The mechanism of ryanodine action in rabbit ventricular muscle evaluated with Ca-selective microelectrodes and rapid cooling contractures

Abstract

Cellular Ca uptake and efflux in rabbit ventricular muscle was measured using double-barreled Ca microelectrodes in the extracellular space. When repetitive stimulation was stopped there was a slow loss of cellular Ca. Upon resumption of stimulation Ca was taken up by the cells. These Ca movements are thought to represent the loss of Ca from the sarcoplasmic reticulum and the cell during rest and the refilling of the sarcoplasmic reticulum during stimulation. Ryanodine (100 nM) greatly enhanced both the efflux of Ca during rest and the uptake of Ca induced by stimulation. These results are consistent with the conclusions drawn below, but they are dependent upon the interpretation that these extracellular Ca depletions are indicative of sarcoplasmic reticulum Ca movements. To examine further this process, contractures induced by rapid cooling to 0 °C were used as an independent assay of sarcoplasmic reticulum Ca content. These rapid cooling contractures were smaller after longer rest intervals (declining with a half time of 1.5 min). In the presence of ryanodine, the rapid cooling contracture immediately after a contraction was greater than that seen under control conditions. However, in the presence of ryanodine these rapid cooling contractures decline as a function of rest duration with a half time of about 1 s. These results suggest that in the presence of ryanodine the sarcoplasmic reticulum can still take up Ca, but that it also loses this Ca very rapidly at the onset of rest. Caffeine (20 mM) inhibits both the extracellular Ca depletions and the rapid cooling contractures that are attributed to the sarcoplasmic reticulum Ca content changes (in the presence or absence of ryanodine). These results suggest the following two actions of ryanodine: (i) inhibition of sarcoplasmic reticulum Ca release into the cytoplasm upon activation, and (ii) enhancement of Ca loss from the sarcoplasmic reticulum upon the termination of stimulation and extrusion from the cell without activation of appreciable tension.