Effects On Electrophysiology Of Rat Ventricular Myocytes And Intracellar Calcium Concentrations Of Cardiocytes And Vascular Smooth Muscle Cells By Enantioselective Amlodipines

Objective Amlodipine (Aml) is a calcium channel blocker of dihydropyridine commonly used in clinical practice. It is widely utilized to deal with cardiovascular diseases. Aml has three enantiomers, i.e., S-Aml, R-Aml, and R, S-Aml; however, the mechanisms of these three enantiomers are still not clear completely. The study was to investigate Aml effects on resting potentials (RP), action potentials (AP), ionic currents of rat ventricular myocytes and effects on intracellar calcium concentrations of rat cardiocytes and vascular smooth muscle cells by patch clamp technique and fluorescent assay technique. Investigating Aml mechanisms from the level of cell, molecule, and ion can provide theoretical evidences for applying different enantiomers rationally in clinical practice.Methods (1) RP, AP, sodium current (I_Na), L-type calcium current (I_Ca-L), transient outward potassium current (I_to), delayed rectification potassium current (I_k), and inwardly rectified potassium current (I_k1) of normal rat ventricular myocytes were respectively recorded by patch clamp after"four-step"enzyme digestion method, i.e., perfusion with Tyrode's solution without Ca2+, perfusion with 50μmol/L Ca2+, perfusion with 200μmol/L Ca2+, incubation with KB solution in room temperature. Effects on RP, AP, I_Na, I_Ca-L, I_to, I_k and I_k1 were observed by addition of 0.1, 0.5, 1, 5 and 10μmol/L different enantiomers of Aml respectively.(2) Intracellar calcium concentrations of ventricular myocytes were measured by fluorescent probe Fura-2/AM after ventricular myocytes were isolated by enzyme digestion. Effects on intracellar calcium concentrations were observed by addition of 0.1, 0.5, 1, 5 and 10μmol/L different enantiomers of Aml respectively. (3) Intracellar calcium concentrations of smooth muscle cells (SMCs) were assayed by fluorescent probe Fura-2/AM after SMCs were isolated by"two-step"enzyme digestion, i.e., digested in enzyme I for about 30 min, and then for about 15 min in enzyme II. Effects on intracellar calcium concentrations were studied by addition of 0.1, 0.5, 1, 5 and 10μmol/L S-Aml, R-Aml, and R, S-Aml respectively.Results (1) Isolation of rat ventricular myocytes:If the whole heart remained red in the process of isolation, the numbers of total cell and calcium-tolerant cell were more than 90% and about 70%. The numbers of total cell and calcium-tolerant cell in local red parts were more than 80% and about 60% respectively, however, there were a great varieties in number in pale part, which were generally less than 50% and calcium-tolerant cells were less. If the whole heart remained pale in the process of isolation, the numbers of total cell were less than 30% and almost no calcium-tolerant cells.(2) RP, AP and ionic currents of normal rat ventricular myocytes: a. RP in epicardial, mid-cardial and endocardial ventricular myocytes were -75.8±9.5 mV (n=87), -76.3±8.4 mV (n=75) and–75.4±7.8 mV (n=68) respectively, which was no significance (P>0.05). b.25%, 50% and 90% of action potential durations (APD) in epicardial cardiocytes were 3.6±1.2ms, 10.3±2.1ms and 46.3±4.8ms (n=50). APD₂₅, APD₅₀ and APD₉₀ in mid-cardial cardiocytes were 6.4±1.8ms, 14.7±2.4ms and 69.4±8.3ms(n=58). APD₂₅, APD₅₀ and APD₉₀ in endocardial ventricular myocytes were 13.8±2.1ms, 45.3±10.2ms and 152.1±33.4ms (n=62) respectively. APD in epicardial, mid-cardial and endocardial ventricular myocytes showed significant differences (P<0.05). c. Peak currents and current densities of I_to in epicardial, mid-cardial and endocardial ventricular myocytes were different. I_to currents at +70mV were 8925.0±2399.3pA (n=57), 4373.1±830.2pA (n=51) and 1843.3±542.4pA (n=60) in epicardial, mid-cardial and endocardial ventricular myocytes, and their corresponding current densities were 59.50±15.99pA/pF, 29.15±5.53pA/pF and 12.29±3.62 pA/pF (P<0.05), however, half-activated voltage, half-inactivated voltage and recovered time from inactivation were all no distinction in epicardial, mid-cardial and endocardial ventricular myocytes (P>0.05, n=6⁸). d.Peak currents, current densities, I-V curves, stably activated curves, stably inactivated curves and recovered curves from inactivation of I_Na,I_Ca-L,I_k and I_k1 were no remarkable significance in epicardial, mid-cardial and endocardial ventricular myocytes (P>0.05, n=6¹0).(3) Effects on RP, AP and ionic currents of cardiocytes by Aml: a. RP, AP maximal velocity, AP amplitude and AP overshoot were not significant (P>0.05, n=8¹0), but APD changed after application 0.1, 0.5, 1, 5 and 10μmol/L S-Aml. APD₂₅ were 11.0±1.7ms, 10.4±1.6ms, 9.2±1.4ms, 6.9±1.0ms and 4.6±0.7ms respectively (P<0.05, n=12). APD₅₀ were 36.2±8.2ms, 33.9±7.7ms, 30.2±6.8ms, 22.6±5.1ms and 15.1±3.4ms respectively (P<0.05, n=7). APD₉₀ were 121.6±26.7ms, 114.0±25.0ms, 101.4±22.2ms, 76.1±16.7ms and 50.7±11.1ms respectively (P<0.05, n=7). APD also changed after application 0.1, 0.5, 1, 5 and 10μmol/L of R, S-Aml. APD₂₅ were 12.4±1.9ms, 10.6±1.6ms, 9.8±1.5ms, 8.0±1.2ms and 6.9±1.0ms respectively (P<0.05, n=10). APD₅₀ were 39.2±9.2ms, 36.7±7.9ms, 33.8±7.2ms, 25.4±5.9ms and 21.7±5.2ms respectively (P<0.05, n=10). APD₉₀ were 138.9±30.1ms, 125.4±25.8ms, 110.5±23.6ms, 88.7±19.5ms and 76.8±14.7ms respectively (P<0.05,n=8). On the contrary, APD did not change remarkably with different concentrations of R-Aml (P>0.05, n=9). b. I_Ca-L were gradually blocked, I-V curves were upward, stably activated curves and stably inactivated curves were shifted to the left, and recovered time from inactivation was prolonged with augmentation of S-Aml and R, S-Aml (P<0.05, n=6¹0). I_Ca-L was blocked to 1.5±0.2%, 25.4±5.3%, 65.2±7.3%, 78.4±8.1% and 94.2±5.0% with 0.1, 0.5, 1, 5 and 10μmol/L of S-Aml under holding potential (HP) equal to -40mV (P<0.05, n=10), and 50% inhibition concentration (IC₅₀) of S-Aml was 0.62±0.12μmol/L. I_Ca-L was blocked to 0.9±0.1%, 10.4±3.2%, 69.1±5.3%, 75.2±7.0% and 81.6±6.4% with 0.1, 0.5, 1, 5 and 10μmol/L of R, S-Aml (P<0.05, n=8), and IC₅₀ of R, S-Aml was 1.32±0.04μmol/L. I_Ca-L was blocked to 1.2±0.2%, 20.4±4.3%, 61.2±6.4%, 75.1±8.2% and 90.1±5.0% with 1, 5, 10, 50 and 100μmol/L of S-Aml under HP equal to -80mV (P<0.05, n=8), and IC₅₀ of S-Aml was 7.26±1.5μmol/L. I_Ca-L was blocked to 0.8±0.1%, 10.8±2.1%, 20.5±5.3%, 67.2±6.1% and 76.1±6.9% with 1, 5, 10, 50 and 100μmol/L of R, S-Aml (P<0.05, n=8), and IC₅₀ of R, S-Aml was 15.01±2.1μmol/L. Effects of S-Aml on I_Ca-L and channel dynamics were more significant than those of R, S-Aml at the same concentrations, but R-Aml at different concentrations did not have any effect on I_Ca-L and channel dynamics (P>0.05, n=6¹0). c. S-Aml, R-Aml and R, S-Aml at different concentrations did not have any effect on I_Na, I_to, I_k, I_k1 and their channel dynamics (P>0.05, n=6¹0). d. S-Aml and R,S-Aml from 0.1μmol/L to 10μmol/L had no effects on I_Na, peak current, I-V curve, stably activated curve, stably inactivated curve, recovered curve from inactivation and channel dynamic parameters (P>0.05, n=10¹5). 40⁵0% peak currents of I_Na could be blocked when applications of 100μmol/L S-Aml or and 200μmol/L R,S-Aml (P<0.05,n= 8¹0).(4) Effects on intracellar calcium concentrations of cardiocytes by Aml: 1.1±0.1%, 15.3±3.8%, 58.1±5.2%, 73.5±5.7% and 88.9±7.2% intracellar calcium concentrations were reduced by addition of 1, 5, 10, 50and 100μmol/L S-Aml respectively, and IC₅₀ of S-Aml on ventricular myocytes was 8.13±1.02μmol/L (P<0.05, n=10). 0.6±0.1%, 5.1±2.0%, 15.2±3.7%, 65.8±4.1% and 72.1±5.2% were reduced by addition of R, S-Aml with same concentrations, and IC₅₀ was 16.19μmol/L (P<0.05, n=8). However, R-Aml at different concentrations did not have any effect on intracellar calcium concentrations of ventricular myocytes (P>0.05, n=8).(5) Effects on intracellar calcium concentrations of SMCs by Aml: 10.3±1.2%,35.2±3.5%,60.1±5.0%,78.9±6.1% and 91.2±7.6% intracellar calcium concentrations of SMCs were reduced by addition of 0.1, 0.5, 1, 5 and 10μmol/L S-Aml respectively, and IC₅₀ of S-Aml on SMCs was 0.72±0.10μmol/L (P<0.05, n=8). 3.4±0.8%,20.7±2.1%,36.4±3.3%,68.9±5.4% and 81.3±6.2% intracellar calcium concentrations of SMCs were reduced by addition of R, S-Aml with same concentrations, and IC₅₀ was 1.51±0.21μmol/L (P<0.05, n=6). However, R-Aml at different concentrations did not have any effect on intracellar calcium concentrations of SMCs (P>0.05, n=6).Conclusion (1) A large number of calcium-tolerant cells with normal electrophysiology can be obtained, if this"four-step"enzyme digestion method is adopted strictly in the process of isolation. (2) AP and I_to are different in epicardial, mid-cardial and endocardial ventricular myocytes of normal rat. APDs are gradually prolonged and I_to currents are shortened by degrees from epicardium to endocardium. However, there are no differences of I_Na,I_Ca-L,I_k and I_k1 from epicardium to endocardium.(3) a. S-Aml and R, S-Aml have blocked effects on I_Ca-L, and S-Aml has more powerful effects on I_Ca-L and channel dynamics than those of R, S-Aml at the same concentrations, while R-Aml has no effects on I_Ca-L and channel dynamics. b. S-Aml and R, S-Aml decurtate APDs by blocking I_Ca-L, but R-Aml has no effects on APDs because it has not any blocked effects on I_Ca-L. c. S-Aml and R, S-Aml with low concentrations do not block I_Na, while they can block I_Na with high concentrations. R-Aml with different concentrations has no effects on I_Na. d. S-Aml, R-Aml and R, S-Aml with different concentrations all have no effects on I_to, I_k, I_k1 and their channel dynamics.(4) S-Aml and R, S-Aml can reduce intracellar calcium concentrations of rat ventricular myocytes by blocking L-type calcium channel. R-Aml has no effects on intracellar calcium concentrations because it can not block L-type calcium channel.(5) a. A lot of rat aortic smooth muscle cells with good quality can be obtained by"two-step"enzyme digestion method. b. Compared with rat ventricular myocytes, S-Aml and R, S-Aml can reduce intracellar calcium concentrations of rat SMCs by blocking L-type calcium channel more easily. R-Aml has no effects on intracellar calcium concentrations of SMCs, because it can not block L-type calcium channel of SMCs either.