Wang B et al / Acta Pharmacol Sin 2002 Dec; 23 (12): 1121-1126

Effect of tetrandrine on free intracellular calcium in cultured calf basilar artery smooth muscle cells¹

WANG Bin², XIAO Ji-Gao

Department of Pharmacology, Nanjing Medical University, Nanjing 210029, China

¹ Project supported by the National Natural Science Foundation of China (No 30000207), the Natural Science Foundation of Education Department, Jiangsu Province (No KJB310001), and Commercialization of Science and Technology Foundation of Education Department, Jiangsu Province (No JH01-050).

² Correspondence to Dr WANG Bin. Phn/Fax 86-25-666-2884. E-mail Binwang@njmu.edu.cn

Received 2002-07-18 Accepted 2002-10-18

KEY WORDS tetrandrine; calcium; basilar artery; vascular smooth muscle; cultured cells; caffeine; phenylephrine

ABSTRACT

AIM: To study the effects of tetrandrine (Tet) on extracellular Ca²⁺ influx and intracellular Ca²⁺ release in cultured calf basilar artery smooth muscle cells. METHODS: Free intracellular calcium was examined by a system of measurement of AR-CM-MIC, using Fura 2-AM as a fluorescent indicator. RESULTS: In the presence of extracellular Ca²⁺ 1.3 mmol/L, no significant effect of Tet on resting [Ca²⁺]_i was found. KCl 20, 40, and 60 mmol/L triggered a sustained rise in [Ca²⁺]_i, pretreatment with Tet inhibited the elevation of [Ca²⁺]_i induced by KCl in concentration-dependent manner, Tet at high concentration (100 µmol/L) almost abolished the rise of [Ca²⁺]_i evoked by KCl. Caffeine 10 mmol/L only produced a transient increase of [Ca²⁺]_i, which spontaneously declined back to resting levels. Tet 10-30 µmol/L had no effect on caffeine-induced [Ca²⁺]_i transient peak. Tet at high concentration (100 µmol/L), however, reduced the [Ca²⁺]_i transient peak induced by caffeine. Phenylephrine (PE) 10 mmol/L produced a rapid transient peak and a distinct sustained elevation in [Ca²⁺]_i in the presence of extracellular Ca²⁺. In the absence of extracellular Ca²⁺ containing egtazic acid (EGTA), PE only produced a rapid transient peak in [Ca²⁺]_i. Pretreatment of Tet (10-100 µmol/L) inhibited the sustained elevation in [Ca²⁺]_i induced by PE in a concentration-dependent manner. However, only 100 µmol/L of Tet inhibited the transient peak in [Ca²⁺]_i induced by PE both in the presence of extracellular Ca²⁺ 1.3 mmol/L and in the absence of extracellular Ca²⁺ containing EGTA. CONCLUSION: Tet inhibited the Ca²⁺ influx from the extracellular site via voltage-activated Ca²⁺ channel and PE-receptor-operated Ca²⁺ channel. At a high concentration, Tet may inhibit the Ca²⁺ release from sarcoplasmic reticulum (SR) or refilling of intracellular calcium store in cerebral artery smooth muscle cells.

INTRODUCTION

Tetrandrine (Tet) is the major active component in the root of Stephania tetrandra S. Tet has been used for the treatment of various diseases, including hypertension, cardiac arrhythmia, angina and inflammation^[1-3]. Recently, it was reported that Tet had a protective effect on the brain ischemia/reperfusion model in rats and gerbils^[4,5]. A series of studies have revealed that Tet blocked Ca²⁺ channel in cardiac cells, neuroblastoma cells, neurohypophysial nerve terminals, and mesenteric artery, which largely accounted for its antihypertensive, antiarrhythmic and antimyocardial ischmeia actions^[1,3]. In addition to its direct effect on neurons, Tet may relax cerebral artery to protect from brain ischmia. However, the effect and mechanism of Tet on cerebral artery remain unclear. In the pesent study, we examined the effect of Tet on Ca²⁺ influx from extracellular space and Ca²⁺ release from sarcoplasmic reticulum (SR) or refilling of intracellular calcium store in cultured calf basilar artery smooth muscle cells.

MATERIALS AND METHODS

Materials Fura 2-AM, dimethyl sulfoxide, caffeine and phenylephrine were purchased from Sigma Chemical Co. Fura 2-AM was dissolved in dimethyl sulfoxide. Tet was obtained from Jin Hua Pharmaceutical Factory, it was dissolved in HCl 0.1 mol/L, then diluted with D-Hanks' medium to desired concentration.Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco. The system of measurement of AR-CM-MIC was the product of Spex Co, USA.

Cell culture Calf basilar artery smooth muscle cells were cultured according to our previous method^[6]. Briefly, the basilar artery of newborn calf (Nanjing Weigang Calf Serum Factory) was isolated rapidly. The connective tissue was removed under a dissecting microscope. The endothelium was removed first by erasing when the blood vessels were cut longitudinally, the pieces (2 mm²) of the blood vessels were placed endosurface downright in culture dishes and were incubated at 37 ºC in a humidified incubator with 95 % air and 5 % CO₂ in DMEM with 10 % (v/v) fetal calf serum(FCS). Cells migrated from the blood vessels in about 7 d. When cells covered the culture dish, small pieces were removed and cells were washed with D-Hanks' solution twice, then incubated at 37 ºC for 10 min in D-Hanks' solution with 0.125 % trypsin. The trypsinization was discontinued by adding ice-cold DMEM 10 mL containing 10 % (v/v) FCS. The cell pellets were resuspended in fresh medium with FCS to 1×10⁸ cells/L and cultured. The medium was changed every 2-3 d. Cells reached confluence in about 5 d and then passaged every 5-6 d. The single cell was cultured in culture dish with a glass cover slip 2 d before experiment. [Ca²⁺]_i was measured in passages 4 and 5.

[Ca²⁺]_i measurement Cells grown on glass coverslip were incubated with DMEM containing Fura 2-AM 3 µmol/L at 37 ºC for 40 min. The loaded cells were washed twice with Hanks' solution or D-Hanks' solution. The fluorescence was recorded in a system of measurement of AR-CM-MIC at _ex=340 nm/380 nm, _em=505 nm, directly after the addition of an agonist. [Ca²⁺]_i of single cell was calculated according to the formula: [Ca²⁺]_i=K_d (sb₁/sb₂) [( R-R_min)/(R_max -R)] nmol/L. Where, the dissociation constant K_d=224 nmol/L at 37 ºC. R was fluorescence intensity, R_max and R_min were obtained by the addition of ionomycin and egtazic acid respectively.

Statistical analysis Date were analyzed with t- test. Differences were considered significant when P<0.05.

RESULTS

Effect of Tet on resting [Ca²⁺]_i The resting [Ca²⁺]_i was (100±9) nmol/L in the presence of extracellular Ca²⁺ 1.3 mmol/L. When 10, 30, and 100 µmol/L of Tet were added, the [Ca²⁺]_i was (101±10), (98±8), and (97±10) nmol/L, respectively (P>0.05 vs 0 µmol/L of Tet). No significant effect of Tet on resting [Ca²⁺]_i was found in cultured calf basilar artery smooth muscle cells.

Effect of Tet on elevation of [Ca²⁺]_i induced by KCl KCl 20, 40, and 60 mmol/L triggered a sustained rise in [Ca²⁺]_i without transient component in the presence of extracellular Ca²⁺ 1.3 mmol/L. In the absence of extracellular Ca²⁺ containing egtazic acid, addition of KCl had no effect on [Ca²⁺]_i. This result suggested that KCl increased [Ca²⁺]_i via Ca²⁺ channel in the plasma membrane rather than in SR in this condition. Pretreatment with Tet inhibited the elevation of [Ca²⁺]_i induced by KCl in a concentration-dependent manner. High concentration of Tet almost abolished the rise of [Ca²⁺]_i evoked by KCl (Fig 1).

Fig 1. Efffect of tetrandrine (Tet) on elevation of [Ca²⁺]_i induced by KCl in the presence of extracellular Ca²⁺ 1.3 mmol/L. n=6. Mean±SD. ^cP<0.01 vs KCl 0 mmol/L. ^eP<0.05, ^fP<0.01 vs 0 µmol/L of Tet.

Effect of Tet on transient elevation of [Ca²⁺]_i induced by caffeine Caffeine 10 mmol/L only produced a transient increase of [Ca²⁺]_i, which spontaneously declined back to resting levels. To clarify whether the SR stores of Ca²⁺ were refilled completely, the repetitive addition and washout of caffeine was performed. When the interval of repetitive addition and washout of caffeine was 5 min, the transient peak in [Ca²⁺]_i evoked by subsequent repetitive addition of caffeine declined, suggesting that the Ca²⁺ store in the SR was depleted. With the interval of washout of caffeine was increased to 25 min, the transient peak in [Ca²⁺]_i evoked by repetitive addition of caffeine was kept stable, indicating that the Ca²⁺ store in the SR was refilled completely within 25 min. We used 25 min as the interval of washout. Tet 10-30 µmol/L had no effect on caffeine-induced [Ca²⁺]_i transient peak. Tet at a high concentration (100 µmol/L) , however, reduced the [Ca²⁺]_i transient peak induced by caffeine (Fig 2).

Fig 2. Effect of tetrandrine on transient elevation of [Ca²⁺]_i induced by caffeine (10 mmol/L) in the presence of extracellular Ca²⁺ 1.3 mmol/L. n=6. Mean±SD. ^cP<0.01 vs 0 µmol/L of Tet.

Effect of Tet on PE-induced elevation of [Ca²⁺]_i PE 10 µmol/L produced an increase in [Ca²⁺]_i , consisting of a rapid transient peak and a distinct sustained component in the presence of extracellular Ca²⁺ 1.3 mmol/L. In the absence of extracellular Ca²⁺ containing egtazic acid, PE only produced a rapid transient peak in [Ca²⁺]_i without the sustained component, which indicated that the rapid transient peak induced by PE represented Ca²⁺ release from Ca²⁺ store. To clarify whether the sustained elevation in [Ca²⁺]_i induced by PE represented Ca²⁺ influx from extracellular site, caffeine was pretreated to deplete Ca²⁺ store. When PE was added 3 min after pretreatment of caffeine (20 mmol/L), only a sustained elevation in [Ca²⁺]_i induced by PE was observed. These data verified that the rapid transient peak and sustained elevation in [Ca²⁺]_i induced by PE represented Ca²⁺ release and Ca²⁺ influx respectively in cultured calf basilar artery smooth muscle cells.

In the presence of extracellular Ca²⁺ 1.3 mmol/L , pretreatment of Tet (10-100 µmol/L) inhibited the sustained elevation in [Ca²⁺]_i induced by PE in a concentration-dependent manner and high concentration of Tet (100 µmol/L) inhibited the transient peak in [Ca²⁺]_i induced by PE (Fig 3). In the absence of extracellular Ca²⁺ containing egtazic acid, 10 and 30 µmol/L of Tet showed no significant effect on the transient peak in [Ca²⁺]_i induced by PE and 100 µmol/L of Tet inhibited the transient peak in [Ca²⁺]_i induced by PE (Fig 4).

Fig 3. Efffect of tetrandrine (Tet) on elevation of [Ca²⁺]_i induced by phenylephrine (PE, 10 µmol/L) in the presence of extracellular Ca²⁺ 1.3 mmol/L. Left: transient peak in [Ca²⁺]_i, Right: sustained component in [Ca²⁺]_i. n=6. Mean±SD. ^cP<0.01 vs Tet 0 µmol/L in [Ca²⁺]_i transient peak. ^fP<0.01 vs Tet 0 µmol/L in [Ca²⁺]_i sustained component.

Fig 4. Efffect of tetrandrine (Tet) on elevation of [Ca²⁺]_i induced by phenylephrine (PE, 10 µmol/L) in the absence of extracellular Ca²⁺ containing egtazic acid. n=6. Mean±SD. ^cP<0.01 vs 0 µmol/L of Tet.

DISCUSSION

The actions of Tet on a series of systems have been investigated, including cardiac cells, cardiac papillary muscle, arotic vessel, mesenteric artery, pulmonary artery, B cells, human leukaemic HL-60 cells, cornea and retina cells^{[3, 7-10]}. However little has been reported on the effect of Tet on cerebral artery. Tet was reported to decrease brain injury on brain ischemia/reperfusion model in rats and gerbils^[4,5]. Neural tissues are highly dependent on glucose and oxygen supplies, an adequate continuous blood supply to the brain is essential to ensure the normal function of the brain. In addition to its direct effect on neurons, Tet was able to inhibit pial artery constriction induced by KCl and PE in rats (unpublished data), which may partially account for its protective effect on cerebral ischemia. However, the effect and mechanism of Tet on cerebral artery remain unclear. In the present study, we demonstrated that Tet blocked voltage-activated Ca²⁺ channel and PE receptor-gated Ca²⁺ channel on the plasma membrane in a concentration-dependent manner and Tet at a high concentration may inhibited Ca²⁺ release or Ca²⁺ uptake via caffeine- and PE-sensitive Ca²⁺ store in cerebral basilar artery smooth muscle cells in rats.

Tet relaxed the high K⁺-induced constrictions in isolated arteries, including pulmonary, coronary, renal arteries^[3,11]. Electrophysiological study revealed that Tet declined a high-voltage-threshold, long-lasting (L-type) Ca²⁺ channel current in GH3 anterior hypophysial cells, cardiac ventricular muscles , which demonstrated that Tet was an L-type Ca²⁺ channel blocker^[3,12]. On the cerebral basilar artery smooth muscle cells (this study), KCl only produced a sustained increase in [Ca²⁺]_i whithout the transient component, indicating that KCl increased Ca²⁺ entry but did not influence the Ca²⁺ store. Tet inhibited markedly KCl-induced rise in [Ca²⁺]_i and high concentration of Tet almost abolished the rise of [Ca²⁺]_i, suggesting that Tet acted as a voltage-activated Ca²⁺ channel antagonist on cerebral artery smooth muscle cells.

Kwang et al^[13] reported that Tet inhibited the binding of ³H-prazosin (an ₁-adrenoceptor antagonist) to dog aortic muscle plasmalemma-rich membranes and Tet was shown as an ₁-adrenoceptor antagonist. In addition to inhibiting voltage-activated Ca²⁺ channel, Tet could inhibit receptor-dependent Ca²⁺ channel, including PE-, norepinephrine-, histamin-, glutamate-sensitive Ca²⁺ channel on plasma membrane^[9,10]. In the present study, Tet concentration-dependently inhibited PE-induced sustained rise in [Ca²⁺]_i in Ca²⁺ -containing medium, indicating that Tet inhibited Ca²⁺ influx via PE-sensitive Ca²⁺ channel in cerebral artery smooth muscle cells. Considering the system difference, whether Tet directly interacted with ₁-adrenoceptor on cerebral artery will require further studies.

The results of studies of Tet on Ca²⁺ release or Ca²⁺ uptake sensitive to caffeine are still confusing: Tet facilitated the contraction induced by caffeine in rabbit aortic strip^[14], however, elicited no effect on caffeine-induced transient contraction in rat aortic rings^[13], it has been also shown that Tet reduced the magnitude of the caffeine-induced [Ca²⁺]_i elevation in rat cardiomyocyte^[15] and the refilling of intracellular calcium stores sensitive to caffeine was inhibited by Tet in rat aortic strips^[16]. The differences of experimental procedures or preparations used might in part account for the results.

The intracellular Ca²⁺ store depletion by caffeine elicited Ca²⁺ entry from extracellular space via specific channels or by passive diffusion or exchange with other ions and intracellular Ca²⁺ can be uptaken by Ca²⁺ store. Tet inhibited the Ca²⁺ uptake to SR in rat cardiomyocyte and the refilling of intracellular calcium stores in rat aortic strips^[15-17], Tet might influence the refilling of intracellular calcium stories via affecting plasma membrane channels indirectly or via affecting intracellular Ca²⁺ store directly, the exact mechanisms remain unclear.

Caffeine inhibited cyclic nucleotide phosphodiesterases and stimulated Ca²⁺ release^[18]. The present study showed that the Ca²⁺ storage sites sensitive to caffeine can not be completely refilled within 5 min with 1.3 mmol/L extracellular Ca²⁺. When caffeine was added again after incubation period of 25 min in extracellular Ca²⁺ 1.3 mmol/L, the magnitude of [Ca²⁺]_i transient was similar to that of the first one, which indicated that Ca²⁺ store was refilled completely. We used 25 min as the interval of washout, and found that 100 µmol/L of Tet had the inhibitory effect on the transient peak in [Ca²⁺]_i evoked by caffeine. Although we can not explain the exact mechanisms, these results also implied that Tet might inhibit refilling of intracellular calcium store in cerebral artery smooth muscle cells.

PE elicited a rapid transient peak companied with a more sustained increase in [Ca²⁺]_i in Ca²⁺ -containing medium. But in absence of Ca²⁺, only rapid transient peak was induced by PE. After caffeine depleted the SR Ca²⁺ store, subsequent addition of PE only elicited a sustained rise in [Ca²⁺]_i without the transient component. These data implied that the caffeine-inducible Ca²⁺ pool and PE-inducible Ca²⁺ pool might overlap in cerebral artery smooth muscle cells. Tet inhibited the transient peak in [Ca²⁺]_i induced by PE both in presence and absence of Ca²⁺. These result indicated that Tet at a high concentration might inhibit Ca²⁺ release or Ca²⁺ uptake via caffeine- or PE-inducible Ca²⁺ pool. The exact mechanism of Tet on Ca²⁺ release or Ca²⁺ uptake in cerebral artery smooth muscle cells will warrant further investigation.

In summary, Tet inhibited the Ca²⁺ influx from the extracellular site via voltage-activated Ca²⁺ channel and PE-receptor-operated Ca²⁺ channel. At high concen-tration, Tet may inhibit the Ca²⁺ release from SR or refilling of intracellular calcium store in cerebral artery smooth muscle cells.

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