Ryanodine releases calcium from sarcoplasmic reticulum in calcium-tolerant rat cardiac myocytes

1. The hypothesis tested in this study is that ryanodine depletes sarcoplasmic reticulum (s.r.) Ca2+ loading in suspensions of single adult rat cardiac myocytes by effecting Ca2+ release into the myoplasm resulting in an increase in myoplasmic free [Ca2+] ([Ca2+]i). The latter was monitored by the fluorescent dye, quin2. 2. The competency of the technique to detect s.r. Ca2+ release was tested by using caffeine to induce Ca2+ release. The addition of 5-10 mM-caffeine to myocytes loaded with quin2 and incubated in a medium containing 1 mM-Ca2+ gives a large, transient increase in fluorescence, which is interpreted as indicating an increase in [Ca2+]i. If the chelating agent EGTA is added to the cell suspension 1-5 min prior to the caffeine, to a concentration sufficient to decrease extracellular Ca2+ to 0.1-0.15 microM, then caffeine again gives a large, transient increase in fluorescence, indicative of the fact that sarcolemmal Ca2+ transport is not necessary for this response. The ionophore ionomycin also raises [Ca2+]i in a transient manner when added after EGTA. The addition of caffeine prior to ionomycin largely diminishes the response to the latter; however, addition of ionomycin prior to caffeine totally abolishes its effect to increase [Ca2+]i. This is taken to indicate that the intracellular store which is releasable by caffeine--and which presumably reflects the s.r.--is also releasable by ionomycin: ionomycin, however, also gives access to another, minor intracellular pool. 3. The plant alkaloid, ryanodine, at concentrations of 10(-8) to 10(-6) M, consistently causes a slow and prolonged increase in [Ca2+]i when added to cell suspensions incubated with 1 mM-extracellular Ca2+. Under conditions precluding net entry of Ca2+ into the cell, viz. 0.1 microM-extracellular Ca2+, ryanodine causes a more limited, partially reversible, increase in [Ca2+]i. 4. When added prior to EGTA, ryanodine attenuates, or prevents, the subsequent response to caffeine: efficacy depends upon the time of pre-incubation (1-10 min) and the concentration of ryanodine (10(-8) to 10(-6) M). When the response to caffeine is largely prevented by ryanodine, the response to ionomycin is also severely attenuated, i.e. there is no evidence that ryanodine causes sequestration of Ca2+ within an ionomycin-sensitive pool.(ABSTRACT TRUNCATED AT 400 WORDS)