M.Dolores Aragonés

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).

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.

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.

Involvement of calcium in phosphoinositide metabolism in the blood-brain barrier

The Ca2+ effect on phosphoinositide metabolism in the blood-brain barrier was studied by using rat cerebral microvessels prelabelled with either [32P]orthophosphate or myo-[3H]inositol and stimulated with Ca2+ ionophore A23187. In radioactivity steady-state conditions, addition of ionophore caused a rapid and marked loss of labelling in both phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2), without significant alterations in phosphatidylinositol (PI) and phosphatidic acid (PA) labelling. These facts were accompanied by a rise in labelling of both inositol 1-monophosphate (IP) and inositol 1,4-bisphosphate (IP2), but not in inositol 1,4,5-trisphosphate (IP3). In addition, a Ca(2+)-dependent inhibition of phosphoinositide kinase activities from isolated membranes was also found. These data suggest that elevated intracellular Ca2+ level evokes a PIP and PIP2 hydrolysis by phosphodiesterasic and phosphomonoesterasic activities respectively, and also partially inhibits the synthesis of these phosphoinositides. Our results constitute evidence that a reciprocal control mechanism between polyphosphoinositide metabolism and mobilization of Ca2+ exists in the blood-brain barrier.

INVOLVEMENT OF CYCLIC GMP IN THE MODE OF ACTION OF A NEW ANTITHROMBOTIC AGENT PCA-4230; INHIBITION OF THE PLATELET CYCLIC GMP DEPENDENT PHOSPHODIESTERASE

The effect of PCA-4230, a new dihydropyridine derivative with a potent antithrombotic activity, on cyclic nucleotide phosphodiesterase in platelets was studied. PCA-4230 inhibited (54%) cyclic GMP hydrolytic activity of a platelet cytosolic fraction, whereas it did not affect that of cyclic AMP. Results suggested that PCA-4230 inhibited a cyclic GMP-dependent phosphodiesterase, known as cGB PDE or type V, on a purified enzyme from rabbit platelets by a non-competitive-uncompetitive type inhibition. In addition, PCA-4230 potentiated the increase in both cyclic GMP and cyclic AMP levels evoked by sodium nitroprusside. Furthermore, PCA-4230 and forskolin caused a synergistic effect in cyclic AMP, and also potentiated the phosphorylation of 50 kDa and 22 kDa proteins, reported as substrates of cyclic GMP- and cyclic AMP-dependent protein kinases that are related to the inhibition of platelet functions. Finally, PCA-4230 also potentiated the forskolin- and sodium nitroprusside-inhibited serotonin release evoked by thrombin, probably related to the increased cyclic nucleotide level.

Protein phosphorylation in the blood-brain barrier. Possible presence of marcks in brain microvessels

The protein phosphorylation in rat brain microvessels has been examined; the major phosphorylated proteins correspond to a doublet of molecular weight 134-141 kDa, and four proteins of approx. 25, 55, 80 and 200 kDa. TPA (12-O-tetradecanoylphorbol-13-acetate) enhanced, in a few minutes, the phosphorylation of three major protein substrates with apparent molecular weights of 17.5, 44.5 and 80 kDa. These effects are inhibited by staurosporine. The 80 kDa protein resulted to be myristoylated alanine-rich C kinase substrate (MARCKS). This work demonstrates that protein kinase C plays an important role in protein phosphorylation in blood-brain barrier (BBB).