title: Extracellular magnesium regulates nuclear and perinuclear free ionized calcium in cerebral vascular smooth muscle cells: possible relation to alcohol and central nervous system injury.
abstract: Quantitative digital imaging microscopy, confocal laser scanning microscopy (CLSM), and multiple molecular fluorescent probes were utilized to test the hypothesis that cerebral vascular muscle cell nuclear ([Ca(2+)](n)), perinuclear ([Ca(2+)](pn)), and cytoplasmic free calcium ([Ca(2+)](i)) levels are regulated by the concentration of extracellular free magnesium ions ([Mg(2+)](o)). Primary cultured canine cerebral vascular smooth muscle cells were loaded with either fura-2/AM, indo-1/AM, or fluo-3/AM, and the subcellular Ca(2+) responses to stepwise reduction in [Mg(2+)](o) (i.e., from 1.36 to 0.17 mM) were analyzed over time. With normal 1.36 mM [Mg(2+)](o)-containing incubation media, basal mean [Ca(2+)](i) was 89.6+/-15 nM. Lowering [Mg(2+)](o) to 1.07, 0.88, 0.48, and 0.17 mM resulted in rapid (<4 min) increments in [Ca(2+)](i) going to 213+/-43, 368+/-67, 471+/-77, and 642+/-98 nM, respectively; the longer the exposure time (up to 30 min) to lowered [Mg(2+)](o), the higher the [Ca(2+)](i). Restoration of [Mg(2+)](o) to normal caused decreases in [Ca(2+)](i) to 215.9+/-42.3 nM, but only complete removal of [Ca(2+)](o) returned [Ca(2+)](i) to basal levels. Results show that basal [Ca(2+)](pn) (282+/-92 nM) exceeds basal cytoplasmic Ca(2+) (61+/-27.8 nM) and [Ca(2+)](n) (20+/-7.6 nM). However, reduction of normal [Mg(2+)](o) to 0.48 mM resulted in dramatic, rapid rises in all subcellular compartments, where [Ca(2+)](pn) (1503+/-102 nM)>cytoplasmic Ca(2+) (688+/-49 nM) approximately equal to [Ca(2+)](n) (674+/-12 nM). Nuclear Ca(2+) rose dramatically (e.g., 35-40 times basal levels). Both verapamil (1 microM) and Ni(2+) (5 mM) prevented, completely, the rises in Ca(2+) in all compartments, suggesting that Mg(2+)-dependent Ca(2+) accumulation may be dependent on nuclear, endoplasmic reticulum-Golgi, and cytoplasmic L-type voltage membrane-regulated Ca(2+) channels. The normally low [Ca(2+)](n) suggests that Ca(2+) does not transport passively across the nuclear membrane in cerebral vascular smooth muscle cells. These results may help to explain much of the impact of hypomagnesemic states on cerebral-central nervous system pathobiology, and, particularly, alcohol-induced strokes.
authors.affiliation: Department of Physiology, Health Science Center, State University of New York at Brooklyn, Brooklyn, NY 11203, USA.
authors.forename: B M
authors.initials: BM
authors.lastname: Altura
authors.forename: A
authors.initials: A
authors.lastname: Zhang
authors.forename: T P
authors.initials: TP
authors.lastname: Cheng
authors.forename: B T
authors.initials: BT
authors.lastname: Altura
mesh_terms: D000437:Alcoholism / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology* / Q000502:physiology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020521:Stroke / Q000378:metabolism
chemical_list: D020746:Calcium Channels, L-Type; D008274:Magnesium; D002118:Calcium