Abstract
Depletion of intracellular Ca2+ stores induces the opening of an unknown Ca2+ entry pathway to the cell. We measured the intracellular free-Ca2+ concentration ([Ca2+]i) at different sarcoplasmic reticulum (SR) Ca2+ content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca2+ in the extracellular medium generated a time-dependent decrement in [Ca2+]i which was proportional to the reduction in the SR-Ca2+ content. This SR-Ca2+ level was indirectly determined by measuring the amount of Ca2+ released by caffeine. Ca2+ restoration at different times after Ca2+-free incubation (2, 4, 6 and 10 min) induced an increment of [Ca2+]i. This increase in [Ca2+]i was considered as Ca2+ entry to the cell. The rate of this entry was slow (~0.3 nM/s) when SR-Ca2+ content was higher than 50% (2 and 4 min in Ca2+-free medium), and significantly (p<0.01) accelerated (>1.0 nM/s) when SR-Ca2+ content was lower than 50% (6 and 10 min in Ca2+-free medium). Thapsigargin significantly induced a higher rate of this Ca2+ entry (p<0.01). Variations in Ca2+ influx after SR-Ca2+ depletion were estimated more directly by a Mn2+ quench approach. Ca2+ restoration to the medium 4 min after Ca2+ removal did not modify the Mn2+ influx. However, when Ca2+ was added after 10 min in Ca2+-free medium, an increment of Mn2+ influx was observed, corroborating an increase in Ca2+ entry. The fast Ca2+ influx was Ni2+ sensitive but was not affected by other known capacitative Ca2+ entry blockers such as La3+, Mg2+, 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 Ca2+ influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca2+ influx after SR-Ca2+ depletion has two rates: A) The slow Ca2+ influx, which occurred in cells with more than 50% of their SR-Ca2+ content, is sensitive to SKF 96365 and appears to be a non-capacitative Ca2+ entry; and B) The fast Ca2+ influx, observed in cells with less than 50% of their SR-Ca2+ content, is probably a capacitative Ca2+ entry and was only Ni2+-sensitive.
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Acknowledgements
This study was supported by grants from DGAPA-UNAM (IN202999) and PUIS-UNAM (394–446/17-X-94) to Dr. Luis M. Montaño.
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Bazán-Perkins, B., Flores-Soto, E., Barajas-López, C. et al. Role of sarcoplasmic reticulum Ca2+ content in Ca2+ entry of bovine airway smooth muscle cells. Naunyn-Schmiedeberg's Arch Pharmacol 368, 277–283 (2003). https://doi.org/10.1007/s00210-003-0806-4
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DOI: https://doi.org/10.1007/s00210-003-0806-4