhibition of hormone-induced cAMP accumulation by vasopressin in liver is not dependent on cellular Ca2+ mobilisation but may involve direct inhibition of adenylate cyclase.     

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.     

Receptor-operated calcium influx in rat hepatocytes. Identification and characterization using manganese

Agonist-stimulated divalent cation entry was studied in fura-2-loaded hepatocytes. In the presence of extracellular Mn2+, the Ca2(+)-mobilizing hormone vasopressin produced a severalfold stimulation of the basal rate of fura-2 fluorescence quenching as a result of Mn2+ influx; this effect was blocked by the presence of Ni2+ in the incubation medium. Half-maximum and maximum stimulation of Mn2+ influx was observed with 0.1 and 0.8 nM vasopressin, respectively. Agonist-stimulated Mn2+ influx was also seen with angiotensin II, ATP, phenylephrine, and the combination of AlCl3 and NaF. The stimulation of Mn2+ influx did not occur immediately after addition of Ca2(+)-mobilizing agents, but was characterized by a latency period of 20-30 s. In contrast to vasopressin, glucagon did not stimulate Mn2+ influx into hepatocytes, but produced both a 3-fold enhancement of the rate of vasopressin-stimulated Mn2+ entry and the abolishment of the latency period. The effects of glucagon were mimicked by forskolin and dibutyryl cAMP. Pretreatment of hepatocytes with pertussis toxin or depolarization of the cells altered neither the basal rate of Mn2+ entry nor the ability of vasopressin to stimulate this rate. Emptying of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store by treatment with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) did not enhance Mn2+ entry into hepatocytes; however, exposure of the cells to tBuBHQ for 2 min markedly enhanced the ability of vasopressin, alone or in combination with glucagon, to increase the rate of Mn2+ influx. Furthermore, pretreatment with tBuBHQ for 2 min abolished the latency of vasopressin-stimulated Mn2+ influx. It is concluded that Ca2(+)-mobilizing hormones stimulate Ca2+ influx in hepatocytes, possibly through receptor-operated Ca2+ channels. The stimulation of divalent cation entry is transduced by a G protein, and the rate of influx appears to be controlled both by the intracellular level of cAMP and the empty state of an intracellular Ca2+ pool that may be inositol 1,4,5-trisphosphate-insensitive.     

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     

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.     

Inhibition of glucagon-stimulated cAMP accumulation and fatty acid oxidation by E-series prostaglandins in isolated rat hepatocytes

E-series prostaglandins have been shown to inhibit hepatic glucagon-stimulated glycogenolysis without inhibiting glycogenolysis stimulated by cAMP analogs. In the present studies, prostaglandin E2 and 16,16-dimethylprostaglandin E2 inhibited glucagon-stimulated cAMP accumulation in isolated rat hepatocytes by 25% and 46%, respectively, without affecting basal cAMP levels. Half-maximal inhibition of glucagon-stimulated cAMP accumulation occurred at approx. 10(-7) M 16,16-dimethylprostaglandin E2. 16,16-Dimethylprostaglandin E2 inhibited glucagon-stimulated palmitate oxidation in intact hepatocytes without affecting basal rates of palmitate oxidation. 16,16-Dimethylprostaglandin E2 had no effect on palmitate oxidation in a liver homogenate system. These studies demonstrate that prostaglandin E antagonizes the effects of glucagon on hepatic metabolism by inhibiting glucagon-stimulated cAMP accumulation.     

Age-related changes in the control of hepatic cyclic AMP levels by alpha 1- and beta 2-adrenergic receptors in male rats

Hepatocytes from juvenile male rats (80-110 g) showed a 12-fold elevation of cAMP in response to epinephrine, which was mediated by beta 2-adrenergic receptors. In these cells, either alpha 1- or beta 2-adrenergic stimulation alone activated phosphorylase and glucose release although the alpha 1-phosphorylase response was 10-fold more sensitive to epinephrine and resulted in more rapid (by 10-20 s) activation of the enzyme. This suggests that the beta 2-adrenergic response is functionally unimportant for glycogenolysis, even in juvenile rats. beta 2-Adrenergic stimulation did, however, produce an increase in the rate of gluconeogenesis from [U-14C] lactate in these cells. Aging in the male rat was associated with attenuation of the beta 2-adrenergic cAMP response coupled with the emergence of an alpha 1-receptor-mediated accumulation of cAMP. The order of potency displayed by the alpha 1-adrenergic/cAMP system to adrenergic agonists and antagonists was identical with that of the alpha 1-adrenergic/Ca2+ system. These data suggest that, in maturity, hepatic alpha 1-receptors become linked to 2 separate transduction mechanisms, namely Ca2+ mobilization and cAMP generation. Calcium depletion of hepatocytes from adult, but not juvenile, male rats increased the alpha 1-component of the cAMP response to epinephrine, but under these conditions, alpha 1-activation of phosphorylase occurred more slowly than in calcium-replete cells. Blockade of alpha 2-adrenergic receptors did not significantly modify catecholamine effects on hepatocyte cAMP or phosphorylase a levels in male rats at any age studied, suggesting a lack of functional significance for these receptors in the regulation of glycogenolysis.     

Calcium-mobilizing hormones and phorbol myristate acetate mediate heterologous desensitization of the hormone-sensitive hepatic Na+/K+ pump.

The Na+/K+ pump in rat hepatocytes is stimulated in response to Ca2+-mobilizing hormones such as [arginine]vasopressin (AVP), angiotensin II and adrenaline, as well as tumour promoters such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The ability of these agents to increase cellular contents of diacylglycerol and activate protein kinase C may be necessary to observe this response. In the present work, ouabain-sensitive 86Rb+ uptake was studied in isolated rat hepatocytes to help to explain why stimulation of the Na+/K+ pump by Ca2+-mobilizing hormones and tumour promoters is not temporally sustained relative to other hormone responses. A transient stimulation (3-4 min) of the Na+/K+ pump was observed in hepatocytes exposed to high (10 nM), but not low (0.1 nM), concentrations of AVP. Experiments with the Ca2+ chelator EGTA and the Na+ ionophore monensin indicate that the rapid secondary decrease in Na+/K+-pump activity which occurs after AVP stimulation is not due to changes in cytosolic Ca2+ and Na+ concentrations. When added after the stimulation and rapid decrease in Na+/K+-pump activity induced in hepatocytes by a high concentration of AVP, a second challenge with AVP or PMA failed to stimulate the pump. Similarly, previous exposure of hepatocytes to angiotensin, adrenaline or PMA attenuated the subsequent Na+/K+-pump responses to AVP and PMA. In contrast, previous exposure to AVP had no significant effect on subsequent stimulation of the Na+/K+-pump by monensin, glucagon, forskolin or 8-p-chlorophenylthio cyclic AMP. In addition, exposure to monensin had no effect on subsequent responses to AVP and PMA. These data indicate that high concentrations of Ca2+-mobilizing hormones and PMA result in heterologous desensitization of the hepatic Na+/K+ pump to subsequent stimulation by Ca2+-mobilizing hormones and PMA, but not by cyclic-AMP-dependent agonists or monensin.     

Control of mitochondrial content of adenine nucleotides by submicromolar calcium concentrations and its relationship to hormonal effects

Rat liver mitochondria were incubated at 30 degrees C with 4 mM ATP in a medium similar in electrolyte composition to that of hepatic cytosol. Under these conditions, a net increase in mitochondrial adenine nucleotides was observed that was dependent on the concentration of free Ca2+ [( Ca2+]) in the incubation medium. At 0.2 microM [Ca2+] or less, there was no demonstrable uptake of adenine nucleotides; at 0.4 microM [Ca2+], or greater, net uptake occurred. The calcium-dependent accumulation of nucleotides by mitochondria required Mg2+ in the incubation medium and was insensitive to carboxyatractyloside. The uptake of adenine nucleotides was enhanced by the addition of antimycin A or antimycin A together with oligomycin. Accumulation of nucleotides appeared to be associated with a small increase in mean mitochondrial volume, but the membrane potential was not affected. No uptake or loss of NAD-NADH by mitochondria was detected. Ruthenium red failed to inhibit the calcium-dependent uptake of adenine nucleotides by the mitochondria, indicating that stimulation of this process by Ca2+ does not involve transport of the cation into mitochondria by the Ca2+ uniporter. Because glucagon acts to elevate cytosolic [Ca2+] from approximately 0.2 microM to 0.6 microM, the same range affecting nucleotide uptake, it is proposed that the increase in mitochondrial adenine nucleotides that follows treatment with glucagon is mediated by the rise in cytosolic [Ca2+] produced by the hormone. This hypothesis was supported by the observation that epinephrine and A23187, agents that raise cytosolic [Ca2+], increased the content of mitochondrial adenine nucleotides in isolated hepatocytes. Furthermore, cells, incubated under calcium-depleting conditions, had a diminished response to glucagon.     

Stimulation of 1,2-diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and angiotensin II

1,2-Diacylglycerol (DAG) was measured in neutral lipid extracts from isolated hepatocytes using high pressure liquid chromatography followed by refractive index detection. Maximally effective doses of epinephrine, angiotensin II, and vasopressin increased DAG by approximately 65, 80, and 180-250%, respectively, with maximal increases being observed at 8-10 min. Depletion of cellular Ca2+ resulted in a 50% decrease in DAG accumulation elicited by vasopressin. Other agents which increased DAG levels were the tumor promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (120% increase at 10(-6) M), the Ca2+ ionophore A23187 (385% increase at 10(-5) M), and ATP (180% increase at 1 mM). The concentration dependence of DAG accumulation in response to epinephrine, angiotensin II, and vasopressin was similar to that found for myoinositol triphosphate accumulation (Charest, R., Prpic, V., Exton, J. H., and Blackmore, P.F. (1985) Biochem. J. 227, 79-90), which was approximately 5-10 times less sensitive to hormone than was phosphorylase activation. Fatty acid analysis revealed that hormonally induced DAG was partially derived from sources other than inositol phospholipids. It is proposed from these studies that Ca2+-mobilizing hormones elicit a prolonged increase in the levels of hepatocyte DAG, which may activate protein kinase C.     

Regulation of hepatic glycogenolysis by glucagon in male and female rats. Role of cAMP and Ca2+ and interactions between epinephrine and glucagon

The effects of 10(-10) to 10(-7) M glucagon on cAMP, phosphorylase a, cell calcium, and glucose production, and glucagon interactions with epinephrine were studied in isolated hepatocytes from adult male and female rats. At physiological concentrations (10(-10) - 10(-9) M), glucagon activated phosphorylase by increasing cAMP and not by raising the cytosolic free calcium. At supra-physiologic concentrations (and in the male only), glucagon slightly increased the cytosolic free calcium, the fractional efflux of calcium, and, after 2 h, decreased the cell calcium content. Exposure of hepatocytes to the simultaneous administration of 10(-9) M glucagon and 10(-7) M epinephrine resulted in a prolongation of the activation of phosphorylase a and a greater release of glucose from glycogen stores than exposure to either agonist alone. In the male, the effects of low concentrations of the two hormones on phosphorylase a activity were additive. Cytosolic free calcium was increased by 10(-6) M epinephrine from 280 to 500 nM while physiological concentrations of glucagon did not change it. In these intact cells, there was no evidence of an alpha 2-adrenergic inhibition of adenyl cyclase and no indication that cAMP depresses the rise in cell calcium induced by alpha-adrenergic stimuli.