Selective translocation of protein kinase c isozymes by PAF in rabbit platelets.

The action of platelet activating factor (PAF) on subcellular distribution and activity of protein kinase C (PKC) isoforms in rabbit platelets was analyzed. The results showed an increase of PKC alpha in membrane fraction, concomitantly with a decrease in cytosolic fraction after 5 min PAF treatment, indicating that a translocation of PKC alpha occurred. In addition, PKC zeta was redistributed in a "reverse" form, from the membrane to cytosolic fraction after PAF treatment. PAF induced an increase of PKC alpha activity, whereas a decrease rather than increase in PKC zeta was observed by using immunoprecipitation assays. In addition, some results indicated that PI3 kinase activation was not involved in PAF-induced PKC zeta translocation as occur in several cells and with other agonists. These actions were time- and concentration-dependent, and were inhibited by the treatment with a PAF antagonist. No translocation was observed when the platelets were incubated with lysoPAF, a PAF related compound. The redistribution of PKC isoforms take place through the activation of high specificity PAF binding sites. The pretreatment of the rabbit platelets with staurosporine, a putative inhibitor of PKC, completely blocked the PAF-evoked aggregation without affecting to PAF-evoked shape change and serotonin release. All together, these data could suggest that the specific translocation of PKC isoforms play an important role in the activation of rabbit platelets.

PAF-stimulated protein tyrosine phosphorylation in hippocampus: involvement of NO synthase.

The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat hippocampal slices. PAF caused an increase in the tyrosine phosphorylation of two phosphoproteins, which we identified by immunoprecipitation assays as the focal adhesion kinase p125FAK and crk-associated substrate p130Ca. The PAF effect was time- and dose-dependent. In addition, the involvement of PAF receptor was demonstrated by using PCA-4248, a specific receptor antagonist. When NO synthase was inhibited by NG-monomethyl-L-arginine (L-NMA), PAF-stimulated protein tyrosine phosphorylation was inhibited. In conclusion, our results indicate that PAF increased the tyrosine phosphorylation of both p125FAK and p130Cas proteins by the production of NO in hippocampus, suggesting that PAF may play a role in the functioning of this cerebral area.

Involvement of phosphatidylinositol 3-kinase in nuclear translocation of protein kinase C zeta induced by C2-ceramide in rat hepatocytes.

In this study we report that protein kinase C zeta (PKC zeta), one of the atypical isoforms of the PKC family located predominantly in cytosol, is redistributed by C2-ceramide treatment in isolated hepatocytes. PKC zeta increased in membrane and nuclear fractions after 30 min of treatment with C2-ceramide in a dose- and time-dependent manner. The action of C2-ceramide was inhibited by wortmannin and LY 294002, indicating that C2-ceramide-induced PKC zeta increase in both nucleus and membrane fractions is mediated by phosphatidylinositol 3-kinase (PI3-kinase) activation. In addition, a significant translocation of PI3-kinase to the nucleus was observed after C2-ceramide treatment.

Sphingolipid derivatives modulate intracellular Ca2+ in rat synaptosomes.

Sphingosylphosphorylcholine (SPC) induces a rapid increase of intracellular Ca2+ concentration in isolated synaptosomes. This effect is dose-dependent and is also dependent on extracellular Ca2+. Sphingosine (SPH) has a smaller effect and treatment with psychosine (PSY) is ineffective, which suggests that phosphorylation of the 1-carbon of SPH is required for the SPC to act as a Ca2+ release agonist in synaptosomes. Experiments performed in the presence of heparin or ryanodine indicate that SPC-elicited Ca2+ release is not mediated by IP3 or ryanodine receptors. Finally, our results show that the effect of SPC on Ca2+ concentration is nimodipine-sensitive, suggesting that SPC possibly activates a specific sphingolipid-gated Ca2+ channel in synaptosomes.

Increase of phosphoinositide hydrolysis and diacylglycerol production by PAF in isolated rat liver nuclei.

When isolated rat liver nuclei were treated with platelet-activating factor (PAF), a rapid increase in the mass of diacylglycerol (DAG) occurred. This effect was dose- and time-dependent. The maximum effect was observed after 1 min of 10(-7) M PAF treatment. A concomitant decrease of polyphosphoinositides and phosphatidic acid (PA) levels was observed. PAF-induced DAG accumulation was inhibited by the treatment with WEB 2086 or PCA-4248, specific PAF-receptor antagonists. This result may suggest that PAF exerts its action in the nucleus through specific nuclear PAF binding sites. The findings described herein are due to the activation of phospholipase C, as the results from experiments using U73122, a phospholipase C inhibitor, indicate. These are the first data on the action of

Differential redistribution of protein kinase C isoforms by cyclic AMP in HL60 cells.

In this study we have analyzed the distribution of protein kinase C isoforms in cytosol, membrane, and nucleus in HL60 cells. Furthermore, we have studied the redistribution of these isoforms after cyclic AMP treatment. Protein kinase C localization and cyclic AMP-induced translocation was demonstrated by Western blot analysis. Cytosol, membrane and nucleus in HL60 cells expressed the abundance of protein kinase C alpha, betaI, betaII, delta, lambda, and zeta isoforms. After cyclic AMP treatment, the amount of protein kinase C betaI and zeta increased only in the nucleus, while protein kinase C delta increased in the three fractions tested. These effects were dependent on the cyclic AMP concentration and duration of action. Our results suggest the existence of cross-talk between the cyclic AMP system and protein kinase C in HL60 cells. Taking into account the processes regulated by protein kinase C, these findings also suggest that cyclic AMP plays a regulatory role in various cellular responses in HL60 cells, such as differentiation and gene expression. The increase observed in PKC delta was due to cyclic AMP-dependent protein kinase C activation, and the synthesis of enzyme was probably activated by the nucleotide.

Endothelin-1 increases isoprenaline-enhanced cyclic AMP levels in cerebral cortex.

We examined the effect of ET-1 on cyclic AMP levels in rat cerebral cortex. The peptide caused a concentration-dependent increase of [(3)H]cyclic AMP accumulation after 10 min of treatment. This effect was due to adenosine accumulation since it was inhibited by the treatment with adenosine deaminase. ET-1, apart from being able to increase cyclic AMP, also potentiated the cyclic AMP generated by isoprenaline in the presence of adenosine deaminase. Experiments performed in the presence of BQ-123 or BQ-788, specific ET(A) or ET(B) receptor antagonists respectively indicated that ET(B) was the receptor involved. This effect was dependent on extracellular and intracellular calcium concentration. These findings suggest that ET-1 plays a modulatory role in cyclic AMP generation systems in cerebral cortex.

Modulation of protein kinase C isoforms by PAF in cerebral cortex.

The effect of platelet activating factor (PAF) on subcellular distribution of protein kinase C isoforms in rat cerebral cortex was investigated. PAF induced an increase in levels of protein kinase C epsilon and gamma in membrane fraction. Results also indicate that PAF induced an increase in protein kinase C delta levels in both cytosolic and membrane fraction. This effect is possibly due to an increase in enzyme synthesis, as indicated by the results obtained from the experiments performed in the presence of cycloheximide and actinomycin. All the effects induced by PAF were time- and dose-dependent, and were mediated through the activation of PAF receptor. These findings indicate that the three isoforms may be involved in signal transduction of PAF in the brain.

Platelet-activating factor stimulation of p125(FAK) and p130(Cas) tyrosine phosphorylation in brain.

The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat brain slices. PAF induced a time- and concentration-dependent increase in tyrosine phosphorylation of a doublet of approximately 125 kDa. These proteins were identified by immunoprecipitation as p125(FAK) and p130(Cas), using monoclonal antibodies. This effect was mediated by PAF receptors, as shown by its inhibition by the action of a PAF antagonist. The tyrosine phosphorylation evoked by PAF was dependent, at least in part, on external calcium. The involvement of protein kinase C was demonstrated by the synergistic effect of TPA on PAF-stimulated tyrosine phosphorylation. The finding that PAF stimulates tyrosine phosphorylation of both focal adhesion protein p125(FAK) and p130(Cas) suggests that PAF might modulate the integrin mediated signal transduction in the brain.

Endothelin stimulates tyrosine phosphorylation of p125FAK and p130Cas in rat cerebral cortex.

Stimulation of rat cerebral cortex with endothelin-1 (ET-1) caused an increase in the tyrosine phosphorylation of several proteins. Two of these phosphoproteins were identified by the immunoprecipitation assays as being the focal adhesion kinase p125FAK and crk-associated substrate p130Cas. This effect was time- and dose-dependent, with an EC50 value of 3.9 x 10(-8) M. In addition, the cerebral cortex ET receptor subtype involved in this action was determined by using BQ-123 and BQ-788, which are ET(A) and ET(B) receptor antagonists respectively. Our results indicate that the ET-1 effect on protein tyrosine phosphorylation occurred through ET(B) receptors. The requirement for extracellular Ca2+ on ET-1 action was also studied. ET-1-stimulated tyrosine phosphorylation of both p125FAK and p130Cas was abolished in the absence of external Ca2+ or in the presence of nimodipine, a Ca2+ channel-blocker. These results suggest that the ET-1-stimulated protein tyrosine phosphorylation was secondary to Ca2+ influx through the dihydropyridine Ca2+-channel. In slices where protein kinase C was inhibited, ET-1-stimulated tyrosine phosphorylation of both proteins was reduced. These results indicate that ET-1 modulates the tyrosine phosphorylation of specific proteins, which may be involved in adhesion processes in the brain.

Sphingosylphosphorylcholine increases calcium concentration in isolated brain nuclei.

Sphingosylphosphorylcholine (SPC) caused a rapid increase of Ca2+ concentration in isolated brain nuclei. This effect was prevented by nimodipine, an inhibitor of L-type Ca2+ channels, and by thapsigargin, an inhibitor of Ca(2+)-ATPase. Neither heparin nor U73122 modified this effect, suggesting that phospholipase C activation and inositol 1,4,5-trisphosphate (IP3) production are not involved. Results also indicated that SPC-induced increase in Ca2+ concentration is not protein kinase C-dependent.

Glutamate release is involved in PAF-increased cyclic GMP levels in hippocampus.

The involvement of glutamate in PAF-increased cyclic GMP levels was studied. Glutamate treatment caused a dose-response increase of cyclic GMP levels in hippocampal slices. The presence of 1 mM glutamate did not modify the effect caused by 10(-7)M PAF. To elucidate the involvement of glutamate in this action, slices were treated with PAF in the presence of MK-801, a NMDA receptor antagonist. Results indicate that PAF-increased cyclic GMP levels were obtained by NMDA receptors activation. Finally, results obtained from the experiments performed with PAF in the presence of riluzole, to inhibit the glutamate release, demonstrated that glutamate release is a stage in the PAF-induced increase of cyclic GMP levels in hippocampus.

Sphingolipids increase calcium concentration in isolated rat liver nuclei.

The sphingolipids, sphingosine (SPH), sphingosylphosphorylcholine (SPC) and psycosine induce a rapid and transient rise in nuclear free Ca2+ concentration in a dose dependent manner. To determine whether these sphingolipids act by a IP3-dependent pathway, we tested the increase of Ca2+ in the presence of heparin, an antagonist of IP3 receptor or U70122, an inhibitor of phospholipase C. Results indicate that the effect of both SPH and SPC, but not that of psychosine, is partially mediated by IP3 production. The sphingolipid-induced Ca2+ mobilization was unaffected by the inhibition of protein kinase C, but was totally abolished in the presence of nimodipine, a L-type Ca2+ channel inhibitor. The results could indicate the existence of a sphingosine-gated Ca2+-permeable channel in liver nuclei.

Protein tyrosine phosphatase activity modulation by endothelin-1 in rabbit platelets.

Protein tyrosine phosphorylation, modulated by the rate of both protein tyrosine kinase and protein tyrosine phosphatase activities, is critical for cellular signal transduction cascades. We report that endothelin-1 stimulation of rabbit platelets resulted in a dose- and time-dependent tyrosine phosphorylation of four groups of proteins in the molecular mass ranges of 50, 60, 70-100 and 100-200 kDa and that one of these corresponds to focal adhesion kinase. This effect is also related to the approximately 60% decrease in protein tyrosine phosphatase activity. Moreover, this inhibited activity was less sensitive to orthovanadate. In the presence of forskolin that increases the cAMP level a dose-dependent inhibition of the endothelin-stimulated tyrosine phosphorylation of different protein substrates and a correlation with an increase in the protein tyrosine phosphatase activity (11.6-fold compared to control) have been found. Further studies by immunoblotting of immunoprecipitated soluble fraction with anti-protein tyrosine phosphatase-1C from endothelin-stimulated platelets have demonstrated that the tyrosine phosphorylation of platelet protein tyrosine phosphatase-1C is correlated with the decrease in its phosphatase activity. As a consequence, modulation and regulation by endothelin-1 in rabbit platelets can be proposed through a cAMP-dependent pathway and a tyrosine phosphorylation process that may affect some relevant proteins such as focal adhesion kinase.

Regulation of PAF-induced platelet responses by cyclic nucleotides.

The existence of cross-talk mechanisms between the cyclic nucleotide system and other transduction systems involved in PAF-activated platelets is described in this study. A protein of 125 kDa, identified as pp 125(FAK), is tyrosine phosphorylated by PAF in a time- and concentration-dependent manner. The presence of a cAMP- or a cGMP-elevating agent, used alone or in combination, together with PAF diminished tyrosine phosphorylation. The sensitivity to cAMP shown by PAF-induced ppl25 phosphorylation on tyrosine residues was similar to PAF-induced phosphorylation of a 47-kDa protein (pp47) on serine and threonine. In contrast, the latter was not affected in the presence of a cGMP-elevating agent, although it was able to enhance synergistically the inhibitory effect of forskolin. Data reported herein also show that pp47 phosphorylation and serotonin secretion are not closely correlated. Accordingly, sodium nitroprusside (SNP) did not have any effect on phosphorylation of pp47, but it was able to inhibit serotonin secretion when added alone, and it showed a synergistic inhibitory action with forskolin.

Mechanism of arachidonic acid-induced Ca2+ mobilization in liver nuclei.

Arachidonic acid treatment in isolated liver nuclei resulted in a rapid and transient increase of Ca2+ concentration in the nucleoplasm which was monitored with the Ca(2+)-sensitive dye fura-2 dextran. This effect was associated with a decrease of Ca2+ concentration in the nuclear envelope as measured with fura-2 AM. Our results indicate that arachidonic acid causes a Ca2+ release from the nuclear envelope to the nucleoplasm similar to that evoked by inositol trisphosphate (IP3). The arachidonic acid-induced Ca2+ mobilization in the nucleus was not due to the metabolites of arachidonic acid. Experiments performed in the presence of ATP and Ca2+ indicate that arachidonic acid-induced Ca2+ mobilization in the nucleus takes place in a non ATP-dependent way. Taken together, these results suggest that arachidonic acid may contribute to the regulation of nuclear Ca2+ mobilization.

Nitric oxide mediates the PAF-stimulated cyclic GMP production in hippocampal slices.

Platelet activating factor (PAF) treatment caused a transient rise in cyclic GMP levels in rat hippocampal slices. The stimulation of cyclic GMP synthesis induced by PAF was dose-dependent and was suppressed after treatment with PCA-4248, a PAF antagonist, a fact that could suggest the involvement of specific PAF receptors. In addition, when slices were incubated in the presence of N-nitro-L-arginine, a nitric oxide (NO) synthase inhibitor, PAF-stimulated cyclic GMP generation was abolished. Therefore, PAF activates guanylyl cyclase most probably via formation of NO. PAF also induced a time-dependent increase of NO synthase activity in hippocampal slices in correlation with the increase observed in cyclic GMP levels.

Endothelin-stimulated phosphoinositide turnover and protein kinase C translocation in rat synaptosomes.

The action of endothelin-1 (ET-1) on phosphoinositide metabolism was studied in rat synaptosomes. ET-1 caused an early and transitory decrease of 32P incorporation into phosphoinositides, concomitantly with an increase into phosphatidic acid (PA). This effect was time-dependent and was not found in the absence of exogenous calcium. Furthermore, ET-1 caused an increase in the generation of inositol phosphates and diacylglycerol (DAG). In addition, the peptide provoked a translocation of protein kinase C from the cytosol to membrane.

Platelet-activating factor inhibits (Na+,K+) ATPase activity in rat brain.

In the present study, experiments were conducted to determine the effect of platelet-activating factor (PAF) on (Na+,K+)-ATPase in rat cerebral cortex. PAF, but not lysoPAF, inhibited (Na+,K+)ATPase activity, in a dose- and time-dependent manner, 10(-7) to 10(6) M being the most effective dose. These effects were abolished in the presence of PCA-4248, a PAF antagonist, indicating that the PAF effect may be mediated by its specific membrane receptors. Omission of external calcium caused an increase in the basal activity and abolished the PAF effect on (Na+,K+)ATPase. The present study demonstrates that PAF inhibits (Na+,K+)ATPase activity in the cerebral cortex and suggests that PAF released during certain pathological conditions, such as ischemia, may act on ATPase. This could be one possible mechanism of PAF action that needs further attention.