Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells

Magnesium Research. Volume 17, Number 1, 20-7, March 2004, ORIGINAL ARTICLE

Author(s) : Kazuo Satake, Jong‐Dae Lee, Hiromasa Shimizu, Hiroyasu Uzui, Yasuhiko Mitsuke, Hong Yue, Takanori Ueda , First Department of Internal Medicine, Fukui University, 23 Shimoaizuki, Matsuoka‐cho, Fukui 910‐1193, Japan .

Summary : This study investigated the effects of extracellular magnesium concentration ([Mg ²⁺ _]e\; 0.3‐3 mM) on intracellular free calcium concentration ([Ca ²⁺ _]i) and prostacyclin (PGI ₂) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist‐stimulated conditions. We used histamine as agonist which increases [Ca ²⁺ _]i and PGI ₂ production in HUVEC, norepinephrine in VSMC. [Mg ²⁺ _]e dose‐dependently increased basal and agonist‐stimulated PGI ₂ production in both cells. [Mg ²⁺ _]e dose‐dependently reduced basal [Ca ²⁺ _]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg ²⁺ _]e reduced agonist‐stimulated [Ca ²⁺ _]i responses. Furthermore, [Mg ²⁺ _]e dose‐dependently reduced agonist‐stimulated [Ca ²⁺ _]i in Ca ²⁺ ‐free buffer, indicating intracellular Ca ²⁺ release. In VSMC, 10 ^‐‐6 M diltiazem and 10 ^‐7 M nifedipine, Ca ²⁺ channel blockers, reduced agonist‐stimulated [Ca ²⁺ _]i as well as 3 mM Mg ²⁺, but did not affect PGI ₂ production. [Mg ²⁺ _]e amplified dose‐dependently arachidonic acid‐induced PGI ₂ production in both cells, suggesting the activation of cyclooxygenase and\\or PGI ₂ synthetase. Our results suggest that [Mg ²⁺ _]e influences intracellular Ca ²⁺ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca ²⁺ influx and intracellular Ca ²⁺ release. [Mg ²⁺ _]e enhances PGI ² production in both types of cells, although the mechanism is likely to be independent from Ca ²⁺ mobilization.

Keywords : magnesium, vascular endothelial cells, vascular smooth muscle cells, intracellular free calcium concentration, prostacyclin

Pictures

Fig. 1. Effects of extracellular Mg²⁺ on basal and agonist-stimulated PGI₂ production by HUVEC. Cultured HUVEC were incubated in Hepes buffer A (see Methods) containing 0.3-3 mM Mg²⁺ in the presence (close circle) or absence (open circle) of 10^–5 M histamine. The supernatant was assayed by RIA. Data are represented as means ± SEM (n = 6). * P < 0.05 compared with 0.3 mM Mg²⁺

Fig. 2. Effects of extracellular Mg²⁺ on basal [Ca²⁺]_i and 10^–5 M histamine-stimulated [Ca²⁺]_i transients in HUVEC. Cultured HUVEC were loaded with 2 µM fura2-AM and resuspended in Hepes buffer B (see Methods) containing 0.3-3 mM MgSO₄ with (Panel A) or without (Panel B) 2.5 mM CaCl₂ Data were obtained in the presence (close circle) or absence (open circle) of 10^–5 M histamine. Data are represented as means ± SEM (n = 8). * P < 0.05 compared with 0.3 mM Mg²⁺.

Fig. 3. Effects of extracellular Mg²⁺ on PGI₂ production by VSMC. Cultured VSMC were incubated in Hepes buffer A (see Methods) containing 0.3-3 mM Mg²⁺ in the presence (close circle) or absence (open circle) of 10^–6 M norepinephrine. The supernatant was assayed by RIA. Data are represented as means ± SEM (n = 8). * P < 0.05 compared with 0.3 mM Mg²⁺.

Fig. 4. Effects of Ca²⁺ channel blockers on 10^–6 M norepinephrine-stimulated [Ca²⁺]_i transients and PGI2 production in VSMC. Panel (A) demonstrates 10^–6 M norepinephrine-stimulated [Ca²⁺]_i transients in Hepes buffer B (see Methods). Panel (B) shows 10^–6 M norepinephrine-stimulated PGI₂ production in Hepes buffer A (see Methods). Data are represented as means ± SEM (n = 8). * Significantly different at P < 0.05. NS indicates no statistically significant difference.

Fig. 5. Effects of extracellular Mg²⁺ on sodium arachidonate-stimulated PGI₂ production by HUVEC and VSMC. HUVEC and VSMC were incubated in Hepes buffer A (see methods) containing 0.3-3 mM Mg²⁺ with 20 µM arachidonic acid (Panel (A): HUVEC, Panel (B): VSMC). The supernatant was assayed by RIA. Data are represented as means ± SEM (n = 8). * Significantly differs (P < 0.05) from corresponding values obtained with 0.3 mM Mg²⁺ * P < 0.05 compared with 0.3 mM Mg²⁺.