Pharmacological Mechanisms Of Magnesium Lithospermate B On Vasoconstriction And Inhibition Of [Ca～(2+)]i Elevation Induced By Activators In Rat Vascular Smooth Muscle Cells

Magnesium lithospermate B (MLB) is an aqueous compound isolated fromtraditional Chinese herb "Dan-shen". In the present paper, we aimed to investigatethe effects of MLB on vasoconstriction of denuded aortic rings in vitro; as well asthe effect of MLB on intracellular free calcium concentration ( [Ca²⁺]i ) in aorticvascular smooth muscle cells (AVSMCs), and discussed the way involved, and thenelucidated the pharmacological mechanisms of MLB on attenuating the cellularcalcium homeostasis in vascular smooth muscle cells.We observed the effects of MLB on vasoconstriction in denuded rat aortic ringsin vitro. MLB had no effects on resting ring tension in the presence or absence ofextracellular Ca²⁺. In rings exposed to 50-200μmol·L^-1 MLB, the isotoniccontractions induced by 1μmol·L^-1 PE were dose-dependently, with decreasesranging from 25.7 to 46.9% in calcium-free K-H solution. However, in the presenceof extracellular Ca2+, KCl 60mmol·L-1 produced isotonic contractions that wereabolished by verapamil 10μmol·L-1 or inhibited by pretreatment of 50-200μmol·L-1MLB in dose-dependent manner, with decreases ranging from 17.9 to 41.2%. Whenadministrated to rings precontracted with 1μmol·L-1 PE in the absence ofextracellular Ca2+, a second extracelluar-calcium-dependent vasoconstrictioninduced by restoration of extracellular Ca2+ were also reduced by pretreatment of 50-200μmol·L-1 MLB dose-dependently. All these results indicated that MLBinhibited vasoconstriction probably resulting from both of the blocking effect on theCa2+ release and Ca2+influx. Measurement of isotonic tension of rat thoracic aortic rings reflected the effectsof MLB on [Ca2+]i only indirectly. So in order to further study the mechanisms,Real-time [Ca2+]i in primary AVSMCs of rat was observed directly usingIntracellular Cation Measurement System, with the help of Ca2+-indicator, Fluo-3.The results suggested that MLB, at concentration of 50–200μmol·L-1, produced littleeffect on AVSMCs [Ca2+]i in the presence or absence of extracellular Ca2+. InAVSMCs exposed to 50, 100, and 200μmol·L-1 MLB, the inhibitory effects of MLBon [Ca2+]i induced by 20μmol·L-1 ATP were dose-dependent, with the inhibitoryrates being 17.4%, 32.4%, and 61.1% respectively in the absence of extracellularCa2+. It was supposed to be the inhibitory effects on Ca2+ release from ER ofVSMCs which lowered the increase of [Ca2+]i. In the presence of extracellular Ca2+and thapsigargin, the increases in AVSMCs [Ca2+]i evoked by 60mmol·L-1 KClwere dose-dependently inhibited by pretreatment with MLB 50, 100, and200μmol·L-1or abolished by pretreatment with verapamil 10μmol·L-1. And theinhibitory rates of MLB were 22.0%, 32.8%, and 52.6% respectively. These resultsindicated that MLB might act as a calcium antagonist blocking voltage-dependentcalcium channels on PM, resulting in inhibition of [Ca2+]i increase. In summary, MLB inhibits vasoconstriction induced by PE, high-K+, andre-Ca2+. Also MLB had inhibitory effects on increase of [Ca2+]i induced by ATP andhigh K+ in AVSMCs. These results indicate that both the blocking effects on calciumrelease and voltage-dependent calcium channels may be responsible for the effectsof MLB on [Ca2+]i in vascular smooth muscle cells.