Toni P.-O. Cheng

Magnesium regulates intracellular free ionized calcium concentration and cell geometry in vascular smooth muscle cells.

Regulatory effects of extracellular magnesium ions ([Mg2+]o) on intracellular free ionized calcium ([Ca2+]i) were studied in cultured vascular smooth muscle cells (VSMCs) from rat aorta by use of the fluorescent indicator fura-2 and digital imaging microscopy. With normal Mg2+ (1.2 mM)-containing incubation media, [Ca2+]i in VSMCs was 93.6 +/- 7.93 nM with a heterogeneous cellular distribution. Lowering [Mg2+]o to 0 mM or 0.3 mM (the lowest physiological range) resulted in 5.8-fold (579.5 +/- 39.99 nM) and 3.5-fold (348.0 +/- 31.52 nM) increments of [Ca2+]i, respectively, without influencing the cellular distribution of [Ca2+]i. Surprisingly, [Mg2+]o withdrawal induced changes of cell geometry in many VSMCs, i.e., the cells rounded up. However, elevation of [Mg2+]o up to 4.8 mM only induced slight decrements of [Ca2+]i (mean = 72.0 +/- 4.55 nM). The large increment of [Ca2+]i induced by [Mg2+]o withdrawal was totally inhibited when [Ca2+]o was removed. The data suggest that: (1) [Mg2+]o regulates the level of [Ca2+]i in rat aortic smooth muscle cells, and (2) [Mg2+] acts as an important regulatory ion by modulating cell shapes in cultured VSMc and their metabolism to control vascular contractile activities.

Low levels of serum ionized magnesium are found in patients early after stroke which result in rapid elevation in cytosolic free calcium and spasm in cerebral vascular muscle cells

Ninety-eight patients admitted to the emergency rooms of three urban hospitals with a diagnosis of either ischemic stroke or hemorrhagic stroke exhibited early and significant deficits in serum ionized Mg2+ (IMg2+), but not total Mg, as measured with a unique Mg2+-sensitive ion-selective electrode. Twenty-five percent of these stroke patients exhibited >65% reductions in the mean serum IMg2+ found in normal healthy human volunteers or patients admitted for minor bruises, cuts or deep lacerations. The stroke patients also demonstrated significant elevation in the serum ionized Ca2+ (ICa2+)/IMg2+ ratio, a sign of increased vascular tone and cerebrovasospasm. Exposure of primary cultured canine cerebral vascular smooth muscle cells to the low concentrations of IMg2+ found in the stroke patients, e.g. 0.30-0.48 mM, resulted in rapid and marked elevations in cytosolic free calcium ions ([Ca2+]i) as measured with the fluorescent probe, fura-2, and digital image analysis. Coincident with the rise in [Ca2+]i, many of the cerebral vascular cells went into spasm. Reintroduction of normal extracellular Mg2+ ion concentrations failed to either lower the [Ca2+]i overload or reverse the rounding-up of the cerebral vascular cells. These results suggest that changes in Mg2+ metabolism play important roles in stroke syndromes and in the etiology of cerebrovasospasm associated with cerebral hemorrhage.

Chronic treatment of cultured cerebral vascular smooth cells with low concentration of ethanol elevates intracellular calcium and potentiates prostanoid-induced rises in [Ca2+]i: Relation to etiology of alcohol-induced stroke

The influence of chronic treatment of cultured canine cerebral vascular smooth muscle cells, with low concentrations of ethanol, on the intracellular concentrations of free calcium ([Ca2+]i) was studied by use of the fluorescent indicator, fura-2, and digital imaging microscopy. The resting level of [Ca2+]i in the cerebral vascular smooth muscle cells was 89 +/- 3.2 nM. Exposure of these cells to 10 and 25 mM ethanol for 5 days resulted in significant elevation of [Ca2+]i (mean rises to 208 +/- 11.4 and 307 +/- 14.0 nM, respectively), and potentiated the transient rise in [Ca2+]i induced by 10(-7) M PGF2 alpha. However, exposure of these cerebral cells to a high-concentration ethanol (100 mM) resulted in only a slight increase of [Ca2+]i (106 +/- 6.9 nM) and lack of effects on the [Ca2+]i response to PGF2 alpha. Irrespective of the different ethanol treatments, the subcellular distribution of [Ca2+]i was heterogeneous in all the cells tested. Our data suggest that chronic exposure of cerebral vascular smooth muscle cells to ethanol, particularly at low concentrations, results in dramatic increases in [Ca2+]i and the responses of these vascular smooth muscle cells to prostanoids. These results support an hypothesis whereby ethanol induces stroke by causing spasm and rupture of cerebral blood vessels as a consequence of large rises in intracellular Ca2+.

Extracellular magnesium regulates intracellular free Mg2+ in vascular smooth muscle cells

SummaryRegulatory effects of extracellular magnesium ions ([Mg2+]o) on intracellular free ionized magnesium ([Mg2+]i) were exmained in cultured vascular smooth muscle cells (VSMCs) fromrat aorta by digital imaging microscopy using the Mg2+ fluorescent probe, Mag-fura-2. With normal Mg2+(1.2 mM)-containing incubation media, [Mg2+]i in VSMCs was 0.63±0.09 mM. The ratio of [Mg2+]i/[Mg2+]o was 0.52±0.07. Elevation of [Mg2+]o up to 4.8 mM induced consistent increments in [Mg2+]i (to a mean values of 1.63±0.08 mM) in 5 min and lowered the ratio of [Mg2+]i/[Mg2+]o to 0.34±0.02. Our data suggest that [Mg2+]o can regulate [Mg2+]i, which may be related to its effects on intracellular Ca2+ ([Ca2+]i) and tone of VSMCs.

Extracellular Mg2+ modulates intracellular Ca2+ in acutely isolated hippocampal CA1 pyramidal cells of the guinea-pig.

Using digital imaging microscopy and fluorescent probes, isolated hippocampal CA1 pyramidal neurons of the guinea-pig were used to examine the roles of [Mg2+]o in regulation of [Ca2+]i and [Mg2+]i. Low extracellular Mg ([Mg2+]o) (0.3 mM) significantly increased [Ca2+]i compared to 1.2 and 4.8 mM [Mg2+]o. In contrast, [Mg2+]i levels remained relatively constant, irrespective of alterations of [Mg2+]o. The sustained rise in [Ca2+]i induced by low [Mg2+]o was reduced 70% by 1 microM verapamil and 42% by 1 mM Ni2+, and completely abolished by 5 mM Ni2+. The data suggest that [Mg2+]o regulates [Ca2+]i in hippocampal neurons, probably by modulating Ca2+ entry via voltage-sensitive Ca2+ channels, which may play important roles in epileptogenesis, memory, learning and brain trauma. Furthermore, the results demonstrate that intracellular Mg2+ concentration does not follow passively the concentration of Mg2+ in the extracellular solution.

Acute cocaine results in rapid rises in intracellular free calcium concentration in canine cerebral vascular smooth muscle cells: possible relation to etiology of stroke

Effects of cocaine on intracellular free calcium concentration ([Ca2+]i) in cultured canine cerebral vascular smooth muscle cells were studied using digital imaging microscopy and the calcium molecular fluorescent indicator, fura-2. Acute treatment of cerebral vascular smooth muscle cells with cocaine HCl, from a low concentration of 10(-9) M up to 10(-5) M, induced significant increases of [Ca2+]i. Irrespective of the changes in [Ca2+]i, the subcellular distribution of [Ca2+]i appeared heterogeneous in both normal and cocaine-treated cells. These results suggest that cocaine induces cerebral vasospasm by a rapid elevation of [Ca2+]i in vascular smooth muscle cells; these ionic events could play a crucial role in the pathogenesis of cocaine-induced cerebral ischemia and stroke.

Cocaine induces rapid loss of intracellular free Mg2+ in cerebral vascular smooth muscle cells

Acute exposure of cultured canine cerebral vascular smooth muscle cells to low concentrations of cocaine HCl (10(-9) to (10(-7) M) resulted in significant, rapid (1 min) loss of intracellular free Mg ions ([Mg2+]i); these reductions (12-25%) in [Mg2+]i were reversible upon exposure to normal, Mg(2+)-containing physiological salt solution. These findings help to provide a rational basis for why cocaine can result in cerebrovasospasm and stroke.

Mg2+ and caffeine-induced intracellular Ca2+ release in human vascular endothelial cells.

Interaction of ionized magnesium ([Mg2+]o) and caffeine in regulation of intracellular free calcium concentration ([Ca2+]i) in human aortic endothelial cells was studied using fura‐2 and digital imaging microscopy. In 1.2 mm [Mg2+]o, basal [Ca2+]i was 73.7 ± 22.4 nm, with a heterogeneous distribution within the cells. No significant changes of basal [Ca2+]i were found either when cells were treated with 10 mm caffeine or when [Mg2+]o was lowered from 1.2 mm to 0.3 mm. However, a combined superfusion of the cells with 0.3 mm [Mg2+]o and 10 mm caffeine resulted in a significant elevation of [Ca2+]i to 382.8 ± 57.1 nm, probably by release of Ca2+ from internal stores, which was attenuated by NiCl2 (1 mm). These results suggest that a Ca2+‐induced Ca2+ release mechanism is involved in regulation of [Ca2+]i in endothelial cells, which may be either regulated or modulated by Mg2+.

Alcohols induce rapid depletion of intracellular free Mg2+ in cerebral vascular muscle cells: relation to chain length and partition coefficient

Acute effects of a series of alcohols (methanol, ethanol, n-butanol) on intracellular free magnesium concentration ([Mg2+]i) in canine cerebral vascular smooth muscle cells was studied using mag-fura-2 and digital imaging microscopy. In 1.2 mM [Mg2+]o, basal [Mg2+]i was 500 +/- 30 microM. Exposure of cells to a low concentration (25 mM) of ethanol, but not methanol, for only 30 s resulted in significant loss of [Mg2+]i. Exposure to 100 mM methanol, ethanol, and butanol for 30 s resulted in a relative order of potency for [Mg2+]i depletion, where butanol >> ethanol > methanol. The heterogeneous and relative subcellular compartmented concentrations of [Mg2+]i, where perinuclear > nuclear >> peripheral (cytosolic) region, was not significantly altered by the alcohols. The degree of cellular depletion of [Mg2+]i was directly a function of each alcohols partition coefficient and chain length. The latter is suggestive of the probability that alcohols promote intracellular depletion of Mg2+ by partitioning in membranes and disordering lipid bilayers.

Exposure of piglet coronary arterial muscle cells to low alcohol results in elevation of intracellular free Ca2+: relevance to fetal alcohol syndrome

Chronic exposure of cultured piglet neonatal coronary arterial smooth muscle cells to low concentrations of ethanol (46-115 mg/dl) for 7 days resulted in concentration-dependent elevation in intracellular free Ca2+ ions ([Ca2+i); these rises (22-56%) in [Ca2+]i were not reversible upon short-term exposure to normal, Ca2(+)-containing physiological salt solution. These findings help to provide a rational basis for why ethanol can result in the well-known fetal alcohol syndrome, including cardiac defects and in-utero death.