Effects of the specific cAMP antagonist, (Rp)-adenosine cyclic 3',5'-phosphorothioate, on the cAMP-dependent protein kinase-induced activity of hepatic glycogen phosphorylase and glycogen synthase

The cAMP-dependent protein kinase-induced effects on phosphorylase and glycogen synthase activities and glucose production were studied in hepatocytes isolated from fed rats in the presence of the diastereomers of adenosine cyclic 3',5'-phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS. Incubation of hepatocytes with (Sp)-cAMPS or glucagon, both of which lead to cAMP-dependent protein kinase activation, resulted in a concentration-dependent increase in glycogen phosphorylase activity and a decrease in glycogen synthase activity. Incubation of hepatocytes with the cAMP-dependent protein kinase antagonist, (Rp)-cAMPS, in the absence of an agonist, had no significant effect on phosphorylase or glycogen synthase activities. Incubation of hepatocytes with a half-maximally inhibitory concentration of (Rp)-cAMPS shifted the agonist-induced activation curves for phosphorylase and the agonist-induced inhibition curves for glycogen synthase to 5-fold higher concentrations for both (Sp)-cAMPS and glucagon. Phosphorylase activity was very sensitive to the rapid, concentration-dependent inhibition by (Rp)-cAMPS of agonist-induced activation of cAMP-dependent protein kinase. The effects on phosphorylase activity were observable in 30 s and were concentration-dependent with half-maximal inhibition at 10 microM, similar to that observed for cAMP-dependent protein kinase. In contrast, glycogen synthase activity was less sensitive to (Rp)-cAMPS inhibition of agonist-induced activation of cAMP-dependent protein kinase. The effects on glycogen synthase activity lagged behind those on phosphorylase activity and the concentration dependence did not parallel the cAMP-dependent protein kinase effect, but was shifted to higher concentrations of (Rp)-cAMPS with half-maximal inhibition at 60 microM. Glucose (10 to 40 mM) increased the sensitivity of glycogen synthase to (Rp)-cAMPS inhibition of cAMP-dependent protein kinase over a narrow range of agonist concentration, but had no significant effect throughout most of the agonist-induced activation range. Thus, the diastereomers, (Sp)- and (Rp)-cAMPS, influence glycogen metabolism and the glycogenolytic enzymes through their modulation of cAMP-dependent protein kinase levels.     

Synergistic inhibition of hepatic glycogenolysis in the presence of insulin and a cAMP antagonist

The effects of insulin on the ability of the specific intracellular cAMP-dependent protein kinase antagonist, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate, to inhibit glycogenolysis induced by the Sp diastereomer was studied in hepatocytes isolated from fed rats. Addition of the cAMP agonist, (Sp)-cAMPS, to hepatocytes resulted in a concentration-dependent increase in glycogenolytic glucose production concomitant with the cAMP-dependent activation of phosphorylase and inhibition of glycogen synthase. Activity curves were shifted to the right in the presence of the cAMP antagonist, (Rp)-cAMPS. Preincubation of the hepatocytes with a maximally effective concentration of insulin did not affect the concentration of (Sp)-cAMPS required for half-maximal activation of phosphorylase but did result in a 10-fold shift in the concentration of (Sp)-cAMPS required for half-maximal inactivation of glycogen synthase. Preincubation of hepatocytes with a combination of the cAMP antagonist, (Rp)-cAMPS, and insulin resulted in synergistic inhibition of (Sp)-cAMPS-induced phosphorylase activation, glycogen synthase inactivation, and glycogenolytic glucose production. Since neither phosphorothioate diastereomer was hydrolyzed significantly during the course of the experiments, the synergistic effects of insulin are postulated to be working through a mechanism subsequent to the phosphodiesterase activation step.     

Synergistic inhibition of glucagon-induced effects on hepatic glucose metabolism in the presence of insulin and a cAMP antagonist

Inhibition of hepatic glycogenolysis by an intracellular inhibitor of cAMP-dependent protein kinase in glucagon-stimulated hepatocytes was potentiated by insulin. When hepatocytes isolated from fed rats were treated with 0.3 nM glucagon, which activates glycogen breakdown half-maximally, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate [Rp-cAMPS), a cAMP antagonist, inhibited glucose production half-maximally at 3 microM. A 10-fold lower concentration of antagonist was required to half-maximally inhibit glucose production in the presence of 10 nM insulin, which alone produced only 15% inhibition. Under the same experimental conditions, the maximal effect of (Rp)-cAMPS was also potentiated. In addition, the increase in the concentration of glucagon required for half-maximal activation of phosphorylase activity and inactivation of glycogen synthase activity in the presence of minimally effective concentrations of insulin and (Rp)-cAMPS were clearly synergistic. It is postulated that the synergism observed is a consequence of action at several enzymatic sites leading to, and including, alteration of the phosphorylation state of the two rate-limiting enzymes in glycogen metabolism.     

Identifying the molecular switches that determine whether (Rp)-cAMPS functions as an antagonist or an agonist in the activation of cAMP-dependent protein kinase I.

Previous investigations revealed that under physiological conditions in the presence of MgATP the phosphorothioate analogue of cAMP, (Rp)-cAMPS, is a competitive inhibitor and antagonist for cAMP for cAMP-dependent protein kinases I and II [DeWit et al., (1984) Eur. J. Biochem. 142, 255-260]. For the type I holoenzyme, the antagonist properties of (Rp)-cAMPS are shown here to be absolutely dependent on MgATP. In the absence of MgATP, (Rp)-cAMPS serves as a weak agonist with a Ka of 7.9 microM. The high-affinity binding of MgATP imposes a barrier on cAMP-induced activation of the homoenzyme--a barrier that both cAMP and (Sp)-cAMPS, but not (Rp)-cAMPS, can overcome. In the absence of MgATP, this barrier no longer exists, and (Rp)-cAMPS functions as an agonist. The holoenzyme also was formed with mutant regulatory subunits. Replacing the essential arginine, predicted to bind the exocyclic oxygens of cAMP, in site A with lysine abolishes high-affinity binding of cAMP to site A. The holoenzyme formed with this mutant R-subunit is activated by (Rp)-cAMPS in both the presence and absence of MgATP. These results suggest that the stereospecific requirements for holoenzyme activation involve this guanidinium side chain. Mutations that eliminate the high-affinity binding of MgATP, such as the introduction of an autophosphorylation site in the autoinhibitory domain, also generate a holoenzyme that can be activated by (Rp)-cAMPS. In the case of the type II holoenzyme, (Rp)-cAMPS is an antagonist in both the presence and absence of MgATP, emphasizing distinct roles for MgATP in these two forms of cAMP-dependent protein kinase.     

Inhibition of choriogonadotropin-activated steroidogenesis in cultured Leydig tumor cells by the Rp diastereoisomer of adenosine 3',5'-cyclic phosphorothioate

The diastereoisomers of adenosine 3',5'-cyclic phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS, have been previously shown to act as agonists and antagonists, respectively, in the activation of several mammalian cAMP-dependent protein kinases. In an effort to characterize further the involvement of cAMP in the activation of Leydig cell steroidogenesis by lutropin/choriogonadotropin (LH/CG), we examined the effects of these cyclic nucleotide analogues on a clonal strain of cultured murine Leydig tumor cells (designated MA-10). Our results show that (i) (Sp)-cAMPS activates and (Rp)-cAMPS inhibits the isolated cAMP-dependent protein kinase of the MA-10 cells; (ii) both analogues inhibit the isolated cAMP phosphodiesterase(s); (iii) (Sp)-cAMPS activates steroid biosynthesis in intact cells, but (Rp)-cAMPS does not; and (iv) (Rp)-cAMPS is a competitive inhibitor of the activation of steroidogenesis by (Sp)-cAMPS, 8-bromo-cAMP, human CG, cholera toxin, and forskolin. However, (Rp)-cAMPS is a more effective inhibitor when steroidogenesis is activated by (Sp)-cAMPS or 8-bromo-cAMP than when it is activated by human CG, cholera toxin, or forskolin. This difference appears to be related to the combined effects of (Rp)-cAMPS on the cAMP-dependent protein kinases and cAMP phosphodiesterase(s). We conclude that cAMP is a quantitatively important mediator of the activation of steroidogenesis by LH/CG even at low concentrations of hormone where an increase in steroid biosynthesis cannot be easily correlated with increased cAMP accumulation. Thus, our data indicate that if other second messengers are involved in the activation of steroidogenesis by LH/CG, they must do so by acting together with, rather than independently of, cAMP.     

Enzymatic synthesis of the cAMP antagonist (Rp)-adenosine 3',5'-monophosphorothioate on a preparative scale

(Rp)-Adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) is a highly specific antagonist of the cAMP-dependent protein kinase from eukaryotic cells and is a very poor substrate for phosphodiesterases. It is therefore a useful tool for investigating the role of cAMP as a second messenger in a variety of biological systems. Taking advantage of stereospecific inversion of configuration around the alpha-phosphate during the adenylate cyclase reaction, we have developed a method for the preparative enzymatic synthesis of the Rp diastereomer of adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) from the Sp diastereomer of adenosine 5'-O-(1-thiotriphosphate) ((Sp)-ATP alpha S). The adenylate cyclase from Bordetella pertussis, partially purified by calmodulin affinity chromatography, cyclizes (Sp)-ATP alpha S approximately 40-fold more slowly than ATP, but binds (Sp)-ATP alpha S with about 10-fold higher affinity than ATP. The triethylammonium salt of the reaction product can be purified by elution from a gravity flow reversed-phase C18 column with a linear gradient of increasing concentrations of methanol. Yields of the pure (Rp)-cAMPS product of a synthesis with 2 mg of substrate are about 75%.     

Inhibition of glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate

The diastereomeric forms of adenosine cyclic 3',5'-phosphorothioate, Rp cAMPS and Sp cAMPS, were studied in isolated hepatocytes from fed rats for their ability to interact with the intracellular cAMP-dependent protein kinase and to affect the phosphorylase kinase-phosphorylase glycogenolytic cascade. Incubation of the cells with increasing concentrations of Sp cAMPS produced a concentration-dependent activation of cAMP-dependent protein kinase with a concomitant increase in the glycogenolytic rate. Half-maximal and maximal velocities of glycogenolysis were reached at 8 X 10(-7) and 1 X 10(-5) M Sp cAMPS, respectively. Incubation of the cells with 10(-9) to 10(-4) M Rp cAMPS had no effect on basal glucose production or on cAMP-dependent protein kinase activity. Incubation of the cells simultaneously with 3 X 10(-6) M Sp cAMPS and increasing concentrations of Rp cAMPS produced half-maximal inhibition of glycogenolysis at 1 X 10(-5) M Rp cAMPS and maximal inhibition at 1 X 10(-4) M. The concentrations of Sp cAMPS required for half-maximal and maximal activation of glycogenolysis were increased 10-fold when 1 X 10(-5) M Rp cAMPS was present. These data imply that Sp cAMPS is a cAMP-agonist while Rp cAMPS is a cAMP-antagonist.     

Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate

The effects of submaximal doses of AlF4- to mobilize hepatocyte Ca2+ were potentiated by glucagon (0.1-1 nM) and 8-p-chlorophenylthio-cAMP. A similar potentiation by glucagon of submaximal doses of vasopressin, angiotensin II, and alpha 1-adrenergic agonists has been previously shown (Morgan, N. G., Charest, R., Blackmore, P. F., and Exton, J. H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4208-4212). When hepatocytes were pretreated with the protein kinase C activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), the effects of AlF4- to mobilize Ca2+, increase myo-inositol 1,4,5-trisphosphate (IP3), and activate phosphorylase were attenuated. Treatment of hepatocytes with PMA likewise inhibits the ability of vasopressin, angiotensin II, and alpha 1-adrenergic agonists to increase IP3 and mobilize Ca2+ (Lynch, C. J., Charest, R., Bocckino, S. B., Exton, J. H., and Blackmore, P. F. (1985) J. Biol. Chem. 260, 2844-2851). In contrast, the ability of AlF4- or angiotensin II to lower cAMP or inhibit glucagon-mediated increases in cAMP was unaffected by PMA. The ability of AlF4- to lower cAMP was attenuated in hepatocytes from animals treated with islet-activating protein, whereas Ca2+ mobilization was not modified. These results suggest that the lowering of cAMP induced by AlF4- and angiotensin II was mediated by the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase, whereas Ca2+ mobilization was not. Addition of glucagon, forskolin, or 8CPT-cAMP to hepatocytes raised IP3 and mobilized Ca2+. Both effects were blocked by PMA pretreatment, whereas cAMP and phosphorylase a levels were only minimally affected by PMA. The mobilization of Ca2+ induced by cAMP in hepatocytes incubated in low Ca2+ media was not additive with that induced by maximally effective doses of vasopressin, angiotensin II, or alpha 1-adrenergic agonists, indicating that the Ca2+ pool(s) affected by agents which increase cAMP is the same as that affected by Ca2+-mobilizing hormones which do not increase cAMP. These findings support the proposal that AlF4- mimics the effects of the Ca2+-mobilizing hormones in hepatocytes by activating a guanine nucleotide-binding regulatory protein (Np) which couples the hormone receptors to a phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphodiesterase. They also suggest that Np, PIP2 phosphodiesterase, or a factor involved in their interaction is activated following phosphorylation by cAMP-dependent protein kinase and inhibited after phosphorylation by protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of glucagon-induced glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate

The ability of the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp cAMPS) to inhibit glucagon-induced glycogenolysis was studied in hepatocytes isolated from fed rats. Preincubation of the cells for 20 min with progressively higher concentrations of Rp cAMPS followed by a 1 X 10(-9) M glucagon challenge resulted in a 50% inhibition of glucose production over a 30-min period at 2-3 X 10(-6) M Rp cAMPS. A maximal inhibition of 50-74% was achieved, the actual value depending upon the length of preincubation with Rp cAMPS. The inhibitory effect did not increase when the concentration of Rp cAMPS was increased from 3 X 10(-6) to 3 X 10(-4) M. Addition of 1 X 10(-5) M Rp cAMPS to the cells followed by 10(-11) to 10(-6) M glucagon shifted the glucagon concentration required for half-maximal glucose production measured at 10 min to 6-fold higher glucagon concentrations and the concentration of glucagon required for apparent maximal glucose production measured at 10 min to greater than 10-fold higher glucagon concentrations. The cAMP-dependent protein kinase activation curve was similarly shifted to higher concentrations of glucagon. These data show that Rp cAMPS acts as a cAMP antagonist capable of opposing the glucagon-induced activation of cAMP-dependent protein kinase and the concomitant activation of the glycogenolytic cascade.     

Starfish oocyte maturation: evidence for a cyclic AMP-dependent inhibitory pathway

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Cyclic AMP seems to play a negative role in maturation since 1-MeAde triggers a decrease of the oocyte cAMP concentration and since intracellular microinjections of cAMP delay or inhibit maturation. Cyclic GMP is also inhibitory but other nucleotides such as cCMP, cIMP, and cUMP are inactive. The involvement of cAMP and cGMP in the control of oocyte maturation has been further investigated by the use of the stereoisomers of the phosphodiesterase-stable adenosine and guanosine 3',5'-phosphorothioates (cAMPS and cGMPS). The Sp isomers of cAMPS and cGMPS respectively activate cAMP-dependent protein kinase and cGMP-dependent kinase, while the Rp isomers inhibit the kinases. Extracellular addition of these cAMPS and cGMPS isomers has no effect on the oocytes. Intracellular microinjection of the kinase-activating (Sp)-cAMPS and (Sp)-cGMPS delays or inhibits 1-MeAde-induced maturation in a concentration-dependent manner (I50, 30 and 300 microM, respectively). Microinjections of (Rp)-cAMPS and (Rp)-cGMPS have no inhibitory effects and neither trigger nor facilitate maturation. Using various analogs, we found that the delaying or inhibiting effect is restricted to the compounds activating cAMP-dependent kinase, while the compounds inactive on or inhibiting the kinase have no effects on maturation. The inhibitory effect of (Sp)-cAMPS can be reversed by comicroinjection of the heat-stable inhibitor of cAMP-dependent protein kinase, by comicroinjection of the antagonist (Rp)-cAMPS, or by an increase in the 1-MeAde concentration. The negative effects of (Sp)-cAMPS or (Sp)-cGMPS are observed only when these isomers are microinjected during the hormone-dependent period. These results suggest that a cAMP-dependent inhibitory pathway participates in the maintenance of the prophase arrest of oocytes and that 1-MeAde acts both by inhibiting this negative pathway (dis-inhibitory pathway) and by stimulating a parallel activatory pathway leading to oocyte maturation. The generality of this mechanism is discussed.     

Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP-dependent protein kinases

A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP-binding sites in mammalian protein kinase type 1. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that cAMP is bound to both sites in a comparable manner: the adenine appears to have no hydrogen-bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2', 3' and 5' positions of cAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation. The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocyclic oxygen atoms may result in a reduced maximal activation level and in one case, (Rp)-adenosine 3', 5'-monophosphorothioate [Rp-cAMPS], in total absence of activation even at concentrations at which the analog saturates both binding sites. Since occupancy of the cAMP-binding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize the activation by cAMP. Indeed (Rp)-cAMPS was found to inhibit cAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type II (Rp)-cAMPS showed antagonistic activity, while with a cAMP-dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed. Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et. al. (1982) Eur. J. Biochem 122, 95-99]. This was based on measurements with impure preparations of (Rp)-cAMPS and the Rp and Sp isomers adenosine 3', 5'-monophosphodimethylamidate. cAMPN(CH3)2. The present work using highly purified compounds suggests the absence of a charge interaction, since the uncharged analog (Sp)-cAMPN(CH3)2 activates the kinase effectively. The data seem compatible with an activation model involving the formation of a covalent bond with phosphorus in both cAMP binding sites.     

Evidence for the activation of the multifunctional Ca2+/calmodulin-dependent protein kinase in response to hormones that increase intracellular Ca2+

The phosphorylation state of six cytoplasmic proteins is increased following treatment of isolated rat hepatocytes with hormones that elevate free intracellular Ca2+ levels (Garrison, J. C. and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Tryptic 32P-phosphopeptide maps of two of the substrates, pyruvate kinase and a 49,000-dalton protein, the major 32P-labeled protein in hepatocytes, were prepared following stimulation of cells with vasopressin, a Ca2+-linked hormone. Peptide maps of the 49,000-dalton protein phosphorylated in vitro with the recently identified multifunctional Ca2+/calmodulin-dependent protein kinase contained phosphopeptides identical to those observed in the intact cell, suggesting that this kinase is activated in response to Ca2+-mobilizing hormones. Similar in vitro phosphorylation experiments with pyruvate kinase suggested that the Ca2+/calmodulin-dependent protein kinase can phosphorylate not only the serine residues observed following vasopressin stimulation of the intact cell but also additional threonine residues. Both pyruvate kinase and the 49,000-dalton protein are also phosphorylated in the hepatocyte in response to glucagon and in vitro by the cAMP-dependent protein kinase. Both vasopressin and glucagon appear to stimulate the phosphorylation of identical serine residues in pyruvate kinase but only vasopressin enhances the phosphorylation of certain sites in the 49,000-dalton protein. Comparison of the tryptic phosphopeptide maps of these substrates phosphorylated in vitro with either the Ca2+/calmodulin-dependent protein kinase or the cAMP-dependent protein kinase suggests that the Ca2+-dependent kinase can phosphorylate unique sites in both substrates. It appears to share specificity at other sites with the cAMP-dependent protein kinase. Overall, the results suggest that the multifunctional Ca2+/calmodulin-dependent protein kinase plays an important role in the response of the hepatocyte to a Ca2+ signal.     

In Vitro Phosphorylation of Microtubule-Associated Protein 2: Differential Effects of Cyclic AMP Analogues

Microtubules purified from brain tissue contain endogenous cyclic AMP (cAMP)-dependent protein kinase activity, and microtubule-associated protein 2 (MAP2) is the major substrate. Beef brain microtubules were prepared and used as a model system to study the differential effects of rationally selected cyclic nucleotide analogues on microtubule receptor protein kinase. Data are presented to indicate that the following molecular interactions are essential for activation of the phosphorylation of MAP2: (a) hydrogen bond formation toward the 2', 3', or 5' position, (b) interaction with phosphorus, and (c) no hydrogen bonds but hydrophobic interactions at the base moiety. Thus, the activation mechanism of the type II protein kinase associated with brain microtubules resembles the mechanism found in protein kinases of other systems. In addition, we have studied the effect of the two diastereomers of adenosine 3',5'-monophosphorothioate (cAMPS). The (Sp)-cAMPS isomer was found to activate MAP2 protein kinase, whereas the (Rp)-cAMPS isomer had no activating effect. In contrast, this compound was able to inhibit cAMP-stimulated MAP2 phosphorylation and thus acts as an antagonist of the Sp diastereomer and cAMP. Hence, this analogue provides a useful means to clarify further the effect of cAMP-dependent phosphorylation on functional properties in microtubules in general.     

The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells

Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.     

Glucagon-stimulated phosphorylation of rat liver glycogen synthase in isolated hepatocytes

Addition of glucagon (20 nM) to the isolated hepatocytes from 24-h starved male rats results in an inactivation of glycogen synthase. The A0.5 for glucose-6-P is increased 2-fold over the control but the S0.5 for UDP-glucose is not significantly affected. The glucagon-stimulated inactivation of glycogen synthase is also accompanied by a 60-120% increase in the phosphorylation of the synthase. Glycogen synthase labeled with 32P by incubation of the hepatocytes with [32P] PO4(3-) was recovered by immunoprecipitation and the resulting immunoprecipitate was subjected to tryptic digestion. Analysis of the 32P-labeled peptides reveals that the sites corresponding to those phosphorylated by cAMP-dependent protein kinase and glycogen synthase (casein) kinase-1 (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057) are rapidly phosphorylated in response to glucagon. These results demonstrate that glucagon not only triggers the activation of cAMP-dependent protein kinase through an increase in the intracellular level of cAMP but also, by an unknown mechanism, activates a Ca2+- and cAMP-independent protein kinase.     

Adenosine 3', 5'-monophosphorothioate (Rp-isomer) induces down-regulation of surface cyclic AMP receptors without receptor activation in Dictyostelium discoideum

cAMP induces the activation and subsequent desensitization of adenylate cyclase in Dictyostelium discoideum. cAMP also induces down-regulation of surface cAMP receptors. Desensitization of adenylate cyclase is composed of a rapidly reversible component (adaptation) and a slowly reversible component related to down-regulation of surface cAMP receptors (Van Haastert, P.J.M. (1987) J. Biol. Chem. 262, 7700-7704). The agonistic and antagonistic activities of the cAMP derivative adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) for these responses were investigated. (Rp)-cAMPS competes with cAMP for binding to different receptor forms with an apparent Ki = 5 microM. (Rp)-cAMPS does not activate adenylate cyclase and antagonizes the cAMP-induced activation with an apparent Ki = 5 microM. (Rp)-cAMPS induces down-regulation of surface cAMP receptors with EC50 = 5 microM. (Rp)-cAMPS induces desensitization of adenylate cyclase, which is not rapidly reversible. These results indicate that desensitization of adenylate cyclase by (Rp)-cAMPS is due to down-regulation of surface cAMP receptors and not to adaptation. We conclude that down-regulation of surface cAMP receptors does not require their activation or modification involved in adaptation.     

Correlation between cAMP, activation of cAMP-dependent protein kinase(s), and rate of glycogenolysis in isolated rat hepatocytes.

We have studied the correlation between cAMP-dependent protein kinase activation and rates of glycogenolysis in hepatocytes isolated from fed rats. With doses of 20 μM glucagon, the protein kinase was activated to a -cAMP/+cAMP ratio of 0.8 within 10 min and remained activated for up to 2 hours. A dose-response relationship between protein kinase activation and rates of glycogenolysis can be demonstrated to 0–20 μM glucagon. Glycogenolysis was stimulated greater than 2-fold after 2 hours of incubation with the higher doses of glucagon. Protein kinase activity ratios correlated well with the rates of glycogenolysis as the ratios varied from control levels of about 0.25 to the stimulated values of 0.5–0.6. However, as the ratios increased from 0.6 to 0.8, with higher doses of glucagon, there were no corresponding increases in the rates of glycogenolysis. These data may indicate (1) that activation of all of the protein kinase present in the liver cells is not necessary for maximal stimulation of glycogenolysis, or (2) that a specific protein kinase is involved in the intracellular control of glycogen breakdown in isolated rat hepatocytes.     

Modulation of Ca2+ fluxes in isolated platelet vesicles: effects of cAMP-dependent protein kinase and protein kinase inhibitor on Ca2+ sequestration and release

In the present study, we investigated the role of cAMP-dependent protein kinase in the process of Ca2+ uptake and release from platelet-derived membrane vesicles enriched in the dense tubular system. It was found that these membrane vesicles contain endogenous cAMP-dependent protein kinase and that stimulation of protein kinase by cAMP resulted in the phosphorylation of a single protein band (22 kDa). Addition of cAMP-dependent protein kinase produced effects on vesicle Ca2+ accumulation which were dependent on the Ca2+ concentration in the incubation medium. Specifically, at low extravesicular Ca2+ concentrations, cAMP-dependent protein kinase (10-100 micrograms/ml) produced a dose-dependent stimulation of Ca2+ uptake, however, a similar stimulation was not observed at high extravesicular Ca2+ concentrations. When endogenous protein kinase was blocked by the addition of protein kinase inhibitor, (2-160 nM) there was a dose-dependent inhibition of Ca2+ uptake at both low and high concentrations of extravesicular Ca2+. Furthermore, the addition of protein kinase inhibitor at steady state caused a rapid and dose-dependent release of vesicle-accumulated Ca2+. Studies on the phosphorylation profile of vesicle protein indicated that protein kinase inhibitor (80 and 160 nM) was capable of inhibiting the phosphorylation of the 22-kDa protein within 15 s. Finally, the ability of thromboxane A2 to cause Ca2+ release was inhibited by the addition of cAMP-dependent protein kinase (1 mg/ml). These findings suggest that cAMP-dependent protein kinase is not only a major determinant in the accumulation of Ca2+ by the dense tubular system, but may play an important role in the process of intraplatelet Ca2+ release by physiologic agents such as thromboxane A2.     

Inhibition of cGMP-dependent protein kinase by (Rp)-guanosine 3',5'-monophosphorothioates

The activation of the cGMP-dependent protein kinase and cAMP-dependent protein kinase by the diastereomers of guanosine 3',5'-monophosphorothioate, (Sp)-cGMPS and (Rp)-cGMPS, and 8-chloroguanosine 3',5'-monophosphorothioate, (Sp)-8-Cl-cGMPS and (Rp)-8-Cl-cGMPS, was investigated using the peptide Kemptide as substrate. The (Sp)-diastereomers, which have an axial exocyclic sulfur atom, bound to the cGMP-dependent protein kinase and stimulated its phosphotransferase activity. In contrast, the (Rp)-isomers, which have an equatorial exocyclic sulfur atom, bound to the enzyme without stimulation of its activity. (Rp)-cGMPS and (Rp)-8-Cl-cGMPS antagonized the activation of the cGMP-dependent protein kinase with a Ki of 20 microM and 1.5 microM, respectively. (Rp)-cGMPS also antagonized the activation of cAMP-dependent protein kinase with a Ki of 20 microM. In contrast, (Rp)-8-cGMPS ws a weak inhibitor of the cAMP-dependent protein kinase with a Ki of 100 microM. (Rp)-8-Cl-cGMPS appears to be a rather selective inhibitor of the cGMP-dependent protein kinase and may be a useful tool for studying the role of cGMP in broken and intact cell systems.     

Posttranslational modification of hepatic cytochrome P-450. Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIB1) and PB-5 (IIB2) in isolated rat hepatocytes and in vivo

Phosphorylation of hepatic cytochrome P-450 was studied in isolated hepatocytes incubated in the presence of agents known to stimulate protein kinase activity. Incubation of hepatocytes isolated from phenobarbital-induced adult male rats with [32P]orthophosphate in the presence of N6,O2'-dibutyryl-cAMP (diBtcAMP) or glucagon resulted in the phosphorylation of microsomal proteins that are immunoprecipitable by polyclonal antibodies raised to the phenobarbital-inducible P-450 form PB-4 (P-450 gene IIB1). Little or no phosphorylation of these proteins was observed in the absence of diBtcAMP or glucagon or in the presence of activators of Ca2+-dependent protein kinases. Two-dimensional gel electrophoresis revealed that these 32P-labeled microsomal proteins consist of a mixture of P-450 PB-4 and the closely related P-450 PB-5 (gene IIB2), both of which exhibited heterogeneity in the isoelectric focusing dimension. Phosphorylation of both P-450 forms was markedly enhanced by diBtcAMP at concentrations as low as 5 microM. In contrast, little or no phosphorylation of P-450 forms reactive with antibodies to P-450 PB-1 (gene IIC6), P-450 2c (gene IIC11), or P-450 PB-2a (gene IIIA1) was detected in the isolated hepatocytes under these incubation conditions. Phosphoamino acid analysis of the 32P-labeled P-450 PB-4 + PB-5 immunoprecipitate revealed that these P-450s are phosphorylated on serine in the isolated hepatocytes. Peptide mapping indicated that the site of phosphorylation in hepatocytes is indistinguishable from the site utilized by cAMP-dependent protein kinase in vitro, which was previously identified as serine-128 for the related rabbit protein P-450 LM2.(ABSTRACT TRUNCATED AT 250 WORDS)