Hoau-Yan Wang

Enhanced Protein Kinase C Activity and Translocation in Bipolar Affective Disorder Brains

Protein kinase C (PKC) activity and its redistribution were determined in the frontal cortices of postmortem brains of bipolar affective disorder subjects and age-, sex-, and postmortem time-matched controls. Membrane and cytosolic PKC activity was determined by histone phosphorylation using [32P]-adenosine triphosphate as substrate. Specific PKC isozyme levels were assessed by Western blot analysis using antipeptide antibodies. Brain membrane-associated PKC activity was higher in bipolar vs. control tissue. An examination of the specific PKC isozymes in cortical homogenates revealed that cytosolic alpha- and membrane-associated gamma- and zeta PKC isozymes were elevated in cortices of bipolar affective disorder subjects, whereas cytosolic epsilon PKC was found to be reduced. In control brain slices, incubation with 1 mumol/L phorbol 12-myristate 13-acetate (PMA) caused an increase in membrane PKC activity, whereas cytosolic enzyme activity was decreased. This redistribution of the enzyme by PMA was markedly potentiated in brain slices of bipolar subjects. The results suggest that PKC-mediated phosphorylation is increased in brains of subjects with bipolar affective illness.

Attenuated protein kinase C activity and translocation in Alzheimer's Disease brain

Protein kinase C (PKC) activity and its redistribution were determined in postmortem Alzheimers disease (AD) and age-matched control brains. Cytosolic and membrane-associated PKC activities were lower in frontal and temporal cortices and hippocampi of AD brains. Increased concentrations of phosphatidyl-L-serine, Ca2+ or phorbol 12-myristate, 13-acetate only weakly increased enzyme activity in AD tissues. Redistribution of cytosolic PKC to the membranous fraction was elicited in control brain slices by 162 nM PMA in the presence of K+ (65 mM). This redistribution of the enzyme was markedly reduced in AD brain slices. In contrast, the immunoreactivity of the alpha- and gamma-PKC isozymes were elevated in cortical tissue from AD subjects. No changes were noted in beta-PKC immunoreactivity. These results suggest that the reduced PKC activity and the attenuated translocation of the enzyme in AD brain tissue may be attributed to down regulation of PKC or to alteration in PKC protein. The increase in PKC immunoreactivity may be a reflection of an altered susceptibility to proteolysis or a compensatory response secondary to the loss in enzyme activity.

Effect of Age on Brain Cortical Protein Kinase C and Its Mediation of 5-Hydroxytryptamine Release

Abstract: The effects of age on the activity and translocation of protein kinase C (PKC) and on the facilitation of 5‐hydroxytryptamine (5‐HT, serotonin) release induced by PKC activation with the phorbol ester phorbol 12‐myristate 13‐acetate were investigated. The activities of cortical PKC and its translocation in response to K+ depolarization and phorbol ester stimulation were reduced during aging in Fischer‐344 rats. Parietal cortical brain slices from 6‐, 12‐, and 24‐month‐old animals were preloaded with [3H]5‐HT and release was evoked by 65 mM K+ or the calcium ionophore A23187. 5‐HT release induced by either K+ or A23187 was found to be reduced in 12‐ and 24‐month‐old as compared to 6‐month‐old animals. This decrease was not reversed by high extracellular Ca2+. Activation of PKC resulted in a facilitated transmitter release in tissue from 6‐ and 12‐month‐old animals but reduced [3H]5‐HT release in slices from 24‐month‐old animals. These responses were prevented by the putative PKC inhibitor 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H‐7), but not by increasing extracellular or intracellular Ca2+. The results demonstrate an age‐related change (1) in brain PKC activity and translocation and (2) in a physiological response to PKC stimulation. These results may have implications for other PKC‐mediated functions that are altered during senescence.

Lithium inhibition of protein kinase C activation-induced serotonin release

The effect of lithium on protein kinase C (PKC) stimulation-induced serotonin and norepinephrine release facilitation was examined in [3H]5-HT and [3H]NE preloaded superfused rat brain slices. The facilitation of K+-evoked [3H]5-HT release elicited by the active phorbol esters PMA and PDBu was inhibited by 4 mM but not by 1 mM in vitro lithium. In experiments performed in cortical, hippocampal and hypothalamic slices obtained from animals treated for 3 weeks with lithium-containing diet, PMA-induced facilitation of K+-elicited [3H]5-HT release was found to be inhibited by the treatment (serum lithium <1 mEq/l). Basal [3H]5-HT efflux, which was enhanced by PMA in control animals, was also inhibited by lithium treatment. In parietal cortical slices, PMA elicited increase in K+-evoked [3H]NE release was prevented in slices taken from lithium-treated (3 weeks) animals. Lithium treatment did not affect the activity and distribution of protein kinase C in cortical tissue. However, 3 weeks of treatment reduced the PMA-induced translocation of the enzyme. These results suggest that lithium treatment interferes with serotonin and norepinephrine release facilitation which results from the stimulation of PKC by phorbol esters. These actions of the ion may be mediated by its ability to inhibit PMA induced PKC translocation.

Receptor-mediated activation of G proteins is reduced in postmortem brains from Alzheimer's disease patients

The effects of Alzheimers disease (AD) on [35S]GTP gamma S binding to G proteins was examined in postmortem cerebrocortex. Stimulation of the beta-adrenergic or muscarinic cholinergic receptors in control tissue with selective agonists resulted in increases in [35S]GTP gamma S binding to G alpha proteins in a receptor-specific fashion. The responses were markedly reduced in brain tissues from AD patients. In contrast, basal [35S]GTP gamma S binding to the G alpha proteins was relatively intact in AD brains. Immunoblot analyses reveal that levels of cerebrocortical G alpha proteins in AD are not altered. The results suggest that in AD the decrease in agonist-stimulated [35S]GTP gamma S binding to G proteins may be a result of decoupling between receptors and their associated G proteins.

Chronic lithium: desensitization of autoreceptors mediating serotonin release

The effect of chronic lithium treatment on K+-induced release of preloaded [3H] serotonin ([3H]5-HT) from brain slices and its regulation by the presynaptic serotonin autoreceptors were investigated in superfused cortical, hippocampal and hypothalamic brain slices. Three weeks of treatment with a lithium-containing diet increased stimulation-induced [3H]5-HT overflow in the three brain regions examined. The sensitivity of the inhibitory serotonin autoreceptors was tested by determining K+-elicited release inhibition or potentiation in response to exposure to the agonist, LSD or to the antagonist, methiothepin, respectively. A reduced maximal inhibitory response to LSD was obtained in lithium-treated animals. The potentiation by methiothepin was also markedly diminished in the treated animals. These results suggest that chronic lithium treatment induces a desensitization of serotonin autoreceptors which may result in increased serotonin release from the serotonin nerve terminals.

Effect of Chronic Lithium Treatment on 5‐Hydroxytryptamine Autoreceptors and Release of 5‐[3H]Hydroxytryptamine from Rat Brain Cortical, Hippocampal, and Hypothalamic Slices

Abstract: The effect of acute and chronic lithium treatments on 5‐hydroxytryptamine (5‐HT, serotonin) release and on its regulation by presynaptic 5‐HT autoreceptors was studied in [3H]5‐HT preloaded superfused rat brain slices. The [3H]5‐HT overflow evoked by a 30‐s exposure to 65 mM K+ was increased after 3 weeks of ingestion of lithium‐containing diet in the three brain areas examined. Acute injection of 4 mEq/kg lithium chloride did not affect 5‐HT release. The K+‐induced release observed in both control and chronically lithium‐treated animals was Ca2+‐dependent. Chronic lithium treatment was also found to be associated with a decrease in basal [3H]5‐HT overflow in the cortex and hypothalamus and with an increase in spontaneous hippocampal 5‐HT overflow. The Ca2+‐independent overflow induced by fenfluramine was also decreased in cortical slices from lithium‐treated animals. The sensitivity of the inhibitory 5‐HT autoreceptors was assessed by the response to the 5‐HT agonist 5‐methoxytryptamine. The results indicate a marked reduction in the maximal inhibition of [3H]5‐HT release induced by 5‐methoxytryptamine in slices obtained from animals which have been treated with lithium for 3 weeks. These data suggest that the functional down regulation of the prejunctional 5‐HT sites may be responsible for the increase in K+‐stimulated 5‐HT overflow in brain slices of animals treated chronically with lithium.

Protein kinase C: regulation of serotonin release from rat brain cortical slices

The effects of phorbol esters on serotonin release were examined in an attempt to investigate the role of protein kinase C in the regulation of serotonin release. Rat brain parietal cortical slices were incubated with [3H]5-HT in the presence of pargyline in order to label the serotonin stores. Potassium stimulated (30 s) release and spontaneous [3H]5-HT efflux were examined in slices during superfusion with Krebs-Ringer solution containing chlorimipramine. Repeated K+ stimulations elicited reproducible responses with release ratios of approximately 1.0. Introduction of phorbol 12-myristate, 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu) 20 min prior to S2, or S3 resulted in dose-related increases in [3H]5-HT or [3H]NE release. PMA was slightly more potent (93% increase) than PDBu in potentiating K+-stimulated [3H]5-HT release. Phorbol and 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) which do not activate protein kinase C did not alter serotonin release. In contrast, basal [3H]5-HT and [3H]NE release were altered to a far lesser extent which was not always dose related. The response to the phorbol esters was reversible, Ca2+-dependent and reached maximal effect after 20 min of superfusion. The putative protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibited K+-induced [3H]5-HT release significantly (11%) but did not alter basal efflux. The PMA facilitation of serotonin release was, however, markedly prevented by the enzyme inhibitor. The effect of PMA on release was found not to be directly mediated through the prejunctional serotonin autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Guanine nucleotide regulatory proteins, Gq and Gi1/2, mediate platelet-activating factor-stimulated phosphoinositide metabolism in immortalized hippocampal cells.

Abstract: Platelet‐activating factor (PAF) may be a neuromodulator involved in neural cell differentiation, cerebral inflammation, and ischemia. The PAF receptor is a member of the G protein‐coupled receptor superfamily. In the present study, we sought to define the specific G protein(s) that mediate PAF‐stimulated phosphoinositide (PI) metabolism in an immortalized hippocampal cell line, HN33.11. PAF increased the production of 3H‐labeled inositol phosphates (IPs) with EC50 values of 1.2–1.5 nM. The effect of PAF on 3H‐IPs formation was completely blocked by the PAF antagonist BN 50739 at a concentration of 300 nM. Pertussis toxin pretreatment attenuated PAF‐stimulated 3H‐IPs production by 20–30% (p < 0.05). Consistent with a role for Gi1/2 in this response, antiserum against Gαi1/2 blocked the response to a similar degree. Pretreatment of permeabilized cells with Gαq/11 antiserum attenuated the response by 70% (p < 0.05), suggesting a role for Gq/11 in mediating the PAF response in this cell line. Stimulation with PAF increased [α‐32P]‐GTP binding to both Gαq and Gαi1/2 proteins. Moreover, specific [3H]PAF binding sites coprecipitated with Gαq and Gαi1/2 proteins. The results suggest that PAF‐stimulated PI metabolism in HN33.11 cells is mediated by both Gq and Gi1/2 proteins.

Protein kinase C translocation in human blood platelets

Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 microM concentrations did not induce redistribution of platelet PKC. Serotonin (0.5-100 microM) and the specific 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (10-100 microM) but not the 5-HT1A or 5-HT1B agonists, (+/-) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT2 receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT2 receptors.