Mobilization of intracellular calcium by glucagon and cyclic AMP analogues in isolated rat hepatocytes

Separate or combined addition of cyclic AMP-dependent and Ca2+-linked hormones to isolated rat hepatocytes suspended in a low Ca2+ medium reduced the total cellular Ca. When the hormones were administered together, their effects were not additive. This suggests that both types of hormones mobilize Ca2+ from a common intracellular pool. In the presence of 1.8 mM extracellular Ca2+, the Ca2+ influx counterbalanced or even exceeded the hormone-induced Ca2+ loss, depending on the ability of the hormones to stimulate the Ca2+ influx.     

Subcellular calcium and magnesium mobilization in rat liver stimulated in vivo with vasopressin and glucagon

The total Ca2+ content of the endoplasmic reticulum and the total Ca2+ and Mg2+ content of mitochondria were determined by electron probe microanalysis of rat liver rapidly frozen in vivo following brief (5-15 s) stimulation with vasopressin or prolonged (10-12 min) stimulation with vasopressin + glucagon. Brief vasopressin injection into the anterior mesenteric vein released 1.8 +/- 0.3 (S.D.) mmol of Ca2+/kg dry weight, from the rough endoplasmic reticulum (p less than 0.01), reducing Ca2+ content of the endoplasmic reticulum from 4.4 +/- 0.2 (S.E.) (controls) to 2.6 +/- 0.2 mmol of Ca2+/kg dry weight. Following vasopressin injection, endoplasmic reticulum Ca2+ was also significantly (p less than 0.025) lower than that in brief sham injected animals (3.5 +/- 0.2 mmol/kg dry weight). Mitochondrial Ca2+ was between 1.0 and 2.3 (+/-0.2) mmol/kg dry weight of mitochondrion, under all conditions studied, and no significant differences were observed. Both hormonal and brief sham injection into the anterior mesenteric vein increased mitochondrial Mg2+ from 42 (+/-0.8) to 49 (+/-1.8) mmol/kg dry weight (p less than 0.05). Hormonal stimulation of Mg2+ uptake was further confirmed by injection of vasopressin + glucagon into the jugular vein (to avoid any stimulation of the liver by the anterior mesenteric vein injection itself); mitochondrial Mg2+ increased from 43 (+/-0.9) (10-min sham) to 57 (+/-1.3) mmol/kg dry weight, with 10-min vasopressin + glucagon injection (p less than 0.01). These results demonstrate that hormones can release Ca2+ from the endoplasmic reticulum and modulate mitochondrial Mg2+ content in vivo without causing detectable changes in mitochondrial Ca2+.     

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.     

Hepatocyte growth factor induces calcium mobilization and inositol phosphate production in rat hepatocytes.

The effects of hepatocyte growth factor (HGF) on intracellular Ca2+ mobilization were studied using fura-2-loaded single rat hepatocytes. Hepatocytes microperfused with different amounts of HGF responded with a rapid concentration-dependent rise in the cytosolic free Ca2+ concentration with a maximum increase of 142% at 80 ng/ml of HGF. The lag period of the Ca2+ response was decreased with increasing HGF concentrations, being 64 +/- 12 s, 42 +/- 6 s, and 14 +/- 2 s, respectively, with 8, 20, and 80 ng/ml of HGF. The detailed pattern of Ca2+ transients, however, was variable. Out of 16 cells tested using 20 ng/ml of HGF, 68% showed sustained oscillatory responses, whereas other cells showed a sustained increase in the cytosolic-free Ca2+ upon exposure to HGF, which was dependent on the presence of extracellular Ca2+. HGF also induced Ca2+ entry across the plasma membrane. Mobilization of Ca2+ by HGF was accompanied by a rapid accumulation of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3). The effects of HGF and epidermal growth factor (EGF) were comparable and partly additive for Ins 1,4,5-P3 production and for the sustained phase of Ca2+ mobilization. Preincubation of cells with 10 microM of genistein to inhibit protein tyrosine kinases abolished the HGF-induced Ca2+ response and also inhibited HGF-induced Ins 1,4,5-P3 production in rat liver cells. These data indicate that early events in the signal transduction pathways mediated by HGF and EGF have in common the requirements for tyrosine kinase activity, Ins 1,4,5-P3 production, and Ca2+ mobilization.     

Evidence for localized calcium mobilization and influx in single rat gonadotropes.

Dynamic video-imaging microscopy was used to investigate the spatial and temporal nature of Ca2+ mobilization and Ca2+ influx in acutely dissociated, fura-2-loaded, rat gonadotropes. Addition of luteinizing hormone-releasing hormone (LHRH) to an isolated gonadotrope stimulated a wave of Ca2+ originating from a specific locus of the cell. This probably reflects Ca2+ mobilization from an intracellular store, since this response was unaffected by the removal of extracellular Ca2+. Application of the dihydropyridine-sensitive Ca2+ channel agonist Bay K 8644 (Bay K) stimulated a rise in cytosolic free Ca2+ concentration in the rat gonadotrope. This response was blocked by the removal of extracellular Ca2+ and probably reflects the influx of Ca2+ across the cell membrane. High speed (30 frames.s-1) imaging of the Bay K-induced Ca2+ influx revealed a wave of Ca2+ originating from a localized part of the cell membrane, which, in general, was spatially distinct from the LHRH-induced Ca2+ wave produced in the same cell. This suggests that Ca2+ channels in the cell membrane may be clustered in a specific area of the cell membrane. The velocity of the LHRH-induced Ca2+ mobilization wave was faster (mean = 79 +/- 5 microns.s-1, n = 9) than the Bay K-induced Ca2+ influx wave (39 +/- 7 microns.s-1, n = 9) (p less than or equal to 0.01, Wilcoxon signed rank test) measured in the same cells. Thus, both Ca2+ mobilization from intracellular stores and Ca2+ influx through the cell membrane appear to be spatially localized in the rat gonadotrope. These findings may have important implications in the intracellular regulation of Ca(2+)-dependent cell functions such as hormone biosynthesis and secretion.     

Inhibitory action of cyclic GMP on secretion, polyphosphoinositide hydrolysis and calcium mobilization in thrombin-stimulated human platelets

The effect of cyclic GMP (cGMP) on human platelet activation was investigated, using its metabolically stable analogue, 8-bromo cGMP (8-bcGMP). Thrombin-induced serotonin secretion was inhibited by pretreatment with 8bcGMP in a dose-dependent manner. Production of inositol trisphosphate (IP3), a Ca2+ releaser was inhibited by 8bcGMP pretreatment of platelets. Preincubation of platelets with 8bcGMP was without effect on the basal level of cytosolic free Ca2+, measured by fluorescent indicator quin2, but suppressed its thrombin-induced enhancement independently of extracellular Ca2+. These results indicate that cGMP may be implicated in phospholipase C activation and Ca2+ mobilization (both influx through the plasma membrane and efflux from internal stores) in thrombin-activated human platelets.     

Pertussis toxin blocks both cyclic AMP-mediated and cyclic AMP-independent actions of somatostatin. Evidence for coupling of Ni to decreases in intracellular free calcium

The neuropeptide somatostatin inhibits hormone release from GH4C1 pituitary cells via two mechanisms: inhibition of stimulated adenylate cyclase and a cAMP-independent process. To determine whether both mechanisms involve the guanyl nucleotide-binding protein Ni, we used pertussis toxin, which ADP-ribosylates Ni and thereby blocks its function. Pertussis toxin treatment of GH4C1 cells blocked somatostatin inhibition of both vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation and prolactin secretion. In membranes prepared from toxin-treated cells, somatostatin inhibition of VIP-stimulated adenylate cyclase activity was reduced and 125I-Tyr1-somatostatin binding was decreased more than 95%. In contrast, pertussis toxin did not affect the biological actions or the membrane binding of thyrotropin-releasing hormone. These results indicate that ADP-ribosylated Ni cannot interact with occupied somatostatin receptors and that somatostatin inhibits VIP-stimulated adenylate cyclase via Ni. To investigate somatostatin's cAMP-independent mechanism, we used depolarizing concentrations of K+ to stimulate prolactin release without altering intracellular cAMP levels. Measurement of Quin-2 fluorescence showed that 11 mM K+ increased intracellular [Ca2+] within 5 s. Somatostatin caused an immediate, but transient, decrease in both basal and K+-elevated [Ca2+]. Consistent with these findings, somatostatin inhibited K+-stimulated prolactin release, also without affecting intracellular cAMP concentrations. Pertussis toxin blocked the somatostatin-induced reduction of [Ca2+]. Furthermore, the toxin antagonized somatostatin inhibition of K+-stimulated and VIP-stimulated secretion with the same potency (ED50 = 0.3 ng/ml). These results indicate that pertussis toxin acts at a common site to prevent somatostatin inhibition of both Ca2+- and cAMP-stimulated hormone release. Thus, Ni appears to be required for somatostatin to decrease both cAMP production and [Ca2+] and to inhibit the actions of secretagogues using either of these intracellular messengers.     

Vanadate counteracts glucagon effects in isolated rat hepatocytes

The incubation of isolated rat hepatocytes with vanadate increased the concentration of fructose 2,6-bisphosphate without modifying 6-phosphofructo-2-kinase activity. Vanadate also reverted and prevented the decrease of fructose 2,6-bisphosphate levels, of the "active" form of the 6-phosphofructo 2-kinase and of the pyruvate kinase activity ratio produced by glucagon, by probably counteracting the increase in cyclic AMP concentration.     

Inactivation of phosphofructokinase by glucagon in rat hepatocytes.

Kinetic evidence of a time- and dose-dependent inactivation of phosphofructokinase by glucagon in isolated rat hepatocytes is reported. This inactivation, which persists after gel filtration of a cell-free extract on Sephadex G-25 and after 400-fold purification of the enzyme on agarose-ATP, is observed when the enzyme activity is measured at subsaturating concentrations of fructose 6-phosphate, while there is no change in Vmax. Phosphofructokinase inactivation by glucagon parallels the known inactivation of pyruvate kinase L and activation of glycogen phosphorylase alpha. Exogenous cyclic AMP mimics the effect of this hormone. Half-maximal effect for both phosphofructokinase and pyruvate kinase L is caused by a similar dose of glucagon (1 x 10(-10) M). The inactivation of phosphofructokinase by nonsaturating concentration of glucagon is reversed spontaneously within 40 min of incubation and this reversion is accelerated by insulin.     

Heterologous desensitization of the cyclic AMP-independent glycogenolytic response in rat liver cells.

Vasopressin and alpha-adrenergic agonists are known to be potent cyclic AMP-independent Ca2+-dependent activators of liver glycogen phosphorylase. When hepatocytes are pre-incubated with increasing concentrations of vasopressin or of the alpha-agonist phenylephrine, they become progressively unresponsive to a second addition of the respective agonist. The relative abilities of six vasopressin analogues and of five alpha-agonists to activate glycogen phosphorylase and to cause subsequent desensitization are highly correlated, indicating that the same vasopressin and alpha-adrenergic receptors are involved in both responses. About 5-times-higher peptide concentrations are needed to desensitize the cells than to activate their glycogen phosphorylase, whereas the concentrations of alpha-agonists required for the desensitization are only twice those needed for the activation of phosphorylase. The desensitization is not mediated by a perturbation in the agonist-receptor interaction. It is clearly heterologous, i.e. it is not agonist-specific, and must therefore involve a mechanism common to both series of agonists. The evidence for a role of Ca2+ movements or phosphatidylinositol turnover is briefly discussed.     

Activation of phospholipid methyltransferase by glucagon in rat hepatocytes

Glucagon produces a time- and dose-dependent activation of phospholipid methyltransferase activity in isolated rat hepatocytes. Half-maximal effect is caused by a dose of glucagon of 1 x 10(-10) M. This activation is due to an increase of the Vmax value of the enzyme, without affecting the Km value for S-adenosylmethionine. Exogenous cyclic AMP added to isolated rat hepatocytes mimics the effect of glucagon, and the activation of phospholipid methyltransferase by a nonsaturating concentration of glucagon is spontaneously reversible within 40 min of incubation.     

Isolation and characterization of cyclic AMP-independent glycogen synthase kinase from rat skeletal muscle

Glycogen synthase kinase was isolated from rat skeletal muscle. This kinase, which is cyclic nucleotide-independent and calcium-independent, was separated from phosphorylase kinase, cyclic AMP-dependent protein kinase and phosvitin kinase by phosphocellulose chromatography. Gel filtration on Sephadex G-100 resolved the glycogen synthase kinase into two fractions with apparent molecular weights of 68 000 (peak I) and 52 000 (peak II). This step also separated glycogen synthase kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase, which had an apparent molecular weight of 39 000. Peak II glycogen synthase kinase activity was not affected by the addition of calcium, EGTA or a number of cyclic nucleotides. In addition to ATP, dATP would serve as the phosphate donor. Other trinucleotides tested were either poor or ineffective substrates. Activity was about 5-fold greater with Mg2+ than with Mn2+. Glycogen stimulated activity about 25%. Modifications of the methods of Soderling et al. ((1970) J. Biol. Chem. 245, 6317--6328) and Nimmo et al. ((1976) Eur. J. Biochem. 68, 21--30) were developed for purification of glycogen synthease (UDPglucose:glycogen 4-alpha D-glucosyltransferase, EC 2.4.1.11) to specific activity of 35 units/mg of protein. Using this preparation of glycogen synthase as substrate, the phosphorylation and inactivation catalyzed by glycogen synthase kinase was compared to that catalyzed by cyclic AMP-dependent protein kinase or phosphorylase kinase. Each of the kinases had different specificities for phosphorylation sites on glycogen synthase.     

The influence of ammonium and calcium lons on gluconeogenesis in isolated rat hepatocytes and their response to glucagon and epinephrine

The use of n-butylmalonate as an inhibitor of malate transport from mitochondria and of aminooxyacetate as an inhibitor of glutamate-aspartate transaminase indicated that rat liver hepatocytes employ the aspartate shuttle for gluconeogenesis from lactate which supplies reducing equivalents to the cytosolic NAD system. In contrast, malate is transported from mitochondria to cytosol for gluconeogenesis from pyruvate. This conclusion is corroborated by the finding that the addition of ammonium ions enhances gluconeogenesis from lactate but inhibits glucose formation from pyruvate. In hepatocytes, glucagon and epinephrine have relatively little effect on glucose synthesis from lactate. Ammonium ions permit both of these hormones to exert their usual stimulation of gluconeogenesis from lactate.Calcium ions (1.3 mm) enhance gluconeogenesis from lactate and from lactatepyruvate mixtures (10:1). The stimulatory effects of Ca²⁺ and NH₄⁺ are additive and, when lactate is the substrate, the rates of gluconeogenesis achieved are so high as to preclude further stimulation by glucagon.     

The action of vasopressin and calcium on palmitate metabolism in hepatocytes and isolated mitochondria from rat liver

Vasopressin inhibits fatty acid oxidation and stimulates fatty acid esterification, glycogenolysis, and lactate production in hepatocytes from fed rats. In cells from fasted rats, the effect of the hormone on palmitate oxidation was absent, while gluconeogenesis was stimulated. The inhibitory action of vasopressin on palmitate oxidation was not due to the increased lactate production. Neither was it correlated to glycogen content or stimulation of glycogenolysis, which were restored earlier than the vasopressin effect on palmitate oxidation when previously fasted rats were refed a carbohydrate diet. The level of malonyl-CoA was moderately increased by vasopressin. Isolated mitochondria from rat liver were incubated in the presence of [U-14C]palmitate, ATP, CoA carnitine, glycerophosphate, ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, and varying amounts of calcium. The oxidation of palmitate was inhibited when the concentration of free calcium was increased from about 0.1 to 10 microM. Simultaneously, palmitate esterification was stimulated. This effect of calcium was observed also with mitochondria from fasted rats and with octanoate as well as palmitate as the substrate. Carnitine acylation was not affected by calcium. The possibility that the observed effects of calcium on mitochondrial fatty acid utilization is part of the mechanism of action of vasopressin on hepatocyte fatty acid metabolism is discussed.