Short-term magnesium deficiency upregulates ceramide synthase in cardiovascular tissues and cells: cross-talk among cytokines, Mg2+, NF-κB, and de novo ceramide.

The present study tested the hypotheses that 1) short-term dietary deficiency (MgD) of magnesium (21 days) would result in the upregulation of ceramide synthase (CS) in left ventricular (LV), right ventricular, atrial, and aortic smooth muscle, as well as induce a synthesis/release of select cytokines and chemokines into the LV and aortic smooth muscle and serum; 2) exposure of primary cultured vascular smooth muscle cells (VSMCs) to low extracellular Mg concentration would lead to the synthesis/release of select cytokines/chemokines, activation of N-SMase, and the de novo synthesis of ceramide; and 3) inhibition of CS by fumonisin B1 (FB1) or inhibition of neutral sphingomyelinase (N-SMase) by scyphostatin (SCY) in VSMCs exposed to low Mg would result in reductions in the levels of the cytokines/chemokines and lowered levels of ceramide concomitant with inhibition of NF-κB activation. The data indicated that short-term MgD (10% normal dietary intake) resulted in the upregulation of CS in ventricular, atrial, and aortic smooth muscles coupled to the synthesis/release of 12 different cytokines/chemokines, as well as activation of NF-κB in the LV and aortic smooth muscle and sera; even very low levels of water-borne Mg (e.g., 15 mg·l(-1)·day(-1)) either prevented or ameliorated the upregulation and synthesis of the cytokines/chemokines. Our experiments also showed that VSMCs exposed to low extracellular Mg resulted in the synthesis of 5 different cytokines and chemokines concomitant with synthesis/release of ceramide. However, inhibition of the synthesis and release of ceramide by either FB1 or SCY attenuated, markedly , the generation of ceramide, release of the cytokines/chemokines, and activation of NF-κB (as measured by activated p65 and cRel).

Sphingolipids regulate [Mg2+]o uptake and [Mg2+]i content in vascular smooth muscle cells: potential mechanisms and importance to membrane transport of Mg2+.

Sphingolipids have a variety of important signaling roles in mammalian cells. We tested the hypothesis that certain sphingolipids and neutral sphingomyelinase (N-SMase) can regulate intracellular free magnesium ions ([Mg2+]i) in vascular smooth muscle (VSM) cells. Herein, we show that several sphingolipids, including C2-ceramide, C8-ceramide, C16-ceramide, and sphingosine, as well as N-SMase, have potent and direct effects on content and mobilization of [Mg2+]i in primary cultured rat aortic smooth muscle cells. All of these sphingolipid molecules increase, rapidly, [Mg2+]i in these vascular cells in a concentration-dependent manner. The increments of [Mg2+]i, induced by these agents, are derived from influx of extracellular Mg2+ and are extracellular Ca2+ concentration-dependent. Phospholipase C and Ca2+/calmodulin/Ca2+-ATPase activity appear to be important in the sphingolipid-induced rises of [Mg2+]i. Activation of certain PKC isozymes may also be required for sphingolipid-induced rises in [Mg2+]i. These novel results suggest that sphingolipids may be homeostatic regulators of extracellular Mg2+ concentration influx (and transport) and [Mg2+]i content in vascular muscle cells.

Magnesium deficiency upregulates serine palmitoyl transferase (SPT 1 and SPT 2) in cardiovascular tissues: relationship to serum ionized Mg and cytochrome c.

The present work tested the hypothesis that a short-term dietary deficiency of magnesium (Mg) (21 days) in rats would result in the upregulation of the two major subunits of serine palmitoyl-CoA-transferase, serine palmitoyl transferase (SPT 1) and SPT 2 (the rate-limiting enzymes responsible for the de novo biosynthesis of ceramides) in left ventricular, right ventricular, and atrial heart muscle and abdominal aortic smooth muscle, as well as induce a reduction in serum sphingomyelin concomitant with the release of mitochondrial cytochrome c (Cyto c) in these tissues. Our data indicate that short-term Mg deficiency (MgD) resulted in an upregulation of SPT 1 and SPT 2, concomitant with a very significant release of Cyto c in left ventricular, right ventricular, atrial, and abdominal aortic smooth muscle. Short-term MgD also produced a lowering of serum sphingomyelin and ionized Mg. The greater the reduction in serum ionized Mg, the greater the upregulation of SPT 1 and 2 and the more the increase in free Cyto c. The data suggest that MgD, most likely, causes a biosynthesis of ceramides via two pathways in cardiovascular tissues, viz., via the activation of serine palmitoyl-CoA-transferase and sphingomyelinase, which lead to apoptotic events via intrinsic (present study) and extrinsic pathways (previous studies). Low levels of drinking water Mg were cardio- and vasculoprotective.

Short-term magnesium deficiency upregulates sphingomyelin synthase and p53 in cardiovascular tissues and cells: relevance to the de novo synthesis of ceramide.

The present study tested the hypotheses that 1) short-term dietary deficiency of magnesium (21 days) in rats would result in the upregulation of sphingomyelin synthase (SMS) and p53 in cardiac and vascular (aortic) smooth muscles, 2) low levels of Mg(2+) added to drinking water would either prevent or greatly reduce the upregulation of both SMS and p53, 3) exposure of primary cultured vascular smooth muscle cells (VSMCs) to low extracellular Mg(2+) concentration ([Mg(2)](o)) would lead to the de novo synthesis of ceramide, 4) inhibition of either SMS or p53 in primary culture VSMCs exposed to low [Mg(2+)](o) would lead to reductions in the levels of de novo ceramide synthesis, and 5) inhibition of sphingomyelin palmitoyl-CoA transferase (SPT) or ceramide synthase (CS) in primary cultured VSMCs exposed to low [Mg(2+)](o) would lead to a reduction in the levels of de novo ceramide synthesis. The data indicated that short-term magnesium deficiency (10% normal dietary intake) resulted in the upregulation of SMS and p53 in both ventricular and aortic smooth muscles; even very low levels of water-borne Mg(2+) (e.g., 15 mg·l(-1)·day(-1)) either prevented or ameliorated the upregulation in SMS and p53. Our experiments also showed that VSMCs exposed to low [Mg(2+)](o) resulted in the de novo synthesis of ceramide; the lower the [Mg(2+)](o), the greater the synthesis of ceramide. In addition, the data indicated that inhibition of either SMS, p53, SPT, or CS in VSMCs exposed to low [Mg(2+)](o) resulted in marked reductions in the de novo synthesis of ceramide.

Short-term magnesium deficiency results in decreased levels of serum sphingomyelin, lipid peroxidation, and apoptosis in cardiovascular tissues.

The present study tested the hypothesis that short-term dietary deficiency of magnesium (Mg) (21 days) in rats would 1) result in decreased serum(s) [the present study tested the levels of Mg, sphingomyelin (SM), and phosphatidylcholine (PC)]; 2) promote DNA fragmentation, lipid peroxidation (LP), and activation of caspase-3 in cardiac (ventricular and atrial) and vascular(aortic) muscle; and 3) low levels of Mg(2+) added to drinking water would either prevent or greatly ameliorate these manifestations. The data indicate that short-term Mg deficiency (10% normal dietary intake) resulted in profound reductions in serum-ionized Mg and total Mg with an elevation in serum-ionized calcium (Ca(2+)), significant lowering of serum SM and serum PC, with concomitant LP, DNA fragmentation, and activation of caspase-3 in ventricular (right and left chambers), atrial (right and left chambers) and abdominal aortic smooth muscle. The greater the reduction in serum-ionized Mg, the greater the effects on DNA fragmentation, LP, and caspase-3 activity. The intake of water-borne Mg(2+) at all levels greatly attenuated or inhibited the reductions in serum SM and serum PC, activation of LP, DNA fragmentation, and the activation of caspase-3; even very low levels of Mg(2+) in drinking water (i.e., 15 parts.million(-1).day(-1)) were cardio- and vascular protective. In addition, we demonstrate that short-term dietary deficiency of Mg probably results in a downregulation of SM synthase and a decreased synthesis of PC.

Expression of the nuclear factor-kappaB and proto-oncogenes c-fos and c-jun are induced by low extracellular Mg2+ in aortic and cerebral vascular smooth muscle cells: possible links to hypertension, atherogenesis, and stroke.

Proto-oncogene (c-fos, c-jun) and nuclear factor-kappa B (NF-kappaB) expression, as well as DNA synthesis, in aortic and cerebral vascular smooth muscle cells (VSMCs) were upregulated by a decrease in extracellular magnesium ions ([Mg2+]o). Upregulation of these transcriptional factors was inversely proportional to the [Mg2+]o and occurred over the pathophysiologic range of serum Mg2+ found in patients presenting with hypertension, ischemic heart disease, and stroke. Removal of extracellular Ca2+ ([Ca2+]o), use of nifedipine or protein kinase C (PKC) inhibitors prevented the upregulation of the proto-oncogenes and DNA synthesis in VSMCs. These data show that [Mg2+]o may be an important, heretofore, overlooked natural modulator of proto-oncogene and NF-kappaB expression in VSMCs and that Ca2+ and PKC may play critical roles in induction of c-fos and c-jun in VSMCs induced by a decrease in [Mg2+]o. These results point to a role for low serum Mg2+ in potential development of hypertension, atherogenesis, vascular disease, and stroke.

Role of leukocytes in ethanol-induced microvascular injury in the rat brain in situ: potential role in alcohol brain pathology and stroke.

Effects of acute and chronic alcohol ethanol administration on in vivo microvascular-leukocyte dynamics was studied in brains of naive and leukocyte-depleted rats by direct, quantitative intravital high-resolution TV microscopy, fluorescence microscopy and myeloperoxidase staining. Administration of alcohol produced dose-dependent venular vasospasm, and rolling and adherence of leukocytes to venular walls; leukocyte velocity concomitantly decreased. Intermediate to high doses of ethanol resulted in infiltration of leukocytes and macrophages across venular walls, and concentration-dependent increases in myeloperoxidase staining in parenchyma, and rupture of postcapillary venules with focal hemorrhages. Use of phosphorus 31-nuclear magnetic resonance spectroscopy on intact animals revealed that the latter were associated with whole brain losses in intracellular levels of ATP and phosphocreatine with concomitant rises in intracellular inorganic phosphate and hydrogen ion concentration. Vinblastine-depletion of circulating leukocytes prevented or ameliorated greatly the alcohol-induced microvascular damage and proinflammatory-like reactions. These new results, when viewed in light of other recent findings, suggest that alcohol-induced cerebral vascular and brain damage is dependent, to a large extent, on recruitment of leukocytes.

Optical spectroscopy and prevention of deleterious cerebral vascular effects of ethanol by magnesium ions.

Previously, it has been suggested that acute ethanol (alcohol) administration can result in concentration-dependent vasoconstriction and decreased cerebral blood flow. Here, we present in vivo results using rapid (240 nm/min) optical backscatter measurements, with an intact cranial preparation in the rat, indicating that acute infusion of ethanol directly into the rat brain rapidly produces dose-dependent vasoconstriction of the cerebral microcirculation associated with a pronounced reduction in tissue blood content, pronounced rises in deoxyhemoglobin, significantly increased levels of reduced cytochrome oxidase and microvascular damage as the dose increases. Furthermore, we present in vivo experiments demonstrating the capability of magnesium ions (Mg(2+)) to attenuate and prevent these deleterious responses. Optical backscatter spectra (500-800 nm) were obtained by directing a single sending and receiving fiber to a portion of the left parietal cranium (in anesthetized rats), shaved to a translucent appearance to facilitate optical penetration. In the absence of added Mg(2+), infusion of a 10% solution of ethanol at 0.34 ml/min ( approximately 26.8 mg/min) produced prompt vasoconstriction as evidenced by a greater than 90% loss of oxyhemoglobin from the field-of-view and increases in levels of reduced cytochrome oxidase to between 50% and >90%. These effects were partially, to nearly completely, attenuated by the addition of MgCl(2) to the infusate containing added ethanol. Of special interest was the observation that attenuation of the vasoconstrictive effect of ethanol by Mg(2+) persisted despite a subsequent ethanol challenge without added Mg(2+). The results obtained demonstrate that, depending on dose, ethanol can produce prompt and severe vasoconstriction of the intact cerebral microcirculation and that infusion of moderate doses of Mg(2+) can largely attenuate and prevent this response. We conclude that appreciable, graded changes in cerebral cytochrome oxidase aa(3), blood volume and the state of hemoglobin occur at minimal tissue levels of ethanol which can be modulated by Mg(2+).

Cocaine induces apoptosis in cerebral vascular muscle cells: potential roles in strokes and brain damage.

Cocaine abuse is known to induce different types of brain-microvascular damage and many adverse cerebrovascular effects, including cerebral vasculitis, intracranial hemorrhage, cerebral infarction and stroke. A major physiological event leading to these pathophysiological actions of cocaine could be apoptosis. Whether cocaine can cause brain-microvascular pathology and vascular toxicity by inducing apoptosis of cerebral vascular smooth muscle cells is not known. This study, using several different methods to discern apoptosis, was designed to investigate if primary cultured canine cerebral vascular smooth muscle cells can undergo apoptosis when treated with cocaine. After treatment with cocaine (10(-6)-10(-3) M) for 12-24 h, the death rates of cerebral vascular smooth muscle cells increased in a concentration-dependent manner compared with controls. Morphological analysis of cerebral vascular smooth muscle cells using confocal fluoresence microscopy showed that the percentage of apoptotic cerebral vascular smooth muscle cells increased after cocaine (10(-6)-10(-3) M) treatment in a concentration-dependent manner. TUNEL assays also showed positive results for cerebral vascular smooth muscle cells treated with cocaine. These results clearly demonstrate that cerebral vascular smooth muscle cells can undergo rapid apoptosis in response to cocaine in a concentration-dependent manner. Cocaine-induced apoptosis may thus play a major role in brain-microvascular damage, cerebral vascular toxicity and strokes.

Mechanisms of hydroxyl radical-induced contraction of rat aorta.

The present study was designed to investigate the effects of hydroxyl radicals (*OH), generated via the Fe2+-mediated Fenton reaction, on isolated rat aortic rings with and without endothelium. In the absence of any vasoactive agent, generation of *OH alone elicited an endothelium-independent contraction in rat aortic rings in a concentration-dependent manner. Hydroxyl radical-induced contractions of denuded rat aortic rings appeared, however, to be slightly stronger than those on intact rat aortic rings. The contractile responses to *OH were neither reversible nor reproducible in the same ring; even small concentrations of *OH radicals resulted in tachyphylaxis. Removal of extracellular calcium ions (Ca2+) or buffering intracellular Ca2+ with 10 microM acetyl methyl ester of bis(o-aminophenoxy) ethane-N,N,N',N',-tetraacetic acid (BAPTA-AM) significantly attenuated the contractile actions of *OH radicals. The presence of 1 microM staurosporine, 1 microM bisindolylmaleimide I, 1 microM Gö6976 [inhibitor of protein kinase C (PKC)], 2 microM PD-980592 (inhibitor of ERK), 10 microM genistein, and 1 microM wortmannin significantly inhibited the contractions induced by *OH. Proadifen (10 microM), on the other hand, significantly potentiated the hydroxyl radical-induced contractions. Exposure of primary cultured aortic smooth muscle cells to *OH produced significant, rapid rises of intracellular free Ca2+ ([Ca2+]i). Several, specific antagonists of possible endogenously formed vasoconstrictors did not inhibit or attenuate either hydroxyl radical-induced contractions or the elevation of [Ca2+]i. Our new results suggest that hydroxyl radical-triggered contractions on rat aortic rings are Ca2+-dependent. Several intracellular signal transduction systems seem to play some role in hydroxyl radical-induced vasoconstriction of rat aortic rings.