Vascular endothelial cells undergo morphogenesis into capillary networks in response to angiogenic factors. We show here that sphingosine-1-phosphate (SPP), a platelet-derived bioactive lipid, activates the EDG-1 and -3 subtypes of G protein-coupled receptors on endothelial cells to regulate angiogenesis. SPP induces the Gi/mitogen-activated protein kinase/cell survival pathway and the small GTPase Rho- and Raccoupled adherens junction assembly. Both EDG-1-and EDG-3-regulated signaling pathways are required for endothelial cell morphogenesis into capillary-like networks. Indeed, SPP synergized with polypeptide angiogenic growth factors in the formation of mature neovessels in vivo. These data define SPP as a novel regulator of angiogenesis.

Local anesthetics such as dibucaine, QX572, tetracaine, and phenacaine, as well as other drugs with local anesthetic-like properties (e.g., mepacrine, propranolol, and SKF 525A) inhibit the specific calmodulin-dependent stimulation of erythrocyte Ca2+-ATPase (ATP phosphohydrolase, EC 3.6

Addition of pertussis toxin to rabbit neutrophils inhibits the fMet-Leu-Phe-induced increases in Na+ influx and in intracellular pH. In addition, pretreatment of the cells with the toxin inhibits the decrease in the levels of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and the enhanced production of phosphatidic acid produced by the chemotactic factor fMet-Leu-Phe. Furthermore, the fMet-Leu-Phe-induced changes in the phosphorylation of a 46-kDa protein and of several other proteins are also inhibited by the toxin. On the other hand, the phorbol 12-myristate 13-acetate (PMA)-induced increases in the phosphorylation of several proteins are not inhibited by the toxin. PMA, but not its inactive analogue 4a-phorbol 12,13-didecanoate, was also found to stimulate Na+ influx and to increase the intracellular pH in rabbit neutrophils. These ionic effects, like those produced by fMet-Leu-Phe, are inhibited by amiloride. The stimulated Na+ influx and H+ efflux produced by the phorbol ester, on the other hand, are not inhibited by pertussis toxin. The results reported here suggest (i) that the activity of the Na+/H+ antiport in neutrophils is regulated by protein kinase C; (ii) that the G-protein system, either directly or indirectly, is involved in the stimulus-response coupling sequence in these cells; and (iii) that the toxin acts at, or prior to, the steps responsible for the activation of phospholipase C, and it does not affect the sequence of reactions initiated by the activation of the protein kinase C.which provide the guanine nucleotide binding sites, are different, whereas the /8 subunit is the same (15). Pertussis toxin causes an NAD-dependent ADP-ribosylation of aj, and this ribosylation blocks the dissociation of a, from 13 and inhibits its action (15). The idea that Gi may be closely involved in signal transduction is suggested, albeit indirectly, by several recent observations. First, it was found that the addition of pertussis toxin to rabbit neutrophils inhibits the fMet-Leu-Phe-induced increase in the intracellular concentration of free Ca2' and other biochemical changes (refs. 16 and 17; unpublished observations). Second, the incorporation of guanine nucleotides into permeabilized mast cells causes secretion in response to the subsequent addition of Ca2+ (18,19), and this release is inhibited by pertussis toxin (19).The present studies were undertaken to investigate the role of protein kinase C in the regulation of the activity of the Na+/H+ antiport and to examine the effect of pertussis toxin on the stimulated biochemical changes in rabbit neutrophils. The results reported here clearly show that (i) phorbol 12-myristate 13-acetate (PMA) activates the Na+/H+ antiport system, and (ii) the addition of pertussis toxin inhibits the fMet-Leu-Phe but not the PMA-induced increase in Na+-influx, the increase in intracellular pH, and the changes in lipid metabolism and protein phosphorylation.The addition of the chemotactic factor fMet-Leu-Phe to neutrophils activates sev...

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