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.     

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.     

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.     

Hormonal regulation of the tricarboxylic acid cycle in the isolated perfused rat liver

The effect of Ca2+-mobilizing hormones, vasopressin, angiotensin II and the alpha-adrenergic agonist phenylephrine, on the metabolic flux through the tricarboxylic acid cycle was investigated in isolated perfused rat livers. All three Ca2+-mobilizing agonists stimulated 14CO2 production and gluconeogenesis in livers of 24-h-fasted rats perfused with [2-14C]pyruvate. Prazosin blocked the phenylephrine-elicited stimulation of 14CO2 and glucose production from [2-14C]pyruvate whereas the alpha 2-adrenergic agonist, BHT-933, did not affect the rates of 14CO2 and glucose production from [2-14C]pyruvate indicating that the phenylephrine-mediated response involved alpha 1-adrenergic receptors. Phenylephrine, vasopressin and angiotensin II stimulated 14CO2 production from [2-14C]acetate in livers derived from fed rats but not in livers of 24-h-fasted rats. In livers of 24-h-fasted rats, perfused with [2-14C]acetate, exogenously added pyruvate was required for an increase in the rate of 14CO2 production during phenylephrine infusion. This last observation suggests increased pyruvate carboxylation as one of the mechanisms involved in stimulation of tricarboxylic acid cycle activity by the Ca2+-mobilizing agonists, vasopressin, angiotensin II and phenylephrine.     

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.     

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.     

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.     

Hormonal regulation of the alpha-ketoglutarate dehydrogenase complex in the isolated perfused rat liver

The metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in perfused livers was monitored by measuring the rate of 14CO2 production from [1-14C]alpha-ketoglutarate. The rates of 14CO2 production and glucose production from [1-14C]alpha-ketoglutarate were increased with increasing perfusate alpha-ketoglutarate concentrations. Vasopressin, angiotensin II, and the alpha 1-adrenergic agonist phenylephrine stimulated transiently by 2.5-fold the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in the presence and absence of Ca2+ in the perfusion medium. High concentrations of glucagon (1 x 10(-8) M) and 8-p-chlorophenylthio-cAMP (100 microM) (data not shown) also stimulated transiently the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction. However, lower glucagon concentrations (1 x 10(-9) M) stimulated the rate of 14CO2 production from [1-14C]alpha-ketoglutarate only under conditions optimized to fix the cellular oxidation-reduction state at an intermediate level, when glucagon (1 x 10(-9) M)-mediated elevation of cAMP content was greater than that observed under highly oxidizing and reducing conditions. These data indicate that agonists which increase cytosolic free Ca2+ levels stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase complex. Furthermore, the data presented here demonstrate for the first time that physiological glucagon concentrations stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction only under conditions known to be optimal for glucagon-mediated Ca2+ mobilization in the isolated perfused rat liver.     

P2-purinergic control of liver glycogenolysis.

Purinergic agonists cause a dose-dependent activation of glycogen phosphorylase in isolated rat hepatocytes. Half-maximally effective concentrations are 5 X 10(-7)M for ATP, 2 X 10(-6)M for ADP, and about 5 X 10(-5) M for AMP and adenosine. This potency series indicates the presence of P2-purinergic receptors. The mode of action of ATP appears to be identical with that of the Ca2+-dependent glycogenolytic hormones angiotensin, vasopressin and alpha 1-adrenergic agonists. (1) They all require Ca2+ for phosphorylase activation; (2) they do not increase cyclic AMP levels; (3) they are susceptible to heterologous desensitization by vasopressin and phenylephrine; (4) they lower cyclic AMP concentrations in hepatocytes stimulated by glucagon, most probably mediated by an enhanced phosphodiesterase activity.     

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.     

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 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)     

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...     

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+.     

Multiple second messenger pathways of alpha-adrenergic receptor subtypes expressed in eukaryotic cells

The alpha-adrenergic receptors mediate the effects of epinephrine and norepinephrine on cellular signaling systems via guanine nucleotide binding regulatory proteins (G-proteins). Three alpha-adrenergic receptor subtypes have been cloned: the alpha 1, the alpha 2-C10, and the alpha 2-C4 adrenergic receptors. To investigate functional differences between the different subtypes, we assessed the ability of each to interact with adenylyl cyclase and polyphosphoinositide metabolism by permanently and transiently expressing the DNAs encoding the alpha 1, the alpha 2-C10, and the alpha 2-C4 adrenergic receptors in cells lacking endogenous alpha-adrenergic receptors. Both alpha 2-C10 and alpha 2-C4 couple primarily to inhibition of adenylyl cyclase and to a lesser extent to stimulation of polyphosphoinositide hydrolysis. alpha 2-C10 inhibits adenylyl cyclase more efficiently than alpha 2-C4. Effects of the alpha 2-adrenergic receptors on adenylyl cyclase inhibition and on polyphosphoinositide hydrolysis are both mediated by pertussis toxin-sensitive G-proteins. The major coupling system of the alpha 1-adrenergic receptor is activation of phospholipase C via a pertussis toxin-insensitive G-protein. alpha 1-Adrenergic receptor stimulation can also increase intracellular cAMP by a mechanism that does not involve direct activation of adenylyl cyclase. As with the muscarinic cholinergic receptor family our results show that each of the alpha-adrenergic receptor subtypes can couple to multiple signal transduction pathways and suggest several generalities about the effector coupling mechanisms of G-protein-coupled receptors.     

Interaction of clonidine and clonidine analogs with human platelet alpha 2-adrenergic receptors

Several new clonidine analogs were synthesized and their ability to inhibit [3H]phentolamine binding to human platelet alpha 2-adrenergic receptors was tested. The order of potency and calculated dissociation constants for clonidine and its analogs were as follows: clonidine (0.020 +/- 0.005 microM) greater than p-aminoclonidine (0.100 +/- 0.010 microM) greater than hydroxy-phenacetyl-aminoclonidine (0.20 +/- 0.03 microM) greater than p-dansyl clonidine (1.00 +/- 0.20 microM) greater than t-boc-tyrosine clonidine (1.80 +/- 0.60 microM). Thus, p-amino substitution reduces alpha 2-adrenergic affinity in the platelet system. The effects of clonidine and its p-amino analogs on platelet adenylate cyclase were also evaluated. This enzyme is inhibited by epinephrine acting via alpha 2-adrenergic receptors. Both clonidine and p-aminoclonidine cause slight inhibition of basal adenylate cyclase and reverse the inhibition induced by epinephrine. These observations indicate that clonidine is a partial agonist for platelet alpha 2-adrenergic receptors.     

Essential role of calcium influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes

The role of Ca2+ in the adrenergic stimulation of pinealocyte cAMP and cGMP was investigated. In this tissue alpha 1-adrenoceptor activation, which by itself is without effect, potentiates beta 1-adrenergic stimulation of cAMP and cGMP 30- to 100-fold. The present results indicate that chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ influx with inorganic Ca2+ channel blockers (La3+, Co2+, Mn2+) markedly reduces the cyclic nucleotide response to norepinephrine, a mixed alpha 1- and beta-adrenergic agonist, but not to isoproterenol, a beta-adrenergic agonist. In addition, the potentiating effects of alpha 1-adrenergic agonists were mimicked by agents which elevate cytosolic Ca2+, including K+ (EC50 = 2 X 10(-2) M), ouabain (EC50 = 2 X 10(-6) M), ionomycin (EC50 = 3 X 10(-6) M), and A23187 (EC50 = 2 X 10(-6) M); each potentiated the effects of beta-adrenergic stimulation but had no effect alone. Together these results indicate that an alpha 1-adrenoceptor-stimulated Ca2+ influx is essential for norepinephrine to increase pinealocyte cAMP and cGMP.     

Phorbol esters inhibit alpha 1-adrenergic effects and decrease the affinity of liver cell alpha 1-adrenergic receptors for (-)-epinephrine

4 beta-Phorbol 12-myristate 13-acetate (PMA) modified the metabolic actions of three calcium-dependent hormones in different ways. The stimulations of glycogenolysis ureogenesis and phosphatidylinositol labeling produced by alpha 1-adrenergic agonist was blocked by the phorbol ester. In contrast, PMA slightly increased the stimulation of ureogenesis produced by low concentration of angiotensin II without modifying the maximal response. No effect of PMA was observed on the stimulation of ureogenesis induced by vasopressin. The stimulation of phosphatidylinositol labeling induced by vasopressin was decreased by PMA, whereas that induced by angiotensin II was not affected. In intact freshly isolated hepatocytes, [3H]prazosin binds with high affinity to a site which displays the characteristics of alpha 1-adrenergic receptor. Competitive inhibition studies with (-)-epinephrine reveal two different sites for this agonist: a high affinity site (Kd 9 nM) and a low affinity site (Kd 2 microM). In the presence of phorbol esters, (-)-epinephrine binding data now show the presence of a single class of low affinity sites, with similar affinity to those present in control cells. Thus, the inhibition of hepatocyte alpha 1-adrenergic action by PMA may be related to the loss of high affinity binding sites caused by the tumor promoter.     

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.     

α1-Adrenergic Receptor-Mediated Downregulation of Angiotensin II Receptors in Neuronal Cultures

Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4-8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding.