Acetylcholine and bradykinin relax intrapulmonary arteries by acting on endothelial cells: role in lung vascular diseases.

Acetylcholine and bradykinin produced potent relaxation of isolated canine intrapulmonary arteries contracted by serotonin, norepinephrine, or phenylephrine-provided the endothelium was left intact. Selective mechanical destruction of the endothelium transformed the activity of these substances from vasodilatation to vasoconstriction. Acetylcholine-induced relaxations, in the presence of intact endothelium, could be selectively inhibited competitively by atropine, but could not be inhibited by cyclooxygenase inhibitors, a lipoxygenase inhibitor, adrenergic blocking drugs, or histaminergic antagonists. RElaxations produced by prostacyclin, prostaglandin E1, isoproterenol, papaverine, or histamine H2-receptor agonists were not modified, or attenuated, by selective destruction of pulmonary endothelial cells. These observations might provide insight into the etiology of the increased pulmonary resistance observed in pulmonary hypertension and shock lung.

Phencyclidine, lysergic acid diethylamide, and mescaline: cerebral artery spasms and hallucinogenic activity.

Phencyclidine (PCP), lysergic acid diethylamide (LSD), and mescaline produced potent contractile responses on isolated basilar and middle cerebral arteries, where, in terms of potency, LSD greater than mescaline greater than PCP. All three drugs produced cerebrovasospasm in a concentration range which parallels that needed for their psychotomimetic and intoxicating actions. Specific receptors for PCP, which subserve contraction and differ from those for LSD and mescaline, are found in cerebral arteries. Concentrations of PCP that produced near-maximum contractile responses on cerebral arteries were similar to those in the blood and brain of human subjects who had died from PCP overdoses. A specific calcium antagonist, verapamil, readily prevented (and reversed) PCP-induced vasospasm. This study provides direct evidence for PCP receptors in cerebral blood vessels, the biologic action of which can be reversed by a calcium antagonist; the clinical use of the latter could prove invaluable in treating PCP-intoxicated victims.

Magnesium deficiency-induced spasms of umbilical vessels: relation to preeclampsia, hypertension, growth retardation.

Isolated umbilical arteries and veins, obtained from normal women at the end of pregnancy, were incubated in krebs-Ringer bicarbonate solution and exposed to magnesium at concentrations ranging from 0 to 9.6 millimoles per liter. The basal tension of the vessels increased when magnesium was withdrawn and decreased when the concentration of magnesium was raised. Absence of magnesium in the medium significantly potentiated the contractile response of the vessels to bradykinin, angiotensin II, serotonin, and prostaglandin F2 alpha. It appears that magnesium deficiency may be responsible for spasms of umbilical and placental vasculature. Our findings may provide a rationale for why magnesium sulfate is an effective therapy in preeclamptic syndromes in pregnant women.

Alcohol-induced spasms of cerebral blood vessels: relation to cerebrovascular accidents and sudden death.

Ethyl alcohol produced graded contractile responses in rat cerebral arterioles and venules in vivo and in isolated canine basilar and middle cerebral arteries at a concentration range (10 to 500 milligrams per deciliter) which parallels that needed for its graded effects of euphoria, mental haziness, muscular incoordination, stupor, and coma in humans. Two specific calcium antagonists, nimodipine and verapamil, prevented or reversed the alcohol-induced cerebrovasospasm and thus may prove valuable in treating the hypertension and stroke observed in heavy users of alcohol.

Magnesium deficiency produces spasms of coronary arteries: relationship to etiology of sudden death ischemic heart disease.

Isolated coronary arteries from dogs were incubated in Krebs-Ringer bicarbonate isolation and exposed to normal, high, and low concentrations of magnesium in the medium. Sudden withdrawal of magnesium from the medium increased whereas high concentrations of magnesium decreased the basal tension of the arteries. The absence of magnesium in the medium significantly potentiated the contractile responses of both small and large coronary arteries to norepinephrine, acetylcholine, serotonin, angiotensin, and potassium. These data support the hypothesis that magnesium deficiency, associated with sudden death ischemic heart disease, produces coronary arterial spasm.

Magnesium deficiency and hypertension: correlation between magnesium-deficient diets and microcirculatory changes in situ.

Rats maintained for 12 weeks on diets moderately or more severely deficient in magnesium showed significant elevations in arterial blood pressure compared to control animals. Examination of the mesenteric microcirculation in situ revealed that dietary magnesium deficiency resulted in reduced capillary, postcapillary, and venular blood flow concomitant with reduced terminal arteriolar, precapillary sphincter, and venular lumen sizes. The greater the degree of dietary magnesium deficiency the greater the reductions in microvascular lumen sizes. These findings may provide a rationale for the etiology, as well as treatment, of some forms of hypertensive vascular disease.

Cocaine induces intracellular free Mg deficits, ischemia and stroke as observed by in-vivo 31P-NMR of the brain.

31P-NMR spectroscopic studies were performed in vivo on brains of rats administered cocaine. Cocaine.HCl (1-5 mg/kg) administered systemically to lightly anesthetized rats resulted in significant and progressive deficits in whole brain intracellular free Mg ([Mg2+]i). Intracellular pH (pHi) also fell in a progressive manner but only after a significant fall in brain [Mg2+]i was noted. Both [Mg2+]i and pHi returned to normal in most rats. Brains of rats that exhibited stroke-like events, however, demonstrated continued intracellular acidosis associated with progressive loss of phosphocreatine and elevation of Pi up until death. These observations are consistent with the tenet that injection of cocaine can result in severe cerebral vasospasm, ischemia and rupture of cerebral blood vessels as a consequence of depletion of brain [Mg2+]i.

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.

Tris(hydroxymethyl)aminomethane inhibits calcium uptake in vascular smooth muscle.

This report demonstrates that the commonly used buffering agent Tris(hydroxymethyl)aminomethane (Tris) in concentrations of 5 and 30 mM inhibits calcium (Ca2+) uptake in both rat aortic and portal venous smooth muscle. The data indicates that total exchangeable Ca2+ in portal vein is reduced by about 35% in 5 or 30 mM Tris, while the intracellular exchangeable Ca2+ is not significantly altered. On the other hand, in aortic smooth muscle, while 30 mM Tris reduces total exchangeable Ca2+ by about 20%, intracellular Ca2+ is reduced by 44% in 5 mM Tris and by 55% in 30 mM Tris. The present studies, thus, reveal that Tris exerts significant inhibitory effects on exchangeability and transmembrane movement of Ca2+ in at least two different types of smooth muscle.

Pentobarbital sodium inhibits calcium uptake in vascular smooth muscle.

This report demonstrates that the commonly used anesthetic agent, pentobarbital sodium, in concentrations of 1 . 10(-4) to 2 . 10(-3) M inhibits calcium (Ca2+) uptake in both rat aortic and portal venous smooth muscle. The data indicate that total exchangeable Ca2+ in portal vein is reduced by about 15% in 1 . 10(-4) M pentobarbital sodium, while the intracellular exchangeable Ca2+ is reduced by 24%. On the other hand, in aortic smooth muscle, while 5--20 . 10(-4) M pentobarbital sodium reduces total exchangeable Ca2+ by about 15%, intracellular Ca2+ is reduced by 22% in 5 . 10(-4) M pentobarbital sodium and by 38% in 2 . 10(-3) M pentobarbital sodium. The present studies thus reveal that concentrations of pentobarbital sodium known to be present during induction of surgical anesthesia can exert significant inhibitory effects on exchangeability and transmembrane movement of Ca2+ in at least two different types of blood vessels.

Low extracellular magnesium induces intracellular free Mg deficits, ischemia, depletion of high-energy phosphates and cardiac failure in intact working rat hearts: a 31P-NMR study.

Hemodynamic and 31P-NMR spectroscopic studies were performed on intact, perfused working rat hearts exposed to low (0.3 mM) extracellular Mg([Mg2+]o). Low [Mg2+]o perfusion resulted in rapid and significant falls in cardiac output, coronary flow, stroke volume, developed pressure and the rate-pressure product. Concomitant with this O2 consumption decreased and lactate production increased. Hearts perfused with 0.3 mM, instead of 1.2 mM, [Mg2+]o exhibited significant reductions in [ATP], [PCr], intracellular free Mg ([Mg2+]i), and pHi; a marked rise in intracellular Pi corresponding to a precipitous fall in the cytosolic phosphorylation potential was seen. Reintroduction of 1.2 mM [Mg2+]o failed to reestablish either normal hemodynamics, or high-energy phosphates and intracellular Pi, suggesting irreversible myocyte injury. These observations are consistent with the tenet that low [Mg2+]o can result in marked reduction in oxygen and substrate delivery to the cardiac myocytes, probably as a result of coronary vasoconstriction.

Ferrylmyoglobin formation induced by acute magnesium deficiency in perfused rat heart causes cardiac failure.

The oxidation states of intracellular myoglobin and cytochrome oxidase aa3 were monitored by reflectance spectrophotometry in isolated perfused rat hearts subjected to an acutely magnesium deficient environment. After exposure to low extracellular [Mg2+]o (i.e., 0.3 mM) for 30 min, more than 80% of the oxymyoglobin converted to its deoxygenated form. The level of reduced cytochrome oxidase aa3 also increased about 80% in low [Mg2+]o. The deoxymyoglobin was converted further to a species identified as ferrylmyoglobin by its reaction with Na2S to form ferrous sulfmyoglobin which was optically visible. This process, set into motion by acute Mg deficiency, resulted from a direct accessibility of the exogenous peroxide to the cytosolic protein. The results suggest that a pathway leading to cardiac tissue damage, induced by magnesium deficiency, is probably involved in the generation of a ferrylmyoglobin radical which could be prevented by addition of ascorbate, which is known to be a one-electron reductant of this hypervalent form of myoglobin. In further studies, we also investigated whether addition of different concentrations of ascorbic acid (AA) to the perfusate could enhance myocardial function after exposure to low [Mg2+]o perfusion. Four concentrations of AA (0.5, 1, 5, 10 mM) were tested, and the results indicate that they exert their effects in a concentration-dependent manner; 1 mM AA was the most effective dose in improving aortic output in a Mg-deficient heart. Ferrylmyoglobin formation was found to be formed considerably before intracellular release of either creatine phosphokinase or lactic dehydrogenase. These studies may have wide implications as a new mechanism by which low extracellular Mg2+ can induce myocardial injury and subsequent cardiac failure.

Short-term reduction in dietary intake of magnesium causes deficits in brain intracellular free Mg2+ and [H+]i but not high-energy phosphates as observed by in vivo 31P-NMR.

31P-NMR spectroscopic studies were performed in vivo on brains of rats fed 30-35% normal dietary Mg intake for 6 weeks. Within 2 weeks of the moderately restricted Mg diet serum Mg fell 50%, and brain intracellular free [Mg2+]i fell 15%; within 3 weeks of restricted diet, brain [Mg2+]i fell 40% and remained at this level for the additional 3 weeks. Intracellular pH (pH[i]) progressively rose in a reciprocal manner for 4 weeks. At no interval of time did brain phosphocreatine (PCr), [ATP], or inorganic phosphate change despite the fall in brain [Mg2+]i, brain pH(i) and serum Mg. The Mg-deficiency-induced cytosolic loss of protons (resulting in an alkaline cytosol) could be a compensatory mechanism to stabilize [PCr], [ATP] and [ADP] levels via creatine kinase, thus maintaining cytosolic phosphorylation potential. The rise in pH(i) associated with Mg-deficiency would also account for increased cerebral vascular muscle contractility under these conditions. Lastly, these studies indicate that brain [Mg2+]i may change without a concomitant change in cell [ATP], and that brain [Mg2+]i may be a useful marker for total body Mg2+ status.

Effect of moderate improvement in metabolic control on magnesium and lipid concentrations in patients with type 1 diabetes.

OBJECTIVE: To evaluate the effect of clinically obtainable improvements in metabolic control in patients with type 1 diabetes on biochemical cardiovascular risk factors. RESEARCH DESIGN AND METHODS: Blood and 24-h urinary samples were obtained from 49 patients with type 1 diabetes before and after a run-in period and after 3 months of intervention, with frequent adjustment of insulin dosage according to measured blood glucose concentrations. RESULTS: The intervention caused a mean insulin dosage increment of 10%, a 20% decrease in fasting plasma glucose concentration, a 10% decrease in albumin corrected serum fructosamine, and a somewhat lesser decrease in HbAlc.A 14% decrease in the renal excretion of magnesium (Mg) was observed, but without a change in average serum Mg concentration. Serum HDL cholesterol increased 4%, and serum triglycerides decreased 10% as an average. Looking at individual patients, the decrease in serum triglycerides correlated with both the change in serum total Mg concentration and with the increase in insulin dosage. Using the change in serum total Mg concentration and in insulin dosage as independent variables in a multiple regression analysis, the coefficient of correlation with the decrease in serum triglycerides was 0.52. CONCLUSIONS: Moderate but clinically obtainable improvement of metabolic control in patients with type 1 diabetes seems to reduce the loss of Mg, increase serum HDL cholesterol, and decrease serum triglycerides. The decrease in serum triglycerides was associated with the change in serum total Mg concentration. These reductions in Mg loss and serum triglycerides might reduce the risk of developing cardiovascular disease in patients with type 1 diabetes.

Ethanol-induced contractions in cerebral arteries: role of tyrosine and mitogen-activated protein kinases.

BACKGROUND AND PURPOSE: The relationship between alcohol consumption and stroke appears complex; moderate ingestion is associated with reduced stroke risk, while heavy intake is associated with increased stroke risk. Ethanol has been shown both experimentally and epidemiologically to induce hemorrhagic and ischemic strokes, which are associated with cerebral vasoconstriction. Ethanol is known to induce contraction in isolated cerebral arteries and intact microvessels from diverse mammalian animals. The relationships between ethanol-induced contractions in cerebral arteries, intracellular free Ca(2+) ([Ca(2+)](i)), tyrosine kinases (including the src family), and mitogen-activated protein kinases (MAPK) were investigated in the present study. METHODS: Cerebral arterial muscle tension and [Ca(2+)](i) were quantified by an isometric contraction technique and direct visualization of Ca(2+) in single cells. RESULTS: Ethanol induces concentration-dependent contractions in intact canine basilar arteries, which are attenuated significantly by pretreatment of the arteries with low concentrations of an antagonist of protein tyrosine kinases (genistein); an src homology 2 (SH2) domain inhibitor peptide; a highly specific antagonist of p38 MAPK (SB-203580); a potent, selective antagonist of MEK1/MEK2 (U0126); and a selective antagonist of mitogen-activated protein kinase kinase (MAPKK) (PD-98059). IC(50) levels obtained for these 5 antagonists are consistent with reported K:(i) values for these tyrosine kinase, MAPK, and MAPKK antagonists. Ethanol induces transient and sustained increases in [Ca(2+)](i) in primary single smooth muscle cells from canine basilar arteries, which are markedly attenuated in the presence of genistein, an SH2 domain inhibitor peptide, SB-203580, U0126, and PD-98059. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the ethanol-induced contractions or the elevation of [Ca(2+)](i). CONCLUSIONS: The present study suggests that activation of protein tyrosine kinases (including the src family) and MAPK appear to play important roles in the ethanol-induced contractions and the elevation of [Ca(2+)](i) in smooth muscle cells from canine basilar arteries. The results could be used to suggest that selective antagonists of protein tyrosine kinases and MAPK may be useful both prophylactically and therapeutically in alcohol-induced strokes.

Modulatory effect of extracellular Mg2+ ions on K+ and Ca2+ currents of capillary endothelial cells from rat brain.

Using whole-cell patch-clamp recording, we demonstrate that exposure of single rat brain capillary endothelial cells to different extracellular Mg2+ concentrations (0.3, 4.8 and 9.6 mM) affects the conductance of K+ and Ca2+ currents elicited under control conditions (1.2 mM). Extracellular Mg2+ concentrations ([Mg2+]o) of 4.8 and 9.6 mM reversibly depress outward K+ currents by about 30 +/- 12% (n = 10) and 34 +/- 13% (n = 10), at all activating potentials, respectively. Using identical concentrations reversibly depressed the Ca2+ current by about 40 +/- 16% (n = 8) and 46 +/- 18% (n = 6), respectively. Using a low Mg2+ concentration of 0.3 mM, the K+ current activation was unexpectedly and mildly increased by about 15 +/- 5% (n = 5), and the inward Ca2+ current was attenuated. When studying this effect of low [Mg2+]o on 'pure' Ca2+ currents, free of outward currents, we found that this inward current was depressed by about 38 +/- 16%(n = 8), and its threshold for activation, in the current-voltage relationship, was shifted to more negative potentials. It is concluded that high [Mg2+]o hinders the entry of Ca2+ through low-voltage activated Ca2+ channels and thereby attenuates a Ca2+-regulated K+ conductance. At a low [Mg2+]o (0.3 mM), Mg2+ shifts the steady-state inactivation of the voltage-activated Ca2+ channel to more negative potentials by about 8 mV (n = 6), probably due to a negative screening effect, i.e. a reduction of positive charges on the cell membrane. This may contribute to an apparent increase in K+ conductance by an, as yet, unknown mechanism.

Mg2+ modulates membrane lipids in vascular smooth muscle: a link to atherogenesis.

Epidemiological studies associate low dietary magnesium intake with an increased incidence of ischemic heart disease and sudden cardiac death. We have used proton-magnetic resonance (1H-NMR) techniques and Mg2+-selective electrodes to monitor changes in lipid extracts of aortic and cerebrovascular smooth muscle as extracellular ionized magnesium ion concentration ([Mg2+]o) is lowered. We have found that, within the pathophysiological range of Mg2+ concentrations, fatty acid chain length and double bond content are progressively reduced as [Mg2+]o is lowered. In contrast, the plasmalogen content is progressively increased. A concomitant decrease in fatty acid chain length and double bonds indicates oxidation of double bonds resulting in truncation of the fatty acids. A decrease in lipid oxidation in the presence of elevated Mg2+ could contribute to the apparent protective role of increased Mg2+ intake on vascular function in humans.

Mg2+ modulates membrane sphingolipid and lipid second messenger levels in vascular smooth muscle cells.

In vitro studies with smooth muscle cells from rat aorta and dog cerebral blood vessels indicate that variation in free Mg2+, within the pathophysiological range of Mg2+ concentrations, found in human serum, causes sustained changes in membrane phospholipids and lipid second messengers. Incorporation of [3H]palmitic acid into phosphatidylcholine (PC) and sphingomyelin (SM) was altered within 15-30 min after modifying the extracellular Mg2+ ion level ([Mg2+]o). Decreased Mg2+ produced a fall in both [3H]SM and [3H]PC over the first 2 h. After an 18-h incubation, the [3H]PC/[3H]SM ratio changed from about 20:1 to about 50:1. Increased [Mg2+]o resulted in a 2- to 3-fold increase in [3H]SM compared to only a small increase in [3H]PC over the same period. There was a reciprocal relationship between [3H]ceramide and [3H]1,2-DAG levels with highest [3H]ceramide and lowest [3H]-1,2-DAG levels seen at lowest [Mg2+]o. The results indicate that a fall in extracellular ionized Mg2+ concentration produces a rapid and sustained decrease in membrane sphingomyelin and a moderate rise in intracellular ceramide. A major effect of lowering [Mg2+]o appears to be a down-regulation of SM synthase. The increased membrane SM content and a concomitant decrease in cell ceramide, in the presence of elevated [Mg2+]o, may be relevant to the apparent protective role of adequate Mg intake on vascular function in humans.

Elevation of extracellular magnesium rapidly raises intracellular free Mg2+ in human aortic endothelial cells: is extracellular Mg2+ a regulatory cation?

Extracellular magnesium ions [Mg2+]o are known to regulate functions of endothelial cells, but whether [Mg2+]o can alter intracellular free ionized magnesium [Mg2+]i in these cells remains unknown. The present studies, using digital imaging microscopy and the Mg2+ fluorescent probe, mag-fura-2, determined effects of elevation of [Mg2+]o on [Mg2+]i in cultured human aortic endothelial cells. With normal Mg2+(1.2 mM)-containing incubation media, [Mg2+]i was 0.51+/-0.04 mM with a heterogeneous distribution. The ratio of [Mg2+]i/[Mg2+]o was 0.52+/-0.07. Elevation of [Mg2+]o up to 4.8 mM increased [Mg2+]i to 0.80+/-0.07 mM in 2-10 min and lowered the ratio of [Mg2+]i/[Mg2+]o to 0.16+/-0.02. Irrespective of the observed increments of [Mg2+]i, a subcellular heterogeneous distribution of [Mg2+]i was always evident in all cells tested. Our results suggest that [Mg2+]o can regulate [Mg2+]i more rapidly than heretofore believed, supporting the hypothesis that extracellular Mg2+ can exert regulatory effects on endothelial cell functions and probably act as extracellular regulatory cations

Magnesium protects against cocaine-induced hemorrhagic stroke in a rat model: a 31P-NMR in-vivo study.

In-vivo 31P-nuclear magnetic resonance (NMR) studies were undertaken with anesthetized rats to determine: a. whether systemic administration of MgCl2 could protect animals against cocaine-induced hemorrhagic stroke, and b. whether a relationship exists between basal levels of brain intracellular free magnesium ions ([Mg2+]i), phosphometabolites, and stroke risk. Repeat 31P-NMR spectra were obtained at various intervals of time (3-120 min, or up until death) after administration of cocaine (5 + 30 mg/kg). Ion selective electrodes were used to measure plasma Mg2+, K+, Na+ and Ca2+. Forty percent of animals died in the absence of Mg2+ infusion following high dosage of cocaine. Only 13% died with cocaine following Mg2+ infusion (p <0.005). In the Mg2+-protected animals, neither brain [Mg2+]i,intracellular pH (pHi), [phosphocreatine-PCr]/[ATP], nor brain [inorganic phosphate-Pi]/[ATP] fell when toxic and lethal doses of cocaine were given. Low basal brain [Mg2+]i (275 +/- 24 vs. 466 +/- 35 microM, p <0.01) and low basal brain [PCr] (3.36 +/- 0.35 vs. 4.26 +/- 0.24 mM, p <0.01) were found to be associated with a 3-fold increased incidence of stroke. A positive correlation (r = 0.31, p <0.03) between brain [Mg2+]1 and [PCr]/[ATP] was found. It is possible that both brain [Mg2+]i and [PCr] may be useful as important predictors of susceptibility to cocaine-induced hemorrhagic stroke.