The effect of intracellular calcium ions on adrenaline-stimulated adenosine 3'':5''-cyclic monophosphate concentrations in pigeon erythrocytes, studied by using the ionophore A23187.

1. The bivalent cation ionophore A23187 was used to increase the intracellular concentration of Ca2+ in pigeon erythrocytes to investigate whether the increase in cyclic AMP content caused by adrenaline might be influenced by a change in intracellular Ca2+ in intact cells. 2. Incubation of cells with adrenaline, in the concentration range 0.55--55 muM, resulted in an increase in the concentration of cyclic AMP over a period of 60 min. The effect of adrenaline was inhibited by more than 90% with ionophore A23187 (1.9 muM) in the presence of 1 mM-Ca2+. This inhibition could be decreased by decreasing either the concentration of the ionophore or the concentration of extracellular Ca2+, and was independent of the concentration of adrenaline. 3. The effect of ionophore A23187 depended on the time of incubation. Time-course studies showed that maximum inhibition by ionophore A23187 was only observed when the cells were incubated with the ionophore for at least 15 min before the addition of adrenaline. 4. The inhibition by ionophore A23187 depended on the concentration of extracellular Ca2+. In the absence of Mg2+, ionophore A23187 (1.9 muM) inhibited the effect of adrenaline by approx. 30% without added Ca2+, by approx. 66% with 10 muM-Ca2+ and by more than 90% with concentrations of added Ca2+ greater than 30 muM. However, even in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate](0.1--10 mM), ionophore A23187 caused an inhibition of the cyclic AMP response of at least 30%, which may have been due to a decrease in cell Mg2+ concentration. 5. The addition of EGTA after incubation of cells with ionophore A23187 resulted in a partial reversal of the inhibition of the effect of adrenaline. 6. Inclusion of Mg2+ (2 mM) in the incubation medium antagonized the inhibitory action of ionophore A23187. This effect was most marked when the ionophore A23187 was added to medium containing Mg2+ before the addition of the cells. 7. The cellular content of Mg2+ was decreased by approx. 50% after 20 min incubation with ionophore A23187 (1.9 muM) in the presence of Ca2+ (1 mM) but no Mg2+. When Mg2+ (2 mM) was also present in the medium, ionophore A23187 caused an increase of approx. 80% in cell Mg2+ content. Ionophore A23187 had no significant effect on cell K+ content. 8. Ionophore A23187 caused a decrease in cell ATP content under some conditions. Since effects on cyclic AMP content could also be shown when ATP was not significanlty lowered, it appeared that a decrease in ATP in the cells could not explain the effect of ionophore A23187 on cyclic AMP. 9. Ionophore A23187 (1.9 muM), with 1 mM-Ca2+, did not enhance cyclic AMP degradation in intact cells, suggesting that the effect of ionophore A23187 on cyclic AMP content was mediated through an inhibition of adenylate cyclase rather than a stimulation of cyclic AMP phosphodiesterase. 10. It was concluded that in intact pigeon erythrocytes adenylate cyclase may be inhibited by intracellular concentrations of Ca2+ in the range 1-10 muM.     

Effect of H-7 on the modulation of glucagon actions by activators of protein kinase-C

The stimulations of ureagenesis and cyclic AMP accumulation induced by glucagon were inhibited by 10 nM vasopressin or 100 nM phorbol 12-myristate 13-acetate (PMA). The maximal accumulation of cyclic AMP induced by glucagon was clearly diminished by these agents without change in the EC50 for the peptide hormone suggesting a non-competitive type of inhibition. H-7 blocked the inhibition of glucagon-stimulated ureagenesis induced by PMA and vasopressin and diminished their effect on the accumulation of cyclic AMP induced by glucagon. It is concluded that activation of protein kinase C inhibits the stimulation of ureagenesis and the accumulation of cyclic AMP induced by glucagon in liver cells from hypothyroid rats; H-7 inhibits the effects of protein kinase C activation.     

Epidermal growth factor mimics insulin effects in rat hepatocytes.

Epidermal growth factor (EGF) mimicked the effect of insulin to activate glycogen synthase and stimulate glycogen synthesis in isolated rat hepatocytes. Both agents required glucose (greater than 5 mM) and had similar time courses of action. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were 9 nM and 4 nM respectively. Combinations of the two agents produced additive responses. EGF also resembled insulin in its ability to inhibit the effects of 0.1-1.0 nM-glucagon on cyclic AMP and glycogen phosphorylase in hepatocytes. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were approx. 5 nM and 0.5 nM respectively. EGF and insulin inhibited phosphorylase activation by exogenous cyclic AMP, and inhibited cyclic AMP accumulation induced by forskolin. They also inhibited phosphorylase activation provoked by phenylephrine, but not by vasopressin. EGF added alone rapidly activated phosphorylase and increased cytosolic [Ca2+], but the effects were no longer apparent at 5 min and were smaller than those of vasopressin. Insulin did not induce these changes. In hepatocytes previously incubated with myo-[3H]inositol, EGF did not significantly increase myo-inositol 1,4,5-trisphosphate. However, its ability to increase cytosolic [Ca2+] was blocked by neomycin, an inhibitor of phosphatidylinositol bisphosphate hydrolysis. It is concluded that some, but not all, of the effects of EGF in liver are strikingly similar to those exerted by insulin, suggesting that these agents may have some similar mechanisms of action in this tissue.     

Amino acid transport in isolated rat thymocytes. Effects of divalent cations and ethanol.

In dispersed rat thymocytes neither basal alpha-aminoisobutyric acid influx nor influx stimulated by insulin, prostaglandin theophylline, or butyryl adenosine 3':5'-monophosphate (cyclic AMP) depended on extracellular calcium or magnesium. The divalent cation ionophore A23187 inhibited both basal and stimulated alpha-aminoisobutyric acid influx. The extent to which influx was inhibited depended on ionophore concentration, extracellular calcium concentration, and time but did not depend on extracellular magnesium. Significant inhibition could be detected at an ionophore concentration of 1 muM and maximal inhibition occurred with 6 muM A23187. A23187 increased cellular uptake of calcium and there was good agred calcium uptake and that for ionophore inhibition of alpha-aminoisobutyric acid influx. Incubating cells with A23187 and then adding ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid completely reversed ionophore-stimulated cellular calcum uptake but did not reverse inhibition of alpha-aminoisobutyric acid influx. Thus, A23187 produces irreversible inhibition of alpha-aminoisobutyric acid transport in dispersed rat thymocytes. Ethanol abolished insulin-stimulated alpha-aminoisobutyric acid influx but did not alter basal influx or that stimulated by prostaglandin E1, theophylline, or N6,O2'-dibutyryl adenosine 3':5'-monophosphate. Inhibition could be detected with 0.2% (v/v) ethanol and insulin-stimulated alpha-aminoisobutyric influx was abolished with 1% ethanol. The effect of ethanol occurred immediately and could be reversed completely. This ability of ethanol to inhibit selectively insulin-stimulated alpha-aminoisobutyric acid influx indicates that the mechanism through which insulin stimulates alpha-aminoisobutyric acid influx is functionally distinct from the stimulation produced by cyclic AMP.     

Stimulation of hamster adipocyte cyclic 3':5'-nucleotide phosphodiesterase activity by ionophore A23187 and calcium.

Incubation of hamster isolated fat cells with the ionophore A23187 and calcium for 20 minutes caused 30-40% increases in the cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) activity of adipocyte homogenates when either 0.6 micron cyclic AMP or 0.6 micron cyclic GMP was the enzyme substrate. The stimulation of adipocyte cyclic AMP phosphodiesterase activity by A23187 and calcium was not antagonized by the adrenergic receptor blocking agents phentolamine and propranolol. The changes in enzyme activity produced by the ionophore and calcium were not associated with elevated intracellular cyclic AMP levels. Furthermore, A23187 and calcium acted to enhance adipocyte phosphodiesterase activity before, but not after, homogenization of the fat cells. These data suggest that the phosphodiesterase activity of hamster isolated fat cells may, at least in part, be regulated by fluctuations in intracellular calcium concentrations.     

Hormonal control of cyclic 3':5'-AMP levels and gluconeogenesis in isolated hepatocytes from fed rats.

Glucagon can stimulate gluconeogenesis from 2 mM lactate nearly 4-fold in isolated liver cells from fed rats; exogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP) is equally effective, but epinephrine can stimulate only 1.5-fold. Half-maximal effects are obtained with glucagon at 0.3 nM, cyclic AMP at 30 muM and epinephrine at 0.2 muM. Insulin reduces by 50% the stimulation by suboptimal concentrations of glucagon (0.5 nM). A half-maximal effect is obtained with 0.3 nM insulin (45 microunits/ml). Glucagon in the presence of theophylline (1 mM) causes a rapid rise and subsequent fall in intracellular cyclic AMP with a peak between 3 and 6 min. Some of the fall can be accounted for by loss of nucleotide into the medium. This efflux is suppressed by probenecid, suggesting the presence of a membrane transport mechanism for the cyclic nucleotide. Glucagon can raise intracellular cyclic AMP about 30-fold; a half-maximal effect is obtained with 1.5 nM hormone. Epinephrine (plus theophylline, 1 mM) can raise intracellular cyclic AMP about 2-fold; the peak elevation is reached in less than 1 min and declines during the next 15 min to near the basal level. Insulin (10 nM) does not lower the basal level of cyclic AMP within the hepatocyte, but suppresses by about 50% the rise in intracellular and total cyclic AMP caused by exposure to an intermediate concentration of glucagon. No inhibition of adenylate cyclase by insulin can be shown. Basal gluconeogenesis is not significantly depressed by calcium deficiency but stimulation by glucagon is reduced by 50%. Calcium deficiency does not reduce accumulation of cyclic AMP in response to glucagon but diminishes stimulation of gluconeogenesis by exogenous cyclic AMP. Glucagon has a rapid stimulatory effect on the flux of 45Ca2+ from medium to tissue.     

Effect of epinephrine and insulin on adenosine 3'5'-cyclic monophosphate--dependent protein kinase in human skeletal muscle in vivo.

Cyclic AMP dependent protein kinase has beeen identified in human skeletal muscle tissue. In crude muscle extracts the enzyme was 3--5 fold activated by cyclic AMP. The cyclic AMP-dependent activity (corresponding to the inactive holoenzyme) was completely inhibited by the heat stable inhibitor of protein kinase. Reciprocal changes of the cyclic AMP-dependent activity in skeletal muscle were observed after administration of epinephrine and insulin in vivo. Infusion of epinephrine in healthy volunteers increased the level of cyclic AMP and decreased the activity of the cyclic AMP-depenent form (i.e. the inactive form) of protein kinase. These changes were reversible after cessation of epinephrine administration. The results are consistent with an activation of protein kinase in vivo due to an epinephrine mediated increase of the concentration of cyclic AMP. I.v. injection of insulin had the opposite effect on the enzyme in skeletal muscle, leading to increased activity of the cyclic AMP-dependent form of protein kinase. Insulin had no effect on the level of cyclic AMP, but promoted a transient increase of cyclic GMP 1 min. after insulin injection. The effect by insulin on protein kinase cannot be related to the level of cyclic AMP or cyclic GMP.     

Cyclic-AMP inhibits neither A23187-stimulated [14C]-arachidonic acid release from prelabelled lipids nor phospholipase A2 activity in resident rat peritoneal macrophages

8-Bromo cyclic AMP inhibited A23187-stimulated PGE2 production in adherent resident rat peritoneal macrophages by 50% when this was assessed by radioimmunoassay. In contrast, neither exogenous 8-bromo cyclic AMP nor elevation of endogenous cyclic AMP with cholera toxin inhibited 14C-arachidonic acid release or labelled prostaglandin formation by [1-14C]-arachidonic acid-prelabelled macrophages stimulated with either A23187 or melittin. Inhibition by cyclic AMP appears to be confined to PGE2 originating from a pool of endogenous phosphoglyceride that does not readily exchange with isotopically-labelled arachidonic acid. Phospholipase A2 activity, assessed as calcium-dependent [1-14C]-arachidonic acid release from exogenous 1-stearoyl, 2-[1-14C]-arachidonyl phosphatidyl choline at pH 8.6, was activated by melittin but not by A23187 in 1000 x g supernates from sonicated cells. Neither melittin nor calcium activation of phospholipase A2 was inhibited by preincubation of the cells prior to breakage with 8-bromo-cyclic AMP, nor by inclusion of either 8-bromo cyclic AMP or the catalytic subunit of cyclic AMP-dependent kinase in the assay. The results are inconsistent with the hypothesis that inhibition of A23187-stimulated PGE2 production by cyclic AMP in peritoneal macrophages is due to inhibition of a calcium-stimulated phospholipase A2.     

GTP-dependent inhibition of insulin secretion by epinephrine in permeabilized RINm5F cells. Lack of correlation between insulin secretion and cyclic AMP levels

The mechanism by which alpha 2-adrenergic agonists inhibit exocytosis was investigated in electrically permeabilized insulin secreting RINm5F cells. In this preparation alpha 2-adrenoceptors remain coupled to adenylate cyclase, since basal- and forskolin-stimulated cyclic AMP production was lowered by epinephrine and clonidine by 30-50%. Cyclic AMP levels did not correlate with the rate of insulin secretion. Thus, at low Ca2+, forskolin enhanced cyclic AMP levels 5-fold without eliciting secretion, and Ca2+-stimulated secretion was associated with decreased cyclic AMP accumulation. Epinephrine (plus propranolol) inhibited Ca2+-induced insulin secretion in a GTP-dependent manner. The maximal inhibition (43%) occurred at 500 microM GTP. Clonidine also inhibited Ca2+-stimulated secretion. Replacement of GTP by GDP or by the nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate as well as treatment of the cells with pertussis toxin prior to permeabilization abolished epinephrine inhibition of insulin secretion. Pertussis toxin did not affect Ca2+-stimulated secretion. Insulin release stimulated by 1,2-didecanoyl glycerol was also lowered by epinephrine suggesting an effect distal to the activation of protein kinase C (Ca2+/phospholipid-dependent enzyme). These results taken together with the ability of epinephrine to inhibit ionomycin-induced insulin secretion in intact cells suggest that alpha 2-adrenergic inhibition is distal to the generation of second messengers. A model is proposed for alpha 2-adrenoceptor coupling to two effector systems, namely the adenylate cyclase and the exocytotic site in insulin-secreting cells.     

Subcellular localization and hormone sensitivity of adipocyte cyclic AMP phosphodiesterase.

Treatment of intact adipocytes with either or both insulin and adrenaline stimulated membrane cyclic AMP phosphodiesterase activity only in the endoplasmic reticulum subfraction. The cyclic GMP-inhibited cyclic AMP phosphodiesterase activity was also found in this fraction. Quantitative Western blotting using a specific polyclonal antibody, raised against the homogeneous 'dense-vesicle' cyclic AMP phosphodiesterase from rat liver, identified a single 63 kDa species which was localized in the adipocyte endoplasmic reticulum fraction. The ability of adrenaline to stimulate adipocyte membrane cyclic AMP phosphodiesterase was shown to be mediated via beta-adrenoceptors and not alpha 1-adrenoceptors. Membrane cyclic AMP phosphodiesterase was stimulated by glucagon but not by vasopressin, A23187 or 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment of adipocytes with either chloroquine or dansyl cadaverine failed to affect the ability of insulin to stimulate cyclic AMP phosphodiesterase activity. Treatment of an isolated adipocyte endoplasmic reticulum membrane fraction with purified protein kinase A increased its cyclic AMP phosphodiesterase activity some 2-fold. When this fraction was treated with purified protein kinase A and [32P]ATP, label was incorporated into a 63 kDa protein which was specifically immunoprecipitated with the antiserum against the liver 'dense-vesicle' cyclic AMP phosphodiesterase.     

The relationship between the concentration of adenosine 3′:5′-cyclic monophosphate and the anti-lipolytic action of insulin in isolated rat fat-cells

The relationship between cyclic AMP content and lipolysis, as measured by glycerol formation, was studied in isolated rat fat-cells. Inhibition of lipolysis by insulin in the presence of a low concentration of adrenaline was accompanied by little or no lowering of cyclic AMP content, measured after 15min incubation. The time-course of cyclic AMP content after addition of adrenaline showed that the effect of insulin in lowering cyclic AMP content measured after 2-5min was gradually lost over the next hour, mainly because of the fall in cyclic AMP content after an early peak in the presence of adrenaline alone. There was a 44% loss of immunoreactive insulin, from an initial concentration of 0.3nm, during a 1h incubation with fat-cells. Insulin did not affect partitioning of cyclic AMP between cells and incubation medium. When the correlation between cyclic AMP content and rate of lipolysis was investigated for a wide range of adrenaline concentrations, it was found that the lowering of cyclic AMP content by insulin was much less than that required to account for the amount of inhibition of lipolysis. It is concluded that inhibition of adrenaline-stimulated lipolysis by insulin involves factors in addition to a decrease in intracellular cyclic AMP concentration.     

Influence of extracellular phosphate concentrations on the regulation of hepatic glucose output

Experiments were carried out to investigate the role of extracellular phosphate in the hormonal regulation of glycogenolysis in perfused fed-rat liver. Omission of phosphate from the perfusate did not affect the ATP, ADP and AMP contents of the tissue and the basal glucose output from the perfused liver. However, it inhibited significantly the glycogenolysis induced by glucagon, cyclic AMP, phenylephrine and vasopressin but not that induced by 2,4-dinitrophenol. In the absence of perfusate phosphate, the increase in phosphorylase a activity caused by the addition of glucagon, phenylephrine and vasopressin was significantly less than that observed in the presence of perfusate phosphate. Insulin inhibition of the glucagon- or cyclic AMP-induced glycogenolysis was abolished when the perfusion was carried out with the phosphate-free buffer. However, the inhibitory effect of insulin on phenylephrine-induced glycogenolysis was clearly demonstrated even when the perfusate contained no phosphate. These data indicate that in the phosphate-depleted liver, the hormonal control of phosphorylation and dephosphorylation of phosphorylase is impaired. The difference in the phosphate dependency of insulin action on glucagon-and alpha-adrenergic agonist-induced glycogenolysis suggests that the mechanism or site of insulin action on glucagon and phenylephrine is different.     

Regulation by insulin of gluconeogenesis in isolated rat hepatocytes.

Insulin (10nM) completely suppressed the stimulation of gluconeogenesis from 2 mM lactate by low concentrations of glucagon (less than or equal to 0.1 nM) or cyclic AMP (less than or equal to 10 muM), but it had no effect on the basal rate of gluconeogenesis in hepatocyctes from fed rats. The effectiveness of insulin diminished as the concentration of these agonists increased, but insulin was able to suppress by 40% the stimulation by a maximally effective concentration of epinephrine (1 muM). The response to glucagon, epinephrine, or insulin was not dependent upon protein synthesis as cycloheximide did not alter their effects. Insulin also suppressed the stimulation by isoproterenol of cyclic GMP. These data are the first demonstration of insulin antagonism to the stimulation of gluconeogenesis by catecholamines. Insulin reduced cyclic AMP levels which had been elevated by low concentrations of glucagon or by 1 muM epinephrine. This supports the hypothesis that the action of insulin to inhibit gluconeogenesis is mediated by the lowering of cyclic AMP levels. However, evidence is presented which indicates that insulin is able to suppress the stimulation of gluconeogenesis by glucagon or epinephrine under conditions where either the agonists or insulin had no measurable effect on cyclic AMP levels. Insulin reduced the glucagon stimulation of gluconeogenesis whether or not extracellular Ca2+ were present, even though insulin only lowered cyclic AMP levels in their presence. Insulin also reduced the stimulation by epinephrine plus propranolol where no significant changes in cyclic AMP were observed without or with insulin. In addition, insulin suppressed gluconeogenesis in cells that had been preincubated with epinephrine for 20 min, even though the cyclic AMP levels had returned to near basal values and were unaffected by insulin. Thus insulin may not need to lower cyclic AMP levels in order to suppress gluconeogenesis.     

Islet cyclic AMP levels are not lowered during alpha 2-adrenergic inhibition of insulin release

Epinephrine-induced changes in insulin release and cyclic AMP levels were measured simultaneously in isolated rat islets. Forskolin was used to enhance islet cyclic AMP levels. Forskolin (30 microM) stimulated adenylate cyclase activity 10-fold in islet homogenates and raised cyclic AMP levels 5-fold in intact islets (both at low and high glucose). Insulin release was enhanced by forskolin only at high glucose. Epinephrine (0.1 microM) inhibited glucose- and forskolin-induced insulin release to basal rates. At the same time epinephrine potentiated forskolin-elevated cyclic AMP levels. In contrast epinephrine attenuated forskolin-stimulated adenylate cyclase activity in islet homogenates. At low glucose, both alpha 2- and beta-adrenergic blockade counteracted the epinephrine potentiation, each by 50%. At high glucose the effect was mainly beta-adrenergic in nature. The actions of epinephrine in the presence of a beta-blocker were mimicked by the alpha 2-agonist clonidine. Despite the variations in cyclic AMP levels stimulated insulin release was always inhibited by activation of alpha 2-receptors. Finally, insulin release stimulated by exogenous cyclic AMP was abolished by epinephrine. These results suggest that epinephrine inhibits insulin release at a step distal to the generation of cyclic AMP.     

Rapid changes in the activities of the enzymes of cyclic AMP metabolism after addition of A23187 to macrophages

The ionophore A23187 stimulated adenylate cyclase activity in intact macrophages within 1 min. This action was blocked by pretreatment with indomethacin (25 μmol/l) suggesting the involvement of a prostaglandin (PG). PGE₂ (500 nmol/l) also stimulated adenylate cyclase activity in intact cells, but this was not prevented by indomethacin pretreatment. Colchicine (100 μmol/l) potentiated the increases in macrophage cyclic AMP production seen after addition of PGE₂ or A23187. The high affinity form of cyclic AMP phosphodiesterase (PDE) was activated within 1 min of the addition of A23187 to intact macrophages. The data suggest that the increase in macrophage cyclic AMP production after A23187 is a consequence of adenylate cyclase activation and not PDE inhibition. The endogenous production of a prostaglandin probably mediates this effect of A23187, emphasizing the importance of arachidonic acid metabolites in the regulation of macrophage functions.     

Correlation of the biosynthesis of prostaglandin and cyclic AMP in monolayer cultures of rabbit articular chondrocytes

We have utilized ionophores to test whether stimulation of chondrocyte prostaglandin biosynthesis is accompanied by an increase in cyclic nucleotide levels in these cells. Radioimmunoassay of prostaglandin E2, 6-oxo-prostaglandin F1 alpha (the stable metabolite of prostaglandin I2) and prostaglandin F2 alpha showed that synthesis of each was stimulated by the divalent-cation ionophore, A23187 after short-term incubation (1-7 min) in serum-free medium. No stimulation of thromboxane B2 was detected. Two monovalent ionophores, lasalocid and monensin failed to stimulate prostaglandin biosynthesis after short-term incubation. Ionophore A23187-stimulated prostaglandin biosynthesis was variably and partially inhibited by sodium meclofenamate, indomethacin and aspirin, but not by sodium salicylate. Ionophore A23187-stimulated prostaglandin biosynthesis was accompanied by a 7.5-fold increase in cyclic AMP levels after 15 min. Sodium meclofenamate, indomethacin and aspirin which inhibited prostaglandin E2 biosynthesis also reduced cyclic AMP levels. Exogenous prostaglandin E2 (1 microgram/ml) stimulated cyclic AMP biosynthesis, which was not inhibited by aspirin. These results indicated that prostaglandins can be considered as one of the local effectors controlling cyclic AMP production in articular cartilage.     

Insulin stimulates tyrosine phosphorylation and inactivation of protein-tyrosine phosphatase 1B in vivo

Protein-tyrosine phosphatase (PTP) 1B has been implicated in negative regulation of insulin action, although little is known of the ability of insulin to regulate PTP1B itself. The ability of insulin to regulate phosphorylation and activation of PTP1B was probed in vivo. Challenge with insulin in vivo provoked a transient, sharp increase in the phosphotyrosine content of PTP1B in fat and skeletal muscle that peaked within 15 min. Insulin stimulated a decline of 60--70% in PTP1B activity. In mouse adipocytes, the inhibition of PTP1B activity and increased tyrosine phosphorylation of the enzyme were blocked by the insulin receptor tyrosine kinase inhibitor AG1024. Phosphoserine content of PTP1B declined in response to insulin stimulation. Elevation of intracellular cyclic AMP provokes a sharp increase in PTP1B activity and leads to increased phosphorylation of serine residues and decreased tyrosine phosphorylation. Suppression of cyclic AMP levels or inhibition of protein kinase A leads to a sharp decline in PTP1B activity, a decrease in phosphoserine content, and an increase in PTP1B phosphotyrosine content. PTP1B appears to be a critical point for insulin and catecholamine counter-regulation.     

Synergistic activation of rat hepatocyte glycogen phosphorylase by A23187 and phorbol ester

The combination of 1.6 microM 4 beta phorbol, 12 beta myristate, 13 alpha acetate (PMA) and 1 microM A23187 produced a five-fold greater stimulation of rat hepatocyte glycogen phosphorylase activity than was seen with PMA alone. Vasopressin activation of glycogen phosphorylase was comparable to that seen with PMA plus A23187. Glycogen phosphorylase activity due to PMA plus A23187 was increased significantly after 30 sec, maximal at 120 and sustained at elevated levels for 240 sec. In contrast, activation due to vasopressin was maximal at 30 sec followed by a decrease. The addition of PMA 5 min prior to the A23187 abolished the synergism between these two agents. These data are compatible with the hypothesis that diacylglycerol and Ca2+ synergistically increase glycogen phosphorylase activity in rat hepatocytes.     

Protein kinase C enhances growth hormone releasing factor (1-40)-stimulated cyclic AMP levels in anterior pituitary. Actions of somatostatin and pertussis toxin

The brain peptide human growth hormone releasing factor (1-40) (GRF), which stimulates adenylate cyclase activity in the anterior pituitary, is the predominant hormone signal for pituitary growth hormone (GH) release. Activators of protein kinase C such as teleocidin and 4 beta-phorbol 12-myristate 13-acetate (PMA) double the cyclic AMP accumulation induced by GRF, with no apparent effect on GRF potency; an inactive 4-alpha-PMA has no such action in cultured anterior pituitary cells. This PMA potentiation can be measured as early as 60 s, is maximal by 15 min, and wanes such that by 3-4 h there is no such amplifying effect of PMA. PMA, phorbol 12,13-dibutyrate, and teleocidin ED50 values for potentiating GRF activity are similar to those obtained for direct protein kinase C activation. The major inhibitory peptide somatostatin reduced both GRF- and GRF + PMA-stimulated cyclic AMP accumulation. Pertussis toxin totally blocked this somatostatin action without affecting the degree of maximal GRF potentiation achieved with PMA. Thus, the pertussis toxin target(s) are required for somatostatin inhibition of the cyclic AMP generating system, but may not be involved in the PMA potentiation of GRF-stimulated cyclic AMP accumulation.