Ryanodine releases calcium from sarcoplasmic reticulum in calcium‐tolerant rat cardiac myocytes.

R. G. Hansford, Edward Lakatta

Research output: Contribution to journalArticle

Abstract

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)

Original languageEnglish (US)
Pages (from-to)453-467
Number of pages15
JournalThe Journal of Physiology
Volume390
Issue number1
DOIs
StatePublished - Sep 1 1987
Externally publishedYes

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Ryanodine
Sarcoplasmic Reticulum
Ionomycin
Caffeine
Cardiac Myocytes
Calcium
Egtazic Acid
Suspensions
Fluorescence
Ionophores
Chelating Agents
Fluorescent Dyes
Alkaloids
Muscle Cells

ASJC Scopus subject areas

  • Physiology

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Ryanodine releases calcium from sarcoplasmic reticulum in calcium‐tolerant rat cardiac myocytes. / Hansford, R. G.; Lakatta, Edward.

In: The Journal of Physiology, Vol. 390, No. 1, 01.09.1987, p. 453-467.

Research output: Contribution to journalArticle

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N2 - 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)

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