Measurement of ATP released from a single cell and its application

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1) ATP caused increases in inward currents, intracellular Ca^<2+> concentration and catecholamine secretion in adrenal chromaffin cells of the guinea-pig. On the other hand, it inhibited voltage-dependent Ca^<2+> currents through G protein activation. 2)N,L,P/Q and R types of Ca^<2+> channels were present in the adrenal chromaffin cell of the guinea-pig and N,L and P/Q but not R types of Ca^<2+> channels in the cultured chromaffin cell of the pig. 3) ATP photoluminescence was continuously monitored by an ATP photometer. The detection limit for ATP was l-2nM.The pig adrenal chromaffin cells cultured on coverslips were put in a chamber (0.5ml) and superfused at a flow rate of 2ml/min. The effluent was divided into two, the one was applied to the ATP photometer to measure ATP and the other to an electrochemical detector to measure catecholamine continuously. Acetylcholine, 60mMKC1 and 5mMBaCl_2 caused increases in the release of catecholamine and ATP.The time courses of catecholamine secretion induced by these stimuli were consistent with those of ATP secretion. The molar ratio of catecholamine to ATP appearing in the effluent was 7-12.4) The content of adenine nucleotides (ATP,ADP,AMP) measured with HPLC was almost the same in the effluent, and the molar ratio of catecholamine to adenine nucleotides in the effluent was 4-5. These values resembled the molar ratio present in the chromaffin granules. 5) The quantal release of catecholamine from a chromaffin cell was measured with a micro-carbon electrode in connection with a patch clamp amplifier. Acetylcholine elicited a dose-dependent increase in the frequency of spike currents responsible for the oxidation of catecholamine near the surface of the carbon electrode. 6) We could detect catecholamine secretion from a single adrenal chromaffin cell but not ATP secretion so far.