Effect of Brain Ischemia on Protein Kinase C

We examined the influence of brain ischemia on the activity and subcellular distribution of protein kinase C (PKC). Two different models of ischemic brain injury were used: postdecapitative ischemia in rat forebrain and transient (6-min) cerebral ischemia in gerbil hippocampus. In the rat forebrain model, at 5 and 15 min postdecapitation there was a steady decrease of total PKC activity to 60% of control values. This decrease occurred without changes in the proportion of the particulate to the soluble enzyme pools. Isolated rat brain membranes also exhibited a concomitant decrease of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding with an apparent increase of the ligand affinity to the postischemic membranes. On the other hand, the ischemic gerbil hippocampus model displayed a 40% decrease of total PKC activity, which was accompanied by a relative increase of PKC activity in its membrane-bound form. This resulted in an increase in the membrane/total activity ratio, indicating a possible enzyme translocation from cytosol to the membranes after ischemia. Moreover, after 1 day of recovery, a statistically significant enhancement of membrane-bound PKC activity resulted in a further increase of its relative activity up to 162% of control values. In vitro experiments using a synaptoneurosomal particulate fraction were performed to clarify the mechanism of the rapid PKC inhibition observed in cerebral tissue after ischemia. These experiments showed a progressive, Ca(2+)-dependent, antiprotease-insensitive down-regulation of PKC during incubation. This down-regulation was significantly enhanced by prior phorbol (PDBu) treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translocation and Activation of Protein Kinase C in Striatal Neurons in Primary Culture: Relationship to Phorbol Dibutyrate Actions on the Inositol Phosphate Generating System and Neurotransmitter Release

The actions of the tumor-promoting phorbol ester phorbol dibutyrate were examined, under identical physiological conditions, on three distinct cellular processes in striatal neurons: the distribution of protein kinase C, the carbachol-stimulated generation of [3H]inositol monophosphate, and the KCl-evoked release of gamma-[3H]aminobutyric acid ([3H]GABA). Phorbol dibutyrate induced a rapid (complete in 5 min), dose-dependent, entirely reversible (t0.5 = 15 min) translocation of protein kinase C from cytosol to membrane. On longer exposure to phorbol dibutyrate, membrane-associated protein kinase C returned toward the control level, and total cellular enzyme activity declined markedly. Phorbol dibutyrate also induced the dose-dependent attenuation of carbachol-stimulated [3H]inositol monophosphate production and potentiation of KCl-evoked release of [3H]GABA. The translocation of protein kinase C and the potentiation of KCl-evoked [3H]GABA release were both rapidly reversed following washout of phorbol dibutyrate. In addition, for both processes, the effect of a 1-h exposure to phorbol dibutyrate was markedly less than that observed following a 5-min exposure to the agent. In direct contrast, inhibition of carbachol-stimulated [3H]inositol monophosphate production was not rapidly reversed following washout of phorbol dibutyrate and was actually more pronounced following a 1-h exposure, compared with a 5-min exposure. These findings indicate that some, but not all, of the actions of phorbol dibutyrate are closely associated with the translocation of protein kinase C in striatal neurons in primary culture.     

Synaptosomal Protein Kinase C Subspecies: B. Down-Regulation Promoted by Phorbol Ester and Its Effect on Evoked Norepinephrine Release

The effect of phorbol esters was investigated on the down-regulation of protein kinase C (PKC) and on the release of [3H]norepinephrine (NE) in synaptosomes from the rat cerebrum. Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) promoted the translocation of PKC activity in a P2 fraction from the cytosol to the membrane fraction and then its down-regulation, in a dose-dependent manner. TPA induced a rapid down-regulation of the type II(beta) and type III(alpha) subspecies, but did not change the activity of the type I(gamma) subspecies in the cytosolic fraction for at least 15 min. The gamma-subspecies was subsequently decreased at a slower rate. In the synaptosomes thus having only the gamma-subspecies, a subsequent dose of TPA could not enhance K(+)-evoked NE release, although, in the original synaptosomes, TPA was able to enhance K(+)-evoked NE release. Pretreatment with TPA did not alter the K(+)-evoked NE release itself. TPA was also found to enhance the K(+)-evoked NE release from synaptosomes prepared from both hippocampus, which express the gamma-subspecies of PKC at a negligible level, and cerebral cortex, which have a significant level of the gamma-subspecies, to the same degree. These results suggest that the gamma-subspecies of PKC does not participate in the TPA-enhanced K(+)-evoked NE release from synaptosomes.     

Evidence that Brain-Derived Neurotrophic Factor Neuroprotection Is Linked to Its Ability to Reverse the NMDA-Induced Inactivation of Protein Kinase C in Cortical Neurons

Abstract : Several lines of evidence indicate that a rapid loss of neuronal protein kinase C (PKC) activity is a characteristic feature of cerebral ischemia and is a necessary step in the NMDA-induced death of cultured neurons. Exposing embryonic day 18 primary rat cortical neurons to 50 μ M NMDA or 50 μ M glutamate for 10 min caused ~80% cell death over the next 24 h, but excitotoxic death was largely averted, i.e., by 70-80%, in cells pretreated with brain-derived neurotrophic factor (BDNF). An 8-h preexposure to BDNF (50-100 ng/ml) maximally protected cortical cells from the effects of NMDA and glutamate, although the transient application of BDNF between 8 and 4 h before NMDA was equally protective. These effects of BDNF were abolished at supralethal, i.e., >100 μ M , NMDA concentrations. It is significant that BDNF pretreatment prevented the inactivation of PKC in cortical cells normally seen 30 min to 2 h following lethal NMDA or glutamate exposure. This BDNF effect did not arise from changes in NMDA channel activity because neither whole-cell NMDA current amplitudes nor increases in intracellular free Ca²⁺ concentration were altered by the 8-h BDNF pretreatment. Furthermore, BDNF offered no neuroprotection to cells treated with the PKC inhibitors staurosporine (10-20 n M ), calphostin C (1-2.5 μ M ), or GF-109203X (100 n M ) at the time of NMDA addition. These results underscore the importance of PKC inactivation in glutamate-induced neuronal death. They also suggest that BDNF neuroprotection arises, at least in part, via its ability to block the mechanism by which pathophysiological Ca²⁺ influx through the NMDA receptor causes membrane PKC inactivation.     

Decreased Protein Kinase C Activity During Cerebral Ischemia and After Reperfusion in the Adult Rat

The possible activation of protein kinase C (PKC) during total cerebral ischemia was investigated in the rat. Translocation of PKC activity from the soluble to the particulate fraction was used as an index of PKC activation. There was a drop in the proportion of particulate PKC activity from 30% for controls to 20% by 30 min of ischemia (p less than 0.01). By 20 min of cardiac arrest, there was a 40% decline of the total cellular PKC activity (p less than 0.01). This was not accompanied by an increase in activator-independent activity, a finding indicating PKC was not being converted to protein kinase M. These data suggest that PKC was not activated during ischemia, but rather that ischemia causes a reduction in cellular PKC activity. Translocation of PKC activity to the particulate fraction was not observed in the cerebral cortex or hippocampus of reperfused brain for up to 6 h of recovery following 11-13 min of total cerebral ischemia. The level of total, soluble, and particulate PKC activity in the cerebral cortex was reduced (p less than 0.05), corresponding to the decrease observed by 15 min of ischemia without reflow. A similar decline in activity was also observed in the hippocampus. No increase in activator-independent activity was observed. These data suggest that PKC was inhibited during cerebral ischemia and that this reduced level of PKC activity was maintained throughout 6 h of recovery. We conclude that pathological activation of PKC was not responsible for the evolution of ischemic brain damage.     

Dopamine Release via Protein Kinase C Activation in the Fish Retina

Calcium-dependent phospholipid-sensitive protein kinase [protein kinase C (PKC)] was partially purified from the carp (Cyprinus carpio) retina through DE 52 ion exchange and Cellulofine gel filtration chromatography. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) activated PKC in the nanomolar range. A major 38-kDa protein in the retinal supernatants (105,000 g) was phosphorylated in vitro by PKC during a short period (3 min). Other phosphoproteins also appeared during a further prolonged period (greater than 15 min). Rod-bipolar and dopamine (DA) interplexiform cells in the fish retina were immunoreactive to a monoclonal antibody to PKC (alpha/beta-subtype). The PKC antibody recognized a 78-kDa native PKC enzyme by means of an immunoblotting method. Subsequently, the effects of two kinds of PKC activators were investigated on [3H]DA release from retinal cell fractions containing DA cells that had been preloaded with [3H]DA. A phorbol ester (TPA) induced a calcium- and dose-dependent [3H]DA release during a short period (2 min), with the minimal effective dose being approximately 1 nM. Other phorbols having no tumor-promoting activity, such as 4 beta-phorbol and 4 alpha-phorbol 12,13-didecanoate, were ineffective on [3H]DA release. A synthetic diacylglycerol [1-oleoyl-2-acetylglycerol (OAG)], which is an endogenous PKC activator, was also able to induce a significant release of [3H]DA. Furthermore, TPA was found to release endogenous DA from isolated fish retina by a highly sensitive HPLC with electrochemical detection method. The OAG- or TPA-induced [3H]DA or DA release was completely blocked by inhibitors of PKC, such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of Two Distinct Populations of Protein Kinase C in Rat Brain Membranes

The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane-associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein-lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein-lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident.     

Protein Kinase C Activity in Rat Brain Cortex

The procedure used to obtain cerebral tissue for analysis of protein kinase C (PKC) activity may affect the subcellular distribution of the enzyme. We compared different methods of tissue preparation and found that the proportion of PKC activity associated with the particulate fraction of the cerebral cortex was only 30% when the brain was frozen in situ while the animal was on life support or after decapitation followed by delayed freezing. Other methods of obtaining cerebral tissue resulted in 49-56% of the PKC activity in the particulate fraction. Freezing per se had no apparent effect on the activity or subcellular distribution of PKC. In addition, whenever the particulate PKC activity was high (greater than 48%), there was also a significant increase in the proportion of particulate protein (from 51 to approximately 63%, p less than 0.05).     

Differential Effects of Ipsapirone on 5-Hydroxytryptamine Release in the Dorsal and Median Raphe Neuronal Pathways

Abstract: Serotonergic neurons of the dorsal and median raphe nuclei are morphologically dissimilar. Recent results challenge previous evidence indicating a greater inhibition of dorsal raphe neurons after 5-hydroxytryptamine_1A (5-HT_1A) autoreceptor activation. As both nuclei innervate different forebrain territories, this issue is critical to understanding the changes in brain function induced by anxiolytic and antidepressant drugs. Using microdialysis, we examined the modifications of 5-HT release induced by the selective 5-HT_1A agonist ipsapirone in both neuronal pathways. Maximal and minimal basal 5-HT values (in the presence of 1 µ M citalopram) were 45.0 ± 4.8 fmol/fraction in the median raphe nucleus and 8.4 ± 0.4 fmol/fraction in the dorsal hippocampus. Ipsapirone (0.3, 3, and 10 mg/kg s.c.) reduced dose-dependently 5-HT in the two raphe nuclei and four forebrain areas. Maximal reductions (to ∼25% of predrug values) were observed in cortex and striatum and in median raphe nucleus. The effects were more moderate in dorsal and ventral hippocampus (to 66 and 50% of baseline, respectively). These results are consistent with a higher sensitivity of dorsal raphe neurons to 5-HT_1A autoreceptor activation. Yet the differential reduction of 5-HT release in the median raphe nucleus and hippocampus suggests the presence of complex mechanisms of control of 5-HT release in these neurons.     

Effect of Electroconvulsive Shock on the Uptake and Release of Noradrenaline and 5-Hydroxytryptamine in Rat Brain Slices

Abstract: The uptake and release of [³H]noradrenaline and [³H]-5-hydroxytryptamine (5-HT) were studied in cerebral cortex slices from rats 30 min and 24 h after a single electroconvulsive shock (ECS) and 24 h after a series of five shocks given over 10 days. Both the K _m and V _max for 5-HT uptake were lower than controls 24 h after a single ECS, whereas after 5 ECS spread over 10 days both parameters remained depressed, though only the fall in V_max was significant. Noradrenaline uptake was not altered after a single ECS, but the V_max and K _m were elevated following chronic ECS treatment. Neither ECS treatment schedule had any effect on the potassium-stimulated release of either transmitter. It is possible that the changes in monoamine uptake seen following ECS are an adaptive response to alterations in the synaptic cleft concentration of these transmitters.     

Activation of Protein Kinase C Augments Peptide Release from Rat Sensory Neurons

Abstract: To determine whether protein kinase C (PKC) mediates release of peptides from sensory neurons, we examined the effects of altering PKC activity on resting and evoked release of substance P (SP) and calcitonin gene-related peptide (CGRP). Exposing rat sensory neurons in culture to 10 or 50 n M phorbol 12,13-dibutyrate (PDBu) significantly increased SP and CGRP release at least 10-fold above resting levels, whereas the inactive 4α-PDBu analogue at 100 n M had no effect on release. Furthermore, 100 n M bradykinin increased peptide release approximately fivefold. Down-regulation of PKC significantly attenuated the release of peptides evoked by either PDBu or bradykinin. PDBu at 1 n M or 1-oleoyl-2-acetyl- sn -glycerol at 50 µ M did not alter resting release of peptides, but augmented potassium- and capsaicin-stimulated release of both SP and CGRP approximately twofold. This sensitizing action of PKC activators on peptide release was significantly reduced by PKC down-regulation or by pretreating cultures with 10 n M staurosporine. These results establish that activation of PKC is important in the regulation of peptide release from sensory neurons. The PKC-induced enhancement of peptide release may be a mechanism underlying the neuronal sensitization that produces hyperalgesia.     

Noradrenaline Release from Streptolysin O-Permeated Rat Cortical Synaptosomes: Effects of Calcium, Phorbol Esters, Protein Kinase Inhibitors, and Antibodies to the Neuron-Specific Protein Kinase C Substrate B-50 (GAP-43)

We studied the molecular mechanism of noradrenaline release from the presynaptic terminal and the involvement of the protein kinase C substrate B-50 (GAP-43) in this process. To gain access to the interior of the presynaptic terminal, we searched for conditions to permeate rat brain synaptosomes by the bacterial toxin streptolysin O. A crude synaptosomal/mitochondrial preparation was preloaded with [3H]noradrenaline. After permeation with 0.8 IU/ml streptolysin O, noradrenaline efflux could be induced in a concentration-dependent manner by elevating the free Ca2+ concentration from 10(-8) to 10(-5) M. Efflux of the cytosolic marker protein lactate dehydrogenase was not affected by this increase in Ca2+. Ca2(+)-induced efflux of noradrenaline was largely dependent on the presence of exogenous ATP. Changing the Na+/K+ ratio in the buffer did not affect Ca2(+)-induced noradrenaline release. Release of noradrenaline could also be evoked by phorbol esters, indicating the involvement of protein kinase C. Ca2(+)- and phorbol ester-induced release were not additive at higher phorbol ester concentrations (greater than 10(-7) M). We compared the sensitivities of Ca2(+)- and phorbol ester-induced release of noradrenaline to the protein kinase inhibitors H-7 and polymyxin B and to antibodies raised against synaptic protein kinase C substrate B-50. Ca2(+)-induced release was inhibited by B-50 antibodies and polymyxin B, but not by H-7; phorbol ester-induced release was inhibited by polymyxin B and by H-7, but only marginally by antibodies to B-50.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ischemia-Triggered Translocation and Inactivation of Protein Kinase C Isoforms in the Fetal Brain

Abstract: The kinetics of protein kinase C (PKC) translocation and down-regulation in the 20-day-old fetal brain following short and long episodes of maternal-fetal blood flow occlusion were examined. Restriction for up to 15 min increased the specific enzymatic activity in the membrane by 73%, indicative of translocation. After a 30-min restriction and a 2.5-h reperfusion the total PKC activity in the cytosol was reduced to ～50%, consistent with down-regulation/inactivation. The total membrane PKC activity remained unchanged. Several PKC isoenzymes, including α, β1, β2, ε, and ζ, but not γ, were identified in the fetal brain on western blots using specific antibodies. Compared with postnatal day 15, a greater proportion of the fetal PKC isoforms, particularly α and ε, were membrane bound. α, β2, ε, and ζ, but not β1, were translocated into the membrane compartment after episodes of ischemia alone or ischemia and reperfusion. There were no major identifiable proteolytic fragments in the 50-kDa region. Major losses in the total enzymatic activity were encountered in both cytosol and membrane fractions after storage of the enzyme for 10 days at 4°C. These losses were less profound in membrane fractions from ischemic than control animals, suggesting a relative sparing of activity in the membrane as a result of the insult. Preincubation of DEAE-purified PKC for 30 min at 50°C resulted in enzyme inactivation. This was accompanied by a size reduction (～2–5 kDa) in the gel migration of several isozymes in both cytosol and membrane fractions. At 42°C, although the molecular size was apparently reduced, limited PKC activity was observed, suggesting either that the two processes are not mutually related or that certain PKC isoforms can act after partial modification. The data suggest that ischemic episodes stimulate two apparently adverse processes in the PKC signal transduction cascade: a decline in the cytosol and a sparing of the membrane-translocated PKC activity. The latter may provide an important regulatory mechanism for PKC long-term activation in nerve cells.     

High-Pressure Extraction of Membrane-Associated Protein Kinase C from Rat Brain

Extraction of rat brain membrane-associated protein kinase C with high specific activity was obtained by applying benzyl alcohol (a membrane fluidizer), EDTA, and high hydrostatic pressures. Approximately 50% of total brain-associated activity was extracted from membranes. The pressure-extracted activity had an eightfold enrichment in the lipid/protein ratio when compared with the cytosolic fraction. This may explain the inability of exogenous diacylglycerol to stimulate endogenous phosphorylation in pressure-extracted activity. The enzyme is extracted at greater than 1,300 atm, a result indicating it most likely has a portion inserted into the hydrophobic portion of the membrane bilayer. Perturbation of the native membrane induces a change in the membrane-associated protein kinase C-lipid interaction that permits extraction under conditions used for the cytosolic species. This is the first report of conversion of the endogenous membrane species to a cytosolic one and may be important in determining the role of protein kinase C in neuronal regulation.     

Pentylenetetrazole-Induced Chemoshock Affects Protein Kinase C and Substrate Proteins in Mouse Brain

Abstract: Protein kinase C (PKC) activity, western blot analysis of PKCα, β, γ, ε, and ζ by isozyme-specific antibodies, and in vitro phosphorylation of endogenous substrate proteins were studied in the mice brain after pentyl-enetetrazole-induced chemoshock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified by DE-52 columns eluted with buffer A containing 100 or 200 m M KCI. This method consistently separates cytosolic and membrane proteins and various PKC isoforms. The 100 m M KCI eluates from DE-52 columns contain more PKC α and β in both cytosol and membrane than the 200 m M KCI eluates, whereas PKCγ, ε, and ζappear in equal amounts in these two eluates. The kinase activity assayed by phosphorylation of exogenous histone was increased in the chemoshocked mice in both the cytosol and membrane of 200 m M KCI eluates. In further analysis by immunoblotting, this increased activity was found to be due to the increase in content of PKC7 isozyme. As for novel-type ε and ζ isozymes, they were not altered in the chemoshocked mice. From autoradiography, the endogenous substrate 17-kDa neurogranin, which was shown below 21 kDa, was mostly eluted by 100 m M KCI from the DE-52 column, whereas 43-kDa neuromodulin, which was also demonstrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, only appeared in the 200 m M KCI eluates. The in vitro phosphorylation of neuromodulin was found to be increased in the chemoshocked mice. Therefore, the increased phosphorylation of neuromodulin and increased content of the PKCγ isoform were involved in the pentylenetetrazole-induced chemoshock.     

Effect of Protein Kinase C Activation on the Release of [3H]Acetylcholine in the Presence of Vesamicol

Abstract: The present work tested whether pharmacological activation of protein kinase C (PKC) influences the release of [³H]-acetylcholine ([³H]ACh) synthesized in the presence of vesamicol, an inhibitor of the vesicular acetylcholine transporter (VAChT). Newly synthesized [³H]ACh was released from hippocampal slices by field stimulation (15 Hz) in the absence of vesamicol, but as expected [³H]ACh synthesized during exposure to vesamicol was not released significantly by stimulation. Treatment of slices with the PKC activator phorbol myristate acetate (PMA) decreased the inhibitory effect of vesamicol on [³H]ACh release. The effect of PMA was dose-dependent, was sensitive to calphostin C, a PKC-selective inhibitor, and could not be mimicked by α-PMA, an inactive phorbol ester. PMA did not alter the release of [³H]ACh in the absence of vesamicol, suggesting that the site of PKC action could be related to the VAChT. In agreement with this observation, immunoprecipitation of VAChT from ³²P-labeled synaptosomes showed that phosphorylation occurs and that incorporation of ³²P in the VAChT protein increases in the presence of PMA. We suggest that PKC alters the output of [³H]ACh formed in the presence of vesamicol and also provide circumstantial evidence for a role of phosphorylation of VAChT in this process.     

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

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 but not in hippocampus [corrected]. 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.     

Effect of Latent Iron Deficiency on 5-Hydroxytryptamine Metabolism in Rat Brain

Eight weeks of latent iron deficiency in weaned rats maintained on an experimental low iron content diet (18-20 mg/kg) did not significantly alter the packed cell volume and hemoglobin concentration; however, the hepatic and brain nonheme iron contents decreased by 66% and 21% (p less than 0.001), respectively. The tryptophan concentration decreased by 31% and 34% in liver and brain, respectively, in rats on experimental diet (p less than 0.01). The brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid contents were reduced by 21% and 23% (p less than 0.01 and p less than 0.02), respectively. However, in the brain, weight, protein, DNA, and the activities of monoamine oxidase, aldehyde dehydrogenase, and liver tryptophan oxygenase were found to remain unaltered. When rehabilitated with a diet containing 390 mg/kg iron, rats previously maintained on the experimental diet for 2 weeks showed partial recovery in tryptophan levels both in liver and brain. However, brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid levels remained unaltered. The hepatic iron content improved without any change in brain iron content. The latent iron deficiency produced significant alterations in the metabolism of 5-hydroxytryptamine and brain iron content that could not be recovered 2 weeks after the iron rehabilitation.     

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.     

Activation of Protein Kinase C in Permeabilized Human Neuroblastoma SH-SY5Y Cells

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.