| Background:Danshen, the dried root of Salvia miltiorrhiza, has been widely used in both Asian and western countries for the treatment of various cardiovascular diseases. With at least 50 hydrophilic and 30 lipophilic constituents identified, danshen has been shown to possess various biological properties, such as the improvement of microcirculation, dilation of the coronary arteries, anti-oxidation, anti-inflammatory, anti-myocardial ischemia and anti-apoptosis. Among these constituents, tanshinoneⅡA is the most abundant and representative lipophilic diterpenoid quinine, and sodium tanshinoneⅡA sulfonate (STS), is a water-soluble derivative of tanshinoneⅡA after sulfonation. Previous studies revealed the vasorelaxant effect of tanshinoneⅡA or other bioactive constituents of danshen on femoral, mesenteric, coronary, renal and aortic arteries from different species of animals. The results suggested that the vasorelaxant actions were produced by inhibition of Ca2+ influx, activation of K+ channels in the vascular smooth muscle cells, release of calcitonin gene-related peptide from sensory nerves, and nitric oxide (NO) and cytochrome P450 metabolites. However, the effects of tanshinoneⅡA on pulmonary arteries and the underlying mechanisms remain unclear.Hypoxic pulmonary hypertension is a severe syndrome and complication characterized by sustained vasoconstriction and structural remodeling of pulmonary artery, and it still lacks ideal therapies. Our previous study suggested that STS had protective effects on rat with hypoxic pulmonary hypertension through decreasing mean pulmonary artery pressure and inhibiting structural remodeling of distal pulmonary arteries. However, there are at present no published reports about the effects of tanshinoneⅡA on normal pulmonary artery rings under acute hypoxia condition and on structure–remodeled pulmonary arteries from hypoxic pulmonary hypertension rats.In the present study, therefore, we investigated the effects and underlying mechanisms of STS on isolated rat pulmonary arteries. Moreover, we investigated whether STS modulated acute hypoxic pulmonary vasoconstriction and vasoreactivity of structure-remodeled pulmonary arteries from hypoxic pulmonary hypertension rats.Objective:(1) To explore the vascular effects of STS on phenylephrine or high-K+ precontracted rat pulmonary arteries under normoxic condition.(2) To investigate the underlying mechanisms of the vascular effects of STS on rat pulmonary arteries.(3) To explore the effects of STS on acute hypoxic pulmonary vasoconstriction and structure-remodeled pulmonary arteries from hypoxic pulmonary hypertension rats.Methods:(1) The third-division (external diameter <300μm) pulmonary arteries were isolated carefully and were cut into 3-mm-length rings. Phenylephrine (1μmol/L) or high-K+ (60 mmol/L) Krebs-Henseleit solution was added to establish a stable contractile tone. When the plateau was attained, concentration-response curve was constructed by cumulative addition of STS (0.01~600μmol/L) under normoxic condition.(2) Suspended pulmonary artery rings were studied in parallel. To examine the role of endothelium in STS-induced vasoreaction, endothelium was removed by gently rubbing the lumen with closed forceps tips in some rings. To test the involvement of nitric oxide (NO), carbon monoxide (CO) and prostaglandins of the vasoreactions, some rings were subjected to pretreatment with the following agents for 30 min before precontraction by phenylephrine (1μmol/L): Nω-nitro-L-arginine methyl ester (L-NAME, NO-synthase inhibitor, 100μmol/L), zinc protoporphyrinⅨ(ZnPPⅨ, heme oxygenase-1 inhibitor, 10μmol/L), and indomethacin (prostaglandins synthesis inhibitor, 10μmol/L). To determine the role of potassium (K+) channels, tetraethylammonium (TEA, 10 mmol/L), 4-aminopyridine (4-AP, 1 mmol/L), BaCl2 (100μmol/L) or glibenclamide (10μmol/L) was applied to some vessel rings for 30 min prior to pre-contraction by phenylephrine. To evaluate whether the vasorelaxant effect of STS was due to the inhibition of intracellular Ca2+ release or extracellular Ca2+ influx, pulmonary arterial rings were equilibrated in Ca2+-free Krebs-Henseleit solution. (3) To investigate the effect of STS on acute hypoxic pulmonary vasoconstriction, pulmonary artery rings primed with phenylephrine (1μmol/L) were exposed to hypoxia, which was induced by changing the aerated gas to 95 % N2 / 5 % CO2, with or without STS pretreatment.(4) To obtain rats with hypoxic pulmonary hypertension, hypobaric hypoxia method was intermittently used. Pulmonary arteries were isolated from hypoxic pulmonary hypertension rats to examine the effect of STS on structure-remodeled pulmonary arteries.Results:(1) Cumulative dosing of STS elicited a biphasic effect on the phenylephrine (1μmol/L) or high-K+ (60 mmol/L) - precontracted artery rings. STS, at concentrations lower than 1μmol/L, produced significant constriction on the artery rings with maximum effect at 1μmol/L, whereas it caused significant relaxation at concentrations higher than 1μmol/L with maximum effect at 300μmol/L. The EC50 for relaxation was about 100μmol /L.(2) Removal of endothelium did not modify the vasorelaxant effect of STS, but significantly attenuated its contraction effect. Preincubation of artery rings with L-NAME (100μmol/L) significantly upward shifted the the concentration-response curve but did not affect the vasorelaxant action of STS. However, the pretreatment of artery rings with ZnPPⅨ(10μmol/L) and indomethacin (10μmol/L), did not affect the vascular effects of STS. The pretreatment with TEA (10 mmol/L), significantly upward shifted the concentration (at 0.1μM to 100μmol/L) - response curve of STS. However, TEA pretreatment did not affect the maximum relaxant effect and relaxation EC50 of STS. Pretreatment with 4-AP (1 mmol/L), or glibenclamide (10μmol/L), or BaCl2 (100μmol/L), did not affect the vasoreaction of STS on pulmonary artery rings. Pretreatment with STS (300μmol/L) almost abolished the contraction of the artery rings induced by different extracellular CaCl2 and also decreased the phenylephrine-primed contraction in Ca2+ free bath solution.(3) Pretreatment with 100μmol/L STS, which is close to the EC50 of STS relaxation effect, totally eliminated the hypoxia induced early contraction, potentiated followed vasorelaxation, and attenuated the phaseⅡvasoconstriction. Moreover, hypoxia-induced phaseⅡvasoconstriction of pulmonary artery rings no longer sustained but reversed to sustained relaxation when pretreated with STS.(4) On structure-remodeled pulmonary artery rings from hypoxic pulmonary hypertension rats, pretreatment with 100μmol/L STS had no effects on the early vasoconstriction induced by phenylephrine, but reversed the delayed sustained constriction to sustained vasodilation.Conclusion:STS produced a biphasic effect on isolated rat pulmonary artery. The potent vasodilation of it was induced primarily by inhibiting extracellular Ca2+ influx and partially by inhibiting intracellular Ca2+ release, as well as the activation of KCa channels. Furthermore, the modulation of STS on acute hypoxic pulmonary vasoconstriction and vasoreactivity of structure–remodeled pulmonary arteries from hypoxic pulmonary hypertension rats may provide a new insight for curing hypoxic pulmonary hypertension. |
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