Multiple effects of ryanodine on intracellular free Ca²⁺ in smooth muscle cells from bovine and porcine coronary artery: modulation of sarcoplasmic reticulum function

Abstract

1 The effects of ryanodine and caffeine on intracellular free Ca2+ concentration ([Ca2+] 5 Cells pretreated with caffeine in 0 Ca PSS, which depleted the caffeine-sensitive sarcoplasmic reticulum Ca2" store, showed no increase in [Ca24], when challenged with 10 fLM ryanodine. The ryanodine-associated increase in [Ca2+],, 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 Ca24 efflux.6 Intracellular free Ba2+ ([Ba24],) 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+-Ca24 exchange. In porcine cells pretreated with caffeine in 0 Ca PSS to deplete the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, depolarization with 80 mM K4 in 2 mM external Ba24 caused a 100 ± 6% increase in fura-2 fluorescence ([Ba2+]

Exercise training attenuates coronary smooth muscle phenotypic modulation and nuclear Ca2+ signaling

“…Fura 2 was excited by 340-and 380-nm light, and the emitted fluorescence (510 nm) was collected by a monochrome charge-coupled device (CCD) camera that was attached to a computer for data acquisition by InCa Ratiometric Fluorescence program version 1.2 (Intracellular Imaging). Data are expressed as a ratio (and indicated as ratio units) of the emitted light intensity at 340-and 380-nm excitation rather than Cam concentration because of uncertainties, mainly impaired calcium sensitivity, detailed in previous reports (45). Cells were superfused with physiological saline solution (PSS) containing (in mM) 2 CaCl 2, 143 NaCl, 1 MgCl 2, 5 KCl, 10 HEPES, and 10 glucose; pH 7.4.…”

“…3H. Briefly, single smooth muscle cells were exposed to 80 mM KCl for 3 min to elicit Ca 2ϩ influx and thereby load the SR with Ca 2ϩ (41,42,45). This was followed by a 4-min washout period and a subsequent 4-min exposure to endothelin-1 (5 ϫ 10 Ϫ8 M) to mobilize endothelin-1-sensitive Ca 2ϩ stores (8,43,46).…”

Exercise training enhances multiple mechanisms of relaxation in coronary arteries from ischemic hearts

“…Cells were pipetted into a superfusion chamber and observed using an epifluorescence microscopy system, which permitted evaluation of DAF fluorescence from multiple user-selected endothelial cells simultaneously throughout the experimental protocol (NIS-Elements AR 3.0). Endothelial cells were morphologically distinguishable from other cell types in the dispersion, as characterized previously (46). Cells were excited with a 175-W xenon arc lamp with a 475-nm interference filter for excitation wavelength (Lambda DG-4, Sutter Instruments).…”

“…The enzyme solution was replaced with enzyme-free low-Ca 2ϩ solution, and the arteries were dispersed with gentle trituration by micropipette for isolation of single smooth muscle cells. Smooth muscle cells were morphologically distinguishable from other cell types, as described previously (46). Isolated cells were maintained in low-Ca 2ϩ solution at 4°C until use (0 -6 h).…”

Toxoplasma gondii Microneme Secretion Involves Intracellular Ca2+ Release from Inositol 1,4,5-Triphosphate (IP3)/Ryanodine-sensitive Stores

“…1B). This sustained [Ca 2ϩ ] i elevation in the presence of extracellular Ca 2ϩ could be due either to capacitative Ca 2ϩ entry (17) or to inhibition of Ca 2ϩ efflux by ryanodine as has been postulated to occur in smooth muscle cells (28). Because treatment of tachyzoites with EGTA prevented microneme secretion (Fig.…”