Co-existence of P-2Y- and PPADS-insensitive P-2u-purinoceptors in endothelial cells from adrenal medulla

1 We have studied the effects of purinoceptor stimulation on Ca2+ signals in bovine adrenomedullary endothelial cells. [Ca2+](i) was determined with the fluorescent probe fura-2 both in population samples and in single, isolated, endothelial cells in primary culture and after subculturing. 2 In endothelial cells, maintained in culture for more than one passage, several purinoceptor agonists elicited clear [Ca2+](i) transient peaks that remained in the absence of extracellular Ca2+. Adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) were equipotently active, with EC(50) values of 8.5+/-0.9 mu M and 6.9+/-1.5 mu M, respectively, whereas 2-methylthioadenosine 5'-triphosphate (2MeSATP), adenosine 5'-(alpha,beta-methylene)triphosphate (alpha,beta-MeATP) and adenosine(5')tetraphospho(5')adenosine (Ap(4)A) were basically inactive. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) was a weak agonist. The apparent potency order was UTP=ATP>ADP beta S much greater than 2MeSATP>alpha,beta-MeATP. 3 Cross-desensitization experiments revealed that UTP or ATP, added sequentially at concentrations of maximal effect, could completely abolish the [Ca2+](i) response to the second agonist. ADP beta S exerted only a partial desensitization of the response to maximal ATP, in accordance with its lower potency in raising [Ca2+](i). 4 The effect on [Ca2+](i) of 100 mu M ATP in subcultured cells was reduced by only 25% with 100 mu M suramin pretreatment and was negligibly affected by exposure to 10 mu M pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). The concentration-effect curve for ATP was not significantly affected by PPADS, but was displaced to the right by a factor of 6.5 by 100 mu M suramin. 5 In primary cultures, clear [Ca2+](i) responses were elicited by 2MeSATP. Suramin totally and selectively blocked 2MeSATP responses, whereas UTP-evoked [Ca2+](i) transients were mainly unaffected by suramin or PPADS. Over 80% of cells tested showed responses to both 2MeSATP and UTP. The [Ca2+](i) response to UTP was not desensitized in the presence of 2MeSATP. 6 ATP and UTP stimulated the release of preloaded [H-3]-arachidonic acid ([H-3]-AA), both in the presence and in the absence of extracellular Ca2+ by approximately 135% with respect to basal levels. Suramin and PPADS enhanced, rather than inhibited, the [H-3]-AA releasing effect of ATP by 2.5 times. Suramin also potentiated the effect of the calcium ionophore A23187. 7 These results indicate that endothelial cells from adrenomedullary capillaries co-express both P-2Y- and P-2U-purinoceptors. P-2Y-purinoceptors are lost in culture with the first passage of the cells. The P-2U-purinoceptor subtype present in these cells is insensitive to PPADS and thus similar to that found in aortic endothelial cells.