Yao WX et al / Acta Pharmacol Sin 2002 Dec; 23 (12): 1069-1074

Effects of tetrandrine on cardiovascular electrophysiologic properties

YAO Wei-Xing¹, JIANG Ming-Xing

Department of Pharmacology, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China

¹ Correspondence to Prof YAO Wei-Xing. Phn 86-27-8369-3633.

Received 2002-08-06 Accepted 2002-10-29

KEY WORDS tetrandrine; myocardium; vascular smooth muscle; vascular endothelium; electrophysiology; action potentials; calcium channels; patch-clamp techniques

ABSTRACT

Tetrandrine (Tet) is one of the best characterized Ca²⁺ channel blocker of plant origin. It can affect cardiovascular electrophysiologic properties in following field: inhibit the contractility, ±dt/dp_max, and automaticity of myocardium, prolong the FRP, and exert concentration-dependent negative inotropic and chronotropic effects without altering cardiac excitability.

Tet directly blocks both T-type and L-type calcium current in ventricular cells and vascular smooth muscle cells, but it does not shift the I-V relationship curve of I_Ca. All its effects would be beneficial in the treatment of angina, arrhythmias, and other cardiovascular disorders.

Tet also directly inhibits the activity of BK_Ca channel in endothelial cell line and also inhibits Ca²⁺-release-activated channels in vessel endothelial cells, which might significantly contribute to the change of endothelial cell activity.

INTRODUCTION

Tetrandrine (Tet) is a bis-benzylisoquinoline alkaloid isolated from the Chinese medicinal herb-root of Stephania tetrandra S Moore, which was traditionally used as an anti-inflammatory, antipyretic, and analgesic herb in Chinese medicine. Tet is active principle of the root of Stephania tetrandra. Empirical formula of Tet is C₃₈H₄₂O₈N₂ with molecular weight 622^[1]. The first pharmacological and toxicological study of Tet was published in 1937 which demonstrated its hypotensive and cardiodepressant effects^[2]. Since 1950s, Tet has been used in China as an antihypertensive agent^[3]. Subsequently, Tet has been shown to be a calcium antagonist of plant origin^[4-7]. Recently, it was reported that Tet could protect hepatocytes against CCl₄ injury through anti-hepatic fibrosis effect^[8-9]; ameliorate development of pulmonary vascular and lung tissue injury induced by monocrotaline in rats^[10]; have potent immunosuppressive properties^[11]; be potentially useful in the treatment of silicosis^[12]; and treat hepatocellular carcinoma as chemotherapeutic/chemopreventive agent^[13] .

During the past decade, Tet has received considerable attention mainly owing to its cardiovascular electrophysiologic properties as Ca²⁺ antagonist^[4,14-16,]. It is believed that the inhibitory effect on Ca²⁺ influx is the basis of the cardiovascular action and therapeutic efficacy of Tet. Tet, in addition to its effects of blocking L- and T-type calcium channels, also produced an inhibition of BK_Ca^[17] and the non-selective cation channels, ie characteristic of Ca²⁺ release-activated Ca²⁺ channel present in endothelial cells^[18]. This review focused on cardiovascular electrophysiologic effects of Tet.

EFFECTS ON MYOCARDIUM^[19-21]

Contractility, ±dt/dp_max, automaticity, FRP, and excitability Papillary muscle of cat right ventricle or guinea pig left atrium suspended in Tyrode's solution is stimulated by square wave. Isometric contractions and ±dt/dp_max were recorded. After addition of Tet 32 µmol/L for 5 min the force of contraction began to fall and decreased by 29 % (papillary muscle), 64 % (atrium) of the control level at the end of 15 min, and dt/dp_max also declined coincidentally.

The threshold concentration of adrenaline to induce automaticity was compared before and after administration of Tet 32 µmol/L at 37 ºC. Tet increased the threshold concentration of adrenaline to induce automaticity in guinea pig by 15 fold.

Functional refractory period (FRP) was determined by paired impulses (3 ms, 0.2 Hz, 5 times threshold voltage), After administration of Tet 32 µmol/L, the FRP of guinea pig left atrium and cat papillary muscle were prolonged from (114±9) ms and (326±19) ms to (158±9) ms and (384±38) ms, respectively.

Excitability of myocardial preparation was determined with intensity-duration curves. There was no significant change in excitability after addition of Tet 32 µmol/L. It suggested that Tet had no effect on sodium channels on cell membrane.

Heart rate The effects of Tet 32 µmol/L on guinea pig right atrium showed that it slowed down the rate of spontaneous beating of right atrium to 1/3 of its original level within 15 min, which indicated that Tet had an inhibitory effect on sinus node automaticity.

In isolated rabbit right atrium and cat papillary muscles, Tet could antagonize the positive chronotropic action of both CaCl₂ and isoprenaline in a non-competitive manner^[22]. Another study indicated that Tet also inhibited positive staircase phenomena and post-rest potentiation of contraction^[23].

In the intact rat ECG (II lead), repeated intravenous or continuous infusion of Tet decreased heart rat^[24]. The P wave was first flattened and then gradually disappeared. But only PR and QT interval were prolonged slightly. No significant change in QRS complex was observed at related therapeutic concentrations. In vivo and in vitro experiments revealed that Tet could decrease the heart rate. This may result from a longterm protection of the myocardium through a decrease in oxygen consumption, which is one of the therapeutic goals for treatment of some cardiovascular disorders.

Surface electrogram and action potentials The normal developed tension and surface electrogram were recorded simultaneously^[25]. Sixty minutes after administration of Tet 32 µmol/L, the normal developed tension (contractile force) was decreased distinctly. But the amplitude and duration of the R wave remained unchanged. It indicated that Tet produced excitation-contraction uncoupling.

Standard microelectrode technique was used to investigate the effect of Tet on the action potential of isolated guinea pig papillary muscles in normal and K⁺-rich Tyrode's solution. Intracellular microelectrode technique and mechano-electric transducer was used to record the action potential and electrically driving contractile force simultaneously. Zong et al reported that Tet 1-100 µmol/L produced a concentration-dependent negative inotropic effect and shortened the action potential duration at 20 % and 90 % depolarization, but did not affect resting potential or action potential amplitude, suggesting an effect on excitation-contraction uncoupling ^[26].

In high K⁺ solutions, which inactivated fast Na⁺ channels, Tet 10-100 µmol/L inhibited contractile force, exerted resting as well as frequency-dependent blockage of the maximal upstroke velocity, and decreased action potential amplitude (from 82 mV to 38 mV) as well as resting potential (from -84 mV to -60 mV)^[27]. All these demonstrated its Ca²⁺channel blocking activity. Similar phenomena were observed in rat cardiomyo-cytes.

In rabbit sino-atrial node dominant pacemaker cells^[28], Tet 1-100 µmol/L decreased APA, maximal upstroke velocity (V_max), and spontaneously depolarizing velocity of phase 4 (SP₄); and prolonged the sinus circle length (SCL) in a concentration-dependent manner. I_Ca (L and T) are considered to contribute to the slow diastolic depolarization of SA nodal cells^[29]. These effects were attributed to its ability in inhibiting I_Ca.

Calcium currents Cardiac I_Ca-L and I_Ca-T channels are believed to have different functions. The I_Ca-T is well suited for a role in pacemaker and has been considered to play an important role in late phase 4 depolarization, while the I_Ca-L may contribute to cell depolarization and increased cytosolic Ca²⁺, which can act as a second messenger for intracellular chemical signals. Tet inhibits the I_Ca-L and reduces Ca²⁺ flows into the cytosol; therefore, the action potential is shortened and the contractility of the heart is decreased. Subsequently the effects of Tet on I_Ca-T and I_Ca-L were further investigated.

The effect of Tet on slowing inward current in canine cardiac Purkinje fibers was studied by using two-microelectrode voltage clamp technique. The results showed that Tet inhibited the peak value of the slow inwardly current (I_si PV) in a concentration- and time-dependent manner. After pretreatment with Tet 30 and 100 µmol/L for 10 min respectively, the I_si PV decreased from (48±6) and (38±22) nA to (22±8) nA and (6±8) nA, respectively, which suggested that Tet was a slow channel blocker^[30].

Whole-cell patch-clamp experiments were performed on isolated single ventricular myocytes of guinea pig. Under the current clamp mode, action potentials were recorded at current stimulation of 0.5 Hz, 3 ms. Tet 30 µmol/L greatly shortened APD₅₀ and APD₉₀, while affecting neither resting membrane potential nor amplitude of the action potential^[31]. The obtained results of Tet on action potential in isolated single ventricular myocytes were consistent with those of papillary muscles of guinea pig.

Whole-cell patch-clamp technique with or without -escin-perforated^[32] was performed to examine the effects of Tet on L-type calcium current (I_Ca-L) and T-type calcium current (I_CaT) in isolated single ventricular myocytes of guinea pig.

Calcium currents were separated into T-type and L-type components by different holding potentials (-80 and -40 mV). When a holding potential was -40 mV (most T channels inactivated), depolarizing to 0 mV for 150 ms, a L-type calcium current was elicited. Tet (1-300 µmol/L) inhibited L-type calcium current in a concentration-dependent manner with IC₅₀ of 14.8 µmol/L^. The shape of the I-V curve remained similar before and after administration.

From a holding potential of -80 mV, depolarizing to -35 mV for 150 ms T-type calcium currents were evoked. Tet 3, 30, and 300 µmol/L suppressed the I_CaT in concentration-dependent manner with inhibition rate of (16.2 %±4.8 %), (39.8 %±7.4%), and (74.8 %± 10.8 %), respectively. The inhibitory action of Tet on the I_Ca-T may also account for the anti-arrhythmic effect observed by other investigators.

The effects of Tet on both I_Ca-L and I_Ca-T were in vestigated in primary cultured neonatal rat ventricular cells^[33]. Tet produced a significant reduction in L-type calcium current amplitude but did not shift the I-V relationship curve. The IC₅₀ of Tet was 0.33 µmol/L.

Nifedipine 10 µmol/L was added to abolish the I_Ca-L. After administration of Tet, the I_Ca-T was reduced by almost 40 %. The inhibitory effect of Tet on the I_Ca-T (46 %±12 %) was less than that on I_Ca-L (56 %±5 %). These data clearly demonstrated that in neonatal ventricular cells Tet inhibited both T- and L-Type calcium channel currents in a concentration-dependent manner.

T-type Ca²⁺ channels have properties different from those of the L-type and are involved in cardiac pacemaking and regulation of blood flow^[34]. T-channel-selective calcium channel blocker included a number of agents, for example, nifedipine, amiloride, efonidipine, ethosuximide, tetrandrine, mibefradil, etc, of which mibefradil is most studied. Mibefradil, an effective anti-hypertensive, anti-anginal, and anti-ischemic agent, has been shown to be beneficial to adjunct therapy in chronic heart failure (CHF)^[35], attenuate leukocyte adhesion, and exert anti-inflammatory effects^[36]. However current clinical and experimental studies showed that mibefradil was withdrawn by the manufacturer because of drug interaction with the cytochrome P-450 3A4 enzyme^[37,38].

Clinical research Early clinical study reported that Tet could relieve angina and hypertension, etc. In patients with paroxysmal supraventricular tachycardia, Tet 3-4 mg/kg prolonged sinus cycle length, Atrio-His (A-H) interval, and Atrio-ventricular node effective refractory period, with no change in P-A, His-Ventricular (H-V), QRS, or Q-T intervals. Sinoatrial conduction time and sinus node recovery time were not altered^[39]. These findings may explain the action of Tet in abolishing the reentry to paroxysmal supraventricular tachycardia.

EFFECTS ON VASCULAR SMOOTH MUSCLE CELLS AND ENDOTHELIAL CELLS

Vascular smooth muscle cells The patch-clamp technique was also used to investigate the effect of Tet on voltage-dependent Ca²⁺ channel current in vascular smooth muscle cells isolated from the rat tail artery. The I_Ca-L was activated at -20 mV, and the peak value occurred at +10 mV. Tet 10 µmol/L produced a significant reduction in Ca²⁺current amplitude, but volt age-dependence of current activation was not altered^[40]. It suggested that Tet could inhibit Ca²⁺ influx through Ca²⁺ channels.

The nystatin-perforated whole-cell voltage-clamp technique was employed to examine the effects of Tet on I_Ca-L in cultured rat aortic smooth muscle cells^[41]. Tet 1-100 µmol/L reversibly inhibited L-type voltage-dependent Ca²⁺ current in a concentration-dependent manner with IC₅₀ value of 5 µmol/L and did not cause any change in the overall shape of the current-voltage relationship of I_Ca,L. Inhibitory effects of Tet on I_Ca,L exhibited tonic and use-dependent characteristics. These results indicated that Tet directly inhibited the voltage-dependent L-type Ca²⁺ current in vascular smooth muscle cells, which may predominantly contribute to the vasodilatory actions of Tet.

Vascular endothelial cells The effects of Tet on I_Ca-L in cultured single bovine pulmonary artery endothelial cells (BPAEC) were examined. BPAEC exhibited strong inward-rectifying K⁺ currents upon hyperpolarizing voltage steps, with a reverse potential near at -70 mV. Small outward K⁺ currents dependent on the presence of Ca²⁺ in the pipette can be stimulated in BPAEC by depolarizing voltage steps. Addition of thapsigargin 100 nmol/L caused a significant enhancement of depolarization to evoke Ca²⁺-dependent outward K⁺ current, which could also be abolished by Tet 30 µmol/L. But Tet itself depressed neither outward K⁺ nor inward-rectifying K⁺ currents. The results implied that Tet acted by inhibiting the cation channel characteristic of Ca²⁺ release-activated Ca²⁺ channel present in BPAEC^[18,42]. Tet, in addition with its known inhibitory effects on vascular smooth muscle by virtue of its Ca²⁺ antagonistic actions, also inhibited Ca²⁺ release-activated Ca²⁺ channels in vascular endothelial cells

In endothelial cell line (HUV-EC-C) originally derived from human umbilical vein^[17], Tet 0.5-50 µmol/L reversibly decreased the amplitude of K⁺ outward current, which was believed to be Ca²⁺-activated K⁺ current. The IC₅₀ value was 5 µmol/L. In outside-out configuration, Tet 5 µmol/L did not change the single-channel conductance but significantly reduced the opening probability of large conductance of Ca²⁺-activated K⁺ (BK_Ca) channel. The change in the kinetic behavior of BK_Ca channel by Tet is due to a decrease in mean open time and an increase in mean closed time. The direct inhibition of BK_Ca channels by Tet should contribute to its effect on the functional activities of endothelial cells.

The non-selection cation channels existing in endothelial cells are involved in the regulation of intracellular Ca²⁺concentration and in the control of the activity of BK_Ca channel. The blockade of BK_Ca channels may cause membrane depolarization and vasoconstriction. Tet at therapeutically relevant concentrations can directly inhibit the activity of BK_Ca channels in HUV-EC-C, which contribute to vasoconstriction of microvascular wall.

Tet exerted dual effects on vascular reactivity. As a vasodilator, it acted directly on the smooth muscle cells and as a vasoconstrictor it acted via the endothelial cells.

CONCLUSION

In past decade, Tet attracted more attention for its potential as a Ca²⁺ channel blocker. According to our data and others reports, Tet can block multiple ion channels, such as L-type, T-type calcium channels, BK_Ca, and Ca²⁺ release-activated Ca²⁺ channels, etc. Its cardiovascular effects may particularly be related with its blockage on L-type calcium channels. Tet has been shown its value in the clinical treatment of a wide range of cardiovascular disorders.

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