Phorbol Esters Mimic α-Adrenergic Potentiation of Serotonin N-Acetyltransferase Induction in the Rat Pineal

alpha-Adrenergic stimulation of the rat pineal gland is known to stimulate phosphatidylinositol turnover and to potentiate the induction of serotonin N-acetyltransferase (SNAT) activity evoked by submaximal beta-adrenergic stimulation. In some (other) systems tumor-promoting phorbol esters are known to mimic physiologic stimulation and to enhance specifically the activity of protein kinase C. Here it is shown that phorbol esters specifically mimic the potentiating effect of alpha-adrenergic stimulation on SNAT activity in the rat pineal. These effects contribute to the argument for a role for phosphatidylinositol turnover and protein kinase C in mediating alpha-adrenergic stimulation.     

Lithium Decreases Membrane-Associated Protein Kinase C in Hippocampus: Selectivity for the α Isozyme

We investigated the effects of lithium on alterations in the amount and distribution of protein kinase C (PKC) in discrete areas of rat brain by using [³H]phorbol 12, 13-dibutyrate quantitative autoradiography as well as western blotting. Chronic administration of lithium resulted in a significant decrease in membrane-associated PKC in several hippocampal structures, most notably the subiculum and the CA1 region. In contrast, only modest changes in [³H]phorbol 12, 13-dibutyrate binding were observed in the various other cortical and subcortical structures examined. Immunoblotting using monoclonal anti-PKC antibodies revealed an isozyme-specific 30% decrease in hippocampal membrane-associated PKC α, in the absence of any changes in the labeling of either the β_(I/II) or γ isozymes. These changes were observed only after chronic (4 week) treatment with lithium, and not after acute (5 days) treatment, suggesting potential clinical relevance. Given the critical role of PKC in regulating neuronal signal transduction, lithium's effects on PKC in the limbic system represent an attractive molecular mechanism for its efficacy in treating both poles of manic-depressive illness. In addition, the decreased hippocampal membrane-associated PKC observed in the present study offers a possible explanation for lithium-induced memory impairment.     

Protein Kinase C and Phospholipase C Mediate α- and β-Adrenoceptor Intercommunication in Rat Hypothalamic Slices

These experiments examined the mechanism by which phenylephrine enhances beta-adrenoceptor-stimulated cyclic AMP formation in rat hypothalamic and preoptic area slices. To this end we manipulated phospholipase C. phospholipase A2, and protein kinase C activity in slices and assessed the effects of these manipulations on phenylephrine augmentation of isoproterenol-stimulated cyclic AMP generation. Since previous work indicated that estrogen enhances the alpha 1-component of cyclic AMP formation, we examined slices from both gonadectomized and estrogen-treated animals. The alpha 1-antagonist prazosin eliminated phenylephrine augmentation of the beta-response, suggesting that alpha 1-adrenergic receptors mediate the potentiation of cyclic AMP formation. Inhibition of protein kinase C by H7 attenuated the alpha 1-augmentation of beta-stimulated cyclic AMP formation. Staurosporine, a more potent protein kinase C inhibitor, completely abolished the alpha 1-augmenting response. In addition, phenylephrine potentiation of the isoproterenol response was not observed if protein kinase C was first stimulated directly with a synthetic diacylglycerol (1-oleoyl-2-acetyl-sn-glycerol) or phorbol ester (phorbol 12,13-dibutyrate). Neomycin, an inhibitor of phospholipase C, decreased alpha 1-receptor enhancement of beta-stimulated cyclic AMP formation, whereas quinacrine, an inhibitor of phospholipase A2, did not. The data suggest that the postreceptor mechanism involved in alpha 1-adrenergic receptor potentiation of cyclic AMP generation in hypothalamic and preoptic area slices includes activation of phospholipase C and protein kinase C.     

Protein Kinase A Phosphorylation of the Proteasome: A Contribution to the α-Secretase Pathway in Human Cells

Abstract: The β-amyloid precursor protein undergoes a physiological cleavage by α-secretase that leads to the release of a secreted C-terminally truncated fragment called APPα and likely concomitantly reduces the formation of the amyloidogenic Aβ peptide. Here we demonstrate that APPα secretion is increased by the protein kinase A (PKA) effectors 8-bromo cyclic AMP and forskolin in human embryonic kidney cells (HK293), and that this can be prevented by a proteasome inhibitor. Furthermore, we establish that PKA effectors but not protein kinase C agonists increase the chymotrypsin-like activity and phosphorylation state of the proteasome in vitro and in vivo in HK293 cells. Altogether, this report demonstrates that the α-secretase pathway is under the control of PKA in human cells and that the proteasome likely contributes, either directly or through yet unknown intermediates, to the PKA-stimulated APPα secretion in human cells.     

Inhibition of β-Amyloid Production by Activation of Protein Kinase C

The cellular factors regulating the generation of β-amyloid from the amyloid precursor protein (APR) are unknown. Activation of protein kinase C (PKC) by phorbol ester treatment inhibited the generation of the 4-kDa β-amyloid peptide in transfected COS cells, a human glioma cell line, and human cortical astrocytes. An analogue of diacylglycerol, the endogenous cellular activator of PKC, also inhibited the generation of β-amyloid. Activation of PKC increased the level of secreted APP in transfected COS cells but did not significantly affect the level of secreted APP in primary human astrocytes or in the glioma cell line. Cell-associated APP and the secreted APP derivative, but not β-amyloid, were phosphorylated on serine residues. Activation of PKC did not increase the level of APP phosphorylation, suggesting that PKC modulates the proteolytic cleavage of APP indirectly by phosphorylation of other substrates. These results indicate that PKC activation inhibits β-amyloid production by altering APP processing and suggest that β-amyloid production can be regulated by the phospholipase C-diacylglycerol signal transduction pathway.     

Endogenous δ-Opioid and ORL1 Receptors Couple to Phosphorylation and Activation of p38 MAPK in NG108-15 Cells and This Is Regulated by Protein Kinase A and Protein Kinase C

The p38 mitogen-activated protein kinase (MAPK) cascade transduces multiple extracellular signals from cell surface to nucleus and is employed in cellular responses to cellular stresses and apoptotic regulation. The involvement of the p38 MAPK cascade in opioid- and opioid receptor-like receptor-1 (ORL1) receptor-mediated signal transduction was examined in NG108-15 neuroblastoma x glioma hybrid cells. Stimulation of endogenous delta-opioid receptor (DOR) or ORL1 resulted in activation of p38 MAPK. It also induced the activation of extracellular signal-regulated kinases (ERKs), another member of the MAPK family, with slower kinetics. Activation of p38 MAPK was abolished by selective antagonists of DOR or ORL1, pretreatment with pertussis toxin, or SB203580, a specific inhibitor of p38 MAPK. Inhibition of p38 MAPK had no significant effect on opioid-induced ERK activation, indicating that p38 MAPK activity was not required for ERK activation, though its stimulation preceded ERK activation. Inhibition of protein kinase A (PKA) strongly diminished p38 activation mediated by DOR or ORL1 but had no significant effect on ERK activation, and protein kinase C (PKC) inhibitors potentiated stimulation of p38 while inhibiting activation of ERKs. Taken together, our results provide the first evidence for coupling of DOR and ORL1 to the p38 MAPK cascade and clearly demonstrate that receptor-mediated activation of p38 MAPK both involves PKA and is negatively regulated by PKC.     

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.     

Chronic Sodium Valproate Selectively Decreases Protein Kinase C α and ε In Vitro

Abstract: Valproic acid (VPA) is a fatty acid antiepileptic with demonstrated antimanic properties, but the molecular mechanism or mechanisms underlying its therapeutic efficacy remain to be elucidated. In view of the increasing evidence demonstrating effects of the first-line antimanic drug, lithium, on protein kinase C (PKC), we investigated the effects of VPA on various aspects of this enzyme. Chronic exposure (6–7 days) of rat C6 glioma cells to "therapeutic" concentrations (0.6 m M ) of VPA resulted in decreased PKC activity in both membrane and cytosolic fractions and increased the cytosol/membrane ratio of PKC activity. Western blot analysis revealed isozyme-selective decreases in the levels of PKC α and ε (but not δ or ζ) in both the membrane and cytosolic fractions after chronic VPA exposure; VPA added to reaction mixtures did not alter PKC activity or ³H-phorbol ester binding. Together, these data suggest that chronic VPA indirectly lowers the levels of specific isozymes of PKC in C6 cells. Given the pivotal role of PKC in regulating neuronal signal transduction and modulating intracellular cross-talk between neurotransmitter systems, the specific decreases in PKC α and ε may play a role in the antimanic effects of VPA.     

Modulation of ζ-Protein Kinase C by Cyclic AMP in PC12 Cells Occurs Through Phosphorylation by Protein Kinase A

Abstract: Although cyclic AMP (cAMP) has been reported to cross talk with the protein kinase C (PKC) system, effects of elevated intracellular cAMP on the activities of specific PKC isoforms have not been studied. We report findings from a permeabilized cell assay that was used to examine changes in the activity of the atypical PKC isoforms brought about by exposure of PC12 cells to agents that elevate intracellular cAMP. We found that increases in intracellular cAMP led to rapid stimulation of atypical PKC activity, 40–70% above control, for a sustained period of time, a response that occurred independent of the phorbol 12-myristate 13-acetate (PMA)-sensitive PKC isoforms. Changes in intracellular cAMP levels resulted in a dose-dependent redistribution of ζ-PKC to the cytoplasm with a concomitant increase in the phosphorylation state of the enzyme. Incubation of purified ζ-PKC with increasing concentrations of PKA likewise caused a twofold increase in the phosphorylation state of ζ-PKC. In contrast to the positive effect that PKA-mediated phosphorylation had on the activity of ζ-PKC, the enzyme displayed reduced binding to ras when phosphorylated. Taken together, these findings are consistent with the hypothesis that protein phosphorylation of PKC acts as a positive effector of its enzyme activity and may serve as a negative modulator for interaction with other proteins.     

Role of Protein Kinase C α in Nerve Growth Factor-Induced Arachidonic Acid Release from PC12 Cells

Abstract: Nerve growth factor (NGF) increases arachidonic acid (AA) release by PC12 pheochromocytoma cells. To explore the role of protein kinase C (PKC) in this action of NGF, PKC was down-regulated by long-term treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Such prolonged exposure to PMA (1 µ M ) resulted in the inhibition of NGF-induced AA release. Moreover, pretreatment of PC12 cells with the protein kinase inhibitor staurosporine or with calphostin C, a specific inhibitor of PKC, also blocks the increase of AA release induced by NGF. These data, as well as that PMA alone can induce AA release in PC12 cells, suggest that PKC is necessary for NGF-induced AA release. Immunoblot analysis of whole cell lysates by using antibodies against various PKC isoforms revealed that our PC12 cells contained PKCs α, δ, ε, and ζ. PMA down-regulation depleted PKCs α, δ, and ε, and partially depleted ζ. To see which isoform was involved in NGF-induced AA release, an isoform-specific PKC inhibitor was used. GO 6976, a compound that inhibits PKCs α and β specifically, blocked NGF-induced AA release. In addition, thymeleatoxin, a specific activator of PKCs α, β, and γ, induced AA release from PC12 cells in amounts comparable with those seen with NGF. Taken together, these data suggest that PKC α plays a role in NGF-induced AA release.     

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.     

Activity of α-Secretase as the Common Final Effector of Protein Kinase C-Dependent and -Independent Modulation of Amyloid Precursor Protein Metabolism

The metabolic fate of the amyloid precursor protein (APP) is one of the key factors in the pathogenesis of Alzheimer's disease (AD). A complex cellular mechanism regulates the rate at which the precursor is cleaved by alpha-secretase and released as soluble protein in the extracellular space. We show here that alpha-secretase constitutes the common final effector of several independent means of stimulation of soluble APP (sAPP) release. The release of sAPP by alpha-secretase resembles that of several other membrane-bound proteins with soluble counterparts, a process that is sensitive to matrix metalloprotease inhibitors. The hydroxamic acid-based compound KD-IX-73-4 inhibits phorbol ester-mediated sAPP release from COS cells with an IC50 of 8 microM, consistent with previous data for the same compound against leukocyte L-selectin shedding. Beyond direct protein kinase C (PKC) activation we show that KD-IX-73-4 inhibits also receptor-mediated sAPP release induced by carbachol in SH-SY5Y cells and by bradykinin in human skin fibroblasts, with the latter being a PKC-independent mechanism. Altogether these data suggest that all pharmacological means of stimulating sAPP release converge to a hydroxamic acid-based inhibitor-sensitive proteolytic enzyme. Moreover, because KD-IX-73-4 was effective in the inhibition of stimulated but not constitutive sAPP release, these data suggest the existence of different enzymes regulating the two metabolic pathways leading to sAPP secretion.     

Desensitization of the δ-Opioid Receptor Correlates with Its Phosphorylation in SK-N-BE Cells: Involvement of a G Protein-Coupled Receptor Kinase

Abstract: Phosphorylation of G protein-coupled receptors is considered an important step during their desensitization. In SK-N-BE cells, recently presented as a pertinent model for the studies of the human δ-opioid receptor, pretreatment with the opioid agonist etorphine increased time-dependently the rate of phosphorylation of a 51-kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino-terminal region of the cloned human δ-opioid receptor, revealed that it corresponded to the δ-opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the δ-opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the δ-opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn²⁺, known to act as G protein-coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human δ-opioid receptor, that a close link exists between receptor phosphorylation and agonist-promoted desensitization and that desensitization involves a GRK.     

Synergistic Stimulation of Interleukin 6 Release and Gene Expression by Phorbol Esters and Interleukin 1β in Rat Cortical Astrocytes: Role of Protein Kinase C Activation and Blockade

Abstract: The involvement of protein kinase C and its interaction with interleukin 1β in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C, and by the desensitization of protein kinase C. Interleukin 1β increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1β stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1β (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurosporine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C. Interleukin 1β stimulated interleukin 6 secretion via a mechanism that is also negatively modulated by a protein kinase C isoform or isoforms sensitive to staurosporine and desensitization. Finally, we showed that interleukin 1β and phorbol 12-myristate 13-acetate synergistically stimulated interleukin 6 release and its gene expression, operating in a manner insensitive to protein kinase C blockers and slightly reduced by protein kinase C desensitization.     

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.     

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.     

Phosphorylation of the GABAa/Benzodiazepine Receptor α Subunit by a Receptor-Associated Protein Kinase

Abstract: Partially purified preparations of GABA_a/benzodiazepine receptor from rat brain were found to contain high levels of a protein kinase activity that phosphorylated a small number of proteins in the receptor preparations, including a 50-kilodalton (kD) phosphoprotein that comigrated on two-dimensional electrophoresis with purified, immunolabeled, and photolabeled receptor α subunit. Further evidence that the comigrating 50-kD phosphoprotein was, in fact, the receptor α subunit was obtained by peptide mapping analysis: the 50-kD phosphoprotein yielded one-dimensional peptide maps identical to those obtained from iodinated, purified α subunit. Phosphoamino acid analysis revealed that the receptor α subunit is phosphorylated on serine residues by the protein kinase activity present in receptor preparations. Preliminary characterization of the receptor-associated protein kinase activity suggested that it may be a second messenger-independent protein kinase. Protein kinase activity was unaltered by cyclic AMP, cyclic GMP, calcium plus calmodulin, calcium plus phosphatidylserine, and various inhibitors of these protein kinases. Examination of the substrate specificity of the receptor-associated protein kinase indicated that the enzyme preferred basic proteins as substrates. Endogenous phosphorylation experiments indicated that the receptor α subunit may also be phosphorylated in crude membranes by a protein kinase activity present in those membranes. As with phosphorylation of the receptor in purified preparations, its phosphorylation in crude membranes also appeared to be unaffected by activators and inhibitors of second messenger-dependent protein kinases. These findings raise the possibility that the phosphorylation of the α subunit of the GABA_a/ benzodiazepine receptor by a receptor-associated protein kinase plays a role in modulating the physiological activity of the receptor in vivo.