MULTIPLE EFFECTS OF RYANODINE ON INTRACELLULAR FREE CA2+ IN SMOOTH-MUSCLE CELLS FROM BOVINE AND PORCINE CORONARY-ARTERY - MODULATION OF SARCOPLASMIC-RETICULUM FUNCTION

1 The effects of ryanodine and caffeine on intracellular free Ca2+ concentration ([Ca2+]i) were studied by use of fura-2 microfluorometry in single smooth muscle cells freshly dispersed from bovine and porcine coronary artery. 2 Bovine and porcine cells demonstrated similar sensitivities to 10 min of exposure to ryanodine in physiological salt solution (PSS), as determined by comparable dose-dependent decreases in the subsequent [Ca2+]i transient induced by 5 mM caffeine. 3 Ryanodine (10-mu-M) caused a significant increase in [Ca2+]i to a plateau level 27 +/- 3% and 38 +/- 4% above baseline [Ca2+]i (baseline [Ca2+]i = [Ca2+]i at 0 min) in porcine and bovine cells, respectively, when bathed in PSS. In bovine cells the time required to reach 1/2 the plateau level was only 3 min versus 6 min for porcine cells. 4 The ryanodine-induced plateau increase in [Ca2+]i was 35 +/- 5% above baseline for bovine cells bathed in 0 Ca PSS (PSS including 10-mu-M EGTA with no added Ca2+), but only 7 +/- 3% above baseline in porcine cells during 10 min exposure to 10-mu-M ryanodine. In bovine cells [Ca2+]i showed proportional increases when extracellular Ca2+ was increased from the normal 2 mM Ca2+ PSS to 5 and 10 mM. 5 Cells pretreated with caffeine in 0 Ca PSS, which depleted the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, showed no increase in [Ca2+]i when challenged with 10-mu-M ryanodine. The ryanodine-associated increase in [Ca2+]i, which was sustained in 0 Ca PSS during the 10 min ryanodine exposure in cells not pretreated with caffeine, suggests that ryanodine releases Ca2+ from the sarcoplasmic reticulum, but also inhibits Ca2+ efflux. 6 Intracellular free Ba2+ ([Ba2+]i) was measured with fura-2 microfluorometry to define further the Ca2+ efflux pathway inhibited by ryanodine; specifically, Ba2+ is not transported by the Ca2+ pump, but will substitute for Ca2+ in Na+-Ca2+ exchange. In porcine cells pretreated with caffeine in 0 Ca PSS to deplete the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, depolarization with 80 mM K+ in 2 mM external Ba2+ caused a 100 +/- 6% increase in fura-2 fluorescence ([Ba2+]i). During the 17.5 min 0 Ca PSS recovery from depolarization, exposure to 10-mu-M ryanodine inhibited the removal of [Ba2+]i by 69 +/- 3% when compared with control (0 Ca PSS without ryanodine). 7 It was concluded that in bovine and porcine smooth muscle cells: (a) ryanodine (greater-than-or-equal-to 10-mu-M) releases Ca2+ from the sarcoplasmic reticulum; (b) ryanodine (greater-than-or-equal-to 10-mu-M) decreases Ca2+ efflux, probably by inhibition of Na+-Ca2+ exchange; (c) the sarcoplasmic reticulum Ca2+ store may be larger in bovine than in porcine smooth muscle cells; thus, porcine cells have a relatively greater reliance on Ca2+ influx to increase [Ca2+]i.