Inhibitory effect of gallic acid on voltage-gated Na+ channels in rat cardiomyocytes.

Gallic acid (GA) has a protective effect on the cardiovascular system. To study its cardiac electrophysiological effects, voltage-gated Na+ channel currents (INa ) were recorded in rat cardiomyocytes using whole-cell patch clamp techniques. Moreover, the effects of GA on aconitine-induced arrhythmias were assessed using electrocardiograms in vivo. We found that the current-voltage characteristic curve (I-V curve) of INa significantly shifted in the presence of 1, 3, and 10 µmol/L of GA. The peak sodium current density (INa -Peak) was reduced from -84.02 ± 5.68 pA/pF to -65.78 ± 3.96 pA/pF with 1 µmol/L, -54.45 ± 5.18 pA/pF with 3 µmol/L, and -44.20 ± 4.35 pA/pF with 10 µmol/L, respectively. GA shifted the steady-state activation curve of INa and recovery curve to the right and the steady-state inactivation curve to the left. The observed inhibitory effect was comparable to that of amiodarone. GA pre-treatment significantly prolonged the onset of fatal ventricular fibrillation. Our results indicated that GA inhibited INa in rat ventricular myocytes and aconitine-induced arrhythmias in vivo. These results suggest the potential of GA for development as a novel anti-arrhythmic therapeutic.

[Effects of isovitexin on transient outward potassium current of ventricular myocytes in rats].

To investigate the effects of isovitexin Ⅳ on transient outward potassium current in rat ventricular myocytes. In this study, MTT assay was used to investigate the safe range of isovitexin. The results showed that the IC50 of the drug was in the range of 10-30 µmol•L⁻¹, and the drug concentration of 1-3 µmol•L⁻¹ for the patch clamp test was within the safe range. In addition, the single ventricular myocytes were obtained by single-enzymatic hydrolysis through aortic retrograde perfusion. The transient outward potassium current (Ito) of rat ventricular myocytes was guided and measured by whole-cell patch-clamp technique and the changes of current characteristics were recorded after isovite was applied. When the concentration of IV was less than 0.1 µmol•L⁻¹, there was no significant effect on Ito. However, with the increase in the concentration of IV (≥0.3 µmol•L⁻¹), the peak of Ito was decreased gradually, from (32.32±2.9) pA/pF to （25.83±4.3） pA/pF, 1 µmol•L⁻¹ IV and (19.51±3.5) pA/pF, 3 µmol•L⁻¹ IV respectively, with an inhibition effect in a concentration-dependent manner. In the range of 1-3 µmol•L⁻¹, IV down-regulated the I-V curve of Ito significantly. The activation curve showed that IV can enable the maximum half activation potential (V1/2) to move to the positive direction, and the V1/2 was increased from (19.59±1.6) mV to (22.81±1.7) mV and (28.86±1.4) mV at concentration of 1, 3 µmol•L⁻¹, meanwhile the activation curve moved to the right. However, the maximum half inactivating potential (V1/2) of the steady-state inactivation curve of Ito was significantly decreased from (－51.43±0.99) mV to (－61.81±1.3) mV with concentration of 1 µmol•L⁻¹ and (－71.50±1.4) mV with concentration of 3 µmol•L⁻¹. The inactivation time constant of recovery from inactivation (τ) was up-regulated significantly from (94.89±0.73) ms to (118.5±1.5) ms and (162.4±1.4) ms at concentration of 1, 3 µmol•L⁻¹ respectively. Meanwhile IV could enable the inactivation recovery curve to move to the right, which suggested that it can prolong the recovery time from inactivation of the transient outward potassium channel. In conclusion, isovitexin had a high inhibitory effect on Ito in rat ventricular myocytes.

Huangkui capsule in combination with metformin ameliorates diabetic nephropathy via the Klotho/TGF-ß1/p38MAPK signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Huangkui capsule (HKC), extracted from Abelmoschus manihot (L.) medic (AM), as a patent proprietary Chinese medicine on the market for approximately 20 years, has been clinically used to treat chronic glomerulonephritis. Renal fibrosis has been implicated in the onset and development of diabetic nephropathy (DN). However, the potential application of HKC for preventing DN has not been evaluated. AIM OF THE STUDY: This study was designed to investigate the efficacy and underlying mechanisms of HKC combined with metformin (MET), the first-line medication for treating type 2 diabetes, in the treatment of renal interstitial fibrosis. MATERIALS AND METHODS: A rat model of diabetes-associated renal fibrosis was established by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg) combined with a high-fat and high-glucose diet. The rats were randomly divided into five groups: normal control, DN, HKC (1.0 g/kg/day), MET (100 mg/kg/d), and HKC plus MET (1.0 g/kg/day + 100 mg/kg/d). Following drug administration for 8 weeks, we collected blood, urine, and kidney tissue for analysis. Biochemical markers and metabolic parameters were detected using commercial kits. Histopathological staining was performed to monitor morphological changes in the rat kidney. High-glucose-induced human kidney HK-2 cells were used to evaluate the renal protective effects of HKC combined with MET (100 µg/mL+10 mmol/L). MTT assay and acridine orange/ethidium bromide were used to examine cell proliferation inhibition rates and apoptosis. Immunofluorescence assay and Western blot analysis were performed to detect renal fibrosis-related proteins including Klotho, TGF-ß1, and phosphorylated (p)-p38. RESULTS: Combination therapy (HKC plus MET) significantly improved the weight, reduced blood glucose (BG), blood urea nitrogen (BUN), total cholesterol (T-CHO), triglycerides (TG), low-density lipoprotein (LDL) and increased the level of high-density lipoprotein (HDL) of DN rats. Combination therapy also significantly reduced urine serum creatinine (SCR) and urine protein (UP) levels as well as reduced the degrees of renal tubule damage and glomerulopathy in DN rats. Combination therapy ameliorated renal fibrosis, as evidenced by reduced levels of alpha-smooth muscle actin and fibronectin and increased expression of E-cadherin in the kidneys. Moreover, HKC plus MET alleviated the degree of DN in part via the Klotho/TGF-ß1/p38MAPK signaling pathway. In vitro experiments showed that combination therapy significantly inhibited cell proliferation and apoptosis and regulated fibrosis-related proteins in high-glucose (HG)-induced HK-2 cells. Further studies revealed that combination therapy suppressed cell proliferation and fibrosis by inhibiting the Klotho-dependent TGF-ß1/p38MAPK pathway. CONCLUSIONS: HKC plus MET in combination suppressed abnormal renal cell proliferation and fibrosis by inhibiting the Klotho-dependent TGF-ß1/p38MAPK pathway. Collectively, HKC combined with MET effectively improved DN by inhibiting renal fibrosis-associated proteins and blocking the Klotho/TGF-ß1/p38MAPK signaling pathway. These findings improve the understanding of the pathogenesis of diabetes-associated complications and support that HKC plus MET combination therapy is a promising strategy for preventing DN.

Inhibition of voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells by the class Ic antiarrhythmic agent lorcainide.

Voltage-dependent K+ (Kv) channels play the role of returning the membrane potential to the resting state, thereby maintaining the vascular tone. Here, we used native smooth-muscle cells from rabbit coronary arteries to investigate the inhibitory effect of lorcainide, a class Ic antiarrhythmic agent, on Kv channels. Lorcainide inhibited Kv channels in a concentration-dependent manner with an IC50 of 4.46 ± 0.15 µM and a Hill coefficient of 0.95 ± 0.01. Although application of lorcainide did not change the activation curve, it shifted the inactivation curve toward a more negative potential, implying that lorcainide inhibits Kv channels by changing the channels' voltage sensors. The recovery time constant from channel inactivation increased in the presence of lorcainide. Furthermore, application of train steps (of 1 or 2 Hz) in the presence of lorcainide progressively augmented the inhibition of Kv currents, implying that lorcainide-induced inhibition of Kv channels is use (state)-dependent. Pretreatment with Kv1.5 or Kv2.1/2.2 inhibitors effectively reduced the amplitude of the Kv current but did not affect the inhibitory effect of lorcainide. Based on these results, we conclude that lorcainide inhibits vascular Kv channels in a concentration and use (state)-dependent manner by changing their inactivation gating properties. Considering the clinical efficacy of lorcainide, and the pathophysiological significance of vascular Kv channels, our findings should be considered when prescribing lorcainide to patients with arrhythmia and vascular disease.

Inhibitory effects of aloperine on voltage-gated Na+ channels in rat ventricular myocytes.

Aloperine (ALO), a quinolizidine alkaloid extracted from Sophora alopecuroides L., modulates hypertension, ventricular remodeling, and myocardial ischemia. However, few studies have evaluated the effects of ALO on other cardiovascular parameters. Accordingly, in this study, we used a rat model of aconitine-induced ventricular arrhythmia to assess the effects of ALO. Notably, ALO pretreatment delayed the onset of ventricular premature and ventricular tachycardia and reduced the incidence of fatal ventricular fibrillation. Moreover, whole-cell patch-clamp assays in rats' ventricular myocyte showed that ALO (3, 10, and 30 µM) significantly reduced the peak sodium current density of voltage-gated Na+ channel currents (INa) in a concentration-dependent manner. The gating kinetics characteristics showed that the steady-state activation and recovery curve were shifted in positive direction along the voltage axis, respectively, and the steady-state inactivation curve was shifted in negative direction along the voltage axis, i.e., which was similar to the inhibitory effects of amiodarone. These results indicated that ALO had anti-arrhythmic effects, partly attributed to INa inhibition. ALO may act as a class I sodium channel anti-arrhythmia agent.

[Effect of Compound Rhubarb Mixture on contraction of isolated small intestinal smooth muscle of rabbit].

OBJECTIVE: To investigate the effect of Compound Rhubarb Mixture on contraction of isolated small intestinal smooth muscle of rabbit. METHODS: Serum containing Compound Rhubarb Mixture was prepared. The contraction curves of isolated small intestinal smooth muscle before and after administration of the serum were recorded by Medlab biological information collecting system. RESULTS: The serum containing Compound Rhubarb Mixture amplified the contraction amplitude of isolated small intestinal smooth muscle of rabbit. The rate of change of contraction amplitude was elevated significantly after administration, while the frequency of contraction did not change obviously. CONCLUSION: The serum containing Compound Rhubarb Mixture can enhance the contractive function of isolated small intestinal smooth muscle of rabbit.

Characterization of transient outward K+ current and ultra-rapid delayed rectifier K+ current in isolated human atrial myocytes from patients with congestive heart failure.

AIM: To study the properties of transient outward K+ current (Ito) and ultra-rapid delayed rectifier K+ current (IKur) in isolated human atrial myocytes from patients with congestive heart failure (CHF). METHODS: Single cells were isolated from CHF patients with collagenase and protease. Ito and IKur were recorded using whole cell patch-clamp technique. RESULTS: The activation and inactivation of I(to) were voltage-dependent and time-dependent. The half-activation and half-inactivation voltage were (15 +/- 12) mV and (-45 +/- 4) mV respectively. When membrane potential went up from -40 mV to +60 mV, the activation time constant means decreased from (6.9 +/- 2.3) ms to (1.40 +/- 0.20) ms, while the inactivation time constant means decreased from (69 +/- 17) ms to (21 +/- 14) ms. Otherwise, the mean reactivation time constants was (125 +/- 65) ms when the membrane potential was held at -80 mV, but the recovery was not complete during the interval observed. Ito showed less frequency-dependent reduction at test frequency between 0.2-2 Hz. Compared with Ito, the activation of IKur only showed voltage-dependence, without time-dependence. Its mean current densities was (3.4 +/- 0.7) pA/pF when test potential was +60 mV. The half activation voltage of IKur was (23 +/- 14) mV. No clear frequency-dependence was observed at the same frequency range of Ito either. CONCLUSION: I(to) and IKur are important outward potassium channel currents in isolated human atrial myocytes from CHF patients and they have different kinetic properties.