ROLE OF [NA+](I) AND [CA2+](I) IN NICOTINE-INDUCED NOREPINEPHRINE RELEASE FROM BOVINE ADRENAL CHROMAFFIN CELLS

Intracellular free sodium ([Na+](i)) and calcium ([Ca2+](i)) concentrations were determined by sodium-binding benzofuran isophthalate (SBFI) and fura 2 microfluorimetry, respectively, in bovine adrenal chromaffin cells (BCC). Validation of SBFI microfluorimetry by in vitro and in vive calibration revealed a reliable assessment of [Na+](i) within a range of 1-30 mM in single BCC. Nicotine (0.1-10 mu M) induced concentration-dependent increases of both [Na+](i) (from 3.3 +/- 0.1 to 25.6 +/- 0.4 mM, n = 76, P < 0.001) and [Ca2(+)](i) (from 64 +/- 1 to 467 +/- 16 nM, n = 87, P < 0.001), which were accompanied by an increase in [H-3]norepinephrine (NE) release. Consistent with an exocytotic release mechanism, nicotine-induced increments of [Ca2+](i) and [H-3]NE release were reduced under calcium-free conditions and by gadolinium chloride (40 mu M), whereas [Na+](i) was not affected. In contrast, a parallel attenuation of nicotine-evoked changes in [Na+](i), [Ca2+](i), and [H-3]NE release was observed during reduction of the extracellular sodium concentration. The nicotine-evoked responses were neutralized by the nicotinic receptor antagonist hexamethonium (100 mu M) but not by blockade of voltage-dependent sodium channels (1 mu M tetrodotoxin). In conclusion, the nicotine-induced exocytotic release of [H-3]NE is triggered by an increase in [Ca2+](i), which is facilitated by sodium influx through the nicotinic receptor ionophore.