Ca2+ entry into primary cultured pig coronary smooth muscle cells after previous store depletion by repetitive P2Y purinoceptor stimulation

Store-operated Ca2+ entry, stimulated by depletion of intracellular Ca2+ po ols, has not been fully elucidated in vascular smooth muscle cells of pig c oronary arteries. Therefore, [Ca2+](i) was measured in cultured cells deriv ed from extramural pig coronary arteries using the Fura-2/AM fluorometry. D ivalent cation entry was visualized with the Fura-2 Mn2+-quenching techniqu e. Ca2+ stores were depleted either by repetitive stimulation of P2Y purino ceptors with ATP (10 mu mol/L), or by the sarcoendoplasmic Ca2+-ATPase inhi bitor 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ; 1 mu mol/L) in Ca2+-f ree medium (EGTA 1 mmol/L). Addition of Ca2+ (1 mmol/L) induced refilling o f ATP-sensitive Ca2+ stores and an increase in [Ca2+](i) in the presence of BHQ. Both could be significantly diminished by Ni2+ (5 and 1 mmol/L), La3 (10 mu mol/L), Gd3+ (10 mu mol/L), and Mg2+ (5.1 mmol/L). In contrast to t he BHQ-mediated rise in [Ca2+](i), refilling of ATP-depleted stores was aff ected by neither flufenamate (0.1 mmol/L), nor by nitrendipine, nifedipine, and nisoldipine (each 1 mu mol/L). The data suggest that after store deple tion in pig coronary smooth muscle cells ATP and BHQ may converge on a comm on, Ni2+-, La3+-, Gd3+-, and Mg2+-sensitive Ca2+ entry pathway, i.e. on a s tore-operated Ca2+ entry. An additional contribution of the Na+/Ca2+ exchan ger cannot be excluded. Flufenamate-sensitive nonselective cation channels and dihydropyridine-sensitive L-type Ca2+ channels are not involved in refi lling of Ca2+ stores after previous depletion by repetitive P2Y purinocepto r stimulation. The store-operated Ca2+ entry in-between repetitive purinoce ptor stimulation, i.e. in the absence of the agonist, may be responsible fo r the maintenance of agonist-induced rhythmic Ca2+ responses. (C) 2001 Harc ourt Publishers Ltd.