Effects of Magnesium Lithospermate B and its Analogues on Ca²⁺ Homeostasis in Cultured Rat Thoracic Aorta Vascular Smooth Muscle Cells

Abstract

Magnesium lithospermate B (MLB), sodium rosmarinate (SR), and magnesium lithospermate (ML) are biologically active components isolated from aqueous extracts of the Chinese medicine Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge (Labiatae). These compounds share similar chemical structures of polyphenols and oligomer condensates with caffeic acid. In this study, we compared the effects of MLB, ML, and SR on intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in cultured rat thoracic aorta vascular smooth muscle cells (VSMCs), using the Ca²⁺-sensitive dye, Fluo-3, as an indicator. In our experiments, MLB, ML, and SR did not affect the basal value of [Ca²⁺]_i in VSMCs. In the absence of extracellular Ca²⁺, the [Ca²⁺]_i increase in VSMCs induced by 20 µM ATP was attenuated by MLB, ML, and SR in a dose-dependent manner. Moreover, under this condition, MLB suppressed the increase in [Ca²⁺]_i in VSMCs induced by 1 µM thapsigargin, but not ML or SR. In the presence of extracellular Ca²⁺, the [Ca²⁺]_i increase in VSMCs induced by 60 mM KCl was attenuated by MLB and SR in a dose-dependent manner, but not by ML. These results suggest that MLB, ML and SR regulate Ca²⁺ homeostasis in VSMCs via different pathways. Our findings should aid in elucidating the mechanisms of the vasodilator action of aqueous extracts of Salvia miltiorrhiza.

Key words

magnesium lithospermate B - sodium rosmarinate - magnesium lithospermate - vascular smooth muscle cells - Fluo‐3

References

• 1 Kasimu R, Tanaka K, Tezuka Y, Gong Z N, Li J X, Basnet P, Namba T, Kadota S. Comparative study of seventeen Salvia plants: aldose reductase inhibitory activity of water and MeOH extracts and liquid chromatography-mass spectrometry (LC‐MS) analysis of water extracts.  Chem Pharm Bull. 1998;  46 500-504
• 2 Zhang Y, Akao T, Nakamura N, Duan C L, Hattori M, Yang X W, Liu J X. Extremely low bioavailability of magnesium lithospermate B, an active component from Salvia miltiorrhiza, in rat.  Planta Med. 2004;  70 138-142
• 3 Hase K, Kasimu R, Basnet P, Kadota S, Namba T. Preventive effect of lithospermate B from Salvia miltiorhiza on experimental hepatitis induced by carbon tetrachloride or D-galactosamine/lipopolysaccharide.  Planta Med. 1997;  63 22-26
• 4 Lam F F, Yeung J H, Kwan Y W, Chan K M, Or P M. Salvianolic acid B, an aqueous component of danshen (Salvia miltiorrhiza), relaxes rat coronary artery by inhibition of calcium channels.  Eur J Pharmacol. 2006;  553 240-245
• 5 Yokozawa T, Lee T W, Oura H, Nonaka G, Nishioka I. Effect of magnesium lithospermate B in rats with sodium-induced hypertension and renal failure.  Nephron. 1992;  60 460-465
• 6 Yokozawa T, Chung H Y, Dong E, Oura H. Confirmation that magnesium lithospermate B has a hydroxyl radical-scavenging action.  Exp Toxicol Pathol. 1995;  47 341-344
• 7 Wu X J, Wang Y P, Wang W, Sun W K, Xu Y M, Xuan L J. Free radical scavenging and inhibition of lipid peroxidation by magnesium lithospermate B.  Acta Pharmacol Sin. 2000;  21 855-858
• 8 Somlyo A P, Somlyo A V. Signal transduction and regulation in smooth muscle.  Nature. 1994;  372 231-236
• 9 Wang W, Hu G Y, Wang Y P. Selective modulation of L-type calcium current by magnesium lithospermate B in guinea-pig ventricular myocytes.  Life Sci. 2006;  78 2989-2997
• 10 Luo W B, Wang Y P. Magnesium lithospermate B inhibits hypoxia-induced calcium influx and nitric oxide release in endothelial cells.  Acta Pharmacol Sin. 2001;  22 1135-1142
• 11 Wang W, Wang Y P, Sun W K, Xu Y M, Xuan L J. Effects of magnesium lithospermate B on aggregation and 5-HT release in rabbit washed platelets.  Acta Pharmacol Sin. 2000;  21 859-863
• 12 Luo W B, Dong L, Wang Y P. Effect of magnesium lithospermate B on calcium and nitric oxide in endothelial cells upon hypoxia/reoxygenation.  Acta Pharmacol Sin. 2002;  23 930-936
• 13 Tian L L, Wang X J, Sun Y N, Li C R, Xing Y L, Zhao H B, Duan M, Zhou Z, Wang S Q. Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents 6-hydroxydopamine induced apoptosis in SH-SY5Y cells.  Int J Biochem Cell Biol. 2008;  40 409-422
• 14 Lin Y H, Liu A H, Wu H L, Westenbroek C, Song Q L, Yu H M, Ter Horst G J, Li X J. Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents Abeta(25–35)-induced reduction in BPRP in PC12 cells.  Biochem Biophys Res Commun. 2006;  348 593-599
• 15 Liu C S, Chen N H, Zhang J T. Protection of PC12 cells from hydrogen peroxide-induced cytotoxicity by salvianolic acid B, a new compound isolated from Radix Salviae miltiorrhizae.  Phytomedicine. 2007;  14 492-497
• 16 Liu X, Chen R, Shang Y, Jiao B, Huang C. Lithospermic acid as a novel xanthine oxidase inhibitor has anti-inflammatory and hypouricemic effects in rats.  Chem Biol Interact. 2008;  176 137-142
• 17 Wagner H, Horhammer L, Frank U. Lithospermic acid, the antihormonally active principle of Lycopus europaeus L. and Symphytum officinale. 3. Ingredients of medicinal plants with hormonal and antihormonal-like effect.  Arzneimittelforschung. 1970;  20 705-713
• 18 Abd-Elazem I S, Chen H S, Bates R B, Huang R C. Isolation of two highly potent and non-toxic inhibitors of human immunodeficiency virus type 1 (HIV‐1) integrase from Salvia miltiorrhiza.  Antiviral Res. 2002;  55 91-106
• 19 Zou Z W, Xu L N, Tian J Y. Antithrombotic and antiplatelet effects of rosmarinic acid, a water-soluble component isolated from Radix Salviae miltiorrhizae (Danshen).  Yao Xue Xue Bao. 1993;  28 241-245
• 20 Osakabe N, Takano H, Sanbongi C, Yasuda A, Yanagisawa R, Inoue K, Yoshikawa T. Anti-inflammatory and anti-allergic effect of rosmarinic acid (RA); inhibition of seasonal allergic rhinoconjunctivitis (SAR) and its mechanism.  Biofactors. 2004;  21 127-131
• 21 Swarup V, Ghosh J, Ghosh S, Saxena A, Basu A. Antiviral and anti-inflammatory effects of rosmarinic acid in an experimental murine model of Japanese encephalitis.  Antimicrob Agents Chemother. 2007;  51 3367-3370
• 22 Ahn S C, Oh W K, Kim B Y, Kang D O, Kim M S, Heo G Y, Ahn J S. Inhibitory effects of rosmarinic acid on Lck SH2 domain binding to a synthetic phosphopeptide.  Planta Med. 2003;  69 642-646
• 23 Kang M A, Yun S Y, Won J. Rosmarinic acid inhibits Ca²⁺-dependent pathways of T-cell antigen receptor-mediated signaling by inhibiting the PLC-γ 1 and Itk activity.  Blood. 2003;  101 3534-3542
• 24 Won J, Hur Y G, Hur E M, Park S H, Kang M A, Choi Y, Park C, Lee K H, Yun Y. Rosmarinic acid inhibits TCR-induced T cell activation and proliferation in an Lck-dependent manner.  Eur J Immunol. 2003;  33 870-879
• 25 Tanaka T, Morimoto S, Nonaka G, Nishioka I, Yokozawa T, Chung H Y, Oura H. Magnesium and ammonium-potassium lithospermates B, the active principles having a uremia preventive effect from Salvia miltiorrhiza.  Chem Pharm Bull. 1989;  37 340-344
• 26 Kelley C J, Harruff R C, Carmack M. Polyphenolic acids of lithospermum ruderale. II. Carbon-13 nuclear magnetic resonance of lithospermic and rosmarinic acids.  J Org Chem. 1976;  41 449-455
• 27 Rodriguez A, Fortuno A, Gomez-Ambrosi J, Zalba G, Diez J, Fruhbeck G. The inhibitory effect of leptin on angiotensin II-induced vasoconstriction in vascular smooth muscle cells is mediated via a nitric oxide-dependent mechanism.  Endocrinology. 2007;  148 324-331
• 28 Wong K L, Yang H Y, Chan P, Cheng T H, Liu J C, Hsu F L, Liu I M, Cheng Y W, Cheng J T. Isosteviol as a potassium channel opener to lower intracellular calcium concentrations in cultured aortic smooth muscle cells.  Planta Med. 2004;  70 108-112
• 29 Akata T. Cellular and molecular mechanisms regulating vascular tone. Part 1: basic mechanisms controlling cytosolic Ca²⁺ concentration and the Ca²⁺-dependent regulation of vascular tone.  J Anesth. 2007;  21 220-231
• 30 Lam F F, Yeung J H, Cheung J H, Or P M. Pharmacological evidence for calcium channel inhibition by Danshen (Salvia miltiorrhiza) on rat isolated femoral artery.  J Cardiovasc Pharmacol. 2006;  47 139-145
• 31 Li X C, Yu C, Sun W K, Liu G Y, Jia J Y, Wang Y P. Pharmacokinetics of magnesium lithospermate B after intravenous administration in beagle dogs.  Acta Pharmacol Sin. 2004;  25 1402-1407