M.C. Calcerrada

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

In this study we report that protein kinase C ζ (PKC ζ), one of the atypical isoforms of the PKC family located predominantly in cytosol, is redistributed by C2‐ceramide treatment in isolated hepatocytes. PKC ζ 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 ζ 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.

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

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.

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.

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 p130Cas. 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.

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 10‐7M 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.

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.