pH control of hepatic glutamine degradation. Role of transport

Glutamine uptake is decreased in isolated perfused rat liver when the extracellular pH is lowered. This is also observed in the presence of ammonia concentrations nearly 20-fold above that required for half-maximal stimulation of glutaminase, indicating that the effect is not explained by a submaximal ammonium activation of the enzyme. In livers perfused with a physiological glutamine concentration (0.6 mM), the tissue glutamine but not glutamate content is strongly dependent on the extracellular pH and increases from 2.9 mumol/g to 4.7 mumol/g liver when the extracellular pH is increased from 7.3 to 7.5. Subfractionation of the livers revealed that the mitochondrial glutamine concentration increases from about 15 mM to 50 mM, when the extracellular pH is raised from 7.3 to 7.7, whereas the cytosolic glutamine concentration increases only slightly. Simultaneously the cytosolic and mitochondrial pH values are largely unaffected, being 7.25 and 7.7 respectively. Thus, the pH gradient between mitochondria and cytosol remains unchanged when the extracellular pH varies. Amiloride (2 mM) inhibits glutamine uptake by the liver and abolishes the extra/intracellular pH gradient. With amiloride present, tissue glutamine levels are no longer dependent on extracellular pH and are only about 2 mumol/g liver. It is concluded that pH control of glutaminase flux is also mediated by variations of the mitochondrial glutamine concentration pointing to a regulatory role of the glutamine carrier in the mitochondrial membrane for hepatic glutamine breakdown.     

Stimulation of glycine catabolism in isolated perfused rat liver by calcium mobilizing hormones and in isolated rat liver mitochondria by submicromolar concentrations of calcium.

Glucagon stimulates flux through the glycine cleavage system (GCS) in isolated rat hepatocytes (Jois, M., Hall, B., Fewer, K., and Brosnan, J. T. (1989) J. Biol. Chem. 264, 3347-3351. In the present study, flux through GCS was measured in isolated rat liver perfused with 100 nM glucagon, 1 microM epinephrine, 1 microM norepinephrine, 10 microM phenylephrine, or 100 nM vasopressin. These hormones increased flux through GCS in perfused rat liver by 100-200% above the basal rate. The possibility that the stimulation of flux by adrenergic agonists and vasopressin is mediated by increases in cytoplasmic Ca2+ which in turn could regulate mitochondrial glycine catabolism was examined by measuring flux through GCS in isolated mitochondria in the presence of 0.04-2.88 microM free Ca2+. Flux through GCS in isolated mitochondria was exquisitely sensitive to free Ca2+ in the medium; half-maximal stimulation occurred at about 0.4 microM free Ca2+ and maximal stimulation (7-fold) was reached when the free Ca2+ in the medium was 1 microM. The Vmax (nanomoles/mg protein/min) and Km (millimolar) values for the flux through GCS in intact mitochondria were 0.67 +/- 0.16 and 20.66 +/- 4.82 in the presence of 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and 3.28 +/- 0.76 and 10.98 +/- 1.91 in presence of 0.5 microM free Ca2+, respectively. The results show that the flux through GCS is sensitive to concentrations of calcium which would be achieved in the cytoplasm of hepatocytes stimulated by calcium-mobilizing hormones.     

Hormonal regulation of glutathione efflux

The efflux of GSH has been shown previously to be a saturable process in both isolated rat hepatocytes and perfused liver, suggesting a carrier-mediated transport mechanism. The possibility in hormonal regulation of this process has been raised by recent reports. Our present work examined the role of hormones known to affect intracellular signal transduction mechanisms on GSH efflux in cultured rat hepatocytes and perfused rat livers. We found that cAMP-dependent factors, such as cholera toxin (CT), dibutyryl cAMP, forskolin, and glucagon all stimulated GSH efflux in cultured rat hepatocytes. The efflux kinetics were compared in cultured cells incubated with or without CT; the stimulation of GSH efflux was related to a near doubling of the Vmax while exhibiting no significant alteration of the Km. The increase in intracellular cAMP level associated with the threshold for this stimulatory effect was 25% above control. The stimulatory effect of CT could not be blocked by cyclohexamide pretreatment or reversed by colchicine treatment. The stimulatory effect of glucagon was abolished in the presence of ouabain but not in the presence of barium. On the other hand, hormones which act through Ca2+ and protein kinase C, such as phenylephrine and vasopressin, had no effect on GSH efflux in the cultured cells. In the perfused liver model, glucagon (10 nM) and dibutyryl cAMP (8 microM) stimulated sinusoidal GSH efflux to 130 and 144% of control values, respectively, and increased bile flow while not affecting biliary GSH efflux. Finally, the physiological significance of glucagon-mediated stimulation of sinusoidal GSH efflux was assessed by both plasma GSH and glucose levels in response to in vivo glucagon infusion. The threshold dose of glucagon for significant increase in plasma GSH (5.21 pmol/min) was lower than for glucose (15.61 pmol/min). At the highest glucagon infusion rate (261 pmol/min), plasma GSH level doubled while glucose level increased 80%. In conclusion, increased cAMP stimulates GSH efflux in cultured rat hepatocytes and perfused livers. The stimulatory effect of cAMP is exerted at the sinusoidal pole and appears to be mediated by hyperpolarization of hepatocytes by stimulation of Na(+)-K(+)-ATPase. In vivo studies confirmed the importance of cAMP-mediated stimulation of sinusoidal GSH efflux as it resulted in significant elevation of the plasma GSH level.     

Exposure to depolarizing concentrations of K+ inhibits hormonally-induced calcium influx in rat liver

The exposure of perfused rat livers to depolarizing concentrations of K+ (60 mM) by partial substitution of the NaCl in the medium with KCl induces glycogenolysis, respiratory changes and vasoconstriction. These responses were found to be inhibited 70-80% by 20 microM indomethacin and by 20 microM bromophenacyl bromide. This suggests that eicosanoids, namely prostaglandins, are involved in mediating these effects, and hence that the action of K+ involves primarily an effect on eicosanoid-producing cells (Kupffer and endothelial cells) within the liver. A 5 min pre-exposure of perfused livers to depolarizing concentrations of K+ (in the presence of indomethacin) was found to inhibit (by approx. 85%) the influx of Ca2+ induced by the co-administration of 10 nM glucagon and 10 nM vasopressin. A similar result was observed in isolated hepatocytes. The inhibition was probably not due to a decrease in the concentration of Na+ in the medium since the substitution of 80 mM NaCl with 80 mM choline chloride resulted in significantly less inhibition (30-40%). These results suggest that under these conditions the influx of Ca2+ in liver occurs through a pathway that is inhibited by high K+ concentration and/or a depolarization of the plasma membrane.     

Role of extracellular calcium and calcium efflux in the activation of hepatic glycogenolysis by calcium-dependent hormones

The role of extracellular calcium in the glycogenolytic effects of calcium-dependent hormones was examined in a rat liver perfusion system. Decreasing the perfusate CaCl2 concentration resulted in a concentration-dependent inhibition of glucose output by maximal concentrations of vasopressin (20 nM) and angiotensin II (10 nM), but not of glucagon (1.4 nM), cyclic AMP (100 microM), dibutyryl cyclic AMP (10 microM) or phenylephrine (5 microM). However, the effect of phenylephrine was inhibited when livers were perfused with CaCl2-free perfusate containing 0.5 mM EGTA in a duration-dependent manner. These effects were exerted through the inhibition of the maximal response of each hormone, and were associated with a parallel decrease in phosphorylase activation but not with changes in tissue cyclic AMP concentrations. When livers were preloaded with 45Ca for 45 min and then washed for either 15 min or 45 min, these hormones elicited a rapid and transient 45Ca efflux regardless of the perfusate calcium concentration. The sequential perfusion of two hormones resulted in the loss of 45Ca efflux by the second hormone. These results suggest that the glycogenolytic effects of vasopressin and angiotensin II depend on the extracellular calcium and that of phenylephrine primarily on the cellular calcium. It was also demonstrated that these calcium-dependent hormones mobilize calcium from the same pools. However, the mobilization of cellular calcium does not necessarily correlate directly with the glycogenolytic actions of vasopressin and angiotensin II.     

Hormone-specific combinations of isoforms of adenylyl cyclase and phosphodiesterase in the rat liver

Since many isoforms of adenylyl cyclase and adenosine 3', 5'-monophosphate (cAMP) phosphodiesterase have been cloned, it is likely that receptors of each hormone have a specific combination of these isoforms. Types I, III and VIII adenylyl cyclases are reported to be stimulated by Ca(2+)-calmodulin, type I phosphodiesterase by Ca(2+)-calmodulin, but types IV and VII (cAMP-specific) phosphodiesterases by Co2+. In the present study, we examined different effects of Ca2+ and Co2+ on hormone-induced cAMP response in the isolated perfused rat liver.The removal of Ca2+ from the perfusion medium (0 mM CaCl(2 ) + 0.5 mM EGTA) did not affect glucagon (0.1 nM)-responsive cAMP but reduced secretin (1 nM)-, vasoactive intestinal polypeptide (VIP, 1-10 nM)- and forskolin (1 microM)-responsive cAMP considerably. The addition of 1 mM CoCl2 reduced glucagon- and secretin-responsive cAMP considerably, forskolin-responsive cAMP partly, did not affect 1 nM VIP-responsive cAMP, but enhanced 10 nM VIP-responsive cAMP. Forskolin- and VIP-responsive cAMP was greater in the combination (0 mM CaCl(2) + 0.5 mM EGTA + 3 mM CoCl2) than in the Ca(2+)-free perfusion alone.These results suggest that secretin, VIP1 and VIP2 receptors are linked to Ca(2+)-calmodulin-sensitive adenylyl cyclase; glucagon receptor to Ca(2+)-calmodulin-insensitive adenylyl cyclase; VIP1 receptor to Ca(2+)-calmodulin-dependent phosphodiesterase; glucagon, secretin and VIP2 receptors to cAMP-specific phosphodiesterase, respectively, in the rat liver.     

The effect of glucagon-induced adenosine 3' ,5' -monophosphate concentrations on bile acid synthesis in isolated rat liver cells

The effect of increased intracellular adenosine 3' ,5' -monophosphate (cAMP) concentrations on bile acid synthesis in isolated rat hepatocytes was investigated. When the cells were incubated in the presence of glucagon (0.2 microM) and theophylline (1 mM) the observed rise in the level of cAMP was accompanied by an increase in bile acid production. Hepatocyte cAMP concentrations after 1 h of incubation showed a highly significant positive linear correlation with the amounts of bile acid synthesised by the cells during this time. These results suggest that bile acid production is related to the concentration of cAMP in isolated hepatocytes and provide evidence for a role for the cyclic nucleotide in the regulation of bile acid synthesis.     

Inhibition of the glycogenolytic effects of alpha-adrenergic stimulation and glucagon by cobalt ions in perfused rat liver

Cobalt ions (2 mM) inhibited the glycogenolysis induced by phenylephrine and glucagon in perfused rat liver. Cobalt ions also inhibited 45Ca++ efflux from prelabelled livers induced by phenylephrine and glucagon. In addition, they inhibited the rise in tissue levels of cyclic AMP caused by glucagon, but did not inhibit the stimulation of 45Ca++ efflux or glycogenolysis by cyclic AMP or dibutyryl cyclic AMP. The specific binding of glucagon and alpha-agonist to hepatocytes was not inhibited by cobalt ions. These data suggest that cobalt ions, presumably through their high affinity for calcium binding sites on membranes inhibit the stimulation of glycogenolysis by phenylephrine and glucagon in distinct ways; one by inhibiting calcium mobilization and the other by inhibiting cyclic AMP production. Therefore, it is conceivable that membrane-bound calcium plays an important role in stimulating Ca++ mobilization by phenylephrine, and cyclic AMP production by glucagon.     

Regulation of hepatic glycine catabolism by glucagon

Glucagon stimulates 14CO2 production from [1-14C] glycine by isolated rat hepatocytes. Maximal stimulation (70%) of decarboxylation of glycine by hepatocytes was achieved when the concentration of glucagon in the medium reached 10 nM; half-maximal stimulation occurred at a concentration of about 2 nM. A lag period of 10 min was observed before the stimulation could be measured. Inclusion of beta-hydroxybutyrate (10 mM) or acetoacetate (10 mM) did not affect the magnitude of stimulation suggesting that the effects of glucagon were independent of mitochondrial redox state. Glucagon did not affect either the concentration or specific activity of intracellular glycine, thus excluding the possibilities that altered concentration or specific activity of intracellular glycine contributes to the observed stimulation. The stimulation of decarboxylation of glycine by glucagon was further studied by monitoring 14CO2 production from [1-14C]glycine by mitochondria isolated from rats previously injected with glucagon. Glycine decarboxylation was significantly stimulated in the mitochondria isolated from the glucagon-injected rats. We suggest that glucagon is a major regulator of hepatic glycine metabolism through the glycine cleavage enzyme system and may be responsible for the increased hepatic glycine removal observed in animals fed high-protein diets.     

Effects of neuromedin B on insulin and glucagon release from the isolated perfused rat pancreas

The effect of neuromedin B (NMB) on insulin and glucagon release was studied in isolated perfused rat pancreas. Infusion of NMB (10 nM, 100 nM and 1 microM) did not affect the insulin release under the perusate conditions of 5.5 mM glucose plus 10 mM arginine and 11 mM glucose plus 10 mM arginine, although 10 nM NMB tended to slightly suppress it under the perfusate condition of 5.5 mM glucose alone. The degree of stimulation of insulin release provoked by the addition of 5.5 mM glucose to the perfusate was not affected by the presence of 10 nM NMB. The glucagon release was slightly stimulated by the infusion of 100 nM and 1 microM NMB but not by 10 nM NMB under the perfusate condition of 5.5 mM glucose plus 10 mM arginine. The effect of C-terminal decapeptide of gastrin releasing peptide (GRP-10) was also examined and similar results were obtained; 10 nM and 100 nM GRP-10 did not affect insulin release and 100 nM GRP-10 stimulated glucagon release under the perfusate condition of 5.5 mM glucose plus 10 mM arginine. The present results concerning glucagon release are consistent with the previous results obtained with isolated perfused canine and porcine pancreas. However, the results regarding insulin release are not. Species differences in insulin release are also evident with other neuropeptides such as substance P and the mechanism of such differences remains for be clarified.     

Assessment of the role of Ca2+ mobilization from intracellular pool(s), using dantrolene, in the glycogenolytic action of alpha-adrenergic stimulation in perfused rat liver

To identify the role of Ca2+ mobilization from intracellular pool(s) in the action of alpha-adrenergic agonist, the effects of dantrolene on phenylephrine-induced glycogenolysis were investigated in perfused rat liver. Dantrolene (5 X 10(-5) M) inhibited both glycogenolysis and 45Ca efflux induced by 5 X 10(-7) M phenylephrine. The inhibition by dantrolene was observed in the presence and absence of perfusate calcium. In contrast, dantrolene did not inhibit glycogenolysis induced by glucagon. To confirm the specificity of dantrolene action on calcium release in liver, experiments were also carried out using isolated hepatocytes. Dantrolene did not affect phenylephrine-induced production of inositol 1,4,5-trisphosphate. The compound did inhibit a rise in cytoplasmic Ca2+ concentration induced by phenylephrine both in the presence and absence of extracellular Ca2+. Thus, these results suggest that calcium release from an intracellular pool is essential for the initiation of alpha-adrenergic stimulation of glycogenolysis in the perfused rat liver.     

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

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

The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats

Production of [14C]glucose from [14C]lactate in the perfused livers of 24-h fasted adrenalectomized rats was not stimulated by 1 nM glucagon but was significantly increased by 10 nM hormone. Crossover analysis of glycolytic intermediates in these livers revealed a significant reduction in glucagon action at site(s) between fructose 6-phosphate and fructose 1,6-bisphosphate as a result of adrenalectomy. Site(s) between pyruvate and P-enolpyruvate was not affected. In isolated hepatocytes, adrenalectomy reduced glucagon response in gluconeogenesis while not affecting glucagon inactivation of pyruvate kinase. A distinct lack of glucagon action on 6-phosphofructo-1-kinase activity was noted in these cells. When hepatocytes were incubated with 30 mM glucose, lactate gluconeogenesis was greatly stimulated by glucagon. A reduction in both sensitivity and responsiveness to the hormone in gluconeogenesis was seen in the adrenalectomized rat. These changes were well correlated with similar impairment in glucagon action on 6-phosphofructo-1-kinase activity and fructose 2,6-bisphosphate content in hepatocytes from adrenalectomized rats incubated with 30 mM glucose. These results suggest that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.     

Metabolic responses of perfused rat livers to alpha- and beta-adrenergic agonists, glucagon and cyclic AMP.

1. The mechanism of action of glucagon and epinephrine was studied in perfused rat livers. Hormone-induced transitions from one metabolic steady state to another were followed in a non-recirculating perfusion system. Glucose and lactate production rates, oxygen uptake and K+ redistribution were measured. 2. Glucagon (3 nM), cyclic AMP (0.2 mM) and epinephrine (0.5 muM) had similar effects on K+ concentrations in the perfusate. Glycogenolysis responded more rapidly and O2 uptake was enhanced to a larger extent with epinephrine than with the other agents. alpha- and beta-receptor responses were differentiated by the use of phenylephrine (0.5 muM), isoproterenol (0.5 muM) and adrenergic blocking agents (phentolamine and beta-blocker Ro 3-4787 at 0.1 mM). 3. alpha-receptors mediated an activation of glucose production that was very rapid and was paralleled by a transient decrease of K+ concentrations in the effluent from the liver, lactate production rose gradually. Respiration was also enhanced, but fell again as lactate production increased. 4. beta-receptor stimulation was followed by an increase of glucose production that was less drastic and was paralleled by a K+ release, lactate production and respiration were only slightly enhanced. beta stimulation and glucagon both resulted in an inhibition of the alpha-adrenergic effect on lactate release and simultaneously increased O2 uptake. 5. We concluded that in perfused rat livers alpha- as well as beta-adrenergic receptor stimulation resulted in an activation of glycogenolysis, possibly by two different mechanisms.     

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.     

Role of plasma membrane transport in hepatic glutamine metabolism

In livers of fed rats and in perfused livers supplied with a physiological portal glutamine concentration of 0.6 mM, the mitochondrial and cytosolic glutamine concentrations are 20 mM and 7 mM, respectively, thus, the mitochondrial/cytosolic glutamine concentration gradient is 2-3. Uptake and release of glutamine by periportal and perivenous hepatocytes occurs predominantly by an Na+-dependent transport system (so-called system 'N'). Histidine in near-physiological concentrations inhibits both glutamine uptake by periportal hepatocytes and its release by perivenous hepatocytes. This is not due to an inhibition of glutamine-metabolizing enzymes by histidine or its metabolites. With physiological portal glutamine concentrations (0.6 mM), stimulation of glutaminase flux or of glutamine transaminase flux is followed by a decrease of hepatic glutamine levels to about 80% or 30%, respectively, glutamine levels are further decreased to 50% or 20% in the presence of histidine. When glutamine is synthesized endogenously (no glutamine added), the histidine-induced inhibition of glutamine release is paralleled by a 210% increase of the hepatic tissue level of glutamine. In experiments with and without methionine sulfoximine and in the absence of added glutamine, the glutamine content in the small perivenous hepatocyte population containing glutamine synthetase is estimated to be about 3.5 mumol/g wet weight and that in the periportal hepatocytes as low as 0.1 mumol/g wet weight. In contrast to the prevailing view, it is concluded that glutamine transport across the plasma membrane of hepatocytes is a potential regulatory site in glutamine degradation and synthesis, especially under the influence of effectors like histidine.     

The Ca2+-dependent actions of the alpha-adrenergic agonist phenylephrine on hepatic glycogenolysis differ from those of vasopressin and angiotensin

The stimulation of hepatic glycogenolysis by the Ca2+-dependent hormones phenylephrine, vasopressin and angiotensin II was studied as a function of intracellular and extracellular Ca2+. In the isolated perfused rat liver the decline in glucose formation was monophasic ('half-life' approximately equal to 3 min) with vasopressin (1 nM) or angiotensin II (0.05 microM), but biphasic (half-life of 4.8 min and 17.6 min) in the presence of the alpha-agonist phenylephrine (0.01 mM), indicating either a different mode of mobilization or the mobilization of additional intracellular calcium stores. Under comparable conditions an elevated [Ca2+] level was maintained in the cytosol of hepatocytes for at least 10 min in the presence of phenylephrine, but not vasopressin. Titration experiments performed in the isolated perfused liver to restore cellular calcium revealed differences in the hormone-mediated uptake of Ca2+. The onset in glucose formation above that seen in the absence of exogenous calcium occurred at approximately 30 microM or 70-80 microM Ca2+ in the presence of phenylephrine or vasopressin respectively. The shape of the response curve was sigmoidal for vasopressin and angiotensin II, but showed a distinct plateau between 0.09 mM and 0.18 mM in the presence of phenylephrine. The plateau was also observed at phenylephrine concentrations as low as 0.5 microM. The formation of plateaus observed after treatment of the liver with A 23187, but not after EGTA, is taken as an indication that intracellular calcium stores are replenished. A participation of the mitochondrial compartment could be excluded by pretreatment of the liver with the uncoupler 2,4-dinitrophenol. Differences in the Ca2+ dependence of the glycogenolytic effects of these hormones were also revealed by kinetic analysis. It is concluded that phenylephrine differs from vasopressin and angiotensin II in that, in addition to a more common, non-mitochondrial pool, which is also responsive to the vasoactive peptides, the agonist mobilizes Ca2+ from a second, non-mitochondrial pool. The results are consistent with the proposal that Ca2+ transport across subcellular membranes may be subject to different hormonal control.     

Effect of calcium on the binding in vitro of [3H]cAMP to synaptosomal membranes from rat brain

1. The binding of [3H]cAMP in vitro to synaptosomal membranes from rat brain was resolved in two components; one saturable at 20 nM cAMP with dissociation constant (KD) of 4.7 nM, and another nonsaturable within the 5-133 nM cAMP concentration range with an estimated KD value of 0.26 microM. 2. MgATP at concentration of 0.4 mM effected complete inhibition of the binding of [3H]cAMP to synaptosomal membranes throughout the used concentration range. This and the above finding indicate that the studied binding was focused on to the cAMP kinase on the membrane. 3. Calcium at concentrations of 0.1 and 10 mM stimulated a transient 20-30% increase of [3H]cAMP binding to the membranes which was influenced, as regards its time of appearance, by the concentration of cAMP. 4. The stimulation by calcium of the binding of [3H]cAMP to the membranes was inversely related to the phosphorylation of an Mr = 80,000 membrane protein, indicating stimulation of a negative effector function of cAMP--through cAMP-mediated phosphorylation--in the phosphorylation by calcium of this substrate. Moreover, the temporal displacement by cAMP of the peak of [3H]cAMP binding, produced similar temporal displacement of the inhibitory effect of cAMP on the Mr = 80,000 substrate phosphorylation. 5. These results suggest interaction in vitro of calcium and cAMP in modulation of the activity of cAMP kinase on the synaptosomal membranes.     

Evidence of cyclic AMP-independent action of glucagon on calcium mobilization in rat hepatocytes

Glucagon increases the cytoplasmic free calcium concentration as measured by aequorin bioluminescence. It has been proposed by Wakelam et al. (Nature 323 (1986) 68-71) that low concentrations of glucagon mobilize calcium from an intracellular pool by causing polyphosphoinositide breakdown. To identify whether cyclic AMP mediates changes in the cytoplasmic free calcium concentration ([Ca2+]c) induced by glucagon, the effects of forskolin and exogenous cyclic AMP on [Ca2+]c were compared with that of glucagon in aequorin-loaded hepatocytes. Although the magnitudes of the [Ca2+]c responses to 250 microM forskolin and 1 mM 8-bromo cyclic AMP were identical to that of 5 nM glucagon, these two agents induced a more prolonged elevation of [Ca2+]c. Glucagon-induced elevation of [Ca2+]c was accompanied by a smaller increase in cyclic AMP than that induced by forskolin. When the cyclic AMP response to glucagon was potentiated by an inhibitor of phosphodiesterase, 3-isobutyl-1-methylxanthine, the glucagon-induced increase in [Ca2+]c was not affected. Conversely, when the cyclic AMP response to glucagon was reduced by pretreatment of the cells with angiotensin II, glucagon-induced changes in [Ca2+]c were rather enhanced. Furthermore, vasopressin potentiated glucagon-induced changes in [Ca2+]c despite the reduction of the cyclic AMP response to glucagon. In the presence of 1 microM extracellular calcium, angiotensin II did not enhance glucagon-induced changes in [Ca2+]c. These results suggest that at least part of the action of 5 nM glucagon on calcium mobilization is independent of cyclic AMP.     

Extracellular calcium transients and action potential configuration changes related to post-stimulatory potentiation in rabbit atrium

Extracellular calcium transients were monitored with 2 mM tetramethylmurexide at low calcium (250 microM total, 130 microM free), and action potentials were monitored together with developed tension at normal calcium (1.3 mM) during the production and decay of post-stimulatory potentiation in rabbit left atrial strips. At normal calcium, the contractile potentiation produced by a brief burst of 4 Hz stimulation is lost in three to five post-stimulatory excitations, which correlate with a negative staircase of the late action potential. At low calcium, stimulation at 4 Hz for 3-8 s results in a net extracellular calcium depletion of 5-15 microM. At the subsequent potentiated contraction (1-45 s rest), total extracellular calcium increases by 4-8 microM. The contractile response at a second excitation is greatly suppressed and results in little or no further calcium shift; the sequence can be repeated immediately thereafter. Reducing external sodium to 60 mM (sucrose replacement) enhances post-rest contractions, suppresses the late action potential, nearly eliminates loss of contractility and net calcium efflux at post-rest excitations, and markedly reduces extracellular calcium depletion during rapid stimulation. 4-Aminopyridine (1 mM) markedly suppresses the rapid early repolarization of this preparation at post-rest excitations and the loss of contractility at post-rest stimulation from the rested state; during a post-stimulatory potentiation sequence at low calcium, replenishment of extracellular calcium takes several post-stimulatory excitations. Ryanodine (10 nM to 5 microM) abolishes the post-stimulatory contraction at rest periods of greater than 5 s. If the initial repolarization is rapid, ryanodine suppresses the late action potential, calcium efflux during quiescence is greatly accelerated, and subsequent excitations do not result in an accumulation of extracellular calcium. A positive staircase of the early action potential correlates with the magnitude of net extracellular calcium depletion. These findings demonstrate that negative contractile staircases at post-rest stimulation correspond closely to an accumulation of extracellular calcium at activation and a negative staircase of the late action potential; the correlation of these three events suggests that electrogenic sodium-calcium exchange is the common underlying mechanism.