Homologous and heterologous desensitization of one of the pathways of the alpha 1-adrenergic action. Effects of epinephrine, vasopressin, angiotensin II and phorbol 12-myristate 13-acetate

Activation of protein kinase C blocks the alpha 1-adrenergic action in hepatocytes. Preincubation of hepatocytes (in buffer with or without calcium) with vasopressin, angiotensin II, phorbol myristate acetate (PMA) or epinephrine + propranolol markedly diminished the alpha 1-adrenergic responsiveness of the cells (stimulation of ureagenesis) assayed in buffer without calcium. On the contrary, when the alpha 1-adrenergic responsiveness was assayed in buffer containing calcium no effect of the preincubation with vasopressin, angiotensin II or PMA was observed. Preincubation with epinephrine diminished the alpha 1-adrenergic responsiveness of the cells. In hepatocytes from hypothyroid rats the preincubation with the activators of protein kinase C (vasopressin, angiotensin II, phorbol 12-myristate 13-acetate and epinephrine) reduced markedly the alpha 1-adrenergic responsiveness of the cells, whereas in identical experiments using cells from adrenalectomized rats only the preincubation with epinephrine diminished the responsiveness. It is concluded that activation of protein kinase C induces desensitization of the alpha 1-adrenergic action in hepatocytes and that the calcium-independent pathway of the alpha 1-adrenergic action (predominant in cells from hypothyroid animals) resensitizes more slowly than the calcium-dependent pathway (predominant in cells from adrenalectomized rats). Epinephrine in addition to inducing this type of desensitization (through protein kinase C) leads to a further refractoriness of the cells towards alpha 1-adrenergic agonists.     

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.     

Phorbol esters, vasopressin and angiotensin II block alpha 1-adrenergic action in rat hepatocytes. Possible role of protein kinase C

The effect of vasopressin, angiotensin II and phorbol myristate acetate on the alpha 1-adrenergic action (induced by epinephrine + propranolol), was studied. We selected three conditions: (a) ureagenesis in medium without added calcium and containing 25 microM EGTA; (b) ureagenesis using cells from hypothyroid animals, and (c) gluconeogenesis from dihydroxyacetone. Under these conditions epinephrine + propranolol produces clear metabolic effects, whereas the vasopressor peptides do not (although they stimulate phosphoinositide turnover). It was observed that the vasopressor peptides and the active phorbol ester inhibited in a concentration-dependent fashion the effect of epinephrine + propranolol. It is suggested that activation of protein kinase C by phorbol esters or physiological stimuli (hormones that activate phosphoinositide turnover, such as vasopressin or angiotensin II) modulate the hepatocyte alpha 1-adrenergic responsiveness.     

Hormonal responsiveness of liver cells during the liver regeneration process induced by carbon tetrachloride administration

In control rats most of the ureagenic effect of adrenaline is mediated through alpha1-adrenoceptors with little participation of beta-adrenoceptors. Administration of carbon tetrachloride to rats induces significant changes in the adrenergic responsiveness of their hepatocytes. In rats intoxicated 3-5 days before the experiments were performed there is a marked increase in the beta-adrenergic and a decrease in the alpha-adrenergic responsiveness of the hepatocytes. The alpha1-adrenergic responsiveness increased with time reaching its basal level 15 days after the administration of carbon tetrachloride; simultaneously, the betal-adrenergic responsiveness was decreased. No change in the responsiveness to vasopressin and angiotensin II was observed in intoxicated animals as compared to the controls. In contrast, the responsiveness to glucagon was increased. Increased ability of local anesthetics to decrease urea production was observed in cells from intoxicated animals. It is suggested that changes at the plasma membrane level (lipids, receptors and transducing proteins) might participate in producing these effects.     

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.     

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.     

Effect of insulin on alpha1-adrenergic actions in hepatocytes from euthyroid and hypothyroid rats. Possible involvement of two pathways in alpha1-adrenergic actions

The effect of insulin on the alpha1-adrenergic stimulation of glycogenolysis and ureogenesis, which is very small or undetectable in hepatocytes from control animals, is marked in hepatocytes from hypothyroid rats; the metabolic actions due to alpha1-adrenergic activation, but not those due to glucagon, were nearly blocked by insulin in cells from hypothyroid rats. The alpha1-adrenergic-mediated stimulation of phosphatidylinositol labelling was not affected by insulin in cells from either control or hypothyroid rats. The data suggest that the alpha1-adrenergic action proceeds through two pathways, one of which is very sensitive to insulin and predominates in cells from hypothyroid rats.     

Rapid reconstitution of a transmembrane protein into supported planar lipid membranes

In hepatocytes from control rats, the ureogenic action of epinephrine is mainly mediated through alpha 1-adrenoceptors and the effect is independent of the presence of extracellular calcium. In hepatocytes from adrenalectomized rats, both alpha 1- and beta-adrenoceptors are involved in the action of epinephrine. Furthermore, the alpha 1-adrenergic-mediated stimulation of ureogenesis in these cells is dependent on the presence of extracellular calcium. Our results indicate that glucocorticoids modulate the calcium dependency of alpha 1-adrenergic effects and are consistent with our suggestion that two pathways are involved in the transduction of the alpha 1-adrenergic signal.     

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)     

Glycogenolytic responsiveness to glucagon, epinephrine, vasopressin and angiotensin II in the liver of developing hypothyroid rats. A comparative study of in vitro hormonal binding and in vivo biological response

Both dose-response curves and time-courses of plasma glucose levels after single maximal doses showed that in vivo glycogenolytic responsiveness to glucagon and epinephrine was significantly higher in developing hypothyroid rats, whereas it remained unchanged after vasopressin and angiotensin II injections. In contrast with the decreased basal activity of phosphorylase(a), the glucagon-stimulated activity increased in hypothyroid rats, whereas it was only slightly modified under vasopressin stimulation. Daily thyroxine treatment abolished these abnormalities. Thus, there is a close correlation between glucose output and enzyme activation. The maximal binding capacity of [3H]vasopressin and [125I]glucagon was significantly decreased in hypothyroid rats, without changes in the apparent dissociation constant of hormone from its specific receptor. Daily thyroxine treatment also abolished this deficit, which moreover appeared to be independent of possible changes in plasma hormone levels. With respect to glucagon action, neither basal nor Gpp(NH)p-stimulated adenylate cyclase activities were affected in hypothyroid rats. Glucagon-sensitive adenylate cyclase activity and the apparent activation constant appeared to be unaffected. The apparent discrepancy between the results obtained from in vivo and in vitro experiments is discussed on the basis of different membrane transducing phenomena and related intracellular mechanisms underlying the biological response to hormonal stimulation.     

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.     

Possible involvement of two mechanisms of signal transduction in alpha 1-adrenergic action. Selective effect of cycloheximide

We have previously suggested that the effects of alpha 1-adrenergic agents on hepatocyte metabolism involve two pathways: (a) a calcium-independent, insulin-sensitive process which is modulated by glucocorticoids; and (b) a calcium-dependent, insulin-insensitive process which is modulated by thyroid hormones. Cycloheximide stimulated ureogenesis through a prazosin-sensitive mechanism in liver cells (alpha 1-adrenergic). The effect of cycloheximide was insulin-insensitive and calcium-dependent. Furthermore, a clear effect of cycloheximide was observed in hepatocytes obtained from adrenalectomized animals, whereas no effect was observed in cell from hypothyroid rats. The effects of epinephrine and cycloheximide were blocked by phorbol esters in all the conditions tested. Binding competition experiments indicated that cycloheximide interacts with only a fraction of the alpha 1-adrenergic sites labeled with [3H]prazosin. It is suggested that cycloheximide activates preferentially one of the pathways involved in the alpha 1-adrenergic action in liver cells.     

Insulin-like effect of epidermal growth factor in isolated rat hepatocytes. Modulation of the alpha-1-adrenergic stimulation of ureagenesis

Insulin and epidermal growth factor (EGF) inhibit the stimulation of ureagenesis induced by adrenaline (alpha 1-adrenergic effect) in hepatocytes from control rats incubated in medium without calcium and in cells from hypothyroid rats. In hepatocytes from euthyroid rats incubated in normal buffer neither insulin or EGF diminished the alpha 1-adrenergic stimulation of ureagenesis. No effect of EGF or insulin on the alpha 1-adrenergic stimulation of phosphatidylinositol labeling was observed under any conditions. It is suggested that EGF mimics the action of insulin on one of the pathways of the alpha 1-adrenergic action: the calcium-independent, insulin-sensitive pathway which predominates in hepatocytes from hypothyroid rats.     

Sensitivity of liver cells formed after partial hepatectomy to glucagon, vasopressin and angiotensin II

During the active proliferation which follows partial hepatectomy, the sensitivity of liver cells to glucagon is markedly diminished. In hepatocytes obtained from rats partially hepatectomized 3 days before experiments were performed, the dose-response curves to glucagon were shifted to the right by about two orders of magnitude as compared to those of the control cells. Later on (7 days after surgery) the dose-response to glucagon was still shifted to the right but by only one order of magnitude. These data are consistent with the diminution in the number of glucagon receptors in liver plasma membrane during liver regeneration reported by other authors. No stimulation of glycogenolysis, gluconeogenesis or ureogenesis was produced by vasopressin or angiotensin II in hepatocytes from rats partially hepatectomized 3 days before experimentation. However, phosphatidylinositol labeling was stimulated in these cells to a similar extent as in the controls. The ionophore A23187 was also ineffective in stimulating glycogenolysis in these cells. Later, 7 days after surgery, the hepatic responsiveness to vasopressin and angiotensin II was restored. The data suggest that, during the initial stages of liver regeneration, the enzymatic machinery of the hepatocyte is not sensitive to calcium-signalling.     

Effect of inositol and tri-iodothyronine on the hormonal responsiveness of hepatocytes obtained from partially hepatectomized rats.

Hepatocytes obtained from animals partially hepatectomized (72 h before the experiment) have a diminished responsiveness to alpha 1-adrenergic amines, vasopressin, angiotensin and glucagon and an increased responsiveness to beta-adrenergic amines. Administration of inositol or tri-iodothyronine to the hepatectomized animals induced a recovery in the hepatocyte responsiveness to the Ca2+-dependent hormones and abolished that to beta-adrenergic amines; the response to glucagon was not improved.     

Roles of alpha 1- and beta-adrenergic receptors in adrenergic responsiveness of liver cells formed after partial hepatectomy

The adrenergic receptor involved in the action of epinephrine changed dramatically during the process of active proliferation which follows partial hepatectomy. In control or sham-operated animals, the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by epinephrine was mediated through alpha 1-adrenergic receptors. In contrast, in hepatocytes obtained from animals partially hepatectomized 3 days before experimentation, the receptor involved in the stimulation of these metabolic pathways by epinephrine was of the beta-adrenergic type. Interestingly, the adrenergic receptor involved in the metabolic actions of epinephrine, in hepatocytes from rats partially hepatectomized 7 days before experimentation was again of the alpha 1-subtype. Thus, it appears that during the process of liver regeneration which follows partial hepatectomy there is a transition in the type of adrenergic receptor involved in the hepatic actions of catecholamines from beta in the initial stages to later alpha 1. A similar transition seems to occur as the animal ages. Cyclic AMP accumulation in response to beta-adrenergic stimulation was significantly enhanced in hepatocytes obtained from rats partially hepatectomized 3 days before the experiment, as compared to control hepatocytes or cells obtained from animals operated 7 days before experimentation. This enhanced beta-adrenergic sensitivity is probably related to the increased number of beta-adrenergic receptors observed at this stage. However, a clear dissociation between cyclic AMP levels and metabolic effects was evidenced when the different conditions were compared. The number and affinity (for epinephrine or prazosin) of alpha 1-adrenergic receptors did not change at any stage of the process, which indicates that the markedly diminished alpha 1-adrenergic sensitivity observed in hepatocytes obtained from rats partially hepatectomized 3 days before experimentation is probably due to defective generation or intracellular processing of the alpha 1-adrenergic signal, rather than to changes at the receptor level.     

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.     

The liver plasma membrane Ca2+ pump: hormonal sensitivity

The liver plasma membrane Ca2+ pump is supposed to extrude cytosolic calcium out of the cell. This system has now been well defined on the basis of its plasma membrane origin, its high affinity Ca2+ -stimulated ATPase activity, its Ca2+ transport activity, its phosphorylated intermediate. The liver calcium pump appears to be a target of hormonal action since it has been shown that glucagon and calcium mobilizing hormones namely alpha 1-adrenergic agonists, vasopressin, angiotensin II inhibit this system. The present review details the mechanism of calcium pump inhibition by glucagon and points out its difference from the inhibition process induced by calcium mobilizing hormones. We conclude that the inhibitory action of the Ca2+ mobilizing hormones and glucagon on the liver plasma membrane Ca2+ pump might play a key role in the actions of these hormones by prolonging the elevation in cytosolic free Ca2+.     

Possible involvement of cyclooxygenase products in the actions of platelet-activating factor and of lipoxygenase products in the vascular effects of epinephrine in perfused rat liver

Platelet-activating factor (PAF) stimulates glycogenolysis and induces vasoconstriction in perfused rat liver. The effect of PAF was rapid but transient and it was blocked by indomethacin and bromophenacyl bromide which suggests a role of cyclooxygenase metabolites in its action. The homologous desensitization of glycogenolysis produced by PAF and the sensitivity of its actions to inhibitors of cyclooxygenase and phospholipase A2 markedly differentiate the mechanism of action of this agent with that of alpha 1-adrenergic agents, vasopressin or angiotensin II. No effect of PAF in isolated hepatocytes was observed which suggest that cells other than hepatocytes could be involved in its action in perfused liver. In addition nordihydroguaiaretic acid and bromophenacyl bromide abolished the vascular effect (but not the glycogenolysis) produced by epinephrine which suggest a role for lipoxygenase products in this effect.     

Effect of adrenalectomy on Ca2+ signaling in rat hepatocytes

To pursue our studies of the effects of adrenalectomy on the adrenergic regulation of phosphorylase a, cAMP, cell calcium, and Ca2+ signaling in rat hepatocytes (Studer, R.K., and Borle, A.B. (1984) Biochim. Biophys. Acta 804, 377-385; Freudenrich, C.C., and Borle, A.B. (1988) J. Biol. Chem. 263, 8604-8610), we have further examined the alpha 1-adrenergic pathway in adrenalectomized and sham-operated male rats. We measured the number and affinity of alpha 1-adrenergic receptors, the cytosolic free Ca2+ concentration [(Ca2+]i) of hepatocytes with aequorin, inositol triphosphate (IP3) accumulation, and Ca2+ influx and efflux across the plasma membrane. We also compared the effects of vasopressin with those obtained with epinephrine. We found that the number of alpha 1-adrenergic receptors was slightly depressed (-23%), but that their affinity was unchanged. However, IP3 accumulation evoked by epinephrine was decreased 50%. This is probably the main cause for the depressed peak rise in [Ca2+]i we previously observed and reported. We also found that the basal resting Ca2+ influx was increased after adrenalectomy. Experiments with the beta-blocker propranolol, which abolished the epinephrine-evoked increase in Ca2+ influx, suggest that this effect may be mediated by cAMP, at least in adrenalectomized animals. The effects of vasopressin on IP3 [Ca2+]i and Ca2+ influx and efflux were also significantly decreased after adrenalectomy, indicating that alpha 1-adrenergic-mediated and other IP3-dependent Ca2+ signaling pathways are depressed after adrenalectomy.