R.Edgardo Catalán

Endothelin Stimulates Phosphoinositide Hydrolysis and PAF Synthesis in Brain Microvessels

Treatment of brain microvessels with the three endothelin (ET) isoforms resulted in an increase of phosphoinositide turnover by activation of phospholipase C in a dose- and time-dependent manner. Both ET-1 and ET-2 are maximally effective, whereas the effect evoked by ET-3 was smaller. Concomitantly, there was an enhanced production of a platelet-activating factor (PAF)-like material. This was identified by standard and biological probes in platelets, such as induction of aggregation, phosphatidic acid (PA) production, increase of endogenous protein phosphorylation, and reversal of these responses by a PAF antagonist. The effects evoked by endothelins on phosphoinositide metabolism and PAF production were, to a certain extent, dependent on the presence of extracellular Ca2+. In addition, ET induced changes in Ca2+ dynamics, evoking an initial and rapid intracellular mobilization and influx of Ca2+ and, later, a maintained Ca2+ influx. These findings contribute to the understanding of the pathophysiological role of ET in the blood–brain barrier (BBB).

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.

Platelet-activating factor stimulation of p125FAK and p130Cas 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.

Effect of indomethacin on the cyclic AMP-dependent protein kinase

Indomethacin inhibited cyclic AMP-dependent protein kinase activity in small intestine in in vivo experiments. An inverse pattern of variation was exhibited by acetyl salicylic acid, eterylate and benorylate, acetyl-p-amino-phenol being inactive. Indomethacin, acetyl salicylic acid, eterylate and benorylate increased the protein kinase activity in liver, lung and heart after in vivo administration. The in vivo effect of indomethacin was confirmed by in vitro experiments with small intestine and heart protein kinases. These results support the concept that indomethacin can affect protein kinase activity in a tissue-specific way.

Brain microvessel endothelin type A receptors are coupled to ceramide production.

Treatment of brain microvessels with endothelin-1 evoked an early decrease in the sphingomyelin levels concomitantly with an increase in those of ceramides. These responses were time- and concentration-dependent. Evidence also shows that endothelin type A receptors are involved. This is the first report on the involvement of an agonist in the regulation of the ceramide signal transduction system on blood-brain barrier and shows a new pathway likely involved in the regulation of the cerebral microvascular functioning.

Exercise training-induced changes in sensitivity to endothelin-1 and aortic and cerebellum lipid profile in rats.

The purpose of this work was to study whether exercise training induces changes in the lipid profile of rat aorta and nervous system and in the in vitro intrinsic responsiveness of these tissues to endothelin-1 (ET-1) treatment. The exercise program performed successfully produced the characteristic metabolic alterations of the trained state. Exercise training induced a large and significant increase in the levels of both aortic ethanolamine plasmalogens (PlasEtn) and glucosylceramides. In contrast, a decrease of aortic ceramide and cholesterol levels was evoked by exercise training. ET-1 increased PlasEtn content only in sedentary animals. An exercise-induced increase in cerebellum levels of ceramides and ceramide monohexosides was found. The cerebellum ceramide content was increased by FT-1 more noticeably in sedentary rats than in trained animals. In contrast, cerebral cortex was observed to be largely insensitive to both exercise training and ET-1 treatment. It was concluded that exercise training (i) induces changes in both vascular and cerebellar lipid profiles, the former being much more pronounced than the latter, and (ii) diminishes the aortic and cerebellar sensitivity to ET-1 action.

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.

Endothelin-1 stimulates myristoylated alanine-rich C-kinase substrate (MARCKS) phosphorylation in rat cerebellar slices

Protein phosphorylation induced by endothelins has been studied using [32P]orthophosphate-prelabelled rat cerebellar slices. Endothelin-1 increased phosphorylation of an 87 kDa protein in a time-dependent manner (reaching a maximum effect at about 2.5 min) and with an EC50 equal to 93 +/- 32 nM. Endothelin-3 and sarafotoxin 6c induced similar levels of phosphorylation. Endothelin-1 also promoted [3H]inositol phosphate accumulation with similar EC50 (71 +/- 7.5 nM). The phosphoprotein of 87 kDa seems to be myristoylated alanine-rich C-kinase substrate (MARCKS) as demonstrated by acetic acid extraction. In addition, 12-O-tetradecanoylphorbol-13-acetate (TPA) increased 87 kDa protein phosphorylation while Ro-31-8220, a specific protein kinase inhibitor, inhibited both TPA and endothelin-induced 87 kDa protein phosphorylation. Therefore, it is concluded that protein kinase C is involved in the endothelin action on cerebellum.

PCA-4248, a PAF receptor antagonist, inhibits PAF-induced phosphoinositide turnover

The effect of a new PAF (platelet activating factor; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoryl-choline) receptor antagonist, PCA-4248 (2-phenylthio)ethyl-5-metoxycarbonyl-2,4,6-trimethyl-1, 4-dihydropyridine-3-carboxylate), on phosphoinositide turnover evoked by PAF was investigated. PAF treatment resulted in an increased 32P incorporation into phosphoinositides and phosphatidic acid in rabbit platelets. Treatment with PCA-4248 abolished both effects in a dose-dependent manner, 10 microM being the most effective dose. In thrombin stimulated platelets, phosphoinositide turnover was not influenced by PCA-4248. In addition, PAF caused a rapid and significant increase in protein phosphorylation. Thus, PAF treatment resulted in a marked phosphorylation of two proteins of 47 kDa and 20 kDa. Treatment with PCA-4248 resulted in an inhibition of these actions. Serotonin secretion evoked by PAF was also inhibited by PCA-4248. It is concluded that PCA-4248 antagonizes the PAF effects by acting as a competitive antagonist at the PAF receptor level as evidenced from binding studies.

Dissociation between secretion and protein phosphorylation in agonist-stimulated platelets; action of PCA-4230, a new antithrombotic drug

We have studied the effects of PCA-4230, a new dihydropyridine derivative with antithrombotic activity, on the secretion of dense and alpha-granules from platelets and on the protein phosphorylation in platelets after stimulation by agonists. The drug prevented both dense and alpha-granule secretion evoked by thrombin, platelet-activating factor (PAF) and ionophore A23187, the former secretion being more sensitive than the latter one to the PCA-4230 action. These inhibitory effects on secretion processes did not correlate with the differential action of PCA-4230 on protein phosphorylation. Thus, the 40 kDa protein phosphorylation evoked by thrombin was potentiated whereas that elicited by ionophore A23187 was partially inhibited. The 20 kDa protein phosphorylation was practically insensitive to the drug action. These data, together with previous evidence reported by us on PCA-4230, lead us to suggest the existence of a common and critical step for platelet secretion evoked by agonists with different signal transduction pathways.