Spontaneous sarcoplasmic reticulum calcium release and extrusion from bovine, not porcine, coronary artery smooth muscle.
1. We tested the hypothesis that the Ca(2+)-loaded sarcoplasmic reticulum (SR) of coronary artery smooth muscle spontaneously releases Ca2+ preferentially toward the sarcolemma to be extruded from the cell without increasing the average free myoplasmic [Ca2+] (Ca(im)) concentration. 2. The SR of bovine cells was Ca(2+)-loaded by depolarization-induced Ca2+ influx. Release (unloading) of Ca2+ from the SR during recovery from depolarization was determined by Fura-2 microfluorometry of Ca(im). The SR Ca2+ unloading was maximal following a long (14 min) recovery from depolarization, as shown by the 66% decrease in the peak caffeine-induced Ca(im) transient compared to the Ca(im) transient after a short (2 min) recovery. No increase in Ca(im) occurred during the long recovery. No unloading of the SR Ca2+ store was noted in porcine cells. 3. Approximately 80% of the outward K+ current in bovine and porcine cells was sensitive to subsarcolemmal Ca2+ (Ca(is)) concentrations. Whole-cell voltage clamp using pipette solutions with Ca2+ concentrations clamped between 0 and 1000 nM with Ca(2+)-EGTA or Ca(2+)-BAPTA buffers showed increasing K+ currents (normalized for cell membrane surface area) as a function of both membrane potential and Ca(is). Clamping of Ca(im) and Ca(is) was verified by the lack of changes in K+ current and Fura-2 ratio in response to Ca2+ influx, Ca(2+)-free external solution, or caffeine-induced Ca2+ release. At +30 to +50 mV the K+ current amplitude showed a similar sensitivity to Ca2+ as Fura-2. These data indicate that in this experimental preparation Ca(2+)-activated K+ current is a valid estimate of Ca(is). 4. Simultaneous Ca(im) and Ca(is) measurements in bovine cells which were not Ca(2+)-clamped (2 x 10(-4) M-EGTA pipette solution) showed that during the long recovery period the K+ current (reflecting Ca(is)) increased 55%, while Ca(im) did not change. 5. In quiescent bovine cells the Ca(is) was higher than Ca(im), while the higher resting Ca(is) gradient was not apparent in porcine cells. 6. The Ca(is) concentration was directly related to the amount of Ca2+ in the SR in bovine, but not porcine cells. Depletion of the SR in bovine cells by caffeine resulted in a 58% decrease in K+ current compared to the resting K+ current. 7. Caffeine-induced Ca2+ release caused an increase in Ca(is) which preceded the increase in Ca(im) by approximately 2 s.(ABSTRACT TRUNCATED AT 400 WORDS)
Stehno-Bittel
L
L
Department of Physiology, School of Medicine, University of Missouri-Columbia 65211.
Sturek
M
M
D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D003331:Coronary Vessels / Q000378:metabolism; D016257:Fura-2; D066298:In Vitro Techniques; D017136:Ion Transport; D007700:Kinetics; D008564:Membrane Potentials; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D012508:Sarcolemma / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013045:Species Specificity; D013552:Swine
D002021:Buffers; D011188:Potassium; D002118:Calcium; D016257:Fura-2