title: Measurement of SR free Ca2+ and Mg2+ in permeabilized smooth muscle cells with use of furaptra.
abstract: The concentrations of intrasarcoplasmic reticulum (SR) free Ca2+ ([Ca2+]SR) and Mg2+ ([Mg2+]SR) were measured in furaptra-loaded saponin-permeabilized cultured aortic smooth muscle (A7r5) cells. Ca(2+)-independent fluorescence emitted by furaptra trapped within organelles, excited at 346 nm (isosbestic point), decreased with a half time of 30 min. All Ca2+ measurements appeared to be from SR, because the apparent Ca2+ distribution within permeabilized cells was uniform and therefore inconsistent with furaptra loading into mitochondria. Moreover, thapsigargin-induced SR Ca(2+)-adenosinetriphosphatase inhibition caused near-total depletion of Ca2+, and the metabolic poisons oligomycin and rotenone had no effect. Calibration curves relating 370 nm-to-346 nm ratios to [Ca2+] and to [Mg2+] were calculated in situ; dissociation constants for Ca2+ and Mg2+ binding were 49 microM and 6.8 mM, respectively. Resting [Ca2+]SR was 75-130 microM, with a mean of 97.2 +/- 2.2 microM (n = 376), whereas [Mg2+]SR, estimated in the absence of Ca2+, was 1.0 mM. Stimulation with inositol 1,4,5-trisphosphate resulted in time-dependent declines in [Ca2+]SR, and pretreatment with guanosine 5'-triphosphate caused a large increase in the rate of inositol 1,4,5-trisphosphate-evoked SR Ca2+ release, although guanosine 5'-triphosphate had no effect by itself. These observations indicate that furaptra will be a valuable tool with which to directly study [Ca2+]SR and SR function.
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, USA.
authors.forename: T
authors.initials: T
authors.lastname: Sugiyama
authors.forename: W F
authors.initials: WF
authors.lastname: Goldman
mesh_terms: D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism*; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D010080:Oxazoles; D051381:Rats; D012503:Saponins / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
chemical_list: D001572:Benzofurans; D005456:Fluorescent Dyes; D010080:Oxazoles; D012503:Saponins; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D015544:Inositol 1,4,5-Trisphosphate; D008274:Magnesium; D002118:Calcium; D016257:Fura-2