SINGLE-CELL FURA-2 MICROFLUOROMETRY REVEALS DIFFERENT PURINOCEPTOR SUBTYPES COUPLED TO CA2+ INFLUX AND INTRACELLULAR CA2+ RELEASE IN BOVINE ADRENAL CHROMAFFIN AND ENDOTHELIAL-CELLS

ATP and adenosine(5')tetraphospho(5')adenosine (Ap(4)A), released from adrenal chromaffin cells, are potent stimulators of endothelial cell function. Using single-cell fura-2 fluorescence recording techniques to measure free cytosolic Ca2+ concentration ([Ca2+](i)), we have investigated the role of purinoceptor subtypes in the activation of cocultured chromaffin and endothelial cells. ATP evoked concentration-dependent [Ca2+](i) rises (EC(50) = 3.8 mu M) in a subpopulation of chromaffin cells. Both ATP-sensitive and -insensitive cells were potently activated by nicotine, bradykinin and muscarine. Reducing extracellular free Ca2+ concentration to around 100 nM suppressed the [Ca2+](i) transient evoked by ATP but not the [Ca2+](i) response to bradykinin. ATP-sensitive chromaffin cells were also potently stimulated by 2-methylthioadenosine triphosphate (2MeSATP; EC(50) 12.5 mu M) and UTP, but did not respond to either adenosine 5'-[beta-thio]diphosphate (ADP[beta S]), a P-2Y receptor agonist, adenosine 5'-[alpha,beta-methylene]triphosphate (pp-[CH2]pA), a P-2X agonist or AMP. Adrenal endothelial cells displayed concentration-dependent [Ca2+](i) responses when stimulated with ATP (EC(50) = 0.86 mu M), UTP (EC(50) = 1.6 mu M) and 2MeSATP (EC(50) = 0.38 mu M). 2MeSATP behaved as a partial agonist. Ap(4)A and ADP[beta S] also raised the [Ca2+](i) in endothelial cells, whereas AMP and pp[CH2]pA were ineffective. Lowering extracellular free Ca2+ to around 100 nM did not affect the peak ATP-evoked [Ca2+](i) rise in these cells. It is concluded that different purinoceptor subtypes are heterogeneously distributed among the major cell types of the adrenal medulla. An intracellular Ca2+-releasing P-2U-type purinoceptor is specifically localized to adrenal endothelial cells, while a subpopulation of chromaffin cells expresses a non-P-2X, non-P-2Y subtype exclusively coupled to Ca2+ influx.