Role of sarcoplasmic reticulum Ca2+ content in Ca2+ entry of bovine airway smooth muscle cells.

Depletion of intracellular Ca(2+) stores induces the opening of an unknown Ca(2+ )entry pathway to the cell. We measured the intracellular free-Ca(2+) concentration ([Ca(2+)]i) at different sarcoplasmic reticulum (SR) Ca(2+) content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca(2+) in the extracellular medium generated a time-dependent decrement in [Ca(2+)]i which was proportional to the reduction in the SR-Ca(2+) content. This SR-Ca(2+) level was indirectly determined by measuring the amount of Ca(2+) released by caffeine. Ca(2+) restoration at different times after Ca(2+)-free incubation (2, 4, 6 and 10 min) induced an increment of [Ca(2+)]i. This increase in [Ca(2+)]i was considered as Ca(2+) entry to the cell. The rate of this entry was slow (~0.3 nM/s) when SR-Ca(2+) content was higher than 50% (2 and 4 min in Ca(2+)-free medium), and significantly ( p<0.01) accelerated (>1.0 nM/s) when SR-Ca(2+) content was lower than 50% (6 and 10 min in Ca(2+)-free medium). Thapsigargin significantly induced a higher rate of this Ca(2+) entry ( p<0.01). Variations in Ca(2+) influx after SR-Ca(2+) depletion were estimated more directly by a Mn(2+) quench approach. Ca(2+) restoration to the medium 4 min after Ca(2+) removal did not modify the Mn(2+) influx. However, when Ca(2+) was added after 10 min in Ca(2+)-free medium, an increment of Mn(2+) influx was observed, corroborating an increase in Ca(2+) entry. The fast Ca(2+) influx was Ni(2+) sensitive but was not affected by other known capacitative Ca(2+) entry blockers such as La(3+), Mg(2+), SKF 96365 and 2-APB. It was also not affected by the blockage of L-type Ca2(+) channels with methoxyverapamil or by the sustained K(+)-induced depolarisation. The slow Ca(2+) influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca(2+) influx after SR-Ca(2+) depletion has two rates: A) The slow Ca(2+) influx, which occurred in cells with more than 50% of their SR-Ca(2+) content, is sensitive to SKF 96365 and appears to be a non-capacitative Ca(2+) entry; and B) The fast Ca(2+) influx, observed in cells with less than 50% of their SR-Ca(2+) content, is probably a capacitative Ca(2+) entry and was only Ni(2+)-sensitive.