Particulate Protein Kinase C Activity Research Articles

Entamoeba invadens: Sphingolipids metabolic regulation is the main component of a PKC signaling pathway in controlling cell growth and proliferation

The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917–924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6 h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4–8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous d-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.

Amended Final Report on the Safety Assessment of Glyceryl Dilaurate, Glyceryl Diarachidate, Glyceryl Dibehenate, Glyceryl Dierucate, Glyceryl Dihydroxystearate, Glyceryl Diisopalmitate, Glyceryl Diisostearate, Glyceryl Dilinoleate, Glyceryl Dimyristate, Glyceryl Dioleate, Glyceryl Diricinoleate, Glyceryl Dipalmitate, Glyceryl Dipalmitoleate, Glyceryl Distearate, Glyceryl Palmitate Lactate, Glyceryl Stearate Citrate, Glyceryl Stearate Lactate, and Glyceryl Stearate Succinate1

Amended Final Report on the Safety Assessment of Glyceryl Dilaurate, Glyceryl Diarachidate, Glyceryl Dibehenate, Glyceryl Dierucate, Glyceryl Dihydroxystearate, Glyceryl Diisopalmitate, Glyceryl Diisostearate, Glyceryl Dilinoleate, Glyceryl Dimyristate, Glyceryl Dioleate, Glyceryl Diricinoleate, Glyceryl Dipalmitate, Glyceryl Dipalmitoleate, Glyceryl Distearate, Glyceryl Palmitate Lactate, Glyceryl Stearate Citrate, Glyceryl Stearate Lactate, and Glyceryl Stearate Succinate1

Effects of fenfluramine and antidepressants on protein kinase C activity in rat cortical synaptoneurosomes.

Fenfluramine releases serotonin (5-HT) via the 5-HT transporter (SERT). Previous work has shown that amphetamine increases particulate protein kinase C (PKC) activity in striatal synaptoneurosomes. The increased PKC activity is linked to the outward transport of dopamine, and when release is diminished, the inward transport of amphetamine inhibits PKC instead. Since there is homology among monoamine transporters, this study was undertaken to determine if D-fenfluramine has similar effects on PKC. The role of 5-HT receptors and endogenous 5-HT were also examined. Naive rats and rats pretreated with p-chlorophenylalanine (PCPA), a 5-HT synthesis inhibitor, were sacrificed. Cortical synaptoneurosomes were prepared and incubated with fenfluramine. PKC activity was determined by thiophosphorylation of endogenous substrates. It was found that 5-HT, D/L-fenfluramine, and D-fenfluramine increased PKC activity in a time- and dose-dependent manner. The 5-HT-mediated increase in PKC activity was attenuated by pretreatment with the 5-HT(2) antagonist ketanserin, but not with the SERT inhibitor fluoxetine. The D-fenfluramine-induced increase in PKC activity was completely prevented, however, by pretreatment with SERT inhibitors and partially with ketanserin. It was also attenuated by pretreatment with PCPA, resulting in a dose-dependent inhibition of PKC instead. Thus, when 5-HT release was diminished the uptake of D-fenfluramine inhibited PKC. Similar effects have been observed with amphetamine. Unlike D-fenfluramine, the D/L-fenfluramine-induced increase in PKC activity was partially resistant to PCPA pretreatment but was attenuated with bupropion, a dopamine transporter (DAT) inhibitor. SERT inhibitors (sertraline, paroxetine, citalopram, and fluoxetine) also increased PKC activity. Nefazodone and bupropion increased PKC activity, but mirtazapine was relatively inactive. The SERT inhibitor-induced increase in PKC was unaffected by pretreatment with PCPA but was inhibited by calcium. Similar effects on PKC activity have been observed with DAT inhibitors. These results, showing that D-fenfluramine altered PKC activity similar to D-amphetamine, suggest that the topographic homology between DAT and SERT may extend to their effects on PKC activity.

Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells.

We have investigated the effects of inhibiting sphingomyelin (SM) biosynthesis on cellular diacylglycerol (DAG) content and protein kinase C (PKC) activation during growth initiation in Madin-Darby canine kidney cells. We utilized beta-chloroalanine (BCA) to inactivate serine C -palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway. This inactivation prevented growth, but did not affect viability. When the inhibitor was replaced with fresh culture medium, the cells continued their proliferation in a normal way. BCA (2 mM) inhibited [(32)P]P(i), [(3)H]palmitic acid and [ methyl -(3)H]choline incorporation into SM, but did not influence the synthesis of other major phospholipids. SM synthesis and DAG generation were decreased by 51% and 47.6% respectively. Particulate PKC activity was not observed in cells incubated with BCA, in contrast with a 5-fold increase in control cells. BCA inhibited 75% of the [(3)H]thymidine incorporation, and the cells were arrested before the S phase of the cell cycle. Moreover, exogenous D-erythrosphingosine restored SM synthesis, DAG generation and cell proliferation. These data indicate that the contribution of DAG generated during SM synthesis plays an important role in PKC activation and cell proliferation.

κ -Opioid Receptor Stimulation Induces Arrhythmia in the Isolated Rat Heart via the Protein Kinase C/Na+–H+Exchange Pathway

The present study attempted to determine whether the protein kinase C (PKC)/Na+–H+exchange (NHE) pathway would mediate the arrhythmogenic action ofκ -opioid receptor (OR) stimulation. We first determined the effects of U50,488H, a selective κ -OR agonist, on PKC activity and cardiac rhythm in the isolated perfused rat heart, and intracellular pH (pHi), and Ca2+([Ca2+]i) and Na+([Na+]i) concentrations in the isolated ventricular myocyte. At 5–40μ m U50,488H concentration dependently increased the particulate PKC activity and pHi, and induced arrhythmia. 40 μ m U50,488H also increased [Na+]iand [Ca2+]i. The arrhythmogenic effects of 40 μ m U50,488H were abolished by nor-binaltorphimine, a selective κ -OR antagonist. Blockade of PKC and NHE with respective blockers, 1 μ m bisindolylmaleimide I or 0.5 μ m calphostin C, and 1 μ m 5-[N -methyl- N -isobutyl]amiloride or 1 μ m 5-([N -ethyl- N -isopropopyl]amiloride, abolished and significantly attenuated, respectively, the effects of κ -OR stimulation on pHi, [Na+]iand [Ca2+]i, and arrhythmia. To determine the role of pHi, we observed U50,488H-induced arrhythmia at pHi6.8. At this pHi, the pHiincreased gradually both in the presence and absence of 40 μ m U50,488H to a similar extent. While the increase in response to U50,488H was significantly less at pHi6.8 (from 0.09 to 0.10) than that at pHi7.1 (from 0.01 to 0.18), the arrhythmia induced by the agonist was the same at both high and low pHs. On the other hand, 5μ m monensin, a sodium ionophore, increased [Na+]iand [Ca2+]i, and induced arrhythmia to similar extents as U50,488H. PKC and NHE inhibitors, that significantly attenuated the effects induced by U50,488H, had no effect on those induced by monensin. In conclusion, κ -OR stimulation induces arrhythmia via PKC/NHE. [Na+]iand [Ca2+]i, but not pHi, may be directly responsible for arrhythmia induced byκ -OR stimulation.

Effects of the peroxisome proliferator ciprofibrate and prostaglandin F2 alpha combination treatment on second messengers in cultured rat hepatocytes.

Peroxisome proliferators induce hepatic peroxisome proliferation and hepatic tumors in rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolizes lipids, including eicosanoids. Peroxisome proliferators transiently induce increased cell proliferation in vivo. However, peroxisome proliferators are weakly mitogenic and are not co-mitogenic with epidermal growth factor (EGF) in cultured hepatocytes. Earlier study found that the peroxisome proliferator ciprofibrate is comitogenic with eicosanoids. In order to study possible mechanisms of the comitogenicity of peroxisome proliferator ciprofibrate and eicosanoids, we hypothesized that the co-mitogenicity may result from synergistic or additive increases of second messengers in mitogenic signal pathways. We therefore examined the effect of the peroxisome proliferator ciprofibrate, prostaglandin F2 alpha (PGF2 alpha) and the combination of ciprofibrate and PGF2 alpha with or without growth factors on the protein kinase C (PKC) activity, and inositol-1, 4, 5-triphosphate (IP3) and intracellular calcium ([Ca2+]i) concentrations in cultured rat hepatocytes. The combination of ciprofibrate and PGF2 alpha significantly increased particulate PKC activity. The combination of ciprofibrate and PGF2 alpha also significantly increased EGF, transforming growth factor-alpha (TGF-alpha) and hepatic growth factor (HGF)-induced particulate PKC activity. The combination of ciprofibrate and PGF2 alpha greatly increased [Ca2+]i. However, the increases of PKC activity and [Ca2+]i by ciprofibrate and PGF2 alpha alone were much smaller. Neither ciprofibrate or PGF2 alpha alone nor the combination of ciprofibrate and PGF2 alpha significantly increased the formation of IP3. The combination of ciprofibrate and PGF2 alpha, however, blocked the inhibitory effect of TGF-beta on particulate PKC activity and formation of IP3 induced by EGF. These results show that co-mitogenicity of the peroxisome proliferator ciprofibrate and eicosanoids may result from the increase in particulate PKC activity and intracellular calcium concentration but not from the formation of IP3.

Regulation of renal Na-HCO3 cotransporter: VIII. Mechanism Of stimulatory effect of respiratory acidosis.

We examined the effect of respiratory acidosis on the Na-HCO3 cotransporter activity in primary cultures of the proximal tubule of the rabbit exposed to 10% CO2 for 5 min, 2, 4, 24 and 48 hr. Cells exposed to 10% CO2 showed a significant increase in Na-HCO3 cotransporter activity (expressed as % of control levels, 5 min: 142 +/- 6, 2 hr: 144 +/- 13, 4 hr: 145 +/- 11, 24 hr: 150 +/- 15, 48 hr: 162 +/- 24). The increase in activity was reversible after 48 hr. The role of protein kinase C (PKC) on the stimulatory effect of respiratory acidosis on the cotransporter was examined in presence of PKC inhibitor calphostin C or in presence of PKC depletion. Both calphostin C and PKC depletion prevented the effect of 10% CO2 for 5 min or 4 hr to increase the activity of the cotransporter. 10% CO2 for 5 min or 4 hr increased total and particulate fraction PKC activity. To examine the role of phosphotyrosine kinase (PTK) on the increase in cotransporter activity we studied the effect of two different inhibitors, 2-hydroxy-5-(2,5-dihydroxylbenzyl) aminobenzoic acid (HAC) and methyl 2,5-dihydroxycinnamate (DHC) which inhibit phosphotyrosine kinase in basolateral membranes. Cells were pretreated either with vehicle or HAC or DHC and then exposed to 10% CO2 for 5 min or 4 hr. In cells treated with vehicle, 10% CO2 significantly increased cotransporter activity as compared to control cells exposed to 5% CO2. This stimulation by 10% CO2 was completely prevented by HAC or DHC at 5 min (5% CO2: 1.8 +/- 0.2, 10% CO2: 2.6 +/- 0.2, 10% CO2 + HAC: 1.6 +/- 0.2, 10% CO2: +DHC: 2.0 +/- 0.3 pH unit/min) and also at 4 hr. The protein synthesis inhibitors actinomycin D and cycloheximide appear to prevent the effect of 10% CO2 for 4 hr on the cotransporter. Our results show that early respiratory acidosis stimulates the Na-HCO3 cotransporter through PKC and PTK-dependent mechanisms and the late effect appears to be mediated through protein synthesis.

Role of Ca(2+)-independent PKC in alpha 1-adrenoceptor-mediated inotropic responses of neonatal rat hearts.

We investigated the role of protein kinase C (PKC) in alpha 1-adrenoceptor (alpha 1-AR)-mediated positive inotropic responses of neonatal rat myocardium. Bisindolylmaleimide (BIM), an inhibitor of all PKC isoforms, reduced the positive inotropic responses to phenylephrine and methoxamine but not to isoproterenol. The positive inotropic effect of phenylephrine was not attenuated by Gö-6976, a selective inhibitor of Ca(2+)-dependent isoforms; it was potentiated by the 1,2-diacylglycerol kinase inhibitor R-59949. Phorbol 12,13-dibutyrate, an activator of both Ca(2+)-dependent and-independent PKC isoforms, as well as thymeleatoxin, a selective activator of Ca(2+)-dependent PKC isoforms, inhibited myocardial contractions, which were prevented by BIM and Gö-6976. BIM inhibited the phenylephrine-induced increase in particulate PKC activity but not the increase in phosphatidylinositide turnover. Phenylephrine induced translocation of only Ca(2+)-independent PKC-epsilon and -delta. These results suggest that activation of Ca(2+)-independent PKC isoforms by alpha 1-AR agonists plays a key role in alpha 1-AR-mediated positive inotropic effect on neonatal myocardium.

Activation of protein kinase C during newt limb regeneration: effect of denervation.

Blastema cell proliferation during newt limb regeneration is a nerve-dependent process. The present study was undertaken to determine whether or not that process is mediated by protein kinase C (PKC) activation during limb regeneration in Pleurodeles walt. Analysis included evaluation of PKC activity and its subcellular localization at various stages of regeneration, both in vivo and in vitro. The data reveal an increase in PKC activity in both the cytosol and particulate fractions of whole blastemas reaching a maximum at the mid-bud stage, which correlates with blastema cell proliferation rate. Denervation significantly reduces blastema cell proliferation and also causes a reduction in membrane-associated PKC activity. The effect of PKC activity appears to be restricted to the blastemal mesenchyme, which exhibits a dramatic reduction in activity 96 h after denervation. In contrast, PKC activity in the epidermal cap did not change. Cultured whole blastemas likewise express a decrease in particulate PKC activity and therefore mimic denervated blastemas in this parameter. Co-culture of blastemas with spinal ganglia partially reduces the decline in PKC activity, and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a direct activator of PKC, also prevents the fall in membrane-bound PKC activity while stimulating blastema cell proliferation, in vitro. These data indicate that blastema cell (mesenchyme) proliferation is related to increased PKC activity and that PKC may therefore be involved in the nerve-dependent signalling pathway regulating the early phase of urodele limb regeneration.

The Role of Protein Kinase C in Ischemic/Reperfused Preconditioned Isolated Rat Hearts

Protein kinase C (PKC) has been implicated in the preconditioning-induced cardiac protection in ischemic/reperfused myocardium. We studied the effect of PKC inhibition with calphostin C (25, 50, 100, 200, 400, and 800 nM), a potent and specific inhibitor of PKC, in isolated working nonpreconditioned and preconditioned ischemic/reperfused hearts. In the nonpreconditioned groups, all hearts underwent 30 min of normothermic global ischemia followed by 30 min of reperfusion. In the preconditioned groups, hearts were subjected to four cycles of ischemic preconditioning by using 5 min of ischemia followed by 10 min reperfusion, before the induction of 30 min ischemia and reperfusion. At low concentrations of calphostin C (25, 50, and 100 nM), the PKC inhibitor had no effect on the incidence or arrhythmias or postischemic cardiac function in the nonpreconditioned ischemic/reperfused groups. With 200 and 400 nM of calphostin C, a significant increase in postischemic function and a reduction in the incidence of arrhythmias were observed in the nonpreconditioned ischemic/reperfused groups. Increasing the concentration of calphostin C to 800 NM, the recovery of postischemic cardiac function was similar to that of the drug-free control group. In preconditioned hearts, lower concentrations (< 100 nM) of calphostin C did not change the response of the myocardium to ischemia and reperfusion in comparison to the preconditioned drug-free myocardium. Two hundred and 400 nM of the PKC inhibitor further reduced the incidence of ventricular fibrillation (VF) from the preconditioned drug-free value of 50% to 0 (p < 0.05) and 0 (p < 0.05), respectively, indicating that the combination of the two, preconditioning and calphostin C, affords significant additional protection. Increasing the concentration of calphostin C to 800 nM blocked the cardioprotective effect of preconditioning (100% incidence of VF). The recovery of cardiac function was similarly improved at calphostin C doses of 200 and 400 nM and was reduced at 800 nM (p < 0.05). With 200 and 400 nM of calphostin C, both cytosolic and particulate PKC activity were reduced by approximately 40 and 60%, respectively, in both preconditioned and preconditioned/ischemic/reperfused hearts. The highest concentration of calphostin C (800 nM) resulted in almost a complete inhibition of cytosolic (100%) and particulate (85%) PKC activity correlated with the abolition of preconditioning-induced cardiac protection. In conclusion, calphostin C protects the ischemic myocardium obtained from intact animals, provides significant additional protection to preconditioning at moderate doses, and blocks the protective effect of preconditioning at high concentrations. The dual effects of calphostin C appear to be strictly dose and "enzyme inhibition" related.

Excitatory amino acid regulation of astrocyte proteoglycans

Activity-dependent enduring change in cellular communication is essential for specific connectivity during development of the nervous system and for adaptive responses of the mature nervous system. Here we report that glutamate activation of excitatory amino acid receptors induces the synthesis and release of proteoglycans (PGs) from fetal hippocampal astrocytes in dissociated culture. PG synthesis and release are mediated via kainate and metabotropic receptor activation. Glutamate exposure did not regulate the release of a specific family of PG, but glutamate inhibited the synthesis of heparan sulfate (HS) PGs that appeared within the extracellular environment of the astrocyte. Particulate protein kinase C (PKC) activity was increased by glutamate and the PKC activator phorbol 10-myristate 13-acetate produced a dose-dependent increase in PG release. However, glutamate-induced PG release was not blocked by inhibition of PKC activity. These data suggest that PKC activation can lead to PG release, but is not necessary for it. Activity-dependent influences on a class of substrate-bound molecular species with growth-modulatory properties may be involved in spatial regulation of neuronal growth responses produced by excitatory amino acids.

Involvement of Protein Kinase Cϵ in the Stimulation of Anionic Amino Acid Transport in Cultured Human Fibroblasts

Protein kinase C (PKC) activation stimulates transport system X-AG for anionic amino acids in cultured human fibroblasts (Franchi-Gazzola, R., Visigalli, R., Bussolati, O., and Gazzola, G. C. (1994) FEBS Lett. 352, 109-112). To identify which PKC isoform is responsible for this effect, aspartate transport through system X-AG, PKC activity, and the subcellular distribution of PKC isoforms have been studied before and after treatment with phorbol 12, 13-dibutyrate (PDBu) in fibroblasts maintained at low serum for 1 (control cells) or 7 days (quiescent cells). In control cells aspartate transport and PKC activity in the particulate fraction were stimulated by short term PDBu treatment; both stimulatory effects were down-regulated by a prolonged exposure to the phorbol. In contrast, in quiescent cells aspartate transport and particulate PKC activity were higher than control under basal conditions, unaffected by a short term PDBu treatment, and lowered by a prolonged incubation with the phorbol. In both control and quiescent cells a short term PDBu treatment modified PKCalpha distribution, increasing its membrane-associated fraction. PKCdelta was mostly in the soluble fraction and scarcely sensitive to PDBu. A brief exposure to PDBu increased membrane-associated PKCepsilon in control but not in quiescent cells. In these cells epsilon isoform was found exclusively in the particulate fraction even in PDBu-untreated cells. A prolonged PDBu treatment caused a partial down-regulation of membrane-associated PKCepsilon in control cells and its marked decrease in quiescent cells. It is concluded that PKC-dependent changes in system X-AG activity parallel the behavior of PKCepsilon, thus suggesting a specific role for this isoform in system X-AG regulation.

Relationship among cellular diacylglycerol, sphingosine formation, protein kinase C activity, and parathyroid hormone secretion from dispersed bovine parathyroid cells.

To separate the role of changes in parathyroid diacylglycerol (DG) from other effects of extracellular calcium, we studied the effect of inhibition of DG metabolism on PTH secretion and cellular DG content in acutely dispersed bovine parathyroid cells. R 59 022, an inhibitor of DG kinase, increased cellular DG, but significantly decreased PTH secretion. Particulate protein kinase C (PKC) activity decreased in bovine parathyroid cells incubated at high extracellular calcium or in the presence of R 59 022, which is the opposite of what was observed in the presence of the phorbol ester, phorbol myristate acetate. Sphinganine, a normal cellular product that is a known inhibitor of PKC, significantly inhibited PTH secretion at low extracellular calcium, but had no significant effect at normal or high extracellular calcium. We then measured sphingosine in bovine parathyroid cells incubated with high extracellular calcium or R 59 022. Both conditions were associated with significant elevations of cellular sphingosine. These studies suggest that inhibition of PTH secretion and PKC activity by enhanced cellular DG may result from the activation of an inhibitory second messenger pathway involving the sphingoid lipids.

Modulation of Protein Kinase C-Related Signal Transduction by 2,3,7,8-Tetrachlorodibenzo-p-dioxin Exhibits Cell Cycle Dependence

The modulation of protein kinase C (PKC)-mediated protein phosphorylation in quiescent vascular smooth muscle cells (SMCs) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exhibits a discrete temporal pattern in which early reductions of kinase activity are followed by marked increases in activity. This profile may be accounted for by transcriptional- and/or cell cycle-related effects of TCDD. To test this hypothesis, experiments were conducted to examine the influence of TCDD on PKC activity during different phases of the cell cycle in vascular (aortic) SMCs. Increased PKC activity was observed in the cytosolic and particulate fractions of randomly cycling SMC cultures derived from female rats treatedin vivowith 10 μg/kg TCDD relative to corn oil. Treatment of cycling naive SMC cultures with TCDD (0.1 to 1000 nM) for 0.5 h caused a concentration-dependent increase of particulate PKC activity and no changes in cytosolic counterparts. Extended challenge of SMCs with TCDD for 24 h increased PKC activity in both cellular fractions. Incubation of SMCs with various concentrations of fetal bovine serum for 72 h to differentially regulate cell cycling followed by challenge with 10 nMTCDD for 24 h reduced cytosolic and particulate PKC activity in quiescent cells, but enhanced activity in cycling cells. To determine if this serum-related profile was strictly dependent upon cell cycle-related events, SMCs were synchronized in the G0phase and then pulsed with 10 nMTCDD during different phases of the cell cycle. Differential profiles were observed where reduced C-kinase activity occurred during the G0/G1transition followed by increases during G1/S and no changes during S. Western blot analysis confirmed the patterns of PKC activity observed during the G0/G1and G1/S transitions. PKCα, βII, and δ isoforms were reduced during G0/G1, while only PKCβII and δ were increased during G1/S. These data show that modulation of PKC by TCDD in vascular SMCs exhibits cell cycle dependence and isoform specificity.

Correlations between hippocampal protein kinase C activity and learning abilities in a spatial reference memory task

Partial learning of a reference memory task induces a decrease in hippocampal cytosolic protein kinase C (PKC) activity with no concomitant changes in the particulate fraction. The particulate PKC activity is correlated with performance (Noguès, Micheau, & Jaffard, 1994). The relationship between the level of training and PKC activity was investigated in the present study. Hippocampal PKC activity was measured after mice were trained for 2, 5, or 12 sessions in a spatial discrimination task in an 8-arm radial maze. No increase in membrane-bound fraction or decrease in cytosolic PKC activity was observed at any stage of learning, although performance was correlated with PKC activity in each subcellular fraction. We sought correlations between performance and the magnitude of the decrease in cytosolic PKC activity, with reference to previously obtained data. The extent of the fall in cytosolic PKC was found to be related to learning abilities rather than to the maximal performance level. Causal explanatory models are proposed to explain these somewhat contradictory data.

Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields.

We examined the effects of electric fields (EFs) on the activity and subcellular distribution of protein kinase C (PKC) of living HL60 cells. Sixty Hertz AC sinusoidal EFs (1.5-1,000 mV/cm p-p) were applied for 1 h to cells (10(7)/ml) in Teflon chambers at 37 degrees C in the presence or absence of 2 microM phorbol 12-myristate 13-acetate (PMA). PMA stimulation alone evoked intracellular translocation of PKC from the cytosolic to particulate fractions. In cells that were exposed to EFs (100-1,000 mV/cm) without PMA, a loss of PKC activity from the cytosol, but no concomitant rise in particulate PKC activity, was observed. In the presence of PMA, EFs (33-330 mV/cm) also accentuated the expected loss of PKC activity from the cytosol and augmented the rise in PKC activity in the particulate fraction. These data show that EFs alone or combined with PMA promote down-regulation of cytosolic PKC activity similar to that evoked by mitogens and tumor promoters but that it does not elicit the concomitant rise in particulate activity seen with those agents.

Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields

We examined the effects of electric fields (EFs) on the activity and subcellular distribution of protein kinase C (PKC) of living HL60 cells. Sixty Hertz AC sinusoidal EFs (1.5–1.000 mV/cm p-p) were applied for 1 h to cells (107/ml) in Teflon chambers at 37 °C in the presence or absence of 2 μM phorbol 12-myristate 13-acetate (PMA). PMA stimulation alone evoked intracellular translocation of PKC from the cytosolic to particulate fractions. In cells that were exposed to EFs (100–1,000 mV/cm) without PMA, a loss of PKC activity from the cytosol, but no concomitant rise in particulate PKC activity, was observed. In the presence of PMA. EFs (33–330 mV/cm) also accentuated the expected loss of PKC activity from the cytosol and augmented the rise in PKC activity in the particulate fraction. These data show that EFs alone or combined with PMA promote down-regulation of cytosolic PKC activity similar to that evoked by mitogens and tumor promoters but that it does not elicit the concomitant rise in particulate activity seen with those agents. © 1996 Wiley-Liss, Inc.

Evidence that the modulation of membrane-associated protein kinase C activity by an endogenous inhibitor plays a role in N1E-115 murine neuroblastoma cell differentiation.

Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.

Alterations in splenocyte protein kinase C (PKC) activity by 2,3,7,8-tetrachlorodibenzo- p-dioxin in vivo

The effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on growth factor-coupled activation of nuclear protein kinase C (nPKC) and on the subcellular distribution of PKC activity in rat splenocytes were investigated. Seven days after a single injection of TCDD (50 μg/kg body weight), cytosolic and particulate PKC activity was significantly higher in splenocytes from TCDD-treated rats or pair-fed control rats compared to ad libitum-fed animals. In a separate experiment, purified splenocyte nuclei from TCDD-treated animals and controls were used to study activation of nPKC by growth factors and other trophic agents. Growth factor-stimulated nPKC activation was attenuated in splenic nuclei from TCDD-treated rats compared to vehicle-treated controls. Evidence presented here suggests that the cellular mechanism of TCDD toxicity leading to immunosuppression in rodents may be mediated in part by uncoupling of growth factor receptors linked to PKC activation at the level of the nucleus. However, changes in total splenocyte PKC activity appear to be correlated with hypophagia since cytosolic and particulate PKC levels were elevated in TCDD-treated rats and their pair-fed partners.

Retinoic acid induces translocation of protein kinase C (PKC) and activation of nuclear PKC (nPKC) in rat splenocytes

Retinoic acid (RA), a vitamin A metabolite, has marked effects on growth of normal and malignant cells; however, the exact mechanism of action remains unclear. The effect of two RA analogs, 13- cis-RA and all- trans-RA, on transmembrane signalling processes was investigated in rat splenocytes. Treatment of rat splenic cells with these retinoic acid analogs resulted in translocation of protein kinase C (PKC) from the cytosol to the membrane. Previous studies have described nuclear RA receptors (RARs and RXRs) for several species and the biologic activity of RA has been shown to be mediated by specific interaction with these nuclear receptors. Thus, activation of nuclear pool(s) of protein kinase C (nPKC) by RA analogs was also studied. Rat splenocyte nuclei pure by enzymatic and electron microscope criteria demonstrated a biphasic pattern of bell-shaped curves for both cis- and trans-RA with maximum statistically significant peak of phosphate incorporation into endogenous substrates at 10 −16 M cis-RA and 10 −16-10 −17 M trans-RA. A monoclonal antibody to PKC and the PKC inhibitors, H-7, sphingosine, and staurosporine, blocked the RA-stimulated nuclear phosphorylation. The ability of RA to activate cell membrane PKC resulting in an increase in particulate PKC activity correlates well with the activation of nPKC since the particulate fraction would include nuclear enzyme systems. This ability of RA to activate nPKC and possibly affect the growth status of a cell may provide a missing link to our understanding of the cellular sites of action for this vitamin.