Effect of the removal of extracellular Ca2+ on the response of cytosolic concentrations of Ca2+ ([Ca2+](i)) to ouabain, an Na+/K+ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca2+](i) (mean +/- S.E.M.; 38 +/- 5% increase, n = 16) in 55% of tested cells (n = 29). The ouabain-induced [Ca2+](i) increase was abolished by the removal of extracellular Na+. D600 (50 mu M), an L-type voltage-gated Ca2+ channel antagonist, inhibited the [Ca2+](i) increase by 57 +/- 7% (n = 4). Removal of extracellular Ca2+ eliminated the [Ca2+](i) increase, but subsequent washing out of ouabain in Ca2+-free solution produced a rise in [Ca2+](i) (62 +/- 8% increase, n = 6, P < 0.05), referred to as a [Ca2+](i) rise after Ca2+-free/ouabain. The magnitude of the [Ca2+](i) rise was larger than that of ouabain-induced [Ca2+](i) increase. D600 (5 mu M) inhibited the [Ca2+](i) rise after Ca2+-free/ouabain by 83 +/- 10% (n = 4). These results suggest that ouabain-induced [Ca2+](i) increase was due to Ca2+ entry involving L-type Ca2+ channels which could be activated by cytosolic Na+ accumulation. Ca2+ removal might modify the [Ca2+](i) response, resulting in the occurrence of a rise in [Ca2+](i) after Ca2+-free/ouabain which mostly involved L-type Ca2+ channels. (C) 1999 Elsevier Science B.V. All rights reserved.