Changes In Protein Kinase C Activity Research Articles

Changes in body mass alone explain almost all of the variance in the serum sodium concentrations during prolonged exercise. Has commercial influence impeded scientific endeavour?

In 1991, we provided definitive evidence that exercise-associated hyponatraemia (EAH) is caused by abnormal fluid retention in those who overdrink during prolonged exercise, but this finding was ignored. Instead, in...

Water permeability through aquaporin-4 is regulated by protein kinase C and becomes rate-limiting for glioma invasion

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K +–Cl − cotransporter 1 (KCC1), believed to provide the pathways for Cl − and K + secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process.

Protein kinase C modulates light responses in Neurospora by regulating the blue light photoreceptor WC‐1

The Neurospora protein kinase C (NPKC) is a regulator of light responsive genes. We have studied the function of NPKC in light response by investigating its biochemical and functional interaction with the blue light photoreceptor white-collar 1 (WC-1), showing that activation of NPKC leads to a significant decrease in WC-1 protein levels. Furthermore, we show that WC-1 and NPKC interact in a light-regulated manner in vivo, and that protein kinase C (PKC) phosphorylates WC-1 in vitro. We designed dominant negative and constitutively active forms of PKC which are able to induce either a large increase of WC-1 protein level or a strong reduction respectively. Moreover, these changes in PKC activity result in an altered light response. As WC-1 is a key component of Neurospora circadian clock and regulates the clock oscillator component FRQ we investigated the effect of NPKC-mutated forms on FRQ levels. We show that changes in PKC activity affect FRQ levels and the robustness of the circadian clock. Together these data identify NPKC as a novel component of the Neurospora light signal transduction pathway that modulates the circadian clock.

Involvement of protein kinase C in the adaptive changes of cholinergic neurons to sympathetic denervation in the guinea pig myenteric plexus

Supersensitivity to muscarinic, κ- and μ-opioid agents modulating cholinergic neurons in the guinea pig colon develops after chronic sympathetic denervation. A possible role for protein kinase C (PKC) in contributing to development of these sensitivity changes was investigated. The PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced acetylcholine (ACh) overflow in preparations obtained from normal animals. The facilitatory effect of PMA was significantly reduced after prolonged exposure to the phorbol ester and by the PKC inhibitors, chelerythrine and calphostin C. Subsensitivity to the facilitatory effect of PMA developed after chronic sympathetic denervation. In this experimental condition, immunoblot analysis revealed reduced levels of PKC in myenteric plexus synaptosomes. The facilitatory effect of the muscarininc antagonist, scopolamine, on ACh overflow was significantly reduced by the phospolipase C (PLC) inhibitor, U73122, chelerythrine and calphostin C, both in normal and denervated animals. However, in both experimental groups, PLC antagonists and PKC antagonists did not affect the inhibitory effect of the muscarinic agonist, oxotremorine-M on ACh overflow. The inhibitory effects of U69593 (κ-opioid receptor agonist) and DAMGO (μ-opioid receptor agonist) on ACh overflow significantly increased in the presence of U73122, chelerythrine and calphostin C in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. These results indicate that activation of PKC enhances ACh release in the myenteric plexus of the guinea pig colon. At this level, chronic sympathetic denervation entails a reduced efficiency of the enzyme. In addition, PKC is involved in the inhibitory modulation of ACh release mediated by muscarinic-, κ- and μ-opioid receptors, although with different modalities. Muscarinic receptors inhibit PKC activity, whereas κ- and μ-opioid receptors increase PKC activity. Both the inhibitory and the facilitatory effect on PKC involve modulation of PLC activity. The possibility that the change in PKC activity represents one of the biochemical mechanisms at the basis of development of sensitivity changes to opioid and muscarinic agents after chronic sympathetic denervation is discussed.

Increased expression of protein kinase C isoforms in heart failure due to myocardial infarction.

The activities of cardiac protein kinase C (PKC) were examined in hemodynamically assessed rats subsequent to myocardial infarction (MI). Both Ca(2+)-dependent and Ca(2+)-independent PKC activities increased significantly in left ventricular (LV) and right ventricular (RV) homogenates at 1, 2, 4, and 8 wk after MI was induced. PKC activities were also increased in both LV and RV cytosolic and particulate fractions from 8-wk infarcted rats. The relative protein contents of PKC-alpha, -beta, -epsilon, and -zeta isozymes were significantly increased in LV homogenate, cytosolic (except PKC-alpha), and particulate fractions from the failing rats. On the other hand, the protein contents of PKC-alpha, -beta, and -epsilon isozymes, unlike the PKC-zeta isozyme, were increased in RV homogenate and cytosolic fractions, whereas the RV particulate fraction showed an increase in the PKC-alpha isozyme only. These changes in the LV and RV PKC activities and protein contents in the 8-wk infarcted animals were partially corrected by treatment with the angiotensin-converting enzyme inhibitor imidapril. No changes in protein kinase A activity and its protein content were seen in the 8-wk infarcted hearts. The results suggest that the increased PKC activity in cardiac dysfunction due to MI may be associated with an increase in the expression of PKC-alpha, -beta, and -epsilon isozymes, and the improvement of heart function in the infarcted animals by imidapril may be due to partial prevention of changes in PKC activity and isozyme contents.

Hyperexcitability and changes in activities of Ca 2+/calmodulin-dependent kinase II and mitogen-activated protein kinase in the hippocampus of rats exposed to 1-bromopropane

Chronic inhalation of 1-bromopropane (1-BP), a substitute of ozone-depleting chlorofluorocarbons, has been suspected of having central neurotoxicity (Clinical Neurology and Neurosurgery 101 (1999) 199; Journal of Occupational Health 44 (2002) 1) for humans. In animal experiments, 1-BP inhalation (1500 ppm) caused hyperexcitability in the CA1 and the dentate gyrus (DG) [Journal of Occupational Health 42 (2000) 149, Journal of Occupational Health 44 (2002) 156]. We studied whether the hyperexcitability is associated with changes of Ca 2+/calmodulin-dependent kinase II (CaMKII), mitogen-activated protein kinase (MAPK), and protein kinase C (PKC). Male Wistar rats were exposed to 1-BP for 6 hours in a day in an exposure chamber with a concentration of 700 ppm for 8 weeks. After the inhalation, paired-pulse ratios of field excitatory postsynaptic potentials and population spikes (PSs) were analyzed in the CA1 and DG of hippocampal slices. Control rats were then given fresh air in the inhalation chamber. Semiquantitative immunoblotting analyses of protein kinases using antibodies against active and conventional protein kinases were done using the whole hippocampus. A paired-pulse ratio of PS was increased at the 5 ms interpulse interval in the CA1 and at the 10–20 ms interpulse intervals in the DG. The amount of active MAPK and total amount of CaMKIIα and β were significantly increased by 28, 29, and 46% compared to control, respectively, without any change in PKC activity. In contrast, the amount of active CaMKIIβ was decreased to 78%. These results suggest that modifications of intracellular signaling cascades are associated with hyperexcitability that occurred in the hippocampal formation of rats exposed to the chronic inhalation of 1-BP.

Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IkappaB-alpha.

The possibility that lipid-induced insulin resistance in human muscle is related to alterations in diacylglycerol (DAG)/protein kinase C (PKC) signaling was investigated in normal volunteers during euglycemic-hyperinsulinemic clamping in which plasma free fatty acid (FFA) levels were increased by a lipid/heparin infusion. In keeping with previous reports, rates of insulin-stimulated glucose disappearance (G(Rd)) were normal after 2 h but were reduced by 43% (from 52.7 +/- 8.2 to 30.0 +/- 5.3 micromol. kg(-1). min(-1), P < 0.05) after 6 h of lipid infusion. No changes in PKC activity or DAG mass were seen in muscle biopsy samples after 2 h of lipid infusion; however, at approximately 6 h, PKC activity and DAG mass were increased approximately fourfold, as were the abundance of membrane-associated PKC-betaII and -delta. A threefold increase in membrane-associated PKC-betaII was also observed at approximately 2 h but was not statistically significant (P = 0.058). Ceramide mass was not changed at either time point. To evaluate whether the fatty acid-induced insulin activation of PKC was associated with a change in the IkB kinase (IKK)/nuclear factor (NF)-kappaB pathway, we determined the abundance in muscle of IkappaB-alpha, an inhibitor of NF-kappaB that is degraded after its phosphorylation by IKK. In parallel with the changes in DAG/PKC, no change in IkappaB-alpha mass was observed after 2 h of lipid infusion, but at approximately 6 h, IkappaB-alpha was diminished by 70%. In summary, the results indicated that the insulin resistance observed in human muscle when plasma FFA levels were elevated during euglycemic-hyperinsulinemic clamping was associated with increases in DAG mass and membrane-associated PKC-betaII and -delta and a decrease in IkappaB-alpha. Whether acute FFA-induced insulin resistance in human skeletal muscle is caused by the activation of these specific PKC isoforms and the IKK-beta/IkappaB/NFkappaB pathway remains to be established.

Protein kinase C changes in diabetes: Is the concept relevant to neuropathy?

Protein kinase C (PKC) comprises a superfamily of isoenzymes, many of which are activated by 1,2-diacylglycerol (DAG) in the presence of phosphatidylserine. In order to be capable of DAG activation, PKC must first undergo a series of phosphorylation at three conserved sites. PKC isoforms phosphorylate a wide variety of intracellular target proteins and have multiple functions in signal transduction-mediated cellular regulation. An elevation in DAG levels and an increase in composite PKC activity and/or certain isoforms occurs in several nonneural tissues from diabetic animals, including the vasculature. The ability of isoform-specific PKC inhibitors to antagonize diabetes-induced abnormalities has implicated altered PKC beta activity in the onset of several diabetic complications, In contrast to many other tissues, DAG levels fall in diabetic nerve and a consistent pattern of change in PKC activity has not been observed. Treatments that alter PKC activity affect nerve Na+, K+-ATPase activity, but the mechanism involved is not well understood, Inhibition of PKC beta in diabetic rats appears to correct reduced nerve blood flow and decreased nerve conduction velocity. These and other findings indicate that changes in the neurovasculature exert adverse effects during the pathogenesis of diabetic neuropathy. Still unresolved is a clear-cut role for PKC in the development of abnormalities in neural cell metabolism. Further progress will depend on a more complete understanding of the functions of individual PKC isoforms in nerve. Future investigation could focus profitably on biochemical processes in nerve cells that modulate PKC activity and that are altered in diabetes, such as vascular endothelial growth factor levels and production of reactive oxygen species arising from oxidative stress.

Levels of protein kinase C and nitric oxide synthase activity in rats exposed to sub chronic low level lead.

The intracellular regulation of protein kinase C (PKC) and nitric oxide synthase (NOS) in relation to the accumulation of lead (Pb2+) in various brain regions following low-level lead exposure (50 ppm) for 90 days in rats was investigated. PKC and NOS are important enzymes in mediating cellular transduction mechanisms and in the regulation of neuronal plasticity. Rats exposed to Pb2+ resulted in blood Pb2+ levels similar to those observed in children affected due to Pb2+ exposure. Further, we examined whether Pb2+ accumulation changed the intracellular signaling mechanisms in different brain regions. Results of these experiments indicate that significant region specific Pb2+ accumulation is associated with down regulation of PKC. The down regulation of PKC increased the activity of NOS following Pb2+ exposure. Thus, the change in PKC activity in respect to Pb2+ accumulation increased NOS activity. These results suggest that neuronal toxicity during Pb2+ exposure is linked to the modulation of PKC followed by NOS activation.

Alteration in the expression of protein kinase C isoforms in human hepatocellular carcinoma

This study was designed to investigate the alterations of individual protein kinase C (PKC) isoforms in human liver cancer. Surgical specimens of hepatocellular carcinoma and adjacent normal tissues were extracted into cytosolic and membranous fractions. The level of membrane-bound PKCα in the cancer tissue was significantly lower than that in the adjacent normal tissue and consistent with the change in PKC activity. In addition, there was a significant negative correlation between PKCα and tumor size. In both cytosolic and membrane fractions, levels of PKCδ and PKCζ was significantly higher in the cancer tissue than those in the adjacent normal liver tissue. The alterations in the PKC isoforms signify their roles in the hyperproliferation in liver cancer.

Contribution of protein kinase C and glutamate in Pb 2+-induced cytotoxicity

Activation of protein kinase C (PKC) plays an important role in lead (Pb 2+)-induced cytotoxicity. The effects of low dose exposure to Pb 2+ on cytosolic free calcium (Ca 2+), PKC activity and mechanisms involved in cell death were studied in PC12 cells. Exposure of PC12 cells to low dose Pb 2+ (0.01 μM) increased PKC activity, while exposure to a higher dose (10 μM) led to decreased PKC activity. Additionally, in normal extracellular medium, low concentration of Pb 2+ (0.01 μM) stimulated increase in cytosolic free calcium while the higher concentrations of Pb 2+ (10 μM) did not. However, the effect of low dose Pb 2+ (0.01 μM) was blocked by removing Ca 2+ from external medium. The role of Pb 2+-induced changes in PKC activity and its relationship to oxidative stress and related cytotoxicity was also studied. Pb 2+ alone (0.01–10 μM) produced reactive oxygen species (ROS) dose dependently over the period of 24 h. Pb 2+-induced ROS were potentiated in the presence of 500 μM glutamate. Furthermore, a correlation was observed between ROS generation and the levels of cytotoxicity, which was observed after 24 h exposures to Pb 2+ by trypan blue method, and the cytotoxicity was enhanced by glutamate co-treatment. Pb 2+-induced cell death was blocked partially by staurosporine (PKC inhibitor, 100 nM) and NMDA antagonist, MK-801 (1 μM). It is concluded that, in Pb-induced cytotoxicty, modulation of PKC and intracellular calcium play significant roles in augmenting glutamate receptor mediated oxidative species formation and subsequent cell death.

Correlation between lead-induced changes in cerebral ornithine decarboxylase and protein kinase C activities during development and in cultured PC 12 CELLS

Exposure to lead (Pb) interferes with neurodevelopment and disturbs ornithine decarboxylase (ODC) activity. ODC the key regulatory enzyme of the polyamine pathway, is a potential substrate for protein kinase C (PKC). Therefore, we examined developmental changes in PKC activity and its relationship to ODC activity. Male rats were lactationally exposed to 0.2% Pb-acetate from birth to weaning. PKC and ODC activity were measured on postnatal days (PND) 3, 5, 10, 20 and 30. We found that the basal patterns of ODC and PKC activities resembled each other in both the neocortex and cerebellum and Pb-exposure attenuated both enzymes in a similar manner. To determine whether any link existed between these enzymes, ODC and PKC activities were induced to increase using nerve growth factor (NGF) in the presence and/or absence of ODC (difluoromethylornithine, DFMO) and PKC (staurosporine) inhibitors, in control and Pb-exposed Pheochromocytoma (PC-12) cells. Staurosporine decreased both ODC activity and PKC activity, while DFMO had no effect on PKC activity. These data suggest that ODC may be regulated by PKC and that Pb-induced developmental alterations in ODC activity may be secondary to changes in the integrity of PKC.

Effect of Ageing on the Expression of Protein Kinase C and Its Activation by 1,25(OH) 2-Vitamin D 3 in Rat Skeletal Muscle

To characterize age-induced effects on muscle protein kinase C (PKC) and its regulation by the steroid hormone 1,25(OH) 2-vitamin D 3 [1,25(OH) 2D 3], changes in PKC activity and the expression and translocation of the specific PKC conventional isoforms α and β, novel isoforms δ, ϵ, and θ and atypical isoform ζ were studied in homogenates and subcellular fractions from skeletal muscle of young (3 months) and aged (24 months) rats treated in vitro with 1,25(OH) 2D 3. The hormone (10 −9 M) increased total and membrane PKC activity, within 1 min, and these effects were completely blunted in muscle from aged rats. The presence of PKC isoenzymes was shown by Western blot analysis with the use of specific antibodies. The expression of PKC α, β and δ was greatly diminished in old rats, whereas age-related changes were less pronounced in the isoforms ϵ, θ and ζ. After a short exposure (1 min) of muscle to 1,25(OH) 2D 3, increased amounts of PKC α and β in muscle membranes and reverse translocation (from membrane to cytosol) of PKC ϵ were observed only in young animals. The data indicate that, in rat muscle, ageing impairs calcium-dependent PKC (α and β) and calcium-independent PKC (δ, ϵ, θ and ζ) signal transduction pathways under selective regulation by 1,25(OH) 2D 3.

Protein kinase C regulation of p-glycoprotein-mediated xenobiotic secretion in renal proximal tubule

Fluorescence microscopy, fluorescent substrates [daunomycin and a fluorescent cyclosporin A (CSA) derivative] and digital image analysis were used to examine the role of protein kinase C (PKC) in the control of p-glycoprotein in killifish renal proximal tubules. PKC activators, phorbol ester (phorbol 12-myristate 13-acetate, PMA) and dioctylglycerol, reduced luminal drug accumulation, and protein kinase inhibitors, staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), increased luminal accumulation; a PMA analog that does not activate PKC was without effect. PMA effects were blocked by staurosporine. The increase in luminal fluorescence caused by staurosporine was blocked by the p-glycoprotein substrate, CSA, indicating that this component of transport was indeed mediated by p-glycoprotein. Neither PMA, dioctylglycerol, nor protein kinase inhibitors altered cellular drug accumulation. Finally, in primary cultures of flounder proximal tubule cells, PMA decreased transepithelial [3H]daunomycin secretion. This pharmacological approach demonstrates that in teleost renal proximal tubule, p-glycoprotein-mediated xenobiotic secretion is negatively correlated with changes in PKC activity, a finding that conflicts with results from studies using mammalian tumor cells that express p-glycoprotein.

Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes.

Prior studies have shown that 24,25-(OH)2D3 and 1,25-(OH)2D3 regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)2D3 affecting primarily growth zone (GC) cells and 24,25-(OH)2D3 affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)2D3 has been shown to increase phospholipase A2 activity in GC, while 24,25-(OH)2D3 has been shown to decrease phospholipase A2 activity in RC. Stimulation of phospholipase A2 in GC caused an increase in PKC, whereas inhibition of phospholipase A2 activity in RC cultures increased both basal and 24,25-(OH)2D3-induced PKC activity, suggesting that phospholipase A2 may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A2 inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A2 activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A2 inhibitors decreased both basal and 1,25-(OH)2D3-induced PKC activity in GC. When phospholipase A2 activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)2D3-induced PKC activity in RC and increased 1,25-(OH)2D3-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A2 activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A2 activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A2. These effects are cell maturation- and time-dependent and metabolite-specific.

Arachidonic acid is an autocoid mediator of the differential action of 1,25‐(OH)2D3 and 24,25‐(OH)2D3 on growth plate chondrocytes

Prior studies have shown that 24,25-(OH)2D3 and 1,25-(OH)2D3 regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)2D3 affecting primarily growth zone (GC) cells and 24,25-(OH)2D3 affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)2D3 has been shown to increase phospholipase A2 activity in GC, while 24,25-(OH)2D3 has been shown to decrease phospholipase A2 activity in RC. Stimulation of phospholipase A2 in GC caused an increase in PKC, whereas inhibition of phospholipase A2 activity in RC cultures increased both basal and 24,25-(OH)2D3-induced PKC activity, suggesting that phospholipase A2 may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A2 inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A2 activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A2 inhibitors decreased both basal and 1,25-(OH)2D3-induced PKC activity in GC. When phospholipase A2 activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)2D3-induced PKC activity in RC and increased 1,25-(OH)2D3-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A2 activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A2 activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A2. These effects are cell maturation- and time-dependent and metabolite-specific. J. Cell. Physiol. 176:516–524, 1998. © 1998 Wiley-Liss, Inc.

Nicotine-like effects of cotinine on protein kinase C activity and noradrenaline release in bovine adrenal chromaffin cells.

1. We studied the effect of cotinine, a slowly eliminated metabolite of nicotine, on protein kinase C (PKC) distribution and noradrenaline release in primary cultured bovine adrenal chromaffin cells. Changes in PKC activity were detected by [3H]-phorbol-12,13-dibutyrate binding, histone phosphorylation assay and by Western blot. 2. Cotinine (10-32 mM) increased phorbol binding to chromaffin cells in response to 10 min but not to 24 h exposure. The increased binding was reversed by a nicotinic antagonist hexamethonium (10 microM). 3. Cotinine (10 mM, 30 min) also increased membrane-associated PKC activity and membrane-associated PKC alpha and epsilon immunoreactivity. 4. Cotinine (0.1-32 mM for 10 s to 20 min) dose- and time-dependently increased the release of preloaded [3H]-noradrenaline from the cultured cells. The release increased with increasing duration of the contact period. In treatments lasting 1 min or longer, a peak effect was followed by a reduced response at higher concentrations. 5. We confirm the earlier findings that cotinine is biologically active, and conclude that its effects are at least partly mediated via nicotinic cholinergic receptors and through PKC.

Induction of a Specific Olfactory Memory Leads to a Long-Lasting Activation of Protein Kinase C in the Antennal Lobe of the Honeybee

In this study we investigated the role of protein kinase C (PKC) in associative learning of Apis mellifera. Changes in PKC activity induced by olfactory conditioning were measured in the antennal lobes, a brain structure involved in associative learning. Multiple conditioning trials inducing a memory different from that induced by a single conditioning trial specifically cause an increase in PKC activity. This increase begins 1 hr after conditioning, lasts up to 3 d, and is attributable to an increased level of constitutive PKC. The increased level of constitutive PKC consists of an early proteolysis-dependent phase and a late phase that requires RNA and protein synthesis. Inhibition of the pathways resulting in constitutive PKC selectively impairs distinct phases of multiple-trial induced memory. The inhibition of the proteolytic mechanism has an instant effect on an early phase of multiple-trial induced memory but does not affect acquisition and the late phase of memory. Blocking of the transient PKC activation during conditioning does not affect the induction of memory formation. Thus, the constitutive PKC in the antennal lobe seems to contribute to the early phase of memory that is induced by multiple-trial conditioning.

Temperature and protein kinase C modulate myofilament Ca2+ sensitivity in pressurized rat cerebral arteries.

The effects of pharmacological activation and inhibition of protein kinase C (PKC) and temperature on the relationship between cytoplasmic Ca2+ and lumen diameter were studied in pressurized (50 mmHg) rat posterior cerebral arteries permeabilized with alpha-toxin. Increasing Ca2+ concentrations (30 nM-10 microM, 22 degrees C) induced stable, concentration-dependent constrictions with a half-maximal effective concentration (EC50) of 112 nM. The maximal constriction was 80% of baseline diameter and 157% of that during depolarization of nonpermeabilized vessels with 124 mM KCl. Elevation of temperature to 37 degrees C increased the EC50 to 246 nM and enhanced the steepness of concentration-response curves. Exposure of permeabilized arteries to indolactam V, an activator of PKC, resulted in a significant myofilament Ca2+ sensitization (e.g., EC50 at 5 microM = 126 nM) without changing efficacy. The effects of calphostin C, a PKC inhibitor, on Ca2+ sensitivity were minimal; however, the amplitude of Ca2+-induced constrictions in both control and indolactam-treated arteries was suppressed in a concentration-dependent manner. Thus 1) temperature is an important variable in studies of arterial Ca2+ sensitivity, and 2) changes in PKC activity can significantly alter both myofilament sensitivity to and constrictor efficacy of cytosolic Ca2+.

The effects of acute and repeated cocaine injections on protein kinase C activity and isoform levels in dopaminergic brain regions

The present study was designed to assess the effects of acute and repeated cocaine exposure on protein kinase C (PKC) activity and the levels of calcium-dependent isoforms of PKC in mesocorticolimbic and nigrostriatal dopamine brain regions. Animals received repeated injections of saline or cocaine and were challenged with saline or cocaine 24 h or 7 days after the last of their daily injections. Animals were sacrificed 2, 6 or 24 h after the challenge injection and their brains were dissected and used in PKC studies. The data demonstrated that previously reported cocaine-induced increases in PKC activity in the ventral tegmental area are transient and not associated with changes in the levels of calcium-dependent isoforms of PKC. In addition, there was a decrease in membrane-associated PKC activity, with a concomitant increase in the levels of PKC β I in the medial prefrontal cortex 24 h after the last injection of cocaine. These data suggest that changes in PKC activity in the ventral tegmental area may be involved in the initiation of sensitization whereas changes in PKC activity in the medial prefrontal cortex may be related to the expression of the sensitized response to cocaine.