- Email: [email protected]
Effect of didrovaltrate on l-calcium current in rabbit ventricular myocytes
Online Submissions:http://www.journaltcm.com [email protected]
J Tradit Chin Med 2012 September 15; 32(3): 1-2 ISSN 0255-2922 © 2012 JTCM. All rights reserved.
BASIC INVESTIGATION
Effect of didrovaltrate on l-calcium current in rabbit ventricular myocytes
Qiang Xie, Weihua Li, ZhengRong Huang, Ziguan Zhang aa Qiang Xie, Weihua Li, ZhengRong Huang, Ziguan Zhang, Department of Cardiology, the First Affiliated Hospital of Xiamen University, Xiamen 361003, China Supported by the Chinese National Science Foundation (No. 81170090), the Science Foundation for Distinguished Young Scholars of Fujian Province (No. 2009D015); the Science Foundation for Distinguished Young Scholars of Xiamen (No. 3502Z20116009), and the Science Foundation of Science and Technology of the Bureau of Xiamen (No. 3502Z20094006) Correspondence to: Prof. Zhengrong Huang, Department of Cardiology, the First Affiliated Hospital of Xiamen University, Xiamen 361003, China. [email protected]. cn Telephone: +86-13606028353 Accepted: March 24, 2012
trate did not affect the activation curve. CONCLUSION: Didrovaltrate blocks ICa-L in a concentration-dependent manner and probably inhibits ICa-L in its inactive state, which may contribute to its cardiovascular effect.
© 2012 JTCM. All rights reserved. Key words: Didrovaltrate; Patch-clamp techniques; Calcium channels, L-Type; Myocytes; Cardiac
INTRODUCTION The mildly sedating effects of valerian (Valeriana officinalis) and its many related Valerianaceae were noted by Greek, Roman, Chinese, and Indian scholars for more than 1000 years. The chemical composition of valerian includes sesquiterpenes of the volatile oil (including valeric acid), iridoids (valepotriates), alkaloids, furanofuran lignans, and free amino acids, such as γ-aminobutyric acid (GABA), tyrosine, arginine, and glutamine. Although the sesquiterpene components of the volatile oil are believed to be responsible for most of valerian's biological effects, it is likely that all of the active constituents of valerian act in a synergistic manner to produce a clinical response.1 Research into the physiological activity of individual components of valerian has demonstrated direct sedative effects (valepotriates and valeric acid) and interaction with neurotransmitters, such as GABA (valeric acid and unknown fractions).2,3 In recent years, cardiovascular effects of valerian extract, such as hypotensive, negative inotropic, and antiarrhythmic actions, have been reported in many studies.4-6 However, the mechanisms underlying the beneficial effects of valerian extract on hearts have not been clearly established. It is
Abstract OBJECTIVE: To investigate the effect of didrovaltrate on L-type calcium current (ICa-L) in rabbit ventricular myocytes. METHODS: We used the whole cell patch clamp recording technique. RESULTS: Didrovaltrate at concentrations of 30 μg/ L and 100 μg/L significantly decreased peak ICa-L (ICa-Lmax) from (6.01±0.48) pA/pF to (3.45±0.27) pA/pF and (2.16 ± 0.19) pA/pF (42.6% and 64.1% , n=8, P< 0.01), respectively. Didrovaltrate shifted upwards the current-voltage curves of ICa-L without changing their active, peak and reverse potentials. Didrovaltrate affected the steady-state inactivation of ICa-L. The half activation potential (V1/2) was significantly shifted from (-26 ± 2) to (-36 ± 3) mV (n=6, P<0.05), with a significant change in the slope factor (k) (from 8.8 ± 0.8 to 11.1 ± 0.9, n=6, P<0.05). DidrovalJTCM | www. journaltcm. com
442
September 15, 2012 | volume 32 | Issue 3 |
Xie Q et al. Effect of didrovaltrate on l-calcium current in rabbit ventricular myocytes postulated that valerian extract has effects on the voltage-dependent ICa-L. Therefore, the purpose of the present study was to clarify the effects of valerian extract (didrovaltrate, one of the valepotriates) on the voltage-dependent ICa-L in rabbit cardiac myocytes.
adjusted to 7.4 with KOH. Other chemical reagents were purchased from China. Didrovaltrate was a kind gift from Professor Xue Cunkuan of the Geriatrics Institute of Huazhong University of Science & Technology. Electrical recordings Membrane currents and action potentials were recorded using an EPC-9 patch clamp amplifier (HEAK, Germany). Myocytes adhering to glass coverslips were placed in a small chamber mounted on the stage of an inverted microscope and superfused at 1.5 mL/min at room temperature (20-22°C). Action potential recordings were performed at 37°C. Other experiments were performed at room temperature. Only rod-shaped single cells that were quiescent and exhibiting well defined cross-striations were studied. After establishment of whole-cell configuration and measurement of cell capacitance, series resistance (≤10 MΩ) was compensated by 50% to 70%. Junction potentials under these conditions were -3mV and corrected. (ICa-L) was elicited by a series of depolarization steps of 200 ms duration applied in 10-mV increments from a holding potential of -40 mV. Action potentials were recorded in current-clamp mode. Borosilicate glass electrodes (1.0 mm OD) were pulled with a vertical puller (PB-7, Narishige, Tokyo, Japan) and had a resistance of 2-3 MΩ when filled with electrode internal solution. Data acquisition and analysis were carried out using pClamp software (Axon Instruments Inc., Foster City, CA, USA). Current signals were filtered at 3 kHz and sampled at a frequency of 10 kHz. Peak ICa amplitudes were estimated as the difference between the maximal inward current and zero-current.
MATERIALS AND METHODS Cell isolation Single rabbit ventricular myocytes were isolated by an enzymatic dissociation procedure modified from Tytgat.7. New Zealand White rabbits of either sex, weighing 1.0-2.0 kg (Experimental Animal Centre of Medical College, Xiamen University), were anesthetized with 1 mL/kg intramuscularly injected Hypnorm (10 mg/mL fluanisone and 0.315 mg/mL fentanyl citrate; Janssen Pharmaceutics, Tilburg, The Netherlands) and 0.1 mL heparin sodium (5000 U/mL) was injected intravenously. The heart was rapidly removed and mounted on a Langendorff perfusion apparatus. It was retrogradely perfused through the aorta with normal Tyrode's solution for 10 min, followed by an additional 10 min of perfusion with Ca2 +-free Tyrode's solution. Perfusion was then switched to Ca2 +-free Tyrode's solution containing collagenase II (Sigma-Aldrich, USA) for 15 min. The enzymatic solution was recirculated. All solutions were saturated with 100% O2 and the temperature was maintained at 37° C. Subsequently, the ventricles were minced and gently agitated in a beaker with low-Ca2 + Tyrode's solution to obtain single myocytes. The calcium concentration was then increased stepwise by replacing approximately 75% of the low-Ca2+ Tyrode's solution by normal Tyrode's solution. This procedure was repeated four times at intervals of 10 min. The cells were placed in a high-K + storage solution and gently triturated with a Pasteur pipette. Isolated myocytes were kept in the medium for at least 1 h at room temperature (20-22°C) before use.
Statistical analyses Data are expressed as the mean±SD. Statistical analysis was performed using paired t-tests to determine statistical significance. A probability of P≤0.05 was considered significant.
Solutions and drugs The normal Tyrode's solution contained (mM): NaCl 140, KCl 5.4, MgCl2 1.0, HEPES 5.0, and glucose 10. The pH was adjusted to 7.4 with NaOH. The pipette solution contained (mM): CsOH 110, TEACl 15, CsCl 20, MgCl2 1.0, egtazic acid (EGTA, Sigma-Aldrich, USA) 5.0, pyruvic acid 5.0, MgATP 5.0, and HEPES 5.0 at pH 7.3 (adjusted with Tris). The external solution contained (mM): NMDG-Cl 140, MgCl2 1.0, CaCl2 1.8, HEPES 5.0, glucose 10, tetrodotoxin (TTX; Hebei Aquatic product Research Institute), and 0.002 and 4-aminopyridine (4-AP, Sigma-Aldrich, USA) 2.0. The pH was adjusted to 7.4 with NMDG. KB solution contained (mM): KOH 110, taurine 10, oxalic acid 10, glutamic acid 70, KCl 25, KH2PO4 10, EGTA 5.0, HEPES 5.0, and glucose 10. The pH was JTCM | www. journaltcm. com
RESULTS Effects of didrovaltrate on voltage-dependent ICa-L The effects of didrovaltrate on voltage-dependent ICa-L were examined in single ventricular myocytes (Figure 1). The membrane potential was maintained at -40 mV, and command voltage pulses (200 ms in duration) from – 40 mV to + 60 mV were applied at 0.2 Hz. Didrovaltrate at 30 μg/L and 100 μg/L significantly decreased peak ICa-L (ICa-Lmax) from (6.01±0.48) pA/pF to (3.45 ± 0.27) pA/pF and (2.16 ± 0.19) pA/pF (42.6% and 64.1%, n=8, P<0.01), respectively. After washout, ICa-L gradually returned to near control levels. The inhibitory effect was concentration dependent and was easily recovered after washout with Tyrode's solution for 443
September 15, 2012 | volume 32 | Issue 3 |
Xie Q et al. Effect of didrovaltrate on l-calcium current in rabbit ventricular myocytes +60mv -40mv
A
to its maximal value, V1/2 is the voltage at which the normalized ICa-L amplitude is 0.5, and k is the slope factor. A concentration of 100 μg/L didrovaltrate caused a marked shift of the inactivation curve toward more negative potentials, significantly shifting V1/2 from (-26±2) mV to (-36±3) mV (n=6, P<0.05), with a significant increase in k (from 8.8±0.8 to 11.1±0.9, n=6,
500.pA
-40mv
50.0ms
B
C Figure 2 Effects of didrovaltrate on the current-voltage relationship of ICa-L Step pulses were applied in 10 mV increments from -40 mV to 6 0mV. The holding potential was -40 mV. Each point is the mean±s.d. of the current density (n=7 cells)
D
5 min. Figure 1 Effects of didrovaltrate on voltage-dependent ICa-L Cells were maintained at – 40 mV, and command voltage pulses (200 ms in duration) were applied at 0.2 Hz from – 40 mV to +60 mV. Superimposed original current traces recorded in a single ventricular myocytes are shown. Trace (A): control. Trace (B): in the presence of didrovaltrate 30 μg /L. Trace (C): in the presence of didrovaltrate 100 μg/L. Trace (D): after washout.
P<0.05). A concentration of 100 μg/L didrovaltrate did not affect the activation curve (data not shown).
Effect of didrovaltrate on the current-voltage relationship of ICa-L The current-voltage relationship of ICa-L and the didrovaltrate current-voltage (I-V) curve of L-type current were obtained by a number of depolarizing step pulses (200 ms) from the holding potential of – 40 mV to test potentials between –40 mV and 60 mV. The test step pulses were delivered in 10 mV increments. ICa-L was activated at –30 mV and the peak amplitude occurred at the potential of 0 mV. Didrovaltrate at 30 μg/ L and 100 μg/L shifted the I-V curve upwards without changes in their active, peak and reverse potentials (Figure 2).
Figure 3 Effect of didrovaltrate on the steady-state inactivation curve of ICa-L Currents were elicited by a 300 ms test pulse to +20 mV after 2 s of conditioning prepulses from –80 to +20 mV in 10 mV increments. Curves were obtained by simulating the voltage dependent changes in I/Imax using the Boltzmann equation: I/Imax=1/ [1 + exp (Vm-V1/2/k)]. A concentration of 100 μg/L didrovaltrate shifted V1/2 from (-26±2) mV to(-36± 3mV and k from 8.8±0.8 to 11.1±0.9 (n=6, P<0.05).
DISCUSSION Our findings demonstrated that didrovaltrate produced voltage-dependent blockade of ICa-L in a concentration-dependent manner. The steady-state inactivation kinetics of ICa-L were changed and the inactivation curve of ICa-L shifted towards more negative potentials. These findings indicated that didrovaltrate inhibited ICa-L by its effect on inactivation kinetics.
Effect of didrovaltrate on the steady-state inactivation and activation of ICa-L The effect of didrovaltrate on the steady-state inactivation of ICa-L is shown in Figure 3. The results were well fitted by the Boltzmann equation I/Imax=1/ [1 + exp (Vm-V1/2/k)], where I/Imax is the ratio of ICa-L amplitude JTCM | www. journaltcm. com
444
September 15, 2012 | volume 32 | Issue 3 |
Xie Q et al. Effect of didrovaltrate on l-calcium current in rabbit ventricular myocytes Two types of calcium channel have been distinguished on the basis of their electrophysiological and pharmacological characteristics in ventricular myocytes.8,9 The physiological significance of the L-type calcium channel is better understood than that of the T-type calcium channel. Calcium influx through the L-type calcium channel is responsible for the upstroke of the action potential in the sinoatrial node and atrioventricular node, and plays an important role in determining the plateau and eventual spike-dome appearance of the action potential in other cardiac cells. The L-type calcium channel is further responsible for the coupling between excitation and contraction, it induces release of Ca2+ from the sarcoplasmic reticulum, and regulates intracellular Ca2 + load. In this way, the L-type calcium channel determines activity of a number of mitochondrial and cytoplasmic Ca2+-sensitive enzymes.10 The underground organs of members of the genus Valeriana (Valerianaceae), as well as related genera such as Nardostachys, are used in the traditional medicine of many cultures as mild sedatives and tranquillizers and to aid the induction of sleep.11-13 The anticonvulsive, sedative, and hypnotic effects have been variously attributed to valeric acid, valeranone, and valepotriates.4 The chemical composition of valerian includes sesquiterpenes of the volatile oil (including valeric acid), iridoids (valepotriates), alkaloids, furanofuran lignans, and free amino acids, such as GABA, tyrosine, arginine, and glutamine. Although the sesquiterpene components of the volatile oil are believed to be responsible for most of valerian's biological effects, it is likely that all of the active constituents of valerian act in a synergistic manner to produce a clinical response.1 Research into physiological activity of individual components has demonstrated direct sedative effects (valepotriates and valeric acid) and interaction with neurotransmitters, such as GABA (valeric acid and unknown fractions).2,3 In recent years, cardiovascular effects of valerian extract, such as hypotensive, negative inotropic, and antiarrhythmic effects, have been reported in some studies.4-6 Our findings demonstrate that didrovaltrate
JTCM | www. journaltcm. com
inhibits ICa-L, which may contribute to its cardiovascular effects.
REFERENCES 1 2
3
4 5
6
7 8
9
10
11 12 13
445
Houghton PJ. The scientific basis for the reputed activity of valerian. J Pharm Pharmacol 1999; 51(5): 505-512. Hendriks H, Bos R, Allersma DP, Malingre TM, Koster AS. Pharmacological screening of valerenal and some other components of essential oil of valeriana officinalis. Planta Med 1981(1); 42: 62-68. Ortiz JG, Nieves-Natal J, Chavez P. Effects of valeriana officinalis extracts on [3H] flunitrazeparm binding, synaptosomal [3H] GABA uptake, and hippocampal [3H] GABA release. Neurochem Res 1999; 24(11): 1373-1378. Houghton PJ. The biological activity of valerian and related plants. J Ethnopharmacol 1988; 22(2): 121-142. Zhang BH, Meng H, Wang T, Dai YC, Shen J, Tao C. Effect of Valeriana officinalis L extract on cardiovascular system. Yao Xue Xue Bao 1982; 17(5): 382-384. Wang YL, Xu SG, Fan SF. Electrophysiological studies on the antiarrhythmic effect of the water extract of valerian. Zhonghua Xin Xue Guan Bing Za Zhi 1979; 7(4): 275-282. Tatgat J. How to isolate cardiac myocytes. Cardiovasc Res 1994; 28(2): 280-283. Mcdonald TF, Pelzer S, Trautwein W, Pelzer DJ. Regulation andmodulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol Rev 1994; 74(2): 365-507. Bean BP. Two kinds of calcium channels in canine atrial cells. Differences in kinetics, selectivity, and pharmacology. J Gen Physiol 1985; 86(1): 1-30. Carmeliet E. Cardiac ionic currents and acute ischemia: from channels to arrhythmias. Physiol Rev 1999; 79(3): 917-1017. Upton R. Valeriana officinalis. J Altern Complement Med 2001; 7(1): 15-17. Hadley S, Petry JJ. Valerian. Am Fam Physician 2003; 67 (8): 1755-1758. Barrett B, Kiefer D, Rabago D. Assessing the risks and benefits of herbal medicine: an overview of scientific evidence. Altern Ther Health Med 1999; 5(4): 40-49.
September 15, 2012 | volume 32 | Issue 3 |
J Tradit Chin Med 2012 September 15; 32(3): 1-2 ISSN 0255-2922 © 2012 JTCM. All rights reserved.
BASIC INVESTIGATION
Effect of didrovaltrate on l-calcium current in rabbit ventricular myocytes
Qiang Xie, Weihua Li, ZhengRong Huang, Ziguan Zhang aa Qiang Xie, Weihua Li, ZhengRong Huang, Ziguan Zhang, Department of Cardiology, the First Affiliated Hospital of Xiamen University, Xiamen 361003, China Supported by the Chinese National Science Foundation (No. 81170090), the Science Foundation for Distinguished Young Scholars of Fujian Province (No. 2009D015); the Science Foundation for Distinguished Young Scholars of Xiamen (No. 3502Z20116009), and the Science Foundation of Science and Technology of the Bureau of Xiamen (No. 3502Z20094006) Correspondence to: Prof. Zhengrong Huang, Department of Cardiology, the First Affiliated Hospital of Xiamen University, Xiamen 361003, China. [email protected]. cn Telephone: +86-13606028353 Accepted: March 24, 2012
trate did not affect the activation curve. CONCLUSION: Didrovaltrate blocks ICa-L in a concentration-dependent manner and probably inhibits ICa-L in its inactive state, which may contribute to its cardiovascular effect.
© 2012 JTCM. All rights reserved. Key words: Didrovaltrate; Patch-clamp techniques; Calcium channels, L-Type; Myocytes; Cardiac
INTRODUCTION The mildly sedating effects of valerian (Valeriana officinalis) and its many related Valerianaceae were noted by Greek, Roman, Chinese, and Indian scholars for more than 1000 years. The chemical composition of valerian includes sesquiterpenes of the volatile oil (including valeric acid), iridoids (valepotriates), alkaloids, furanofuran lignans, and free amino acids, such as γ-aminobutyric acid (GABA), tyrosine, arginine, and glutamine. Although the sesquiterpene components of the volatile oil are believed to be responsible for most of valerian's biological effects, it is likely that all of the active constituents of valerian act in a synergistic manner to produce a clinical response.1 Research into the physiological activity of individual components of valerian has demonstrated direct sedative effects (valepotriates and valeric acid) and interaction with neurotransmitters, such as GABA (valeric acid and unknown fractions).2,3 In recent years, cardiovascular effects of valerian extract, such as hypotensive, negative inotropic, and antiarrhythmic actions, have been reported in many studies.4-6 However, the mechanisms underlying the beneficial effects of valerian extract on hearts have not been clearly established. It is
Abstract OBJECTIVE: To investigate the effect of didrovaltrate on L-type calcium current (ICa-L) in rabbit ventricular myocytes. METHODS: We used the whole cell patch clamp recording technique. RESULTS: Didrovaltrate at concentrations of 30 μg/ L and 100 μg/L significantly decreased peak ICa-L (ICa-Lmax) from (6.01±0.48) pA/pF to (3.45±0.27) pA/pF and (2.16 ± 0.19) pA/pF (42.6% and 64.1% , n=8, P< 0.01), respectively. Didrovaltrate shifted upwards the current-voltage curves of ICa-L without changing their active, peak and reverse potentials. Didrovaltrate affected the steady-state inactivation of ICa-L. The half activation potential (V1/2) was significantly shifted from (-26 ± 2) to (-36 ± 3) mV (n=6, P<0.05), with a significant change in the slope factor (k) (from 8.8 ± 0.8 to 11.1 ± 0.9, n=6, P<0.05). DidrovalJTCM | www. journaltcm. com
442
September 15, 2012 | volume 32 | Issue 3 |
Xie Q et al. Effect of didrovaltrate on l-calcium current in rabbit ventricular myocytes postulated that valerian extract has effects on the voltage-dependent ICa-L. Therefore, the purpose of the present study was to clarify the effects of valerian extract (didrovaltrate, one of the valepotriates) on the voltage-dependent ICa-L in rabbit cardiac myocytes.
adjusted to 7.4 with KOH. Other chemical reagents were purchased from China. Didrovaltrate was a kind gift from Professor Xue Cunkuan of the Geriatrics Institute of Huazhong University of Science & Technology. Electrical recordings Membrane currents and action potentials were recorded using an EPC-9 patch clamp amplifier (HEAK, Germany). Myocytes adhering to glass coverslips were placed in a small chamber mounted on the stage of an inverted microscope and superfused at 1.5 mL/min at room temperature (20-22°C). Action potential recordings were performed at 37°C. Other experiments were performed at room temperature. Only rod-shaped single cells that were quiescent and exhibiting well defined cross-striations were studied. After establishment of whole-cell configuration and measurement of cell capacitance, series resistance (≤10 MΩ) was compensated by 50% to 70%. Junction potentials under these conditions were -3mV and corrected. (ICa-L) was elicited by a series of depolarization steps of 200 ms duration applied in 10-mV increments from a holding potential of -40 mV. Action potentials were recorded in current-clamp mode. Borosilicate glass electrodes (1.0 mm OD) were pulled with a vertical puller (PB-7, Narishige, Tokyo, Japan) and had a resistance of 2-3 MΩ when filled with electrode internal solution. Data acquisition and analysis were carried out using pClamp software (Axon Instruments Inc., Foster City, CA, USA). Current signals were filtered at 3 kHz and sampled at a frequency of 10 kHz. Peak ICa amplitudes were estimated as the difference between the maximal inward current and zero-current.
MATERIALS AND METHODS Cell isolation Single rabbit ventricular myocytes were isolated by an enzymatic dissociation procedure modified from Tytgat.7. New Zealand White rabbits of either sex, weighing 1.0-2.0 kg (Experimental Animal Centre of Medical College, Xiamen University), were anesthetized with 1 mL/kg intramuscularly injected Hypnorm (10 mg/mL fluanisone and 0.315 mg/mL fentanyl citrate; Janssen Pharmaceutics, Tilburg, The Netherlands) and 0.1 mL heparin sodium (5000 U/mL) was injected intravenously. The heart was rapidly removed and mounted on a Langendorff perfusion apparatus. It was retrogradely perfused through the aorta with normal Tyrode's solution for 10 min, followed by an additional 10 min of perfusion with Ca2 +-free Tyrode's solution. Perfusion was then switched to Ca2 +-free Tyrode's solution containing collagenase II (Sigma-Aldrich, USA) for 15 min. The enzymatic solution was recirculated. All solutions were saturated with 100% O2 and the temperature was maintained at 37° C. Subsequently, the ventricles were minced and gently agitated in a beaker with low-Ca2 + Tyrode's solution to obtain single myocytes. The calcium concentration was then increased stepwise by replacing approximately 75% of the low-Ca2+ Tyrode's solution by normal Tyrode's solution. This procedure was repeated four times at intervals of 10 min. The cells were placed in a high-K + storage solution and gently triturated with a Pasteur pipette. Isolated myocytes were kept in the medium for at least 1 h at room temperature (20-22°C) before use.
Statistical analyses Data are expressed as the mean±SD. Statistical analysis was performed using paired t-tests to determine statistical significance. A probability of P≤0.05 was considered significant.
Solutions and drugs The normal Tyrode's solution contained (mM): NaCl 140, KCl 5.4, MgCl2 1.0, HEPES 5.0, and glucose 10. The pH was adjusted to 7.4 with NaOH. The pipette solution contained (mM): CsOH 110, TEACl 15, CsCl 20, MgCl2 1.0, egtazic acid (EGTA, Sigma-Aldrich, USA) 5.0, pyruvic acid 5.0, MgATP 5.0, and HEPES 5.0 at pH 7.3 (adjusted with Tris). The external solution contained (mM): NMDG-Cl 140, MgCl2 1.0, CaCl2 1.8, HEPES 5.0, glucose 10, tetrodotoxin (TTX; Hebei Aquatic product Research Institute), and 0.002 and 4-aminopyridine (4-AP, Sigma-Aldrich, USA) 2.0. The pH was adjusted to 7.4 with NMDG. KB solution contained (mM): KOH 110, taurine 10, oxalic acid 10, glutamic acid 70, KCl 25, KH2PO4 10, EGTA 5.0, HEPES 5.0, and glucose 10. The pH was JTCM | www. journaltcm. com
RESULTS Effects of didrovaltrate on voltage-dependent ICa-L The effects of didrovaltrate on voltage-dependent ICa-L were examined in single ventricular myocytes (Figure 1). The membrane potential was maintained at -40 mV, and command voltage pulses (200 ms in duration) from – 40 mV to + 60 mV were applied at 0.2 Hz. Didrovaltrate at 30 μg/L and 100 μg/L significantly decreased peak ICa-L (ICa-Lmax) from (6.01±0.48) pA/pF to (3.45 ± 0.27) pA/pF and (2.16 ± 0.19) pA/pF (42.6% and 64.1%, n=8, P<0.01), respectively. After washout, ICa-L gradually returned to near control levels. The inhibitory effect was concentration dependent and was easily recovered after washout with Tyrode's solution for 443
September 15, 2012 | volume 32 | Issue 3 |
Xie Q et al. Effect of didrovaltrate on l-calcium current in rabbit ventricular myocytes +60mv -40mv
A
to its maximal value, V1/2 is the voltage at which the normalized ICa-L amplitude is 0.5, and k is the slope factor. A concentration of 100 μg/L didrovaltrate caused a marked shift of the inactivation curve toward more negative potentials, significantly shifting V1/2 from (-26±2) mV to (-36±3) mV (n=6, P<0.05), with a significant increase in k (from 8.8±0.8 to 11.1±0.9, n=6,
500.pA
-40mv
50.0ms
B
C Figure 2 Effects of didrovaltrate on the current-voltage relationship of ICa-L Step pulses were applied in 10 mV increments from -40 mV to 6 0mV. The holding potential was -40 mV. Each point is the mean±s.d. of the current density (n=7 cells)
D
5 min. Figure 1 Effects of didrovaltrate on voltage-dependent ICa-L Cells were maintained at – 40 mV, and command voltage pulses (200 ms in duration) were applied at 0.2 Hz from – 40 mV to +60 mV. Superimposed original current traces recorded in a single ventricular myocytes are shown. Trace (A): control. Trace (B): in the presence of didrovaltrate 30 μg /L. Trace (C): in the presence of didrovaltrate 100 μg/L. Trace (D): after washout.
P<0.05). A concentration of 100 μg/L didrovaltrate did not affect the activation curve (data not shown).
Effect of didrovaltrate on the current-voltage relationship of ICa-L The current-voltage relationship of ICa-L and the didrovaltrate current-voltage (I-V) curve of L-type current were obtained by a number of depolarizing step pulses (200 ms) from the holding potential of – 40 mV to test potentials between –40 mV and 60 mV. The test step pulses were delivered in 10 mV increments. ICa-L was activated at –30 mV and the peak amplitude occurred at the potential of 0 mV. Didrovaltrate at 30 μg/ L and 100 μg/L shifted the I-V curve upwards without changes in their active, peak and reverse potentials (Figure 2).
Figure 3 Effect of didrovaltrate on the steady-state inactivation curve of ICa-L Currents were elicited by a 300 ms test pulse to +20 mV after 2 s of conditioning prepulses from –80 to +20 mV in 10 mV increments. Curves were obtained by simulating the voltage dependent changes in I/Imax using the Boltzmann equation: I/Imax=1/ [1 + exp (Vm-V1/2/k)]. A concentration of 100 μg/L didrovaltrate shifted V1/2 from (-26±2) mV to(-36± 3mV and k from 8.8±0.8 to 11.1±0.9 (n=6, P<0.05).
DISCUSSION Our findings demonstrated that didrovaltrate produced voltage-dependent blockade of ICa-L in a concentration-dependent manner. The steady-state inactivation kinetics of ICa-L were changed and the inactivation curve of ICa-L shifted towards more negative potentials. These findings indicated that didrovaltrate inhibited ICa-L by its effect on inactivation kinetics.
Effect of didrovaltrate on the steady-state inactivation and activation of ICa-L The effect of didrovaltrate on the steady-state inactivation of ICa-L is shown in Figure 3. The results were well fitted by the Boltzmann equation I/Imax=1/ [1 + exp (Vm-V1/2/k)], where I/Imax is the ratio of ICa-L amplitude JTCM | www. journaltcm. com
444
September 15, 2012 | volume 32 | Issue 3 |
Xie Q et al. Effect of didrovaltrate on l-calcium current in rabbit ventricular myocytes Two types of calcium channel have been distinguished on the basis of their electrophysiological and pharmacological characteristics in ventricular myocytes.8,9 The physiological significance of the L-type calcium channel is better understood than that of the T-type calcium channel. Calcium influx through the L-type calcium channel is responsible for the upstroke of the action potential in the sinoatrial node and atrioventricular node, and plays an important role in determining the plateau and eventual spike-dome appearance of the action potential in other cardiac cells. The L-type calcium channel is further responsible for the coupling between excitation and contraction, it induces release of Ca2+ from the sarcoplasmic reticulum, and regulates intracellular Ca2 + load. In this way, the L-type calcium channel determines activity of a number of mitochondrial and cytoplasmic Ca2+-sensitive enzymes.10 The underground organs of members of the genus Valeriana (Valerianaceae), as well as related genera such as Nardostachys, are used in the traditional medicine of many cultures as mild sedatives and tranquillizers and to aid the induction of sleep.11-13 The anticonvulsive, sedative, and hypnotic effects have been variously attributed to valeric acid, valeranone, and valepotriates.4 The chemical composition of valerian includes sesquiterpenes of the volatile oil (including valeric acid), iridoids (valepotriates), alkaloids, furanofuran lignans, and free amino acids, such as GABA, tyrosine, arginine, and glutamine. Although the sesquiterpene components of the volatile oil are believed to be responsible for most of valerian's biological effects, it is likely that all of the active constituents of valerian act in a synergistic manner to produce a clinical response.1 Research into physiological activity of individual components has demonstrated direct sedative effects (valepotriates and valeric acid) and interaction with neurotransmitters, such as GABA (valeric acid and unknown fractions).2,3 In recent years, cardiovascular effects of valerian extract, such as hypotensive, negative inotropic, and antiarrhythmic effects, have been reported in some studies.4-6 Our findings demonstrate that didrovaltrate
JTCM | www. journaltcm. com
inhibits ICa-L, which may contribute to its cardiovascular effects.
REFERENCES 1 2
3
4 5
6
7 8
9
10
11 12 13
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September 15, 2012 | volume 32 | Issue 3 |