| Background:Gallbaldder evacuation dysfunction is a key risk factor for gallstone formation. The sphincter of Oddi (SO) is a smooth muscle with autonomic relaxation and contraction function, encirlcling the end of the bile duct. And it plays important roles in the regulation of gallbladder evacuation and maintenance of normal hydraulic pressure of biliary tract. Our previous study has demonstrated that hypercholesterolemia (HC) initially induced SO dysfunction (SOD), leading to increased gallbladder evacuation resistance and cholestasis and that intracellular calcium concentration ([Ca2+]i) overload potentially play important roles in the pathogenesis of SOD; and HC decreased the outward currents of voltage-dependent potassium channel (Kv) and large conductance calcium-activated potassium channel (BKca) in SO cells. However, the underlying mechanism of HC-induced inhibition of Kv and BKca channel activities remains unkown.Kv1.5 and BKca channels play important roles in regulating [Ca2+]i and tuning smooth muscle cell (SMC) contractility, by setting resting membrane potential and thus modulating opening of L-type voltage-dependent calcium channel (L-VDCC). Many factors affect channel activities, such as protein expression level and phosphorylation/dephosphorylation status of channels. By regulating the phosphorylation status, protein kinases such as calmodulin-dependent protein kinaseⅡ(CaMKⅡ), cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG) and protein tyrosine kinase (PTK) increase BKca channel activity, whereas protein kinase C (PKC) inhibits the channel. In addition, there are several theoretical phosphorylation sites of the above-mentioned protein kinases on BKca channel. Activation of CaMKⅡand PKC are closely related with [Ca2+]i, and the previous study showed that SO cells from hypercholesterolemic rabbits were in the state of [Ca2+]i overload. However, whether HC affects expressions of Kv1.5 and BKca channels and modulates the acitivity of BKca channel through regulating the activities of these two protein kinases is not well defined.To date, the available therapy of SOD, including surgery and endoscopic injection therapy, might affect the physiological function of SO and even induce many complications. Therefore, they are not considered to be good and safe enough. It is important for SOD patients to seek for a more security and effective method. Sodium tanshinone IIA sulphonate (STS), a derivative of tanshinoneⅡA, is isolated from the root of Salvia miltiorrhiza known as Danshen in China. Injection of Salvia miltiorrhiza extract was successfully used for cardiac diseases, such as hypertension and pulmonary arterial hypertension, partly based on its beneficial effects on modulation of SMC channel activities. Our previous study suggested that downregulation of Kv and BKca channel activities, which showed similarities as the pathological basis of hypertension or pulmonary arterial hypertension, resulted in SOD in rabbit model under hypercholesterolemic conditions. However, limited information is available on the effect of STS on SOD and its possible involved mechanism in rabbit HC models.Aims: to investigate the underlying mechanism of downregulation of Kv and BKca channel activities in SO cells and to explore the effect of STS on SO muscle rings from hypercholesterolemic rabbits and mechanism involved in it.Methods: rabbit HC model was successfully established by feeding cholesterol- enriched chows for 12 weeks. The mRNA and protein expression of Kv1.5 and BKca channels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting, respectively. Immunoprecipitation and immunoblotting were used to detect the levels of threonine-, serine-and tyrosine-phosphorylated BKca alpha and beta1 subunits. Protein expressions of calmodulin (CaM), CaMKⅡand p-CaMKⅡwere detected by western blotting. The activities of CaMKⅡand PKC were determined by a radioenzymatic technique. In addition, SO muscle rings precontracted with acetylcholine (Ach) were exposed to cumulative increase in STS concentration and tested for relaxation by measurement of isometric tension. To investigate whether the effect of STS on SO muscle rings precontracted with Ach was related with BKca or Kv channels, some functional test on SO muscle rings were performed on Ach-contracted rings in the presence of IbTX (BKca blocker) and 4-AP (Kv blocker).Results:(1) Twelve-week cholesterol treatment significantly increased the serum cholesterol concentrations in rabbits (> 10 mmol/L), satisfying the standard for the rabbit HC model.(2) HC significantly reduced the mRNA and protein expressions of Kv1.5 channels in SO cells. In contrast, it imposed little effect on expressions of BKcaαorβ1 subunits.(3) HC downregulated the threonine-and tyrosine-phosphorylated BKcaαsubunits,whereas it upregulated the serine-phosphorylated BKcaαsubunits. In comparison, threonine-phosphorylated BKcaβ1 subunits did not change in SO cells from hypercholesterolemic rabbits compared to control rabbits.(4) HC significantly increased the PKC activities of SO cells, and this might attributed to HC-induced upregulation of serine-phosphorylated BKcaαsubunits.(5) HC did not affect the protein expression of CaM and CaMKⅡ. However, it significantly upregulated autophosphorylated CaMKⅡin SO cells.(6) NS1619 (BKca opener, 30μmol/L) partly improved the hyperresponsiveness of SO muscle rings from hypercholesterolemic rabbits with the significant descending response to acetylcholine (Ach). Genistein (PTK inhibitor, 10-5 mol/L) further enhanced the contractile response of SO to Ach and made the cumulative dose-response curve of Ach shift to the left, while incubation of Na3VO4 (protein tyrosine phosphatase inhibitor, 10-4 mol/L) significantly caused a decrease of SO responsiveness, and the cumulative dose-response curve of Ach shifted to the right as compared with HC group. Na3VO4 plus NS1619 treatment shifted the cumulative dose-response curve of Ach to the right and decreased the contractile response of SO to Ach. Genistein-induced increased hyperresponsiveness was partly inhibited by preincubation of NS1619.(7) Various concentrations of STS induced a marked relaxation on of SO muscle rings precontracted with 10-5μmol/L Ach from hypercholesterolemic rabbits in a dose-dependent manner. And this relaxative effects of STS on Ach-precontracted SO were significantly reduced in the presence of IbTX (200 nmol/L). Conversely, 4-AP did not affect the concentration-response curve to STS.Conclusions:(1) Downregulation of Kv1.5 expression and modulation of phosphorylated BKca result in the decreased activities of Kv and BKca channel in SO cells under hypercholesterolmica conditions. And this accounts for, at least in part, [Ca2+]i overload in SO cells and the development of SOD under hypercholesterolemic conditions.(2) Modulation of serine-phosphorylated BKca might result from the increased activity of PKC.(3) BKca plays an important role in the occurrence of SOD under hyperchole- sterolemic conditions. BKca tyrosine phosphorylation mediated by PTK and/or PTP is partly associated with the development of SOD under hypercholesterolemic conditions.(4) STS exhibits relaxing properties on SO muscle rings from hypercholesterolemic rabbits precontracted with Ach. And this might be attributed to involvement of of BKca activation. And it makes STS have potential to develop a new method for treatment of SOD.
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