Single-cell fura-2 microfluorometry reveals different purinoceptor subtypes coupled to Ca²⁺ influx and intracellular Ca²⁺ release in bovine adrenal chromaffin and endothelial cells

Abstract

ATP and adenosine(5′)tetraphospho(5′)adenosine (Ap₄A), 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 (EC₅₀=3.8 μM) 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; EC₅₀= 12.5 μM) and UTP, but did not respond to either adenosine 5′-[β-thio]diphosphate (ADP[βS]), a P_2Y receptor agonist, adenosine 5′-[α,β-methylene]triphosphate (pp[CH₂]pA), a P_2X agonist or AMP. Adrenal endothelial cells displayed concentration-dependent [Ca²⁺]_i responses when stimulated with ATP (EC₅₀=0.86 μM), UTP (EC₅₀=1.6 μM) and 2MeSATP (EC₅₀= 0.38 μM). 2MeSATP behaved as a partial agonist. Ap₄A and ADP[βS] 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.