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Background: We previously have proved that sodium tanshinone II-A sulfonate (DS- 201), a derivative of traditional Chinese medicinal herb Danshen (Salvia miltiorrhiza), is an opener and vasodilator of BKCa channel in the vascular smooth muscle cells (VSMCs). Vascular tension is closely associated with Ca2+dynamics and activation of BKCa channel may not be the sole mechanism for the relaxation of the vascular tension by DS-201. Therefore, we hypothesized that the vasorelaxing effect of DS-20 may be also related to Ca2+ channel and cytoplasmic Ca2+ level in the VSMCs. Methods: Arterial tension was measured by Danish Myo Technology (DMT) myograph system in the mesentery vessels of rats, intracellular Ca2+level by fluorescence imaging system in the VSMCs of rats, and L-type Ca2+ current by patch clamp technique in Ca2+ channels transfected human embryonic kidney 293 (HEK-293) cells. Results: DS-201 relaxed the endothelium-denuded artery rings pre-constricted with PE or high K+ and the vasorelaxation was reversible. Blockade of K+ channel did not totally block the effect of DS-201 on vasorelaxation. DS-201 suppressed [Ca2+]i transient induced by high K+ in a concentration-dependent manner in the VSMCs, including the amplitude of Ca2+ transient, the time for Ca2+ transient reaching to the [Ca2+]i peak and the time to remove Ca2+ from the cytoplasm. DS-201 inhibited L-type Ca2+ channel with an EC50 of 59.5 μM and at about 40% efficacy of inhibition. However, DS-201did not significantly affect the kinetics of Ca2+ channel. The effect of DS-201 on L-type Ca2+channel was rate-independent. Conclusion: The effect of DS-201 on vasorelaxation was not only via activating BKCa channel, but also blocking Ca2+ channel and inhibiting Ca2+ influx in the VSMCs of rats. The results favor the use of DS-201 and Danshen in the treatment of cardiovascular diseases clinically. [ABSTRACT FROM AUTHOR]

Vascular wall-resident stem cells (VW-SCs) play a critical role in maintaining normal vascular function and regulating vascular repair. Understanding the basic functional characteristics of the VW-SCs will facilitate the study of their regulation and potential therapeutic applications. The aim of this study was to establish a stable method for the isolation, culture, and validation of the CD34 + VW-SCs from mice, and to provide abundant and reliable cell sources for further study of the mechanisms involved in proliferation, migration and differentiation of the VW-SCs under various physiological and pathological conditions. The vascular wall cells of mouse aortic adventitia and mesenteric artery were obtained by the method of tissue block attachment and purified by magnetic microbead sorting and flow cytometry to obtain the CD34 + VW-SCs. Cell immunofluorescence staining was performed to detect the stem cell markers (CD34, Flk-1, c-kit, Sca-1), smooth muscle markers (SM22, SM MHC), endothelial marker (CD31), and intranuclear division proliferation-related protein (Ki-67). To verify the multipotency of the isolated CD34 + VW-SCs, endothelial differentiation medium EBM-2 and fibroblast differentiation medium FM-2 were used. After culture for 7 days and 3 days respectively, endothelial cell markers and fibroblast markers of the differentiated cells were evaluated by immunofluorescence staining and q-PCR. Furthermore, the intracellular Ca 2+ release and extracellular Ca 2+ entry signaling were evaluated by TILLvisION system in Fura-2/AM loaded cells. The results showed that: (1) High purity (more than 90%) CD34 + VW-SCs from aortic adventitia and mesenteric artery of mice were harvested by means of tissue block attachment method and magnetic microbead sorting; (2) CD34 + VW-SCs were able to differentiate into endothelial cells and fibroblasts in vitro; (3) Caffeine and ATP significantly activated intracellular Ca 2+ release from endoplasmic reticulum of CD34 + VW-SCs. Store-operated Ca 2+ entry (SOCE) was activated by using thapsigargin (TG) applied in Ca 2+ -free/Ca 2+ reintroduction protocol. This study successfully established a stable and efficient method for isolation, culture and validation of the CD34 + VW-SCs from mice, which provides an ideal VW-SCs sources for the further study of cardiovascular diseases.

Background: We previously have proved that sodium tanshinone II-A sulfonate (DS-201), a derivative of traditional Chinese medicinal herb Danshen (Salvia miltiorrhiza), is an opener and vasodilator of BK Ca channel in the vascular smooth muscle cells (VSMCs). Vascular tension is closely associated with Ca 2+ dynamics and activation of BK Ca channel may not be the sole mechanism for the relaxation of the vascular tension by DS-201. Therefore, we hypothesized that the vasorelaxing effect of DS-20 may be also related to Ca 2+ channel and cytoplasmic Ca 2+ level in the VSMCs. Methods: Arterial tension was measured by Danish Myo Technology (DMT) myograph system in the mesentery vessels of rats, intracellular Ca 2+ level by fluorescence imaging system in the VSMCs of rats, and L-type Ca 2+ current by patch clamp technique in Ca 2+ channels transfected human embryonic kidney 293 (HEK-293) cells. Results: DS-201 relaxed the endothelium-denuded artery rings pre-constricted with PE or high K + and the vasorelaxation was reversible. Blockade of K + channel did not totally block the effect of DS-201 on vasorelaxation. DS-201 suppressed [Ca 2+ ] i transient induced by high K + in a concentration-dependent manner in the VSMCs, including the amplitude of Ca 2+ transient, the time for Ca 2+ transient reaching to the [Ca 2+ ] i peak and the time to remove Ca 2+ from the cytoplasm. DS-201 inhibited L-type Ca 2+ channel with an EC 50 of 59.5 μM and at about 40% efficacy of inhibition. However, DS-201did not significantly affect the kinetics of Ca 2+ channel. The effect of DS-201 on L-type Ca 2+ channel was rate-independent. Conclusion: The effect of DS-201 on vasorelaxation was not only via activating BK Ca channel, but also blocking Ca 2+ channel and inhibiting Ca 2+ influx in the VSMCs of rats. The results favor the use of DS-201 and Danshen in the treatment of cardiovascular diseases clinically.

This study was aimed to establish a method to create a stable planar lipid bilayer membranes (PLBMs), in which large conductance calcium-activated potassium channels (BK Ca ) were reconstituted. Using spreading method, PLBMs were prepared by decane lipid fluid consisting of N 2 -weathered mixture of phosphatidylcholine and cholesterol at 3:1 ratio. After successful incorporation of BK Ca channel into PLBMs, single channel characteristics of BK Ca were studied by patch clamp method. The results showed that i) the single channel conductance of BK Ca was (206.8 ± 16.9) pS; ii) the activities of BK Ca channel were voltage dependent; iii) in the bath solution without Ca 2+ , there was almost no BK Ca channel activities regardless of under hyperpolarization or repolarization conditions; iv) under the condition of +40 mV membrane potential, BK Ca channels were activated in a Ca 2+ concentration dependent manner; v) when [Ca 2+ ] was increased from 1 μmol/L to 100 μmol/L, both the channel open probability and the average open time were increased, and the average close time was decreased from (32.2 ± 2.8) ms to (2.1 ± 1.8) ms; vi) the reverse potential of the reconstituted BK Ca was -30 mV when [K + ] was at 40/140 mmol/L (Cis/Trans). These results suggest that the spreading method could serve as a new method for preparing PLBMs and the reconstituted BK Ca into PLBMs showed similar electrophysiological characteristics to natural BK Ca channels, so the PLBMs with incorporated BK Ca can be used in the studies of pharmacology and dynamics of BK Ca channel.