| Chan Su, a traditional Chinese medicine prepared from the dried white secretion of auricular glands and skin glands of Chinese toads(Bufomelanostictus Schneider or Bufo bufo gargarizans cantor), have attracted the attention of many clinicians due to its diverse biological activities, such as cardiotonic, antitumor. The chemical structure of CBG(C26H34O6), the main active ingredient isolated from Chan Su, has been determined(Fig. 1). Chan Su is not only used as a traditional Chinese medicine, but it is also listed in Pharmacopoeia of People’s Republic of China(PRC; State Pharmacopoeia Committee, 2010). Extracts of Chan Su, also known as dried toad venom in English, "toad cake" in China, "Senso" in Japan and "Somsa" in Korea are used in cardiac therapies.Although the pharmacological profile of Chan Su has been extensively characterized, research on Chan Su has mainly focused on its toxicity. Chan Su in high doses causes cardiac arrhythmia, seizure and coma. Death has been reported of one woman after the consumption of Chinese herbal tea and of another woman following ingestion of Lu-Shen-Wan pills containing Chan Su. Therefore, the use of Chan Su by the general population was recommended to be avoided due to its toxicity. Interestingly, formulations of Chan Su have long been widely applied in China, Japan and other Asian countries and are currently used as a major component of other traditional medicines such as Kyushin and Shexiang Baoxin Pill(SBP). Kyushin and SBP have been successfully used for many years in the treatment of numerous ailments, especially coronary artery heart disease(CHD) such as angina, coronary artery spasm and myocardial infarction(MI). In addition, chemical analyses have shown intact CBG and its metabolites in plasma and urine of healthy volunteers receiving a drug Kyushin containing Chan Su by enzyme immunoassay after the separation of these compounds by high performance liquid chromatography(HPLC). CBG also has been detected in SBP by HPLC. SBP has demonstrated synergistic effects in rats with MI with a therapeutic mechanism based on inhibiting dysfunctions in energy metabolism, oxidative injury and inflammation in the development of MI. Therefore, the current clinical evidence for these medicines supports the potential cardioprotective effects of CBG.As a major steroid compound isolated from Chan Su, CBG has been reported by some to exert its pharmacological effects on delayed rectifier potassium channels in primary cultures of rat hippocampus neurons. Nevertheless, direct evidence for the effects of CBG on L-type calcium channels(LTCC) are lacking. Increased contractility of ventricular myocytes is a well-known central feature of the cardiac response to ischemic myocardial diseases. In pathological conditions such as ischemia, the amount of Ca2+ that crosses the sarcolemma exceeds the Ca2+ sequestration and extrusion capacity of the cell, leading to the disturbance of the intracellular Ca2+([Ca2+]i) homeostasis. This Ca2+ overload is characterized by a rise in [Ca2+]i to a level that triggers pathological events in the cell, such as arrhythmias, mechanical dysfunction(e.g., increased diastolic tension, reduced peak force and relaxation rate) and eventually cell death. In addition, the opening of LTCCs is associated with Ca2+ overload, and LTCC blockers have generally proven to be effective when given during the ischemic period, illustrating their energy sparing properties. Since drugs that suppress cardiac LTCC and contractility, such as Ca2+ antagonists and β-adrenoceptor blocking agents, are able to effectively protect the myocardium from ischemic injury, we speculated that CBG exerts its cardioprotective effects via inhibition of LTCCs and cardiac contractility.We examined the effects of CBG on LTCC, Ca2+ transient, and contractility of isolated adult rat ventricular myocytes. Therefore, weused the whole-cell patch-clamp technique and video-based edgedetection and dual excitation fluorescence photomultiplier systems in thisstudy to study the influence of CBG on LTCC, Ca2+ transient, contractility in rat ventricular myocytes. Additional research on the cellular mechanisms of CBG will not only contribute to a better understanding of the efficacies of Chan Su in clinical treatments, but also provide experimental evidence for rational applications of Kyushin and SBP.Objective: To investigate the effect of cinobufagin on L-typecalcium current, Ca2+ homeostasis and contractility.Methods: we used the whole-cell patch-clamp technique and video-based edge detection and dual excitation fluorescence photomultiplier systems in this study to study the influence of CBG on LTCC, Ca2+ transient, contractility in rat ventricular myocytes.Results:1 Confirmation of ICa-LThe recored currents were completely blocked by 10-7 M Verapamil, which indicated that the currents were calcium currents. The calcium currents were blocked by 10-8 M CBG and it could partly recover after washed(P < 0.01 and P < 0.001, respectively).2 Dose-dependent effects of CBG on ICa-LCBG reduced the ICa-L in a concentration-dependent manner.(10-12, 10-11, 10-10, 10-9 and 10-8 M) CBG reduced the ICa-L by 14.8%, 18.8 %, 27.8 %, 38.4% and 47.9 %, respectively. CBG reduced ICa,L in a concentration-dependent manner with an IC50 of 4 × 10-10 M.3 Effects of CBG on current-voltage relationship of ICa-LThe I-V relationship for Ca2+ currents between-60 and 60 m V revealed that the normalized currents decreased from1 p A to-0.72 ± 0.02 p A and-0.53 ± 0.04 p A in the presence of at 10-10 M CBG, 10-8 M CBG, respectively.4 Effects of CBG on ICa-L of ischemic ventricular myocytesThe peak amplitude of ICa-L was decreased by 29.99 ± 2.78% and 51.57 ± 3.12% by CBG derivatives at 10-10 and 10-8 M, respectively(P < 0.05 and P < 0.001, respectively).5 Effects of CBG on steady-state activation and inactivation of ICa-LCBG caused a significant leftward(hyperpolarizing) shift in the voltage dependence of the ICa-L activation curve. The V1/2 value for activation in the control was-46.90 ± 1.21 m V with a slope factor(k) of 7.95 ± 1.07 m V. Meanwhile, V1/2 values for activation with 10-10 and 10-8 M CBG were-52.02 ± 0.80 m V with a k value of 6.77 ± 0.71 m V and-53.44 ± 0.60 m V with a k value of 6.78 ± 0.55 m V, respectively, compared with the control group. CBG also caused a marked hyperpolarizing leftward shift in the voltage dependence of the ICa-L inactivation curve. The result shows the V1/2 value for inactivation was-26.5 ± 0.61 m V with a k value of 3.98 ± 0.45 m V. In the presence of 10-10 and 10-8 M CBG, V1/2 values for inactivation were-29.02 ± 0.50 m V with a k value of 3.82 ± 0.54 m V and-30.70 ± 0.10 m V with a k value of 4.60 ± 0.07 m V, respectively.6 Effects of CBG on Cell Contraction and Ca2+ Transient in Rat Ventricular MyocytesCBG(10-8 M) decreased the amplitudes of myocyte shortening 21.02% and lessened the peak value of Ca2+ transient 23.33% respectively(P < 0.001).7 Effects of CBG on the time parametes of cell shorteningFrom the time parametes of cell shortening, CBG was found to significantly shorten the time to 50% of the baseline(Tr), the time to 10% of the peak(Tp)(P < 0.001).Conclusions: CBG not only significantly inhibited ICa-L in a concentration-dependent manner but also suppressed calcium transient and contraction in rat ventricular myocytes under physiological conditions. |
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