Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) with thapsigargin, on both [Ca2+]i and the sarcoplasmic reticulum (SR) Ca2+ level during caffeine-induced Ca2+ release in single smooth muscle cells. Incubation with 10 μM ryanodine did not inhibit the first caffeine-induced [Ca2+]i response, although it abolished the [Ca2+]i response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca2+ stores, we measured the SR Ca2+ level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca2+ level was not augmented by incubating cells with 1 μM ryanodine. Moreover, on removal of caffeine, the SR Ca2+ levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 μM ryanodine instead of inducing complete depletion of SR Ca2+ stores markedly reduced the caffeine-induced SR Ca2+ response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca2+ stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca2+ stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca2+ levels to determine the effect of ryanodine on the internal Ca2+ stores.