No Role for Phospholipase A2 and Protein Kinase C in the Potentiation by α-Adrenoceptors of β-Adrenoceptor-Mediated Cyclic AMP Formation in Rat Brain

This study was undertaken to examine the role of phospholipase A2 and protein kinase C in the potentiation of beta-adrenoceptor-mediated cyclic AMP formation by alpha-adrenoceptors in rat cerebral cortical slices. Inhibition of arachidonic acid metabolism by a range of cyclooxygenase and lipoxygenase inhibitors had no effect on the potentiation of isoprenaline-stimulated cyclic AMP. Conversely, stimulation of leukotriene formation had no effect on the response to isoprenaline. The phospholipase A2 activator, melittin, stimulated cyclic AMP and potentiated the effect of isoprenaline, but these responses were not influenced by cyclooxygenase or lipoxygenase inhibitors. Indomethacin was also ineffective against the potentiation of vasoactive intestinal peptide-stimulated cyclic AMP by noradrenaline. Phorbol ester potentiated the cyclic AMP response to isoprenaline, and this potentiation was antagonized by three different putative protein kinase C inhibitors. However, the same inhibitors did not affect the alpha-adrenoceptor-stimulated enhancement of the response to isoprenaline. We have found no evidence, therefore, to support the suggestion that arachidonic acid and its metabolites and/or protein kinase C mediate the alpha-adrenoceptor modulation of beta-adrenoceptor function.     

An Examination of the Involvement of Phospholipases A2 and C in the α-Adrenergic and γ-Aminobutyric Acid Receptor Modulation of Cyclic AMP Accumulation in Rat Brain Slices

Experiments were undertaken to define the role of two calcium-associated enzyme systems in modulating transmitter-stimulated production of cyclic nucleotides in rat brain. Cyclic AMP (cAMP) accumulation was examined in cerebral cortical slices using a prelabeling technique. The enhancement of isoproterenol-stimulated cAMP production by alpha-adrenergic and gamma-aminobutyric acid-B (GABAB) agonists was reduced by exposing the tissue to EGTA, a chelator of divalent cations, or quinacrine, a nonselective inhibitor of phospholipase A2. Likewise, chronic (2 weeks) administration of corticosterone decreased the alpha-adrenergic and GABAB receptor modulation of second messenger production. Neither cyclooxygenase nor lipoxygenase inhibitors selectively influenced the facilitating response of alpha-adrenergic and GABAB agonists. Other experiments revealed that although norepinephrine and 6-fluoronorepinephrine stimulated inositol phosphate (IP) production in cerebral cortical slices with potencies equal to those displayed in the cyclic nucleotide assay, selective alpha 1-adrenergic agonists were less efficacious on IP formation and were without effect in the cAMP assay. Conversely, a selective alpha 2-adrenergic receptor agonist facilitated the cAMP response to a beta-adrenergic agonist without affecting IP formation. The rank orders of potency of a series of alpha-adrenergic antagonists suggest that IP accumulation is mediated solely by alpha 1-adrenergic receptors, whereas the augmentation of cAMP accumulation is regulated by a mixed population of alpha-adrenergic sites. The results suggest that the alpha-adrenergic and GABAB receptor-mediated enhancement of isoproterenol-stimulated cAMP formation appears to be more closely associated with phospholipase A2 than phospholipase C and may be mediated by arachidonate or some other fatty acid.     

Accumulation of Cyclic AMP Elicited by Vasoactive Intestinal Peptide Is Potentiated by Noradrenaline, Histamine, Adenosine, Baclofen, Phorbol Esters, and Ouabain in Mouse Cerebral Cortical Slices: Studies on the Role of Arachidonic Acid Metabolites and Protein Kinase C

In mouse cerebral cortical slices, noradrenaline (NA) potentiates cyclic AMP (cAMP) accumulation elicited by vasoactive intestinal peptide (VIP) through alpha 1-adrenergic receptors. This synergism is inhibited by indomethacin, and the prostaglandins E2 and F2 alpha mimic the effect of NA. In the present study, we observed that the synergism between VIP and NA is not inhibited by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) or the diacylglycerol-lipase inhibitor RHC 80267, thus further stressing the role of phospholipase A2 activation. Various neuroactive agents that potentiate the stimulatory effect of VIP on cAMP formation were also examined. As with NA, the potentiation by histamine and adenosine is inhibited by indomethacin. In contrast to NA, histamine, and adenosine, the synergistic interaction between phorbol esters and VIP on cAMP formation is abolished by H-7 but not by indomethacin. The potentiation by baclofen, a gamma-aminobutyric acidB receptor agonist, is partially inhibited by the 5-lipoxygenase inhibitor nafazatrom. The synergism between ouabain and VIP is reduced by H-7 but not by indomethacin and nafazatrom. These data indicate that the stimulation of cAMP formation elicited by VIP is under the modulation of various neuroactive agents that trigger diverse intracellular mechanisms to potentiate the effect of the peptide.     

Down-Regulation of Pinealocyte Protein Kinase C: Effect on α1-Adrenergic Potentiation of β-Adrenoceptor Stimulation of Cyclic AMP Accumulation and Induction of Serotonin N-Acetyltransferase Activity

Treatment of rat pinealocytes with 4 beta-phorbol 12,13-dibutyrate down-regulated protein kinase C (PKC) activity. Loss of activity was concentration-dependent (50% loss at 8 x 10(-7) M after 18 h of treatment) and time-dependent (50% loss after 2 h with 3 x 10(-6) M). Phenylephrine, an alpha 1-adrenergic agonist, and phorbol esters unable to activate PKC did not down-regulate the enzyme. alpha 1-Adrenergic amplification of beta-adrenergic stimulation of cyclic AMP accumulation, a response previously shown to be mediated by PKC activation, was reduced by only 50% in cells in which PKC activity was down-regulated by approximately 95%. These data suggest that there is not a simple proportional relationship between the degree of activation of pinealocyte PKC and the alpha 1-adrenergic amplification of beta-adrenergic cyclic AMP synthesis. In down-regulated cells, alpha 1-adrenergic amplification of beta-adrenergic induction of serotonin N-acetyltransferase activity, a key cyclic AMP-responsive enzyme involved in the nocturnal synthesis of the pineal hormone melatonin, was unchanged. Thus, even though alpha 1-adrenergic amplification of cyclic AMP synthesis is impaired, sufficient cyclic AMP is generated to allow a full induction of serotonin N-acetyltransferase activity. This finding raises the important question of whether the alpha 1-adrenergic amplification mechanism has a physiological role in regulating melatonin synthesis in vivo.     

Mediation of Norepinephrine-Stimulated Cyclic AMP Accumulation by Adrenergic Receptors in Hypothalamic and Preoptic Area Slices: Effects of Estradiol

Adrenergic receptor agonists and antagonists were employed to establish (a) which receptor subtypes mediate the cyclic AMP response to norepinephrine in hypothalamic and preoptic area slices from gonadectomized female rats and (b) which receptor subtypes might be modulated by the steroid hormone estradiol. Slice cyclic AMP levels were elevated by the beta receptor agonist isoproterenol, but not by alpha 1 (phenylephrine, methoxamine) or alpha 2 (clonidine) agonists. However, the alpha agonist phenylephrine potentiated the effect of the beta agonist isoproterenol on slice cyclic AMP accumulation. In slices from rats given no hormone treatment, the beta antagonist propranolol inhibited norepinephrine-stimulated cyclic AMP production, while the alpha 1 antagonist prazosin was without effect. In contrast, the cyclic AMP response to norepinephrine in slices from estradiol-treated rats was blocked more effectively by prazosin than by propranolol. Estradiol treatment also attenuated the production of cyclic AMP by the beta agonist isoproterenol. The data suggest (a) that norepinephrine induction of cyclic AMP accumulation in hypothalamic and preoptic area slices is mediated by beta receptors and potentiated by alpha receptor activation and (b) that estradiol depresses beta and increases alpha 1 receptor function in slices from brain regions associated with reproductive physiology.     

Metabotropic Glutamate Receptor Agonists Potentiate Cyclic AMP Formation Induced by Forskolin or β-Adrenergic Receptor Activation in Cerebral Cortical Astrocytes in Culture

Abstract: The metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (ACPD) potentiated the accumulation of cyclic AMP induced by either β-adrenergic receptor stimulation (isoproterenol) or direct activation of adenylyl cyclase (AC) with forskolin in rat cerebral cortical astrocytes grown in a defined medium. In contrast, ACPD inhibits the cyclic AMP response in astrocytes cultured in a serum-containing medium. Pharmacological characterization indicated that a group I mGluR, of which only mGluR5 is detectable in these cells, is involved in the potentiation of cyclic AMP accumulation. Potentiation was elicited by mGluR I agonists [e.g., ( R,S )-3,5-dihydroxyphenylglycine (DHPG)], but not by mGluR II or III agonists; it was pertussis toxin resistant and abolished by procedures suppressing mGluR5 function (phorbol ester pretreatment or DHPG-induced receptor down-regulation). Nevertheless, it appears that products generated through the mGluR5 transduction pathway, such as elevated [Ca²⁺]_i or activated protein kinase C (PKC), are not involved in the potentiation as it was not influenced by either the intracellular calcium chelator BAPTA-AM or the PKC inhibitor Ro 31-8220. An inhibitor of phospholipase C, U-73122, markedly attenuated mGluR5-activated phosphoinositide hydrolysis but did not significantly affect the DHPG potentiation of the cyclic AMP response. A mechanism is proposed in which the potentiating effect on AC could be mediated by free βγ complex that is liberated after the agonist-bound mGluR5 interacts with its coupled G protein.     

Synergistic Interactions Between α1- and α2-Adrenergic Receptors in Activating 3H-Inositol Phosphate Formation in Primary Glial Cell Cultures

Norepinephrine (NE)-stimulated 3H-inositol phosphate (3H-InsP) formation in primary glial cell cultures is thought to be due to alpha 1-adrenergic receptor activation. Surprisingly, the alpha 1-selective agonists phenylephrine and methoxamine showed only 12-21% of the intrinsic activity of NE in activating this response. Although the alpha 2-selective agonist UK 14,304 was itself inactive, inclusion of UK 14,304 increased the response to the alpha 1-selective agonists by about threefold. This increase was concentration-dependent and occurred at all time points examined. 6-Fluoro-NE and alpha-methyl-NE mimicked the effect of NE in glial cultures, although with lower potencies. However, several partial agonists were ineffective in activating this response, in both the presence and absence of UK 14,304. Synergistic interactions were not observed for alpha 1-mediated responses in slices of rat cerebral cortex, either for formation of 3H-InsPs or potentiation of isoproterenol- or adenosine-stimulated cyclic AMP accumulation. Both UK 14,304 and phenylephrine inhibited NE-stimulated 3H-InsP formation in concentrations similar to those necessary to activate this response directly. These results suggest that NE activates 3H-InsP formation in primary glial cultures by synergistic actions on both alpha 1- and alpha 2-adrenergic receptors. The agonists UK 14,304 and phenylephrine also can act to inhibit the response to NE competitively.     

Phorbol Esters Enhance Neurotransmitter-Stimulated Cyclic AMP Production in Rat Brain Slices

The effect of phorbol esters on cyclic AMP production in rat CNS tissue was examined. Using a prelabeling technique for measuring cyclic AMP accumulation in brain slices, it was found that phorbol 12-myristate, 13-acetate (PMA) enhanced the cyclic AMP response to forskolin and a variety of neurotransmitter receptor stimulants while having no effect on second messenger accumulation itself. A short (15-min) preincubation period with PMA was required to obtain maximal enhancement, whereas the augmentation was lessened by prolonged exposure (3 h) to the phorbol. The response to PMA was concentration dependent (EC50 = 1 microM) and regionally selective, being most apparent in forebrain, and was not influenced by removal of extracellular calcium or by inhibition of phosphodiesterase or phospholipase A2. Only those phorbols known to stimulate protein kinase C augmented the accumulation of cyclic AMP. Moreover, the membrane substrates phosphorylated by endogenous C kinase and by a partially purified preparation of this enzyme were similar. The results suggest that phorbol esters, by activating protein kinase C, modify the cyclic AMP response to brain neurotransmitter receptor stimulation in brain by influencing a component of the adenylate cyclase system beyond the transmitter recognition site.     

Ethanol causes desensitization of receptor-mediated phospholipase C activation in isolated hepatocytes.

The effect of ethanol on receptor-mediated phospholipase C-linked signal transduction processes was investigated in isolated rat hepatocytes. Pretreatment of the cells with ethanol (6-300 mM) markedly inhibited a subsequent stimulation of phospholipase C by vasopressin, angiotensin II, or epidermal growth factor. By contrast, the effects of the alpha 1-adrenergic agonist phenylephrine and of glucagon were not affected by ethanol pretreatment. Ethanol inhibited the agonist-induced decrease in polyphosphoinositides, the formation of inositol phosphates, and the increase in cytosolic free Ca2+ levels, as detected with the intracellular Ca2+ indicator indo-1. The effects of ethanol were concentration dependent and were pronounced at low concentrations of agonists but were not significant at saturating levels. Pretreatment of the cells with the protein kinase C inhibitor H7 partly prevented the inhibition by ethanol of vasopressin-induced phospholipase C activation. By contrast, pretreatment of the cells with (Rp)-adenosine cyclic 3':5'-phosphorothioate [Rp)-cAMP-S), a competitive inhibitor of protein kinase A, potentiated the inhibitory effect of ethanol on the Ca2+ mobilization by vasopressin. (Rp)-cAMP-S similarly potentiated the inhibition of phospholipase C by the protein kinase C-activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The kinase A inhibitor also made the Ca2+ mobilization by phenylephrine sensitive to ethanol, indicating that the formation of cAMP in the cells played a role in suppressing the sensitivity to ethanol. Pretreatment of the cells with ethanol enhanced the inhibitory effects of TPA on the vasopressin-induced phospholipase C activation at all concentrations of the hormone; however, these synergistic effects were prevented when TPA was added prior to ethanol, a condition that prevents the activation of phospholipase C by ethanol. The data indicate that ethanol causes desensitization of the receptor-mediated phospholipase C secondary to the ethanol-induced activation of phospholipase C and activation of protein kinase C. Ethanol treatment also affects the sensitivity of the phospholipase C system to control by protein kinases A and C. The data indicate that ethanol can affect the control of intracellular signal transduction processes in liver cells under physiologically relevant conditions.     

Inhibition of alpha 2-adrenergic receptor-mediated cyclic GMP formation by a phorbol ester, a protein kinase C activator

alpha 2-adrenergic receptor-mediated signal transduction in rat adrenocortical carcinoma cells occurs through the opposing regulation of two second messengers, cyclic GMP and cyclic AMP, in which guanylate cyclase is coupled positively and adenylate cyclase negatively to the receptor signal. We now show that in these cells phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, inhibits the alpha 2-agonist (p-aminoclodine)-dependent production of cyclic GMP in a dose-dependent and time-dependent fashion. The half-maximal inhibitory concentration of PMA was 10(-10) M. A protein kinase C inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H-7), caused the release of the PMA-dependent attenuation of p-aminoclodine-stimulated cyclic GMP formation. These results suggest that protein kinase C negatively regulates the alpha 2-receptor coupled cyclic GMP system in these cells, a feature apparently shared with the other cyclic GMP-coupled receptors such as those of muscarine, histamine, and atrial natriuretic factor.     

Different Mechanisms of β-Adrenoceptor Down-Regulation by Chronic Imipramine and Electroconvulsive Treatment: Possible Role for Protein Kinase C

The aim of this study was to find out how protein kinase C (PKC) is involved in down-regulation of the beta-adrenoceptor in cortical slices of rats subjected to antidepressant treatments. The responses of the cyclic AMP generating system to forskolin, isoproterenol, and noradrenaline were tested in the absence and presence of a PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA). The antidepressive treatments applied were chronic administration of imipramine and electroconvulsive shock. The potentiating effect of the phorbol ester on cyclic AMP response to isoproterenol was retained in imipramine-treated animals and even accentuated in rats subjected to electroconvulsive treatment; the TPA effect on noradrenaline-induced cyclic AMP response was blunted in rats receiving imipramine, but augmented in those receiving electroconvulsive treatment. In imipramine-treated rats the beta-down-regulation was still evident in the presence of TPA; after electroconvulsive treatment the phorbol ester-induced potentiation was so high that no significant beta-down-regulation could be observed. No procedure affected the response to forskolin. The beta-down-regulation that develops during chronic imipramine treatment differs from that caused by chronic electroconvulsive treatment; in both cases it is not related to the direct effect on adenylate cyclase.     

Phorbol esters down-regulate protein kinase C in rat brain cerebral cortical slices

The effect of phorbol esters on cyclic AMP production in rat cerebral cortical slices was studied using a prelabelling technique to measure cyclic nucleotide accumulation. Cholera toxin-stimulated cyclic AMP accumulation was enhanced approximately 2-fold by phorbol 12-myristate, 13-acetate (PMA) which alone had no effect on cyclic AMP production. The augmentation by PMA was maximal within the first hour of incubation, decreasing progressively thereafter. Protein kinase C activity was decreased 80-90% during a 3 hr exposure to PMA, as was 3H-phorbol 12,13-dibutyrate binding. Both phosphatidyl serine and arachidonic acid were found to enhance protein kinase C activity in a concentration-dependent manner, an effect that was attenuated by prolonged incubation of the brain tissue with PMA. The results indicate that exposure of brain slices to phorbol esters causes a down-regulation of rat brain protein kinase C, and that this modification corresponds with a decrease in the ability of PMA to augment cyclic AMP production, suggesting a functional relationship between the two systems in rat brain.     

Thrombin exerts a dual effect on stimulated adenylate cyclase in hamster fibroblasts, an inhibition via a GTP-binding protein and a potentiation via activation of protein kinase C.

Previous studies in Chinese-hamster fibroblasts (CCL39 line) indicate that an important signalling pathway involved in thrombin's mitogenicity is the activation of a phosphoinositide-specific phospholipase C, mediated by a pertussis-toxin-sensitive GTP-binding protein (Gp). The present studies examine the effects of thrombin on the adenylate cyclase system and the interactions between the two signal transduction pathways. We report that thrombin exerts two opposite effects on cyclic AMP accumulation stimulated by cholera toxin, forskolin or prostaglandin E1. (1) Low thrombin concentrations (below 0.1 nM) decrease cyclic AMP formation. A similar inhibition is induced by A1F4-, and both thrombin- and A1F4- -induced inhibitions are abolished by pertussis toxin. (2) Increasing thrombin concentration from 0.1 to 10 nM results in a progressive suppression of adenylate cyclase inhibition and in a marked enhancement of cyclic AMP formation in pertussis-toxin-treated cells. A similar stimulation is induced by an active phorbol ester, and thrombin-induced potentiation of adenylate cyclase is suppressed by down-regulation of protein kinase C. Therefore, we conclude that (1) the inhibitory effect of thrombin on adenylate cyclase is the direct consequence of the activation of a pertussis-toxin-sensitive inhibitory GTP-binding protein (Gi) possibly identical with Gp, and (2) the potentiating effect of thrombin on cyclic AMP formation is due to stimulation of protein kinase C, as an indirect consequence of Gp activation. Our results suggest that the target of protein kinase C is an element of the adenylate cyclase-stimulatory GTP-binding protein (Gs) complex. At low thrombin concentrations, activation of phospholipase C is greatly attenuated by increased cyclic AMP, leading to predominance of the Gi-mediated inhibition.     

Activators of Protein Kinase C Act at a Postreceptor Site to Amplify Cyclic AMP Production in Rat Pinealocytes

Activation of alpha 1-adrenoceptors appears to amplify beta-adrenergic stimulation of cyclic AMP (cAMP) accumulation in rat pinealocytes severalfold by a mechanism involving activation of a Ca2+-, phospholipid-dependent protein kinase (protein kinase C). The mechanism of action of protein kinase C was investigated in this report using intact cells. Activation of protein kinase C with 4 beta-phorbol 12-myristate 13-acetate (PMA; 10(-7) M) or the alpha 1-adrenergic agonist phenylephrine (PE; 10(-6) M) did not inhibit cAMP efflux in beta-adrenergically stimulated cells. The amplification of the beta-adrenergic cAMP response by these agents also occurred in the presence of isobutylmethylxanthine (10(-3) M) and Ro 20-1724 (10(-4) M), an observation suggesting that inhibition of cAMP phosphodiesterase activity is not the mechanism of action. Furthermore, although PMA (10(-7) M) caused a sixfold increase in the magnitude of the cAMP response to isoproterenol, it did not alter the EC50 of the response (1.7 X 10(-8) M), a result indicating that protein kinase C activation does not alter beta-adrenoceptor sensitivity. The cAMP response following cholera toxin pretreatment (60-120 min) was rapidly and markedly enhanced by alpha 1-adrenergic agonists (cirazoline greater than PE greater than methoxamine), by phorbol esters (PMA greater than 4 beta-phorbol 12,13,-dibutyrate much greater than 4 alpha-phorbol 12,13-didecanoate), and by synthetic diacylglycerols (1,2-dioctanoylglycerol greater than 1-oleoyl 2-acetylglycerol much greater than diolein). The cAMP response to forskolin (10(-5)-10(-3) M) was also increased by PE (3 X 10(-6) M) and PMA (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C regulates the activity and stability of serotonin N-acetyltransferase

Effects of protein kinase C on protein stability and activity of rat AANAT were investigated in vitro and in vivo. When COS-7 cells transfected with AANAT cDNA were treated with phorbol 12-myristate 13-acetate (PMA), both the activity and protein level of AANAT were increased. These effects of PMA were blocked by GF109203X, a specific inhibitor of PKC. Moreover, PMA increased the phosphorylation of AANAT and induced the formation of AANAT/14-3-3zeta complex. PMA did not affect the basal level of cAMP and did not involve the potentiation of the cAMP production by forskolin, indicating that PKC-dependent activation of adenylyl cyclase was excluded in transfected COS-7 cells. To identify which amino acids were phosphorylated by PKC, several conserved Thr and Ser residues in AANAT were targeted for site-directed mutagenesis. Mutations of Thr29 and Ser203 prevented the increase of enzymatic activity and protein level mediated by PMA. To explore the nature of AANAT phosphorylation, purified rat AANAT was subjected to in vitro PKC kinase assay. PKC directly phosphorylated the rat recombinant AANAT. The phosphopeptides identified by mass spectrometric analysis, and western blotting indicated that Thr29 was one of target sites for PKC. To confirm the effects of the physiological activation of PKC, rat pineal glands were treated with alpha(1)-adrenergic specific agonist phenylephrine. Phenylephrine caused the phosphorylation of endogenous AANAT whereas GF109203X or prazosin, an alpha(1)-adrenergic-specific antagonist, markedly inhibited it. These results suggest that AANAT was phosphorylated at Thr29 by PKC activation through the alpha(1)-adrenergic receptor in rat pineal glands, and that its phosphorylation might contribute to the stability and the activity of AANAT.     

Potentiation of Agonist-Stimulated Cyclic AMP Accumulation by Tyrosine Kinase Inhibitors in Rat Pinealocytes

Abstract: To study cross-talk mechanisms in rat pinealocytes, the role of tyrosine kinase or kinases in the regulation of adrenergic-stimulated cyclic AMP production was investigated. Both norepinephrine- and isoproterenol-stimulated cyclic AMP accumulation were increased by two distinct tyrosine kinase inhibitors, genistein or erbstatin, in a concentration-dependent manner. A similar increase was observed with two other inhibitors, tyrphostin B44 and herbimycin. In contrast, daidzein, an inactive analogue of genistein, was ineffective; whereas vanadate, a phosphotyrosine phosphatase inhibitor, reduced the adrenergic-stimulated cyclic AMP accumulation. The tyrosine kinase inhibitors were effective in potentiating the cholera toxin-or forskolin-stimulated cyclic AMP accumulation, indicating that their sites of action are at the postreceptor level. Neither an activator nor inhibitors of protein kinase C influenced the potentiation of the cyclic AMP responses by genistein, suggesting that the potentiation effect by tyrosine kinase inhibitors does not involve the phospholipase C/protein kinase C pathway. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein failed to potentiate and vanadate did not inhibit the adrenergic-stimulated cyclic AMP accumulation, indicating that the phosphodiesterase is a probable site of action for these inhibitors. These results suggest that cyclic AMP metabolism in the pinealocytes is tonically inhibited by tyrosine kinase acting on the cyclic AMP phosphodiesterase.     

A role for Gi in control of thrombin receptor-phospholipase C coupling in human platelets

Stimulation of washed human platelets with alpha-thrombin was accompanied by aggregation, formation of inositol phosphates and phosphatidic acid, liberation of arachidonic acid, mobilization of intracellular Ca2+ stores, and influx of Ca2+ from the extracellular medium. Each of these responses was potentiated by a short pretreatment with epinephrine, although alone this agent was ineffective. A prolonged (5 min) stimulation with alpha-thrombin desensitized both phospholipase C and Ca2+ mobilization to a further thrombin challenge. Epinephrine added following thrombin desensitization restored both the ability of thrombin to release Ca2+ stores and stimulate inositol phospholipid hydrolysis. Resensitization was mediated by alpha 2-adrenergic receptors and lasted about 3 min, after which the Ca2+ levels returned again to basal levels. Pretreatment of platelets with phorbol dibutyrate at concentrations which specifically activate protein kinase C increased the rate of desensitization of the thrombin-induced release of Ca2+ stores and abolished the ability of epinephrine to restore the thrombin response. The protein kinase C inhibitor, staurosporine, blocked the inhibitory effect of phorbol ester and also reduced the rate of desensitization of thrombin and subsequent epinephrine action. These results suggest that thrombin activation of protein kinase C phosphorylates and inactivates a signaling protein which is common to both thrombin and alpha 2-adrenergic receptors. This protein is involved in thrombin stimulation of phospholipase C but is not directly stimulatory since epinephrine alone does not activate this enzyme. We searched for a known second messenger protein common to both thrombin and alpha 2-adrenergic receptors which was phosphorylated in intact platelets by protein kinase C in parallel with thrombin-induced desensitization. The alpha subunit of the inhibitory GTP-binding protein, Gi, was the only candidate which fulfilled all of these criteria as shown by immunoprecipitation. Therefore, we suggest that alpha i maintains the thrombin receptor in a state which can couple to phospholipase C when activated with thrombin. This permissive state of alpha i is blocked by phosphorylation by thrombin-activated protein kinase C.     

ACCUMULATION OF CYCLIC ADENOSINE 3', 5'-MONOPHOSPHATE IN CEREBRAL CORTICAL SLICES FROM RAT AND MOUSE: STIMULATORY EFFECT OF α- AND β-ADRENERGIC AGENTS AND ADENOSINE

Abstract— Norepinephrine, epinephrine, isoproterenol, and adenosine elicit enhanced accumulations of cyclic AMP in incubated slices of rat cerebral cortex. Combinations of norepinephrine, epinephrine, isoproterenol, or histamine with adenosine have a greater than additive effect on cyclic AMP levels. The effects of isoproterenol appear to be mediated via a classical β-adrenergic receptor whereas the effects of norepinephrine appear due to interactions with both α- and β-adrenergic receptors. The presence of the phosphodiesterase inhibitor, isobutylmethylxanthine, potentiates the effects of the catecholamines and reveals a histamine-mediated increase in cyclic AMP levels. After an initial stimulation of cyclic AMP formation with norepinephrine, followed by washing of the slices, the cyclic AMP-generating system is unresponsive to norepinephrine but does respond to an adenosine-norepinephrine combination. In mouse cerebral cortical slices, catecholamines appear to elicit an accumulation of cyclic AMP primarily via interaction with a β-adrenergic receptor.     

Alpha 1-adrenergic receptor-mediated activation of phospholipase D in rat cerebral cortex.

We have investigated phospholipase D activity in rat brain cortical slices prelabeled with [32P]orthophosphoric acid. In the presence of ethanol (170 mM), norepinephrine stimulated, in a dose-dependent manner (EC50 = 2.2 microM), the accumulation of [32P]phosphatidylethanol as a result of phospholipase D activity. Norepinephrine-stimulated phospholipase D activity was completely inhibited by prazosin, a specific alpha 1-adrenergic antagonist (Ki = 2.8 nM). However, no accumulation of phosphatidylethanol was observed in the presence of the muscarinic agonist carbachol. The Ca2+ ionophore ionomycin and the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also stimulated [32P]phosphatidylethanol accumulation in cortical slices, in a dose- and time-dependent manner, whereas the inactive phorbol, 4 alpha-phorbol 12,13-didecanoate, did not stimulate phospholipase D activity. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, two potent inhibitors of protein kinase C, inhibited PMA and ionomycin stimulation of phospholipase D activity, but did not affect the response to norepinephrine. Furthermore, the effects of PMA and norepinephrine were additive. Differences between PMA and norepinephrine stimulation of phospholipase D activity were also found with regard to the extracellular Ca2+ requirement and time course of phosphatidylethanol accumulation. No stimulation of phospholipase D activity by norepinephrine was observed in slices from cerebellum, a brain area with a low density of alpha 1-adrenergic receptors, while the effect of PMA was greater in the cerebellum than in cortical or hippocampal slices. These results strongly suggest that activation of phospholipase D in cortical slices by norepinephrine and PMA involve different mechanisms.     

Intracellular pH on Protein Kinase C and Ionomycin Potentiation of Isoproterenol-Stimulated Cyclic AMP and Cyclic GMP Production in Rat Pinealocytes

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.