Abstract
Calcium release from intracellular stores occurs in a graded manner in response to increasing concentrations of either inositol 1,4,5-trisphosphate or caffeine. To investigate the mechanism responsible for this quantal release phenomenon, [Ca2+] changes inside intracellular stores in isolated single smooth muscle cells were monitored with mag-fura 2. Following permeabilization with saponin or alpha-toxin the dye, loaded via its acetoxymethyl ester, was predominantly trapped in the sarcoplasmic reticulum (SR). Low caffeine concentrations in the absence of ATP induced only partial Ca2+ release; however, after inhibiting the calcium pump with thapsigargin the same stimulus released twice as much Ca2+. When the SR Ca(2+)-ATPase was rendered non-functional by depleting its "ATP pool," submaximal caffeine doses almost fully emptied the stores of Ca2+. We conclude that quantal release of Ca2+ in response to caffeine in these smooth muscle cells is largely due to the activity of the SR Ca(2+)-ATPase, which appears to return a portion of the released Ca2+ back to the SR, even in the absence of ATP. Apparently the SR Ca(2+)-ATPase is fueled by ATP, which is either compartmentalized or bound to the SR.
Highlights
Cells respond to increasing concentrations of many hormones and neurotransmitters with graded changes in cytosolic [Ca2ϩ]
Caffeine (25 mM, no ATP), which activates the RyR in these cells [20], decreased the fluorescence ratio of mag-fura 2, and the [Ca2ϩ], in the intracellular stores from 2.42 to 0.58. These stores apparently released their total free calcium in response to this high caffeine concentration as the final fluorescence ratio was virtually identical to Rmin in vitro
Refilling of internal stores following caffeine stimulation was almost completely blocked by the specific inhibitor of the sarcoplasmic reticulum (SR) Ca2ϩ-ATPase thapsigargin (5 min, 2 M; Fig. 2B)
Summary
Cells respond to increasing concentrations of many hormones and neurotransmitters with graded changes in cytosolic [Ca2ϩ]. Caffeine (25 mM, no ATP), which activates the RyR in these cells [20], decreased the fluorescence ratio of mag-fura 2, and the [Ca2ϩ], in the intracellular stores from 2.42 to 0.58.
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