Modulation of the effects of extracellular ATP on [Ca2+]i in rat brain microvascular endothelial cells

This study examined the intracellular regulation of signal transduction initiated by activation of the P2Y(2) purinoceptor in a cultured rat brain microvascular endothelial cell line (RBE4). Intracellular free Ca2+ ([Ca2+](i)) was monitored in single cells, using FURA-2 fluorimetry. As previously described [Nobles, M., Revest, P.A., Couraud, P.-O., Abbott, N.J., 1995. Characteristics of nucleotide receptors that cause elevation of cytoplasmic calcium in immortalized rat brain endothelial cells, RBE4, and in primary cultures. Br. J. Pharmacol., 115, 1245-1252], extracellular ATP (100 mu M, 20 s) evoked a transient increase in intracellular free calcium concentration ([Ca2+](i)). The amplitude of the Ca2+ transient evoked by ATP decreased with successive applications (desensitisation), as expected for a P2 purinoceptor. The modulation of the Ca2+ signal downstream to the activation of the ATP receptor was investigated, using agents selected for their ability to interfere with the intracellular pathways activated by ATP. The amplitude of the Ca2+ transient observed on the second application of ATP was compared in the presence and absence of these agents. The Ca2+ transient triggered by ATP was decreased by the inhibitor of nitric oxide synthesis, N-omega-nitro-L-arginine methyl ester (L-NOARG). The inhibition induced by 100 mu M r-NOARG was reversed by coapplication of the permeant cGMP analogue 8-brcGMP (100 mu M) 8-BrcGMP caused a transient increase in [Ca2+](i) when applied alone, and a dose-dependent inhibition of the increase in [Ca2+](i) elicited by ATP. Indomethacin, an inhibitor of prostaglandin synthesis, inhibited the response to ATP. The inhibition caused by 10 mu M indomethacin was reversed by coapplication of the permeant analogue of cAMP, 8-brcAMP (100 mu M). 8-BrcAMP caused a transient rise in [Ca2+](i) when applied alone, and a dose-dependent inhibition of the Ca2+ response evoked by ATP. The non-permeant cyclic nucleotides cAMP and cGMP did not affect the desensitising response to ATP, nor did they reverse the inhibitory actions of L-NOARG or indomethacin. It is concluded that cyclic nucleotides, nitric oxide, and prostaglandin synthesis pathways are able to interact with the Ca2+ second messenger pathway in rat brain endothelial cells activated by extracellular ATP. (C) 1998 Elsevier Science B.V. All rights reserved.