Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein Phosphorylation in Astrocytes Mediated by Protein Kinase C: Comparison with Phosphorylation by Cyclic AMP-Dependent Protein Kinase

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.     

Evidence for a Single Protein Kinase C-Mediated Phosphorylation Site in Rat Brain Protein B-50

The neuronal protein B-50 may be involved in diverse functions including neural development, axonal regeneration, neural plasticity, and synaptic transmission. The rat B-50 sequence contains 226 amino acids which include 14 Ser and 14 Thr residues, all putative sites for phosphorylation by calcium/phospholipid-dependent protein kinase C (PKC). Phosphorylation of the protein appears to be a major factor in its biochemical and possibly its physiological activity. Therefore, we investigated rat B-50 phosphorylation and identified a single phosphorylated site at Ser41. Phosphoamino acid analysis eliminated the 14 Thr residues because only [32P]Ser was detected in an acid hydrolysate of [32P]B-50. Staphylococcus aureus protease peptide mapping produced a variety of radiolabelled [32P]B-50 products, none of which had the same molecular weights or HPLC retention times as several previously characterized fragments. Indirect confirmation of the results was provided by differential phosphorylation of major and minor forms of B-60 that have their N-termini at, or C-terminal to, the Ser41 residue and are the major products of specific B-50 proteolysis. Only those forms of B-60 that contained the Ser41 residue incorporated phosphate label. The results are discussed with reference to the substrate requirements for B-50 phosphorylation by PKC and the proposed structure of the B-50 calmodulin binding domain.     

Increased Phosphorylation of a 17-kDa Protein Kinase C Substrate (P17) in Long-Term Potentiation

Hippocampal long-term potentiation (LTP) is a persistent increase in the efficacy of synaptic transmission, which is widely thought to be a cellular mechanism that could contribute to learning and memory. Studies on the biochemical mechanisms underlying LTP suggest the involvement of protein kinases in both LTP induction and maintenance. In this report we describe an LTP-associated increase in the phosphorylation in vitro of a 17-kDa protein kinase C (PKC) substrate protein, which we have termed P17, in homogenates from the CA1 region of rat hippocampal slices. This LTP-associated increase in phosphorylation was expressed independent of significant levels of free Ca2+, as phosphorylation reactions were performed in the presence of 500 microM EGTA. The increased phosphorylation of P17 was substantially inhibited by PKC(19-36), a selective inhibitor of PKC. These data support the model that persistent PKC activation contributes to the maintenance of LTP and implicate P17 as a potential target for PKC in the CA1 region of the hippocampus.     

Endothelin Stimulates Phospholipase D in Striatal Astrocytes

Abstract: In primary cultures of mouse striatal astrocytes prelabeled with [³H]myristic acid, endothelin (ET)-1 induced a time-dependent formation of [³H]phosphatidic acid and [³H]diacylglycerol. In the presence of ethanol, a production of [³H]phosphatidylethanol was observed, indicating the activation of a phospholipase D (PLD). ET-1 and ET-3 were equipotent in stimulating PLD activity (EC₅₀ = 2–5 n M ). Pretreatment of the cells with pertussis toxin partially abolished the effect of ET-1, indicating the involvement of a G_i/G_o protein. Inhibition of protein kinase C by Ro 31-8220 or down-regulation of the kinase by a long-time treatment with phorbol 12-myristate 13-acetate (PMA) totally abolished the ET-1-induced stimulation of PLD. In contrast, a cyclic AMP-dependent process is not involved in the activation of PLD, because the ET-1-evoked formation of [³H]phosphatidylethanol was not affected when cells were coincubated with either isoproterenol, 8-bromo-cyclic AMP, or forskolin. Acute treatment with PMA also stimulated PLD through a protein kinase C-dependent process. However, the ET-1 and PMA responses were additive. Furthermore, the ET-1-evoked response, contrary to that of PMA, totally depended on the presence of extracellular calcium. These results suggest that at least two distinct mechanisms are involved in the control of PLD activity in striatal astrocytes. Finally, ET-1, ET-3, and PMA also stimulated PLD in astrocytes from the mesencephalon, the cerebral cortex, and the hippocampus.     

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.     

Purification and Characterization of a Novel Brain-Specific 14-kDa Protein

A new acidic protein specifically present in the brain was purified to homogeneity from bovine brain. The apparent molecular mass was estimated to be 14 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 57 kDa by gel filtration, a finding suggesting that it exists as a tetramer under physiological conditions. The protein had a high content of Glu and Pro, and its pI was 4.3. The first six amino acid residues of the protein were Met-Asp-Val-Phe-Met-Lys, and the amino terminal was blocked. The distribution of the protein examined by Ouchterlony gel immunodiffusion indicates that it is present specifically in brain, including rat, human, and bovine, but could not be detected in 10 other rat tissues examined. The protein was absent in Purkinje cell bodies, as examined by electron microscopic immunocytochemistry, but was present in nerve terminals that make synapse-like contacts with Purkinje cells and in neurons with dark granules in the globus pallidus of the rat.     

Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases

Phosphatidic acid (PA) has been increasingly recognized as an important signaling lipid regulating cell growth and proliferation, membrane trafficking, and cytoskeletal reorganization. Recent studies indicate that the signaling PA generated from phospholipase D (PLD) and diacylglycerol kinase (DGK) plays critical roles in regulating the activity of some members of Ras superfamily of small guanosine triphosphatases (GTPases), such as Ras, Rac and Arf. Change of PA levels regulates the activity of small GTPases by modulating membrane localization and activity of small GTPase regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In addition, PA also targets some small GTPases to membranes by direct binding. This review summarizes the roles of PLD and DGK in regulating the activity of several Ras superfamily members and cellular processes they control. Some future directions and the implication of PA regulation of Ras small GTPases in pathology are also discussed.     

Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D-catalyzed transphosphatidylation, was formed. The half-maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol-treated cells was accompanied by an inhibition of the TPA-induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoyl-glycerol (100 microM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol-treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.     

Phosphorylation by Protein Kinase C of the Muscarinic Acetylcholine Receptor

Muscarinic acetylcholine receptors purified from porcine cerebrum were phosphorylated by protein kinase C purified from the same tissue. More than 1 mol of phosphate was incorporated per mole of receptor, with both serine and threonine residues being phosphorylated. Neither the degree nor the rate of the phosphorylation was affected by the presence or absence of acetylcholine. GTP-sensitive high-affinity binding with acetylcholine was observed for muscarinic receptors reconstituted with GTP-binding proteins (Gi or Go), irrespective of whether muscarinic receptors or the GTP-binding proteins had been phosphorylated by protein kinase C or not. This indicates that the interaction between purified muscarinic receptors and purified GTP-binding proteins in vitro is not affected by their phosphorylation.     

Phosphorylation of the Conditioning-Associated GTP-Binding Protein cp20 by Protein Kinase C

Abstract: The phosphorylation state of cp20, a low molecular weight membrane-associated GTP-binding protein, was previously shown to increase two- to threefold 24 h after associative conditioning. Here, cp20 is shown to be phosphorylated by protein kinase C (PKC) in vitro. Pronounced differences in activity were observed with the three major isoforms of PKC, whereas casein kinase, calcium/calmodulin-dependent protein kinase II, and cyclic AMP-dependent protein kinase produced no detectable phosphorylation of cp20. Phosphorylation of cp20 had no effect on its GTPase or GTP-binding activity but caused a translocation of cp20 from cytosol to the nuclei/mitochondrial particulate fraction. These results suggest that the increase in phosphorylation of cp20 after conditioning may be due to PKC.     

Phosphorylation and Inactivation of Brain Glycogen Synthase by a Multifunctional Calmodulin-Dependent Protein Kinase

Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin-dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin-dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme.     

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.     

Functional Impairment in Protein Kinase C by RACK1 (Receptor for Activated C Kinase 1) Deficiency in Aged Rat Brain Cortex

Abstract: Several laboratories have reported a lack of protein kinase C (PKC) activation in response to various stimuli in the brain of aged rats. It has been suggested that changes in lipid membrane composition could be related to this functional deficit. However, recent evidence has indicated that the translocation of PKC to the different subcellular compartments is controlled by protein-protein interactions. Recently, a class of proteins, termed receptors for activated C kinase (RACKs), have been described that bind PKC. The present study was conducted to determine whether alterations in RACK1, the best-characterized member of RACKs, were associated with changes in translocation and expression of PKC. Quantitative immunoblotting revealed that RACK1 content was decreased by ∼50% in aged rat brain cortex, compared with that in adult and middle-aged animals. The levels of calcium-independent PKCδ and ε, interacting with RACK1, and related calcium-independent PKC activity were not modified by the aging process. By comparison, phorbol ester-stimulated translocation of this activity and of PKCδ and ε immunoreactivity was absent in cortex from aged animals, as well as the translocation of the calcium-dependent PKCβ, also known to interact with RACK1. These results indicate that a deficit in RACK1 may contribute to the functional impairment in PKC activation observed in aged rat brain.     

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.     

Roles for Protein Kinase C and Mitogen-Activated Protein Kinase in Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Abstract: Both the Ca²⁺/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca²⁺ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca²⁺ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC₅₀ 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC₅₀～10 µM) than that induced by nicotine (IC₅₀～30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca²⁺ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.     

Protected-Site Phosphorylation of Protein Kinase C in Hippocampal Long-Term Potentiation

Abstract: One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the α and βII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca²⁺, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a “protected site” model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.     

Activation of Protein Kinase C by Purified Bovine Brain 14-3-3: Comparison with Tyrosine Hydroxylase Activation

Abstract: In the course of the purification of 14-3-3 protein (14-3-3) we found that 14-3-3 isolated from bovine forebrain activates protein kinase C (PKC), rather than the previously reported protein kinase C inhibitory activity (KCIP). We have characterized the 14-3-3 activation of PKC. The physical properties of purified PKC activator are the same as those previously reported for 14-3-3 and KCIP; i.e., (1) it is composed of subunits of molecular weight 32,000, 30,000, and 29,000; (2) it is homogeneous with respect to molecular weight, as judged by native gradient-gel electrophoresis, with a molecular weight of 53,000; and (3) it is composed of at least six isoforms when analyzed by reverse-phase HPLC. The concentration dependence of PKC activation by 14-3-3 is in the same range as that shown previously for KCIP inhibition of PKC, and as that required for 14-3-3 activation of tyrosine hydroxylase; a maximal stimulation of two- to three-fold occurs at 40–100 µg/ml. 14-3-3's activation of PKC is sensitive to α-chymotrypsin digestion but is not heat labile. Activation is specific to PKC; at least two other protein kinases, cyclic AMP- and calcium/calmodulin-dependent protein kinases, are not activated. The activation of PKC by 14-3-3 is independent of phosphatidylserine and calcium and, as such, is an alternative mechanism for the activation of PKC that obviates its translocation to membranes.     

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.     

Loss of Protein Kinase C-αβ in Brain of Heroin Addicts and Morphine-Dependent Rats

Abstract: The biochemical status of human brain protein kinase C (PKC)-αβ during opiate dependence was studied by means of immunoblotting techniques in postmortem brain of heroin addicts who had died by opiate overdose. In the frontal cortex, a marked decrease (53%, p < 0.05) in the immunoreactivity of PKC-αβ was found in heroin addicts compared with matched controls. The loss of PKC-αβ in the brain of human addicts paralleled that observed in the frontal cortex of rats after chronic treatment with morphine (10–100 mg/kg i.p. for 5 days) (PKC-αβ decreased by 34%, p < 0.05). Chronic treatment with naloxone (1 mg/kg i.p. every 12 h for 5 days) did not alter PKC-αβ immunoreactivity in the rat brain. However, in morphine-dependent rats, naloxone-precipitated withdrawal induced a rapid and strong behavioral reaction with a concomitant up-regulation of PKC-αβ immunoreactivity to control values. These results indicated that the decrease of brain PKC-αβ induced by heroin/morphine is a μ-opioid receptor-mediated effect. The chronic administration of opiates has been associated with a marked sensitization of the adenylyl cyclase/cyclic AMP system, although this phenomenon is not exclusive of the opioid system but the general cellular adaptation to chronic inhibition of adenylyl cyclase. In this context, chronic treatment of rats with other inhibitory agonists (e.g., clonidine, 1 mg/kg i.p. every 12 h for 14 days) acting through receptors (e.g., α₂-adrenoceptors) also coupled to adenylyl cyclase did not alter brain PKC-αβ immunoreactivity. Together these findings suggest that the brain PKC system might play a major role in opiate addiction.