Effects of chemical anoxia on adrenergic responses of goldfish hepatocytes and the contribution of alpha- and beta-adrenoceptors

Adrenergic responses during normoxia and chemical anoxia were investigated in anoxia-tolerant hepatocytes from the goldfish, Carassius auratus. Epinephrine-stimulated glucose release was unaltered after 1 hr of chemical anoxia, the concentration of epinephrine required for half maximal stimulation of glucose release (K0.5(GLU)) ranging from 0.62 x 10(-8) to 2.05 x 10(-8) M. Similarly, the maximum rate of glucose release caused by hormonal stimulation was not affected by chemical anoxia. In anoxic goldfish hepatocytes [Ca2+](i) remained constant in nonstimulated cells but could be elevated by addition of epinephrine. The magnitude of this [Ca(2+)](i)-increase was dependent on the concentration of the catecholamine and this dependency was similar under normoxia (K0.5(Ca2+) = 1.17 x 10(-8) M) and chemical anoxia (K0.5(Ca2+) = 1.15 x 10(-8) M), as was the percentage of cells responding (77%) and displaying oscillatory [Ca2+]i response patterns (60%) after epinephrine addition, although the frequency of [Ca2+]i oscillations was significantly lower in anoxic cells. To analyze a possible shift in the importance of alpha- and beta-adrenoceptors during chemical anoxia, the effect of phentolamine and propranolol, alpha- and beta-adrenergic antagonists respectively, on epinephrine-stimulated glucose release was studied. Application of the alpha-antagonist caused a dose-dependent reduction of glucose-release which was similar under both conditions, whereas the sensitivity to the beta-antagonist was lowered after chemical anoxia. Taken together these results provide evidence that during chemical anoxia goldfish hepatocytes remain responsive to adrenergic stimulation and that there is a partial shift regarding the contribution of alpha- and beta-adrenergic pathways to the induction of cellular glucose release stimulated by epinephrine.     

Prostaglandins, steroids and reception (an attempt to model the structure of the active centers of adrenoreception)

On the basis of numerous results of investigations on adrenergic systems, an orientational model of the adrenoreceptor (AR) is postulated. Its active center includes low-molecular-weight components--prostaglandins (PGE, PGF), steroids (cortisone, hydrocortisone), S+-adenosylmethionine, Ca, Mg, and Mn ions. Appraisal of the stereospecific characteristics of such a functional unit of AR explains the difference in the nature and magnitude of the effects of interaction of the catecholamines, their agonists and antagonists will the so-called alpha- and beta-AR. Depending on the organ or tissue in which the AR is located, its protein subunits comprise adenylcyclase (beta-AR) or Na,K-ATPase (alpha-AR). An obligatory component of the AR is catechol-O-methyltransferase. The model elaborated describes satisfactorily the molecular mechanisms of action of many pharmacological agents, explains why attempts to isolate and reconstruct the AR have proved fruitless, and gives grounds for rejecting the hypothesis that there exist steroid, prostaglandin, and purinergic receptors, linking the exceptionally high and diverse activity of these biologically active substances with their participation in adrenoreception among other reasons. A conception of the active centers of the AR as low-molecular-weight entities permits the explanation of such phenomena as the desensitization of the AR, the "interconversion" of beta-AR into alpha-AR with a change in the parameters of the medium, and certain components of the pathogenesis of bronchial asthma, etc.     

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.     

Inosine released after hypoxia activates hepatic glucose liberation through A3 adenosine receptors

Inosine, an endogenous nucleoside, has recently been shown to exert potent effects on the immune, neural, and cardiovascular systems. This work addresses modulation of intermediary metabolism by inosine through adenosine receptors (ARs) in isolated rat hepatocytes. We conducted an in silico search in the GenBank and complete genomic sequence databases for additional adenosine/inosine receptors and for a feasible physiological role of inosine in homeostasis. Inosine stimulated glycogenolysis (approximately 40%, EC50 4.2 x 10(-9) M), gluconeogenesis (approximately 40%, EC50 7.8 x 10(-9) M), and ureagenesis (approximately 130%, EC50 7.0 x 10(-8) M) compared with basal values; these effects were blunted by the selective A3 AR antagonist 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino][1,2,4]-triazolo[1,5-c]quinazoline (MRS 1220) but not by selective A1, A2A, and A2B AR antagonists. In addition, MRS 1220 antagonized inosine-induced transient increase (40%) in cytosolic Ca2+ and enhanced (90%) glycogen phosphorylase activity. Inosine-induced Ca2+ mobilization was desensitized by adenosine; in a reciprocal manner, inosine desensitized adenosine action. Inosine decreased the cAMP pool in hepatocytes when A1, A2A, and A2B AR were blocked by a mixture of selective antagonists. Inosine-promoted metabolic changes were unrelated to cAMP decrease but were Ca2+ dependent because they were absent in hepatocytes incubated in EGTA- or BAPTA-AM-supplemented Ca2+-free medium. After in silico analysis, no additional cognate adenosine/inosine receptors were found in human, mouse, and rat. In both perfused rat liver and isolated hepatocytes, hypoxia/reoxygenation produced an increase in inosine, adenosine, and glucose release; these actions were quantitatively greater in perfused rat liver than in isolated cells. Moreover, all of these effects were impaired by the antagonist MRS 1220. On the basis of results obtained, known higher extracellular inosine levels under ischemic conditions, and inosine's higher sensitivity for stimulating hepatic gluconeogenesis, it is suggested that, after tissular ischemia, inosine contributes to the maintenance of homeostasis by releasing glucose from the liver through stimulation of A3 ARs.     

Absence of alpha-2 adrenergic effects on cAMP production in a genital tract smooth muscle cell line

This study sought to evaluate alpha-2 and beta adrenergic modulation of cAMP production in the DDT1 MF-2 transformed smooth muscle myocyte. After stimulation with forskolin or adrenergic agonists with or without subtype specific antagonists, cAMP production was determined. These experiments confirmed an increase of cAMP in response to forskolin, isoproterenol, epinephrine, and norepinephrine; the adrenergic stimulation was inhibited by propranolol. On the other hand, the alpha-2 agonist clonidine did not inhibit cAMP production. Likewise, alpha-2 receptor blockade did not increase cAMP production in response to epinephrine. These studies, therefore, suggest that the DDT1 MF-2 myocyte does not contain a significant population of functional alpha-2 adrenergic receptors.     

Stimulatory effect of glucose 6-phosphate on the non-mitochondrial Ca2+ uptake in permeabilized hepatocytes and Ca2+ release by inositol trisphosphate

The relationships between Ca2+ transport and glucose-6-phosphatase activity, previously studied in isolated liver microsomes, were investigated in permeabilized hepatocytes in the presence of mitochondrial inhibitors. It was found that the addition of glucose 6-phosphate to the cells markedly stimulates the MgATP-dependent Ca2+ uptake. A progressive increase in the stimulation of Ca2+ uptake was seen with increasing amounts of glucose 6-phosphate up to 5 mM concentrations. Vanadate, when added in adequate concentrations (20-40 microM) to the hepatocytes inhibits both the glucose-6-phosphatase activity and the stimulation of Ca2+ uptake by glucose 6-phosphate, while not affecting the MgATP-dependent Ca2+ uptake. The addition of inositol 1,4,5-trisphosphate to permeabilized hepatocytes in which Ca2+ had been accumulated in the presence of MgATP and glucose 6-phosphate, results in a rapid release of Ca2+.     

G proteins immunodetection and adrenergic transduction pathways in the liver of Anguilla anguilla

G proteins are members of a highly conserved superfamily of GTPases, which includes heterotrimeric (alpha, beta, gamma) proteins acting as critical control points for transmembrane signaling. In ectothermal vertebrates, knowledge about these proteins is scarce, and our work provides the first demonstration that G(s), G(q), and G(i) proteins are all present in the liver of a fish. G(q)alpha subunits of about 42 kDa have been identified in European eel (Anguilla anguilla) liver membranes, supporting previous reports about the existence of hormone transduction pathways coupled to inositol 1,4,5-trisphosphate/Ca(2+) enhancement in fish hepatocytes. Although two G(s)alpha proteins of about 45 and 52 kDa have been reported in mammals, a single isoform of approximately 45 kDa has been recognized in eel liver. G(s)alpha and G(q)alpha proteins are involved in the epinephrine transduction pathway, leading to cAMP and Ca(2+) intracellular increments, respectively. Interestingly, both messengers significantly stimulated glucose release from eel hepatocytes but with a different time course. In fact, the Ca(2+)-dependent glucose output preceded the cAMP-mediated release by about 7 min. G(i)alpha subunits of about 40 kDa were also immunodetected, suggesting the presence of hormone receptors leading to adenylyl cyclase inhibition in eel liver; however, alpha(2)- adrenoreceptor ligands were ineffective on both enzyme activity and glucose release.     

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.     

Control of organelle transport in melanophores: regulation of Ca2+ and cAMP levels.

Melanophores of the cichlid Tilapia mossambica can be induced to aggregate pigment by addition of epinephrine to the medium, suggesting adrenergic control of this transport. The melanophore response to adrenergic stimulation was examined using agonists and antagonists that are highly specific for each alpha-adrenoceptor subclass. The signal transduction mechanism of each subclass is unique: stimulation of alpha 1 receptors results in a rise in intracellular free Ca2+, while alpha 2 stimulation results in decreased cAMP levels [Exton, 1985: Am. J. Physiol. 248:E633-E647]. Each alpha 1 or alpha 2 specific agonist tested showed a dose dependent ability to induce aggregation and each was able to effect complete aggregation of pigment, suggesting that aggregation can be mediated either by elevating Ca2+ or by lowering cAMP. However, in the presence of either an alpha 1 or an alpha 2 receptor antagonist, none of the agonists were able to induce significant aggregation, suggesting that changes in levels of both messengers are required for pigment aggregation in the melanophores. Moreover, experiments in which intracellular levels of Ca2+ or cAMP were perturbed, using BAPTA and forskolin, respectively, indicated that elevating Ca2+ in the presence of high cAMP is not sufficient to induce aggregation and, conversely, that lowering cAMP levels in the presence of reduced Ca2+ is not sufficient to induce pigment aggregation. These data indicate that the concentrations of both cAMP and Ca2+ are important in regulating pigment aggregation in teleost melanophores, and suggest that maximal aggregation of pigment requires altering the levels of both messengers.     

Differential potentiation of arachidonic acid release by rat alpha2 adrenergic receptor subtypes

CHO transfectants expressing the three subtypes of rat alpha2 adrenergic receptors (alpha2AR): alpha2D, alpha2B, alpha2C were studied to compare the transduction pathways leading to the receptor-mediated stimulation of phospholipase A2 (PLA2) in the corresponding cell lines CHO-2D, CHO-2B, CHO-2C. The alpha2B subtype stimulated the arachidonic acid (AA) release after incubation of the cells with 1 microM epinephrine, whereas alpha2D and alpha2C gave no stimulation. Calcium ionophore A23187 (1 microM) increased the release by a factor of 2-4 in the three strains. When cells were incubated with both epinephrine and Ca2+ ionophore, the AA release differed greatly between cell lines with strong potentiation in CHO-2B (2-3 times greater than Ca2+ ionophore alone), moderate potentiation in CHO-2D, and no potentiation in CHO-2C. The three cell lines each inhibited adenylylcyclase with similar efficiencies when 1 microM epinephrine was used as the agonist. The potentiation depended on both alpha2AR and Gi proteins since yohimbine and pertussis toxin inhibited the process. Pretreatment of CHO-2B cells with MAFP which inhibits both cytosolic and Ca2+-independent PLA2, reduced the release of AA induced by epinephrine+Ca2+ ionophore to basal value, whereas bromoenol lactone, a specific Ca2+-independent PLA2 inhibitor, had no effect. Preincubation of the cells with the intracellular calcium chelator BAPTA gave a dose-dependent inhibition of the arachidonic acid (AA) release. In CHO cells expressing the angiotensin II type 1 receptor, coupled to a Gq protein, the agonist (10-7 M) produced maximal AA release: there was no extra increase when angiotensin and Ca2+ ionophore were added together. There was no increase in the amount of inositol 1,4, 5-triphosphate following stimulation of CHO-2B, -2C, -2D cells with 1 microM epinephrine. Epinephrine led to greater phosphorylation of cPLA2, resulting in an electrophoretic mobility shift for all three cell lines, so inadequate p42/44 MAPKs stimulation was not responsible for the weaker stimulation of cPLA2 in CHO-2C cells. Therefore, the stimulation of cPLA2 by Gi proteins presumably involves another unknown mechanism. The differential stimulation of cPLA2 in these transfectants will be of value to study the actual involvement of the transduction pathways leading to maximal cPLA2 stimulation.     

On the mechanism by which hormones induce the release of Ca2+ from mitochondria in the liver cell.

1. The abilities of dinitrophenol, NaCl, Ruthenium Red and the Ca2+-selective ionophore A23187 to release 45 Ca2+ from isolated hepatocytes and liver mitochondria (incubated at 37 degrees C in the presence of 0.1 microM-free Ca2+, Mg2+, ATP and phosphate ions) were compared with the action of adrenaline on 45Ca2+ release from isolated hepatocytes. The effects of adrenaline were most closely described by those of the ionophore A23187. 2. In isolated hepatocytes, a release of 45Ca2+ and stimulation of O2 utilization similar to that induced by adrenaline was observed in the presence of 500 and 20 microM-arachidonic acid respectively. The effect of arachidonic acid on 45Ca2+ release was not specific for this unsaturated fatty acid. 3. Inhibitors of arachidonic acid metabolism, including indomethacin and eicosa-5,811,14-tetraynoic acid, did not block the effects of adrenaline on 45Ca2+ or glucose release from isolated hepatocytes. 4. The ability of adrenaline to stimulate 45Ca2+ release from isolated hepatocytes was rapidly reversed after the subsequent addition of phenoxybenzamine to the cell suspension, and was completely blocked by 0.5 mM-dibucaine. 5. The results are consistent with the action of a Ca2+-selective ionophore in the mechanism by which adrenaline induces the release of Ca2+ from mitochondria in the liver cell and indicate that it is unlikely that arachidonic acid or a metabolite of arachidonic acid is involved in this process.     

Potassium-mediated stimulation of hepatic glycogenolysis

Increased extracellular potassium concentrations ([K+]o) stimulated transient increases in glucose release and 45Ca2+ washout in the perfused rat liver. Stimulated glucose release had a K0.5 of about 26 mM for [K+]o, was not desensitized by successive infusion intervals of increased [K+]o, was not affected by altering the direction of perfusion, was absolutely dependent on the presence of [Ca2+]o, and was blocked by 2 mM cobalt or 10 microM verapamil. The increase in 45Ca2+ washout resulting from increased [K+]o also was blocked by 2 mM cobalt or 10 microM verapamil. Inhibitors of vascular tone (nitroprusside, atriopeptin II), arachidonic acid metabolism (indomethacin, nordihydroguaiaretic acid), and alpha- or beta-adrenergic or muscarinic nerve stimulation/secretion (phentolamine, propranolol, atropine) were unable to inhibit the [K+]o-stimulated glucose release. ATP, ADP, and AMP concentrations in tissue freeze-clamped 2 min after the onset of infusion of 50 mM K+ were not significantly different from control tissue. Glucose release from freshly isolated suspensions or primary cultured monolayers of hepatocytes or from liver slices, all of which responded to glucagon or phenylephrine, did not respond to increased [K+]o. The results indicate that glycogenolysis stimulated by depolarizing gradients of K+ is dependent on an intact perfused vasculature and may be mediated by potential-sensitive Ca2+ channels present in the vascular endothelium of the liver.     

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.     

Exogenous phospholipase enzymes mimic effects of phenylephrine on Ca2+ transport in hepatocytes

Phospholipase C from Clostridium perfringens induced the release of 45Ca2+ from isolated rat hepatocytes incubated at 0.1 mM extracellular Ca2+ with a time course similar to that for the action of phenylephrine. Under the conditions of these experiments, no significant damage to the plasma membrane was detected in the presence of phospholipase C. Little 45Ca2+ release was induced by bee venom phospholipase A2. At 1.3 mM extracellular Ca2+, both phospholipase enzymes stimulated the initial rate of 45Ca2+ exchange. Concentrations of phospholipase C comparable with those that stimulated 45Ca2+ release increased the rates of glucose release and O2 utilization by 70 and 20% respectively. An increase in the rate of O2 utilization but not glucose release was observed after the addition of phospholipase A2 to hepatocytes. The possible role for a cellular phospholipase C in the mechanism by which phenylephrine stimulates glycogenolysis in the liver cell is briefly discussed.     

Adrenergic regulation of glucose metabolism in rat heart. A calcium-dependent mechanism mediated by both alpha- and beta-adrenergic receptors

Epinephrine treatment of the perfused rat heart led to an increase in glucose uptake, detritiation of [5-3H] glucose, glycogenolysis, and the formation of lactate. The change in the rate of formation of 3H2O from [5-3H]glucose was slower to develop (commencing at approximately 30 s) than changes in cyclic AMP concentration, hexose-6-P concentration, and the phosphorylase a/(a + b) ratio which were maximal at 24 s. Epinephrine plus propranolol (alpha-adrenergic combination) treatment of the perfused heart also led to increases in glucose uptake, detritiation of [5-3H]glucose, and the formation of lactate, but these occurred without significant changes in cyclic AMP concentration, hexose-6-P concentration, or the phosphorylase a/(a + b) ratio. Half-maximal stimulation of glucose uptake occurred at 0.2 microM epinephrine, 1.5 microM methoxamine, and 1 microM isoproterenol. The increase in glucose uptake mediated by 1 microM epinephrine was blocked by 10 microM prazosin but unaffected by 10 microM propranolol. The increase in glucose uptake mediated by 10 microM epinephrine plus 10 microM propranolol was partly blocked by yohimbine and completely blocked by prazosin. A role for Ca2+ in the adrenergic regulation of glucose uptake was indicated by the sensitivity of the epinephrine dose curve to Ca2+ and the dependence of epinephrine on Ca2+. In addition the increases in glucose uptake mediated by 1 microM epinephrine, 1 microM epinephrine plus 10 microM propranolol, 1 microM isoproterenol, and by 10 mM CaCl2 were each blocked by the Ca2+ channel blocker nifedipine (1 microM). It is concluded that Ca2+-dependent alpha- and beta-adrenergic receptor mechanisms are present in rat heart for controlling glucose uptake. At submicromolar levels of epinephrine the predominant receptors utilized appear to be alpha 1.     

Function of myocardial alpha-adrenoceptors

In addition to beta-adrenoceptors (beta ARs), cardiac myocytes of animals and man possess alpha 1ARs, but not alpha 2ARs. Norepinephrine and epinephrine have a higher affinity for myocardial alpha 1ARs than for beta ARs. Unlike beta AR stimulation, myocardial alpha 1AR stimulation does not increase the slow inward current. The alpha 1AR-mediated positive inotropic effect seen in isolated heart preparations appears to involve increased Ca sensitivity of myofibrils and production of inositol triphosphate (IP3) and diacylglycerol (DAG), but the functions of IP3 and DAG are not clear. Myocardial alpha 1AR stimulation reduces rate of isolated atria and Purkinje fibers and lengthens refractory period and action potential duration. Hypoxia increases alpha 1AR density in cardiomyocytes. alpha 1AR-mediated arrhythmias occur in isolated Purkinje fibers during hypoxia, following infarction, and in the presence of Ba2+ or high Ca2+. In animals, coronary artery occlusion and/or reperfusion increase myocardial alpha 1AR density and responsiveness, and alpha AR blocking drugs attenuate arrhythmias. However, an antiarrhythmic effect of alpha AR blocking drugs mediated by action on coronary vascular alpha ARs cannot be excluded. Presently available drugs do not differentiate between myocardial and vascular alpha ARs and thus affect the coronary and systemic circulations and, indirectly, the heart. Additional myocardial alpha 1AR-mediated effects include production of cardiac hypertrophy, stimulation of glucose uptake and phosphofructokinase and cyclic AMP phosphodiesterase activity, and release of atrial natriuretic peptide.     

Regulation of the black bullhead hepatic beta-adrenoceptors

Black bullhead catfish (Ameiurus melas) were exposed to air for 1 h to examine the effect of an acute stress on the distribution and function of the hepatic beta-adrenoceptors (beta-ARs). Air exposure significantly reduced both adrenaline (ADR)- and noradrenaline (NADR)-stimulated glucose production in isolated hepatocytes with no effect on either receptor affinity (K(d)) or number of binding sites (B(max)). A 24 h exposure of isolated hepatocytes to the beta-agonist isoproterenol also had no significant impact on either binding parameter. Competition studies using selective agonists and antagonists suggest that the hepatic beta-AR in this species is pharmacologically beta(2)-like. However in addition to the beta(2)-AR, molecular evidence provides support for the existence of hepatic beta-ARs that phylogenetically group with the beta(3)-ARs and the beta(1)-ARs. Despite the presence of several potential phosphorylation sites in the third intracellular loop and cytoplasmic tail of the bullhead beta(2)-AR, no significant changes were observed in the binding parameters. While physiological data supports the presence of only a single subtype, molecular data supports the existence of multiple beta-AR subtypes in this species. The mechanisms thought to regulate mammalian beta-ARs exist in the bullhead ARs reported here but these mechanisms are not as effective in this fish system as in mammals.     

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.     

Epinephrine stimulates the Na+-K+ ATPase in isolated rat jejunal crypt cells

The effect of epinephrine on the activity of the Na+-K+ ATPase was studied in isolated rat jejunal cells. The activity of the pump was assessed by measuring the ouabain inhibitable K+ accumulation by the enterocytes using 86Rb as a tracer. Epinephrine stimulated significantly the Na+-K+ ATPase in crypt cells but not in villus cells. This effect was still apparent in presence of propranolol and prazocin but disappeared in presence of yohimbine. Amiloride did not affect the epinephrine-induced stimulation. Calcium channel blockers and dibutyryl cAMP enhanced the activity of the pump, and exerted respectively overlapping and additive effects with epinephrine, when added simultaneously. The calcium ionophore A23187 inhibited the basal activity of the ATPase and the stimulatory effect of epinephrine disappeared in its presence. These results suggest that epinephrine stimulates the Na+-K+ ATPase in jejunal crypt cells by activating alpha2 receptors and decreasing intracellular calcium, and not by altering cAMP levels.     

Effect of sodium fluoride on cyclic AMP production in rat hepatocytes.

The effect of NaF on cAMP production was studied in hepatocytes isolated from fed and fasted rats. A four-six fold increase in hepatocyte cAMP production was observed in the presence of 10-20 mM NaF in cells isolated from either fed or fasted rats. The maximal stimulation of cAMP production was observed after a 10 min incubation in the presence of 1 mM theophylline. However, as little as 0.05-0.15 mM NaF induced a significant increase in cAMP production. It was also found that NaF would alter the production of glucose in isolated rat hepatocytes. When hepatocytes from fed rats were incubated with 0.05-5 mM NaF there was an increase in amount of glucose released from endogenous sources. Also NaF resulted in a decrease in lactate and pyruvate production. Similarly NaF stimulated glucose production in hepatocytes from fasted rats. The maximal stimulation was observed with about 0.15-0.25 mM NaF. At NaF concentrations greater than 1.5 mM a decrease in glucose production was observed. It is concluded that NaF increases the level of cAMP and alters glucose metabolism in intact hepatocytes.