Magnesium deficiency in vitro enhances free radical-induced intracellular oxidation and cytotoxicity in endothelial cells. (49/320)

The effect of magnesium (Mg)-deficient culture on endothelial cell susceptibility to oxidative stress was examined. Bovine endothelial cells were cultured in either control sufficient (0.8 mM) or deficient (0.4 mM) levels of MgCl2. Oxygen radicals were produced extracellularly by the addition of dihydroxyfumarate and Fe(3+)-ADP. Isolated Mg-deficient endothelial cells produced 2- to 3-fold higher levels of thiobarbituric acid (TBA)-reactive materials when incubated with this free radical system. Additional studies were performed using digitized video microscopy and 2',7'-dichlorofluorescein diacetate (DCFDA) as an intracellular indicator for oxidative events at the single cell level. In response to the exogenous oxidative stress, endothelial cells exhibited a time-dependent increase in fluorescence, suggestive of intracellular lipid peroxidation. The increase in cellular fluorescence began within 1 min of free radical addition; the Mg-deficient cells exhibited a more rapid increase in fluorescence than that of Mg-sufficient cells. In separate experiments, cellular viability was assessed using the Trypan blue exclusion assay. Mg deficiency increased cytotoxicity of the added oxyradicals, but the loss of cellular viability began to occur only after 15 min of free radical exposure, lagging behind the detection of intracellular oxidation products. These results suggest that increased oxidative endothelial cell injury may contribute to vascular injury during Mg deficiency.  (+info)

TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption. (50/320)

Mg2+ is an essential ion involved in a multitude of physiological and biochemical processes and a major constituent of bone tissue. Mg2+ homeostasis in mammals depends on the equilibrium between intestinal Mg2+ absorption and renal Mg2+ excretion, but little is known about the molecular nature of the proteins involved in the transepithelial transport of Mg2+ in these organs. Recently, it was shown that patients with mutations in TRPM6, a member of the transient receptor potential family of cation channels, suffer from hypomagnesemia with secondary hypocalcemia (HSH) as a result of impaired renal and/or intestinal Mg2+ handling. Here, we show that TRPM6 is specifically localized along the apical membrane of the renal distal convoluted tubule and the brush-border membrane of the small intestine, epithelia particularly associated with active Mg2+ (re)absorption. In kidney, parvalbumin and calbindin-D28K, two divalent-binding proteins, are co-expressed with TRPM6 and might function as intracellular Mg2+ buffers in the distal convoluted tubule. Heterologous expression of wild-type TRPM6 but not TRPM6 mutants identified in HSH patients induces a Mg2+- and Ca2+-permeable cation channel tightly regulated by intracellular Mg2+ levels. The TRPM6-induced channel displays strong outward rectification, has a 5-fold higher affinity for Mg2+ than for Ca2+, and is blocked in a voltage-dependent manner by ruthenium red. Our data indicate that TRPM6 comprises all or part of the apical Mg2+ channel of Mg2+-absorbing epithelia.  (+info)

Low serum magnesium predicts neurological events in patients with advanced atherosclerosis. (51/320)

BACKGROUND AND PURPOSE: Magnesium (Mg) deficiency is thought to be a risk factor for cerebrovascular atherosclerosis and complications. We investigated the prognostic impact of Mg serum levels with respect to the occurrence of neurological events in patients with advanced atherosclerosis. METHODS: We prospectively studied 323 patients with symptomatic peripheral artery disease and intermittent claudication (197 men; median age, 68 years). Serum Mg was determined, and patients were followed for a median of 20 months (interquartile range, 12 to 25 months) for the occurrence of neurological events, defined as ischemic stroke and/or carotid revascularization (carotid endarterectomy or carotid stenting). Multivariate Cox proportional hazards analysis was applied to assess the association of serum Mg (in tertiles) and neurological events. RESULTS: Neurological events occurred in 35 patients (11%) (15 patients with stroke, 13 with carotid revascularization, and 7 with stroke and subsequent revascularization). Compared with patients in the highest tertile of Mg serum levels (>0.84 mmol/L), patients with Mg serum values <0.76 mmol/L (lowest tertile) exhibited a 3.29-fold increased adjusted risk (95% CI, 1.34 to 7.90; P=0.009) for neurological events, but patients with Mg serum values of 0.76 mmol/L to 0.84 mmol/L (middle tertile) had no increased risk (adjusted hazard ratio, 1.10; 95% CI, 0.35 to 3.33; P=0.88). Mg serum levels were not associated with all-cause mortality (P=0.87) or coronary events (P=0.67) during follow-up. CONCLUSIONS: Low Mg serum levels indicate an increased risk for neurological events in patients with symptomatic peripheral artery disease, favoring Mg substitution therapy in those patients with advanced atherosclerosis.  (+info)

Bone loss induced by dietary magnesium reduction to 10% of the nutrient requirement in rats is associated with increased release of substance P and tumor necrosis factor-alpha. (52/320)

Dietary Mg intake has been linked to osteoporosis. Previous studies have demonstrated that severe Mg deficiency [0.04% of nutrient requirement (NR)] results in osteoporosis in rodent models. We assessed the effects of more moderate dietary Mg restriction (10% of NR) on bone and mineral metabolism over a 6-mo experimental period in rats. At 2, 4 and 6 mo, serum Mg, Ca, parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D, alkaline phosphatase, osteocalcin and urine pyridinoline were measured. Femurs and tibiae were collected for measurement of mineral content, microcomputerized tomography, histomorphometry, and immunocytochemical localization. By 2 mo, profound Mg deficiency had developed as assessed by marked hypomagnesemia and up to a 51% reduction in bone Mg content. These features continued through 6 mo of study. Serum Ca was slightly but significantly higher in Mg-deficient rats than in controls at all time points. At 2 mo, serum PTH was elevated in Mg-deficient rats but was significantly decreased at 6 mo in contrast to control rats in which PTH rose. Serum 1,25-dihydroxy-vitamin D was significantly lower than in controls at 4 and 6 mo. A significant fall in both serum alkaline phosphatase and osteocalcin suggested decreased osteoblast activity. Histomorphometry demonstrated decreased bone volume and trabecular thickness. This was confirmed by microcomputerized tomography analysis, which also showed that trabecular volume, thickness and number were significantly lower in Mg-deficient rats. Increased bone resorption was suggested by an increase in osteoclast number over time compared with controls as well as surface of bone covered by osteoclasts and eroded surface, but there was no difference in osteoblast numbers. The increased bone resorption may be due to an increase in TNF-alpha because immunocytochemical localization of TNF-alpha in osteoclasts was 199% greater than in controls at 2 mo, 75% at 4 mo and 194% at 6 mo. The difference in TNF-alpha may be due to substance P, which was 250% greater than in controls in mononuclear cells at 2 mo and 266% at 4 mo. These data demonstrated that a Mg intake of 10% of NR in rats causes bone loss that may be secondary to the increased release of substance P and TNF-alpha.  (+info)

Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia. (53/320)

Impaired magnesium reabsorption in patients with TRPM6 gene mutations stresses an important role of TRPM6 (melastatin-related TRP cation channel) in epithelial magnesium transport. While attempting to isolate full-length TRPM6, we found that the human TRPM6 gene encodes multiple mRNA isoforms. Full-length TRPM6 variants failed to form functional channel complexes because they were retained intracellularly on heterologous expression in HEK 293 cells and Xenopus oocytes. However, TRPM6 specifically interacted with its closest homolog, the Mg(2+)-permeable cation channel TRPM7, resulting in the assembly of functional TRPM6/TRPM7 complexes at the cell surface. The naturally occurring S141L TRPM6 missense mutation abrogated the oligomeric assembly of TRPM6, thus providing a cell biological explanation for the human disease. Together, our data suggest an important contribution of TRPM6/TRPM7 heterooligomerization for the biological role of TRPM6 in epithelial magnesium absorption.  (+info)

Pathophysiology of functional mutations of the thiazide-sensitive Na-Cl cotransporter in Gitelman disease. (54/320)

Most of the missense mutations that have been described in the human SLC12A3 gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC, NCC, or NCCT), as the cause of Gitelman disease, block TSC function by interfering with normal protein processing and glycosylation. However, some mutations exhibit considerable activity. To investigate the pathogenesis of Gitelman disease mediated by such mutations and to gain insights into structure-function relationships on the cotransporter, five functional disease mutations were introduced into mouse TSC cDNA, and their expression was determined in Xenopus laevis oocytes. Western blot analysis revealed immunoreactive bands in all mutant TSCs that were undistinguishable from wild-type TSC. The activity profile was: wild-type TSC (100%) > G627V (66%) > R935Q (36%) = V995M (32%) > G610S (12%) > A585V (6%). Ion transport kinetics in all mutant clones were similar to wild-type TSC, except in G627V, in which a small but significant increase in affinity for extracellular Cl(-) was observed. In addition, G627V and G610S exhibited a small increase in metolazone affinity. The surface expression of wild-type and mutant TSCs was performed by laser-scanning confocal microscopy. All mutants exhibited a significant reduction in surface expression compared with wild-type TSC, with a profile similar to that observed in functional expression analysis. Our data show that biochemical and functional properties of the mutant TSCs are similar to wild-type TSC but that the surface expression is reduced, suggesting that these mutations impair the insertion of a functional protein into the plasma membrane. The small increase in Cl(-) and thiazide affinity in G610S and G627V suggests that the beginning of the COOH-terminal domain could be implicated in defining kinetic properties.  (+info)

Gitelman's syndrome with exercise-induced ventricular tachycardia. (55/320)

A 62-year-old female with palpitations was admitted to hospital where she recorded 12,299 monofocal ventricular premature contractions (VPCs) in 24 h and nonsustained ventricular tachycardia (VT) on exertion. She had hypokalemia with renal potassium wasting, a chloride-resistant metabolic alkalosis, elevated plasma renin, elevated plasma aldosterone (relative to the serum K concentration), hypomagnesemia with renal magnesium wasting, decreased urine calcium excretion, and normal blood pressure. The hypokalemia and hypomagnesemia were thought to have precipitated the VT. The coronary angiogram showed normal coronary arteries; however, the left ventriculogram revealed akinesis of the posterolateral wall. Because the VT could not be induced by programmed electrical stimulation either before or during intravenous administration of isoproterenol, the VPC with the same QRS morphology as the VT became the target of radiofrequency catheter ablation (RF-CA). Intracardiac mapping showed that the earliest activation site was situated in the asynergic area of the left ventricle (LV) and radiofrequency catheter ablation directed at the LV asynergy area completely eliminated the VPCs without any complications. During the follow-up period (6 months), she was free from palpitation and VT was not clinically documented.  (+info)

Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis. (56/320)

Because (i). endothelial cells are important players in cardiovascular diseases and (ii). Mg deficiency promotes atherosclerosis, thrombosis and hypertension, we evaluated whether low concentrations of Mg could directly affect endothelial behavior. We found that low Mg concentrations reversibly inhibit endothelial proliferation, and this event correlates with a marked down-regulation of the levels of CDC25B. The inhibition of endothelial proliferation is due to an up-regulation of interleukin-1 (IL-1), since an antisense oligonucleotide against IL-1 could prevent the growth inhibition observed in cells exposed to low concentrations of the cation. We also report the up-regulation of Vascular Cell Adhesion Molecule-1 (VCAM) and Plasminogen Activator Inhibitor (PAI)-1 after Mg deficiency. VCAM is responsible, at least in part, of the increased adhesion of monocytoid U937 cells to the endothelial cells grown in low magnesium. In addition, endothelial migratory response is severely impaired. By cDNA array, we identified several transcripts modulated by exposure to low Mg, some of which-c-src, ezrin, CD9, cytohesin and zyxin-contribute to endothelial adhesion to substrates and migration. In conclusion, our results demonstrate a direct role of low magnesium in promoting endothelial dysfunction by generating a pro-inflammatory, pro-thrombotic and pro-atherogenic environment that could play a role in the pathogenesis cardiovascular disease.  (+info)