On-line measurement of adenosine triphosphate and catecholamine released from adrenal chromaffin cells

Adenosine triphosphate (ATP) and catecholamine (CA) released from cultured porcine adrenal chromaffin cells were continuously measured with an ATP photometer (luciferin-luciferase method) and electrochemical detector, respectively. Application of acetylcholine (ACh, 0.1 mM) or high K+ (60 mM) caused increases of ATP and CA in perfused effluent with the same time course. The peak molar ratio of CA to ATP in the effluent was about 10 for ACh and high K+ stimulation. The high-performance liquid chromatographic (HPLC) analysis of adenine nucleotides in the collected effluent revealed that the relative amounts of ATP, ADP and AMP were almost the same throughout the period of stimulation, suggesting that ATP breakdown in the effluent was constant. Changes in the peak molar ratio of CA to ATP appearing in the effluent did not occur with repetitive high K+ or sustained Ba2+ stimulation (5 mM). The similarity between the time courses of ATP and CA appearing in the effluent suggests that releasable chromaffin granules have a constant molar ratio of CA to ATP. The on-line system developed is a simple and rapid method for examining ATP and CA secretion, simultaneously.