Ca " Dependence of Transverse Tubule - mediated Calcium Release in Skinned Skeletal Muscle Fibers

Isometric force and "Ca efflux from the sarcoplasmic reticulum were measured at 19°C in frog skeletal muscle fibers skinned by microdissection . After Ca" loading, application of the ionophores monensin, an Na+(K+)/ H' exchanger, or gramicidin D, an H+ > K' > Na' channel-former, evoked rapid force development and stimulated release of ^"30% of the accumulated "Ca within 1 min, whereas CCCP (carbonyl cyanide pyruvate p-trichloromethoxyphenylhydrazone), a protonophore, and valinomycin, a neutral, K+specific ionophore, did not. When monensin was present in all bathing solutions, i .e ., before and during Ca" loading, subsequent application failed to elicit force development and to stimulate "Ca efflux . 5 min pretreatment of the skinned fibers with 50,uM digitoxin, a permeant glycoside that specifically inhibits the Na+,K+ pump, inhibited monensin and gramicidin D stimulation of "Ca efflux ; similar pretreatment with 100 AM ouabain, an impermeant glycoside, was ineffective . Monensin stimulation of 45Ca efflux was abolished by brief pretreatment with 5 mM EGTA, which chelates myofilament-space calcium. These results suggest that : (a) monensin and gramicidin Dstimulate Ca" release from the sarcoplasmic reticulum that is mediated by depolarization of the transverse tubules, which seal off after sarcolemma removal and form closed compartments; (b) a transverse tubule membrane potential (myofilament space-negative) is maintained and/or established by the operation of the Na'',K+ pump in the transverse tubule membranes and is sensitive to the permeant inhibitor digitoxin ; (c) the transverse tubule-mediated stimulation of "Ca efflux appears to be entirely Ca" dependent .