Mitsuisa Yoshimura

Relationship Between Insulin Resistance and Endothelium-Dependent Vascular Relaxation in Patients With Essential Hypertension

The infusion of L-arginine induces the production of nitric oxide and stimulates the immediate secretion of insulin. To examine the relationship between insulin resistance and endothelium-dependent vascular relaxation in patients with essential hypertension, we evaluated the renal and insulin responses to L-arginine, 500 mg/kg infused intravenously over 30 minutes, in 23 patients with mild essential hypertension who were neither obese nor diabetic and in 20 normotensive control subjects. We found no difference between the two groups in blood glucose or insulin in the fasting condition. The renovascular relaxation induced by L-arginine was significantly less in patients with essential hypertension than in normotensive control subjects. The increase in plasma cyclic GMP in response to L-arginine was lower in hypertensive patients than in normotensive subjects. Although the serum concentrations of glucose in response to L-arginine were similar in the two groups, the serum insulin response of the essential hypertensives was significantly higher than that of the normotensive subjects. In all subjects, the peak cyclic GMP response to L-arginine was significantly correlated with the peak delta glucose/ delta insulin ratio response to L-arginine (r = .69, P < .001). Findings suggested that an impairment of endothelium-dependent renal vascular relaxation and a reduced sensitivity to insulin are present in patients with essential hypertension. A link may be present between the abnormality of the L-arginine/nitric oxide/cyclic GMP pathway and insulin resistance in patients with essential hypertension.

Extracellular Mg2+ Inhibits Capacitative Ca2+ Entry in Vascular Smooth Muscle Cells

BACKGROUND Agonist-induced Ca2+ entry is thought to be mediated by capacitative Ca2+ entry other than L-type Ca2+ channels in vascular smooth muscle cells (VSMCs). The mechanism for capacitative Ca2+ entry has not been fully elucidated. Our objective was to examine the effect of external Mg2+ on capacitative Ca2+ entry in cultured rat aortic VSMCs. METHODS AND RESULTS Three doses of external Mg2+ concentration (nominally 0, 1, and 5 mmol/L) were used. After exposure to 1 mumol/L, angiotensin II (Ang II) in Ca(2+)-free medium, addition of Ca2+ to the medium caused an increase in cytosolic free Ca2+ concentration ([Ca2+]i), indicating Ang II-induced Ca2+ influx. This Ca2+ influx was attenuated in cells preincubated with high external Mg2+ concentrations or with 1 mumol/L nifedipine. After VSMCs in Ca(2+)-free medium were exposed to 1 mumol/L thapsigargin, which inhibits the sarcoplasmic reticulum Ca(2+)-ATPase and depletes Ca2+ stores, addition of Ca2+ to the medium induced an increase in [Ca2+]i, indicating capacitative Ca2+ entry. This entry pathway was found to be independent of dihydropyridine-sensitive Ca2+ channels and inhibited by increased external Mg2+ concentration. External Mg2+ concentration did not influence Ca2+ efflux across the plasma membrane after stimulation with Ang II plus thapsigargin. CONCLUSIONS Results suggest that in VSMCs, capacitative Ca2+ entry is reduced by external Mg2+. This mechanism may explain in part the inhibitory effect of external Mg2+ on Ca2+ handling.

Abnormal platelet Ca2+handling accompanied by increased cytosolic free Mg2+ in essential hypertension

To test the hypothesis that abnormal platelet Ca2+ handling in essential hypertension results from cellular Mg2+ deficiency, cytosolic free Mg2+ concentration ([Mg2+]i) and Ca2+ metabolism were studied in mag-fura 2 and fura 2-loaded platelets from 30 essential hypertensive patients and 30 sex- and age-matched normotensive controls. Basal cytosolic free Ca2+ concentration ([Ca2+]i) and intracellular Ca2+ discharge capacity were higher in hypertensives than in normotensives (22 ± 5 vs. 18 ± 5 nM, P < 0.05; 743 ± 250 vs. 624 ± 144 nM, P < 0.05, respectively). The thrombin (0.03-1.0 U/ml)-evoked [Ca2+]iresponse was also enhanced in platelets from hypertensives in both the absence and presence of extracellular Ca2+. However, basal [Mg2+]iwas higher in hypertensives than in normotensives (437 ± 110 vs. 353 ± 85 μM, P < 0.05), whereas serum Mg2+ was similar in the two groups. These results oppose the Mg2+ deficiency hypothesis in platelets in essential hypertension.

Differential Effects of Extracellular Mg2+ on Thrombin-Induced and Capacitative Ca2+ Entry in Human Coronary Arterial Endothelial Cells

Receptor-mediated and capacitative Ca2+ entry are the primary Ca2+ entry pathways in endothelial cells (ECs). The mechanisms for Ca2+ entry via these pathways have not been fully elucidated. In this study, the effect of low and high external Mg2+ concentrations on these Ca2+ entry pathways was examined in human coronary arterial ECs. External Mg2+ concentration did not affect cytosolic free Mg2+ concentration. After exposure to thrombin in Ca(2+)-free medium, addition of Ca2+ to the medium caused a rise in cytosolic free Ca2+ concentration ([Ca2+]i), indicating thrombin-induced Ca2+ influx. Thrombin-induced Ca2+ influx was inhibited by not only low but also high external Mg2+ concentrations. After depletion of endoplasmic Ca2+ stores by thapsigargin, addition of Ca2+ to the medium induced an increase in [Ca2+]i, indicating capacitative Ca2+ entry. Capacitative entry was found to be accelerated by low external Mg2+ and inhibited by high external Mg2+ concentration. Results suggest that receptor-mediated Ca2+ influx requires external Mg2+ but is inhibited by increased external Mg2+ concentrations and that capacitative Ca2+ entry is reduced by external Mg2+ in human coronary arterial ECs.

Potentiation of the intracellular Ca2+ response to arginine vasopressin by increased cytosolic-free Mg2+ in rat vascular smooth muscle cells

Although the inhibitory effects of extracellular Mg2+ on Ca2+ influx are well established, little is known about the effects of intracellular Mg2+ on Ca2+ handling. In the present study, the effects of cytosolic-free Mg2+ concentration in the physiological (submillimolar) range on Ca2+ handling were investigated after stimulation of rat vascular smooth muscle cells with arginine vasopressin. Cytosolic Mg2+ was manipulated by culturing cells in medium containing different Mg2+ concentrations. Peak cytosolic-free Ca2+ concentration responses to arginine vasopressin (1 mumol/1) were measured in the presence and absence of external Ca2+. The results suggest that an increase in cytosolic-free Mg2+ concentration increases both Ca2+ discharge from intracellular stores and Ca2+ influx, whereas a decrease in intracellular Mg2+ attenuates Ca2+ influx.

Increased cytosolic free Mg2+and Ca2+ in platelets of patients with vasospastic angina

This study was designed to test the hypothesis that the cellular metabolism of Ca2+ and Mg2+, which is important in platelet function, is abnormal in the platelets of patients with vasospastic angina. Cytosolic free Mg2+ concentration ([Mg2+]i) and Ca2+ handling were determined in the platelets of 24 patients with vasospastic angina and 24 control subjects by use of mag-fura 2 and fura 2. Platelet aggregation was also examined. Basal [Mg2+]i and cytosolic free Ca2+ concentration ([Ca2+]i) in platelets were significantly higher in patients with vasospastic angina than in control subjects. The amplitude of the [Ca2+]i transient induced by thrombin (0.03-1.0 U/ml) was significantly increased in the presence, but not in the absence, of extracellular Ca2+ in patients with vasospastic angina, as compared with controls. Therefore, the influx of Ca2+ across the plasma membrane may be accelerated in vasospastic angina. Thrombin (0.1-1.0 U/ml)-induced maximum aggregation response was significantly greater in patients with vasospastic angina than in controls. Results suggest that increased [Mg2+]i and altered Ca2+ handling by platelets may be associated with coronary vasospasm.This study was designed to test the hypothesis that the cellular metabolism of Ca2+ and Mg2+, which is important in platelet function, is abnormal in the platelets of patients with vasospastic angina. Cytosolic free Mg2+ concentration ([Mg2+]i) and Ca2+ handling were determined in the platelets of 24 patients with vasospastic angina and 24 control subjects by use of mag-fura 2 and fura 2. Platelet aggregation was also examined. Basal [Mg2+]iand cytosolic free Ca2+concentration ([Ca2+]i) in platelets were significantly higher in patients with vasospastic angina than in control subjects. The amplitude of the [Ca2+]itransient induced by thrombin (0.03-1.0 U/ml) was significantly increased in the presence, but not in the absence, of extracellular Ca2+ in patients with vasospastic angina, as compared with controls. Therefore, the influx of Ca2+ across the plasma membrane may be accelerated in vasospastic angina. Thrombin (0.1-1.0 U/ml)-induced maximum aggregation response was significantly greater in patients with vasospastic angina than in controls. Results suggest that increased [Mg2+]iand altered Ca2+ handling by platelets may be associated with coronary vasospasm.

LACK OF EFFECT OF TRANSMEMBRANE GRADIENT OF MAGNESIUM AND SODIUM ON REGULATION OF CYTOSOLIC FREE MAGNESIUM CONCENTRATION IN RAT LYMPHOCYTES

The regulation of the intracellular concentration of Mg2+ ([Mg2+]i) is not fully understood. The level of Mg in lymphocytes is a good predictor of total body Mg status. We measured [Mg2+]i and total Mg in rat lymphocytes by using, respectively, the fluorescent Mg2+ indicator mag-fura-2 and atomic absorption spectrophotometry. The basal [Mg2+]i in rat lymphocytes was 328 +/- 23 micromol/l. An elevation to 5 mmol/l or the removal of extracellular Mg2+ did not affect [Mg2+]i. A reduction in extracellular Na+ did not influence [Mg2+]i for 60 min. The total Mg concentration in lymphocytes also remained stable. Results suggest that the permeability of the plasma membrane to Mg2+ is very low, and that Na+/Mg2+ exchange is not involved in the regulation of [Mg2+]i in rat lymphocytes.