Peng-Yun Li

Function of BKCa channels is reduced in human vascular smooth muscle cells from Han Chinese patients with hypertension.

Chronic hypertension is associated with an impaired vascular relaxation caused by an increased vascular tone; however, the underlying mechanisms are not fully understood in human patients. The present study was to investigate whether large-conductance Ca2+- and voltage-activated K+ (BKCa) channels are involved in dysfunctional relaxation of artery in Han Chinese patients with hypertension using the perforated patch clamp, inside-out single-channel, and macromembrane patch recording techniques to determine whole-cell current, spontaneous transient outward current, open probability, and Ca2+ sensitivity and the reverse transcription polymerase chain reaction and Western blot analysis to examine the gene and protein expression of &agr;-subunit (KCa1.1) and &bgr;1-subunit (KCNMB1) of BKCa channels in isolated human vascular smooth muscle cells and mesenteric arteries from normotensive and hypertensive patients. It was found that whole-cell current density, spontaneous transient outward current, and Ca2+ sensitivity, but not single-channel open probability and slope conductance, were significantly decreased in vascular smooth muscle cells from patients with hypertension. Interestingly, mRNA and protein levels of KCNMB1, but not KCa1.1, were reduced in the arterial tissue from patients with hypertension. These results demonstrate for the first time that whole-cell current, spontaneous transient outward current, and Ca2+ sensitivity of BKCa channels are reduced in human vascular smooth muscle cells, which resulted from downregulation of &bgr;1-subunit of the channel. This may account, at least in part, for the dysfunction of artery relaxation in Han Chinese patients with primary hypertension.

Activation of high conductance Ca2+-activated K+ channels by sodium tanshinoneII-A sulfonate (DS-201) in porcine coronary artery smooth muscle cells

High conductance Ca(2+) activated K(+) channels (BK(Ca)) in vascular smooth muscles play important roles in controlling the vascular tone by determining the level of membrane potential and Ca(2+) influx through voltage gated Ca(2+) channels. Agents that alter the activity of Ca(2+) channels or BK(Ca) thus affect the vascular tone in both physiological and pathological conditions. Danshen, the dried root of Salvia miltiorrhiza, is a commonly used traditional Chinese medicine and is widely used as an effective remedy for cardiovascular and cerebral vascular diseases partly by its vasodilatation. Sodium tanshinoneII-A sulfonate (DS-201) is a water-soluble derivative of Tanshinone IIA, the main active component of Danshen. The purpose of this study was to explore possible mechanisms of vasodilative effects of DS-201 using porcine coronary artery smooth muscle. DS-201 induced relaxation of the coronary smooth muscle which had been contracted with 30 mM KCl, and the relaxation was inhibited by 100 nM iberiotoxin (IbTX), a specific BK(Ca) channel blocker. Using perforated whole-cell recordings and single channel recordings, effects of DS-201 on BK(Ca) were examined. The results showed that DS-201 activated BK(Ca). Extracellular application of DS-201 at 40, 80 microM under the whole-cell configuration induced increases of the BK(Ca) macroscopic currents by 43.6%, 42.1% respectively, and the spontaneous transient outward K(+) currents (STOCs) by 48.7%, 47.4% respectively. In inside-out patches, bath application of 20-150 muM of DS-201 activated BK(Ca) by 5.4-173.2 fold. These results indicate that the vasodilatation by DS-201 is related to activation of BK(Ca).

Unique action of sodium tanshinone II-A sulfonate (DS-201) on the Ca2+ dependent BKCa activation in mouse cerebral arterial smooth muscle cells

Sodium tanshinone II-A sulfonate (DS-201) is a water-soluble derivative of tanshinone IIA, a main active constituent of Salvia miltiorrhiza which has been used for treatments of cardio- and cerebro-vascular diseases. DS-201 activates large conductance Ca(2+)-sensitive K(+) channels (BK(Ca)) in arterial smooth muscle cells, and reduces the vascular tone. Here we investigated the effect of DS-201 on the BK(Ca) channel kinetics by analyzing single channel currents. Smooth muscle cells were freshly isolated from mouse cerebral arteries. Single channel currents of BK(Ca) were recorded by patch clamp. DS-201 increased the total open probability (NPo) of BK(Ca) in a concentration-dependent manner. But this action required intracellular Ca(2+), and the effect depended on the Ca(2+) concentration ([Ca(2+)](free)). DS-201 activated BK(Ca) with the half maximal effective concentration (EC(50)) of 111.5μM at 0.01μM [Ca(2+)](free), and 68.5μM at 0.1μM [Ca(2+)](free.) The effect of DS-201 on NPo was particularly strong in the range of [Ca(2+)](free) between 0.1 and 1μM. Analysis of the channel kinetics revealed that DS-201 had only the effect on the channel closing without affecting the channel opening, which was a striking contrast to the effect of [Ca(2+)](free), that is characterized by changing the channel opening without changing the channel closing. DS-201 may be bound to the open state of BK(Ca), and have an inhibitory effect on the transition from the open to closed state. By this way DS-201 may enhance the activity of BK(Ca), and exhibit a strong vasodilating effect against vasoconstriction in the range of [Ca(2+)](free) between 0.1 and 1μM.

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca(2+)-activated K(+) channel (BKCa) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BKCa channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K(+) currents (STOC, a component pattern of BKCa currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BKCa currents) via an IP3 receptor- and sarcoplasmic Ca(2+) mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BKCa channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BKCa channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BKCa channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca(2+) sensitivity of the channels.

Different Effects of Hypertension and Age on the Function of Large Conductance Calcium‐ and Voltage‐Activated Potassium Channels in Human Mesentery Artery Smooth Muscle Cells

Background Large‐conductance calcium‐ and voltage‐activated potassium channels (BKC a channels) play important roles in the maintenance of vascular tone, and their dysregulation is associated with abnormal vascular relaxation and contraction. We tested the changes in BKC a channel properties in patients at different ages to assess the effects of hypertension and aging on the functional changes of BKC a channels. Methods and Results Patch clamp was performed to detect the activities of BKC a channels in freshly isolated human mesenteric artery smooth muscle cells from younger patients (aged ≤45 years) without hypertension, older patients (aged ≥65 years) without hypertension, and older patients with hypertension. The expression of mRNA and protein from BKC a channels was evaluated by reverse transcription polymerase chain reaction and Western blot analysis, respectively. Results showed that the whole‐cell current density, spontaneous transient outward current, and Ca2+ sensitivity of the artery smooth muscle cells were significantly decreased in the older patients with hypertension; the decreases were insignificant in the older patients without hypertension, although a clear tendency to have spontaneous transient outward current was detected in these patients. The expression of both mRNA and protein of BKC a subunits α and β1 was significantly decreased in the older patients with hypertension but not in the older patients without hypertension compared with the younger patients without hypertension. Conclusions Our findings demonstrate for the first time that hypertension is an important factor for the pathological alteration of the properties of BKC a channels in human mesenteric artery smooth muscle cells, and aging itself may also be a factor in these changes in the cells.

Sodium tanshinone IIA sulfonate prevents lipopolysaccharide-induced inflammation via suppressing nuclear factor-κB signaling pathway in human umbilical vein endothelial cells

Sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of tanshinone IIA, has been demonstrated to have potent anti-inflammatory properties. However, the protective effects of STS on lipopolysaccharide (LPS)-induced inflammation in endothelial cells remain to be elucidated. In the present study, human umbilical vein endothelial cells (HUVECs) were used to explore the effects of STS on LPS-induced inflammation and the molecular mechanism involved. HUVECs were pretreated with STS for 2 h, followed by stimulation with LPS. Then expression and secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and the activation of nuclear factor-κB (NF-κB) were assessed. The results demonstrated that STS significantly decreased LPS-induced TNF-α and IL-1β protein expression in HUVECs. Similarly, the increased levels of TNF-α and IL-1β in cell supernatants stimulated by LPS were also significantly inhibited by STS. Furthermore, STS inhibited LPS-induced NF-κB p65 phosphorylation and nuclear translocation. All the results suggest that STS prevents LPS-induced inflammation through suppressing NF-κB signaling pathway in endothelial cells, indicating the potential utility of STS for the treatment of inflammatory diseases.

Sodium Tanshinone II-A Sulfonate (DS-201) Induces Vasorelaxation of Rat Mesenteric Arteries via Inhibition of L-Type Ca2+ Channel

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.