Introduction

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One of the important functional activities in endothelial cells is the regulation of exchange between blood and tissue. Situated at the interface between blood and muscular media of the vessel, endothelial cells play a dynamic role in the regulation of vascular tone. Endothelial cells are known to lack voltage-dependent Na⁺ or Ca²⁺ channels but to exhibit a Ca²⁺ entry mechanism that depends on the electrochemical driving force and/or Ca²⁺ stores (Nilius et al., 1997). As a result, Ca²⁺ influx may regeneratively couple in a positive feedback relationship with Ca²⁺-activated K⁺ channels present in endothelial cells. The activity of these channels was thought to control K⁺ efflux and affect the degree of membrane hyperpolarization. It has recently been shown that the vascular tone can be modulated by either the change of K⁺ concentration in myoendothelial gap junctions (Edwards et al., 1998) or electrotonic spreading of membrane potential (Bény, 1999). Indeed, the blockade of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels was previously reported to cause membrane depolarization and vasoconstriction in a pressurized vessel preparation (Cabell et al., 1994; Koichi et al., 1997).

Tetrandrine, a bisbenzyltetrahydroisoquinoline alkaloid extracted from the Chinese medicinal herb Radix stephania tetrandrae, is known to possess a wide spectrum of pharmacological activities. Previous studies have demonstrated that tetrandrine can block voltage-dependent Ca²⁺ channels in various cell types (Wang and Lemos, 1992; Weinsberg et al., 1994; Liu et al., 1995; Wu et al., 1997, 1998). Tetrandrine was also reported to inhibit charybdotoxin-sensitive BK_Ca channels present in rat neurohypophysial nerve terminals and expressed in Xenopus oocytes (Wang and Lemos, 1992; Dworetzky et al., 1996; Gribkoff et al., 1996). On the other hand, tetrandrine seemed to produce no effect on charybdotoxin-sensitive BK_Ca channels that were reconstituted from rat fast-twitch muscle microsomes or present in arterial smooth myocytes (Wang and Lemos, 1992, 1995). Several reports showed that tetrandrine could cause the transient vasoconstriction, and this effect seemed to require the presence of functional endothelium (Su, 1993; Kwan et al., 1999). To date, however, none of the studies have demonstrated the underlying mechanism of actions of tetrandrine on ionic currents in endothelial cells.

The objectives of this study reported here were to 1) examine the effect of tetrandrine on Ca²⁺-activated K⁺ currents in a human endothelial cell line (HUV-EC-C) that was originally derived from umbilical vein, 2) study whether this effect is mediated by the change in the level of internal Ca²⁺, and 3) determine whether tetrandrine can affect the activity and gating of BK_Ca channels. Previous observations at our laboratory have demonstrated that BK_Ca channels, which are dependent on voltage and Ca²⁺, are expressed in these cells (Wu et al., 1999). The present results indicate that the direct inhibition by tetrandrine of BK_Ca channels in endothelial cells would lead to the change in membrane potential of endothelial cells and vascular tone.

	Materials and Methods

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Cell Culture. The clonal strain HUV-EC-C cell line, an endothelial cell line originally derived from the vein of a normal human umbilical cord, was obtained from Culture Collection and Research Center (CCRC-60016; Hsinchu, Taiwan, ROC; Wu et al., 1999). Endothelial cells were grown in monolayer culture in 50-ml plastic culture flasks at 37°C in a humidified environment containing 5% CO₂/95% air. Cells were maintained in 5 ml of Ham's F-12K nutrient medium supplemented with 10% fetal bovine serum (v/v), 100 µg/ml heparin, and 30 to 50 µg/ml endothelial cell growth supplement. Cells were passaged once a week, and a new stock line was generated from frozen cells (frozen in 10% dimethyl sulfoxide in cultured medium) every 3 months. The experiments were performed after 5 or 6 days of subcultivation (60-80% confluence).

Electrophysiological Measurements. Immediately before each experiment, the cells were dissociated, and an aliquot of the cell suspension was transferred to a recording chamber mounted on the stage of an inverted microscope (Diaphot 200; Nikon, Tokyo, Japan). The microscope was coupled to a video camera system with magnification up to 1500× to continually monitor cell size during the experiments. Cells were bathed at room temperature (20-25°C) in normal Tyrode's solution containing 1.8 mM CaCl₂. The patch pipettes were prepared from Kimax capillary tubes (Vineland, NJ) using a vertical two-step electrode puller (PB-7; Narishige, Tokyo, Japan), and the tips were fire-polished with a microforge (MF-83; Narishige). The resistance of the patch pipette was 3 to 5 M when it was immersed in normal Tyrode's solution. A programmable stimulator (SMP-311; Biologic, Claix, France) was used to digitally generate voltage pulses. Ionic currents were recorded with glass pipettes in whole-cell or outside-out configuration of patch-clamp technique with an RK-400 patch amplifier (Biologic; Hamill et al., 1981; Wu et al., 1999). All potentials were corrected for liquid junction potential that developed at the tip of the pipette when the composition of pipette solution was different from that of bath. Tested drugs were applied through perfusion or added to the bath to obtain the final concentrations indicated.

Data Recording and Analysis. The signals consisting of voltage and current tracings were monitored with a digital storage oscilloscope (model 1602; Gould, Valley View, OH) and recorded on-line using a digital audio tape recorder (model 1204; Biologic). After the experiments, the stored data were then fed back and digitized at 5 to 10 kHz with a Digidata 1200 analog-to-digital device (Axon Instruments, Foster City, CA) interfaced to a Pentium-grade computer and pClamp 6.03 software package (Axon Instruments). Voltage-activated currents recorded during whole-cell experiments were stored without leakage correction and analyzed using ClampFit subroutine (Axon Instruments) to establish a current-voltage relationship for ionic currents.

Drugs and Solutions. Evans' blue tetrasodium salt [(6,6-[3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo)bis[4-amino-5-hydroxy-1,3-naphthalenedisulfonic acid]] was purchased from Sigma Chemical Co. (St. Louis, MO). 6,6',7,12-Tetramethoxy-2,2'-dimethylberbamam was obtained from Aldrich Chemical Co. (Milwaukee, WI). Glibenclamide, niflumic acid, iberiotoxin, ionomycin, sodium nitroprusside, apamin, and thapsigargin were obtained from Research Biochemical Inc. (Natick, MA). Tissue culture media, penicillin-streptomycin, fungizone, and trypsin were obtained from Life Technologies (Grand Island, NY). Endothelial cell growth supplement was purchased from Upstate Biotechnology (Lake Placid, NY). All other chemicals were commercially available and of reagent grade. The composition of normal Tyrode's solution was 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.53 mM MgCl₂, 5.5 mM glucose, and 5.5 mM HEPES-NaOH buffer, pH 7.4. To record K⁺ currents, the patch pipette was filled with solution consisting of 130 mM K-aspartate, 20 mM KCl, 1 mM MgCl₂, 3 mM Na₂ATP, 0.1 mM Na₂GTP, 0.1 mM EGTA, and 5 mM HEPES-KOH buffer, pH 7.2. In single-channel recording, high K⁺ bathing solution contained 145 mM KCl, 0.53 mM MgCl₂, and 5 mM HEPES-KOH, pH 7.4. The pipette solution contained 145 mM KCl, 2 mM MgCl₂, and 5 mM HEPES-KOH, pH 7.2. The value of free Ca²⁺ concentration was calculated assuming the dissociation constant for EGTA and Ca²⁺ (at pH 7.2) at 10⁷ M (Portzehl et al., 1964).

	Results

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Effect of Tetrandrine on K⁺ Outward Current in Cultured Endothelial Cells of Umbilical Veins (HUV-EC-C). The whole-cell configuration of the patch-clamp technique was used to investigate the effect of tetrandrine on macroscopic ionic currents in these cells. In these experiments, the cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl₂, and the pipette solution contained a low concentration (0.1 mM) of EGTA and 3 mM ATP. As shown in Fig. 1A, when the cell was held at 40 mV, voltage pulses ranging from 30 to +50 mV in 20-mV increments elicited a family of outward currents. The amplitudes of these currents were increased with greater depolarization, and the currents during constant depolarization were not rapidly inactivated. Within 1 min of exposure of the cells to tetrandrine (2 and 5 µM), the amplitude of outward currents was markedly decreased throughout the entire voltage-clamp step (Fig. 1). For instance, when a cell was depolarized from 40 to +50 mV, tetrandrine (5 µM) significantly decreased the current amplitude to 247 ± 47 pA from a control value of 655 ± 78 pA (n = 8, P < .05). This inhibitory effect was readily reversed on the washout of tetrandrine. The averaged current-voltage relations for these currents in the absence and presence of tetrandrine (2 and 5 µM) are shown in Fig. 1B. Figure 1C shows the relationships between the concentration of tetrandrine and the percentage inhibition of K⁺ outward current. The half-maximal concentration required for the inhibitory effect of tetrandrine on K⁺ outward current was 5 µM, and 50 µM tetrandrine almost completely suppressed the current amplitude. These results indicated that tetrandrine reduced the amplitude of K⁺ outward current in a concentration-dependent manner in these cells.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Inhibitory effect of tetrandrine on K+ outward current in cultured endothelial cells of human umbilical veins (HUV-EC-C). A, superimposed current traces in control and during the exposure to 2 and 5 µM tetrandrine (tet). The cells, bathed in normal Tyrode's solution containing 1.8 mM CaCl2, were held at 40 mV, and voltage pulses from 30 to +50 mV in 20-mV increments were then applied at 0.1 Hz. A, top, voltage protocol. Arrows, zero current level. B, averaged current-voltage relations of outward currents measured at the end of voltage pulses in control (), during exposure to 2 µM () and 5 µM tetrandrine (), and during washout of tetrandrine (; mean ± S.E.; n = 6-12 for each point). C, concentration-dependent inhibition of K+ outward current by tetrandrine. The relation between the percentage inhibition of K+ outward current and the concentration of tetrandrine is illustrated. Each cell was depolarized from 40 to +50 mV with a duration of 300 ms. Various concentrations of tetrandrine (0.5-50 µM) were applied. The amplitude of outward current during the application of tetrandrine red was compared with the control value (i.e., in the absence of tetrandrine; mean ± S.E.; n = 6-10 for each point). The percentage inhibition of tetrandrine on outward currents was plotted. The smooth line represents the best fit to the Hill equation as described in Materials and Methods. The value of IC50 and maximally inhibited percentage of K+ outward current in the presence of tetrandrine were 5 µM and 99%, respectively. The Hill coefficient was 1.1.

Comparison between Effect of Tetrandrine and Those of Iberiotoxin, Glibenclamide, and Apamin on K⁺ Outward Current. The effects of glibenclamide, apamin, and iberiotoxin on K⁺ outward current in human umbilical vascular endothelial cells were examined and compared. Iberiotoxin is a selective blocker of BK_Ca channels, whereas glibenclamide and apamin are considered to be inhibitors of ATP-sensitive K⁺ (K_ATP) and small-conductance Ca²⁺-activated K⁺ channels, respectively. As shown in Fig. 2, neither glibenclamide (10 µM) nor apamin (200 nM) produced any effect on the amplitude of K⁺ outward current, whereas iberiotoxin (200 nM) significantly suppressed K⁺ outward current. In addition, the presence of ionomycin (1 µM) significantly enhanced the amplitude of K⁺ outward current (data not shown). These findings suggest that the effect of tetrandrine or iberiotoxin on this current was more potent that that of glibenclamide or apamin. The K⁺ outward current observed in the present study is referred to as Ca²⁺-activated K⁺ current.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Comparison of the effect of tetrandrine and those of iberiotoxin, glibenclamide, and apamin on the amplitude of K+ outward current. Each cell was held at 40 mV, and the voltage pulses to +50 mV (300 ms in duration) were applied. A, the original current traces showing the effect of glibenclamide, apamin, or iberiotoxin on outward current. Arrows, the zero current level. B, effects of glibenclamide, apamin, iberiotoxin, and apamin on K+ outward current. The amplitude of K+ outward current in the control was considered to be 1.0, and the relative amplitude of K+ outward current after application of each agent was plotted. *, significant difference from control group (P < .05). Each point represents the mean ± S.E. (n = 8-10). Ctrl, control; Glib, glibenclamide (10 µM); Apa, apamin (200 nM); IbTx, iberiotoxin (200 nM); Tet, tetrandrine (5 µM).

Effect of Evans' Blue and Niflumic Acid on Tetrandrine-Mediated Inhibition of K⁺ Outward Current. To assess whether K⁺ outward current inhibited by tetrandrine is a component of Ca²⁺-activated K⁺ current, the effect of Evans' blue and niflumic acid on its inhibition of current amplitude was studied. Both Evans' blue and niflumic acid have previously been reported to stimulate the activity of BK_Ca channels (Gribkoff et al., 1996; Wu et al., 1999). As shown in Fig. 3, both Evans' blue (30 µM) and niflumic acid (30 µM) can significantly reverse the inhibitory effect of tetrandrine (20 µM) on K⁺ outward current. There was a significant difference in the current amplitudes between tetrandrine alone and tetrandrine plus Evans' blue (145 ± 25 pA, n = 7, versus 475 ± 65 pA, n = 6, P < .05). Likewise, the current amplitudes observed in tetrandrine alone groups were significantly lower than those in tetrandrine-plus-niflumic acid groups (145 ± 25 pA, n = 7, versus 480 ± 72 pA, n = 7, P < .05). These findings suggested that the blockade of these currents by tetrandrine was reduced in the presence of Evans' blue or niflumic acid. In addition, because there appeared to be a partial reversal of inhibition, tetrandrine might not be displaced by these two agents.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of Evans' blue or niflumic acid on tetrandrine-induced inhibition of K+ outward current. Each cell was depolarized from 40 to +50 mV at a rate of 0.1 Hz. A, time course in change of current amplitudes measured at the end of voltage pulses. Inset, original current traces from the depolarizing pulses marked by a, b, and c. Arrow, zero current level. Horizontal bars denote the application of tetrandrine (20 µM) and Evans' blue (30 µM). a, control. b, in the presence of tetrandrine (20 µM). c, Evans' blue (30 µM) but still in the continued presence of tetrandrine. B, summary of data depicting the effect of tetrandrine in the absence and presence of Evans' blue (30 µM) or niflumic acid (30 µM). *, significant difference from control group (P < .05). **, significant difference between tetrandrine-alone group and tetrandrine-plus-Evans' blue or -plus-niflumic acid group (P < .05). Each point represents the mean ± S.E. (n = 6-10). Ctrl, control; Tet, tetrandrine (20 µM); EB, Evans' blue (30 µM); Nifl, niflumic acid (30 µM).

Effect of Tetrandrine on K⁺ Outward Current in Cells Preincubated with Thapsigargin or Sodium Nitroprusside. It is previously reported that the effect of tetrandrine on vascular tone is related to the function of Ca²⁺ stores or the production of nitric oxide (Leung et al., 1994; Kwan et al., 1999). The effect of tetrandrine on K⁺ outward current was also assessed in cells treated with thapsigargin (1 µM) or sodium nitroprusside (10 µM) for 5 h. Thapsigargin is an inhibitor of Ca²⁺-ATPase, whereas sodium nitroprusside is nitric oxide donor and may enhance the activity of BK_Ca channels (Yamakage et al., 1996). However, as depicted in Fig. 4, in these endothelial cells preincubated with thapsigargin or sodium nitroprusside for 5 h, the inhibitory effect of tetrandrine on the current-voltage relationship of K⁺ outward current was unaltered. There was no significant difference in the magnitude of tetrandrine-induced inhibition on K⁺ outward current between control cells and cells treated with thapsigargin or sodium nitroprusside. Thus, the inhibitory effect of tetrandrine on this current seems to be unrelated to the function of Ca²⁺ stores or the production of nitric oxide.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of tetrandrine on the averaged current-voltage relations of K+ outward currents in human umbilical vascular endothelial cells treated with thapsigargin or sodium nitroprusside. Endothelial cells were preincubated with thapsigargin (1 µM) or sodium nitroprusside (10 µM) for 5 h. Each cell was depolarized from 40 to +50 mV in 20-mV increments. Each point represents the mean ± S.E. (n = 6-10). , Control. , In the presence of tetrandrine (5 µM).

Inhibitory Effect of Tetrandrine on Activity of BK_Ca Channels in Outside-Out Patches. To further characterize the effect of tetrandrine on ionic current, we also performed single-channel experiments with an outside-out membrane patch in which the patch pipette contained 3 µM Ca²⁺ and the holding potential was set at +40 mV. As shown in Fig. 5, when applied to the extracellular surface of membrane patch, tetrandrine (20 µM) produced a significant decrease in the channel activity. The P_o of BK_Ca channels in control was found to be 0.842 ± 0.023 (n = 8). Within 1 min of exposure of the membrane patch to tetrandrine (20 µM), the P_o was significantly decreased to 0.126 ± 0.008 (n = 7, P < .01). The inhibitory effect was reversed after the removal of tetrandrine. The current-voltage relations of BK_Ca channels in the absence and presence of tetrandrine (5 µM) were also constructed and plotted (Fig. 6). The single-channel conductance of the BK_Ca channels in control was 170 ± 9 pS (n = 10), a value that was not different from that (169 ± 8 pS; n = 9, P > .05) measured in the presence of tetrandrine (5 µM). These results indicated that tetrandrine produced no significant change in the single-channel conductance but suppressed the activity of BK_Ca channels in these cells.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effect of tetrandrine on BKCa channels in human umbilical vascular endothelial cells. The single-channel experiments in an outside-out membrane patch were conducted with symmetrical K+ concentration (145 mM). The patch pipette contained 3 µM Ca2+. The membrane potential was held at +40 mV. A, the original current trace showing the change in the activity of BKCa channels after the addition of tetrandrine (20 µM). Channel openings are shown as upward deflection. Bottom, current traces obtained in expanded time scale corresponding to those labeled a, b, and c in the top and in B. Of note, the unitary currents show only a few, brief openings in the presence of tetrandrine. B, Po for the activity of BKCa channels shown in A plotted against time of recording. Bin width, 0.5 s. Horizontal bars indicate the application of tetrandrine (20 µM).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of tetrandrine on the current-voltage relation of BKCa channels in human umbilical vascular endothelial cells. The cells were bathed in symmetrical K+ solution (145 mM), and the single-channel experiments were conducted under outside-out configuration. A, examples of BKCa channels in the absence (left) and presence (right) of tetrandrine (5 µM) measured from cells at various membrane potentials. Tetrandrine was applied to the bathing solution. The numbers shown at the beginning of each current trace mark the voltage applied to the patch pipette. Upward deflections are the opening events of the channel. B, the current-voltage relations of BKCa channels in the absence () and presence () of tetrandrine (5 µM). Of note, the single-channel conductance in the absence and presence of tetrandrine is nearly identical. Each point represent mean ± S.E. (n = 9-10). C, the relationship between Po of BKCa channels and membrane potential in the absence () and presence () of 5 µM tetrandrine. The smooth lines represent the best fit to the Boltzmann equation as described in Results.

Effect of Tetrandrine on Activation Curve of BK_Ca Channels. To examine the voltage dependence of tetrandrine effect on BK_Ca channels, the activation curves of BK_Ca channels in the absence and presence of tetrandrine (5 µM) were constructed. In these experiments, the outside-out configuration was performed, and the patch pipette contained 1 µM Ca²⁺. As shown in Fig. 6C, the relationships between membrane potentials and P_o of BK_Ca channels with or without the application of tetrandrine (5 µM) were plotted and well fit by the Boltzmann equation using a nonlinear regression analysis: N P_o = n/{1 + exp[  (V  a)/b]}, where N is the number of channels in the patch, n is the maximal N P_o level, V is the membrane potential in mV, a is the membrane potential for half-maximal activation, and b is the slope factor of the activation curve. In control, a is 65.1 ± 1.8 mV, b is 6.8 ± 0.2, and n is 1.20 ± 0.12 (n = 5), whereas in the presence of tetrandrine (5 µM), a is 80.4 ± 2.1 mV, b is 6.5 ± 0.2, and n is 0.58 ± 0.08 (n = 4). Thus, the presence of tetrandrine was found to shift the activation curve to more positive membrane potentials by approximately 15 mV, as well as to inhibit the maximal P_o of BK_Ca channels. However, there was no significant difference in the slope (i.e., b value) of the activation curve between the absence and presence of tetrandrine. Thus, tetrandrine can suppress the activity of BK_Ca channels in a voltage-dependent fashion in these cells.

Effect of Tetrandrine on Kinetic Behavior of BK_Ca Channels. Because tetrandrine produced no effect on single-channel conductance, the effect of tetrandrine on the gating of these channels was further analyzed. As shown in Fig. 7, in an outside-out patch of control cells (i.e., in the absence of tetrandrine), both open-time and closed-time histograms of BK_Ca channel at the level of +60 mV can be fitted by a two-exponential curve. The time constants for the fast and slow components of open-time histogram were 2.1 ± 0.3 and 16.1 ± 1.2 ms (n = 5), respectively, whereas those in closed-time histogram were 1.5 ± 0.3 and 38.9 ± 2.4 ms (n = 5), respectively. The addition of tetrandrine (5 µM) significantly decreased the fast and slow and time constants of the open state to 1.4 ± 0.2 and 9.1 ± 0.9 ms (n = 5, P < .05) and increased the mean closed time to 5.2 ± 0.7 and 82.8 ± 4.3 ms (n = 5, P < .05). Thus, it is clear that the presence of tetrandrine can alter the gating behavior of BK_Ca channels expressed in these cells and that its inhibitory effect on the channel activity is due to both a decrease in mean open time and an increase in mean closed time.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Effect of tetrandrine on mean open and closed time of BKCa channels in human umbilical vascular endothelial cells. Under symmetrical K+ condition, the membrane patch was held at +60 mV in an outside-out membrane patch, and the patch pipette contained 1 µM Ca2+. The open-time (top) and closed-time (bottom) histograms in the absence (left) and presence (right) of tetrandrine (5 µM) are shown. Open- and closed-time histograms were fitted by a two-exponential function with the time constants shown in the graph. Note that the abscissa and ordinate show the logarithm of the open or closed time and the square root of the number of events, respectively. The smooth curves indicate two exponential fitting using the least-squares method. In control, data were obtained from a measurement of 1032 channel openings with a total record time of 30 s, and in the presence of tetrandrine, data were measured from 784 channel openings with a total record time of 1 min. o(f) and o(s) represent fast and slow time constants of mean open time, respectively, whereas c(f) and c(s) represent fast and slow time constants of mean closed time, respectively.

Lack of Effect of Internal Ca²⁺ Concentration on Tetrandrine-Induced Inhibition of BK_Ca Channels. Whether the reduced activity of BK_Ca channels caused by tetrandrine is related to internal Ca²⁺ concentration was also determined. In this series of experiments, the outside-out configuration was performed, and various concentrations of Ca²⁺ were included in the recording pipette. As shown in Fig. 8, increasing the concentrations of internal Ca²⁺ was found to significantly enhance the activity of BK_Ca channels. At a given concentration of tetrandrine (5 µM), the magnitude of tetrandrine-induced inhibition on BK_Ca channels was also increased as internal Ca²⁺ was elevated. However, we failed to find that the inhibitory effect of tetrandrine on BK_Ca channels was related to the change in the level of intracellular Ca²⁺ concentration. For instance, at the holding potential of +60 mV, tetrandrine (5 µM) caused a decrease in the P_o of BK_Ca channels at internal Ca²⁺ concentrations of 0.1, 1, and 10 µM to a similar extent (i.e., about 50% decrease).

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Lack of dependence on internal Ca2+ concentrations of tetrandrine-induced decrease of BKCa channels. In this series of experiments, an outside-out configuration with symmetrical K+ concentrations was used. The patch pipettes were filled with various concentrations of Ca2+, and the holding potential was set at +60 mV. Tetrandrine (5 µM) was applied to the extracellular surface of membrane patch. A, the original current traces showing BKCa channel activity in the absence (left) and presence of 5 µM tetrandrine (right) at various concentrations of internal Ca2+. The numbers shown on the left mark the internal Ca2+ concentration. Upward deflections represent channel openings. B, summary of the effect of tetrandrine on channel activity in which patch pipettes contained different concentrations of Ca2+. Each point represents the mean ± S.E. (n = 5-8). *, significant difference from controls (P < .05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The major findings of this study were as follows. First, in cultured endothelial cells of human umbilical veins, tetrandrine suppressed K⁺ outward current in a concentration-dependent manner. Second, either Evans' blue or niflumic acid could reverse tetrandrine-mediated inhibition of the K⁺ outward current. Third, tetrandrine did not change single-channel conductance of BK_Ca channels but significantly suppressed channel activity by decreasing mean open time and increasing mean closed time. Fourth, tetrandrine produced a rightward shift in the activation curve of BK_Ca channels. Finally, tetrandrine-induced block in BK_Ca channels was independent of intracellular Ca²⁺ concentration. Our study allows us to suggest that tetrandrine-induced decrease in the activity of BK_Ca channels in endothelial cells may involve its vasoconstrictor action in endothelium-intact blood vessels.

The results presented in the present study are consistent with a previous finding that tetrandrine produced an inhibition of BK_Ca channels (Wang and Lemos, 1995). However, the concentration required for the inhibition of BK_Ca in our study is higher than that of the report of Wang and Lemos (1995). The IC₅₀ value of tetrandrine required for the inhibition of K⁺ outward current in endothelial cells was 5 µM, whereas in rat neurohypophysial nerve terminals, tetrandrine inhibited charybdotoxin-insensitive BK_Ca with an IC₅₀ value of 0.5 µM. On the other hand, by comparison, the concentration of tetrandrine required for the inhibition of Ca²⁺ currents is similar to that used in the present study (Weinsberg et al., 1994; Liu et al., 1995; Wang and Lemos, 1995; Wu et al., 1997, 1998). In addition, in rat tail artery, tetrandrine inhibited KCl-induced contraction with an IC₅₀ value of 6 µM (Liu et al., 1995). This value is comparable to the IC₅₀ value reported in our study. Thus, BK_Ca channels expressed in endothelial cells are likely to be a relevant "target" for the action of tetrandrine.

In our study, tetrandrine had no effect on single-channel conductance of BK_Ca channels in human umbilical vascular endothelial cells; thus, the reduction in the conductance of whole-cell current shown in Fig. 1 must be due to a decrease in channel P_o. In addition, the present results showed that tetrandrine shifted the activation curve of BK_Ca channels to the right but did not affect the slope factor. Thus, tetrandrine is likely to produce the inhibition of BK_Ca via a direct effect on the channel or a closely associated site, although the exact mechanisms by which this action occurs remain to be determined. The present results demonstrating the voltage dependence of tetrandrine-mediated inhibition of the activity of BK_Ca channels suggested that the magnitude of its inhibition would be dependent on the preexisting level of resting membrane potential, the concentration of tetrandrine used, or both, assuming that tetrandrine action in endothelial cells in vivo is the same as that on these cells shown in the present study.

It was reported that tetrandrine could increase the level of intracellular cAMP in neutrophils (He et al., 1989). Recently, many studies have found that tetrandrine elevated intracellular Ca²⁺ and blocked Ca²⁺ entry that had been induced by emptying of Ca²⁺ stores in leukemic HL-60 cells, vascular smooth myocytes, and endothelial cells (Leung et al., 1994; Liu et al., 1995; Low et al., 1996). However, in our study, we examined the channel activity in the outside-out configuration and found that tetrandrine was capable of significantly suppressing the activity of BK_Ca channels in these endothelial cells. It thus seems unlikely that tetrandrine-mediated inhibition of these channels requires the cytosolic-diffusible substances (e.g., cAMP). In addition, the present results showing that its inhibitory action was independent on internal Ca²⁺ concentration suggest that the inhibition of channel activity is not related to the decreased availability of cytosolic Ca²⁺, from either a decrease in Ca²⁺ influx or reduced Ca²⁺ release from internal stores. In fact, in endothelial cells preincubated with thapsigargin, tetrandrine was also found to effectively suppress the amplitude of K⁺ outward currents.

A recent report found that tetrandrine-mediated vasoconstriction in endothelium-intact preparations seemed to be due to the reduced production of nitric oxide (Kwan et al., 1999). However, in our study, in cells preincubated with sodium nitroprusside, the tetrandrine-mediated inhibition of outward currents was not significantly affected. Therefore, it is likely that the inhibition by tetrandrine on the generation of nitric oxide in endothelial cells is not responsible for the suppression of BK_Ca channels observed in the present study.

The single-channel conductance of BK_Ca channels measured with the use of 145 mM K⁺ on both sides of the membrane was 170 ± 9 pS (n = 10). This value is similar to those of typical BK_Ca channels previously reported in bovine aortic endothelial cells (Fichtner et al., 1987), rabbit endothelial cells (Rusko et al., 1992), and EAhy926 endothelial cells (Haburcák et al., 1997). However, BK_Ca channels observed in this study have properties different from those reported in neurohypophysial nerve terminals (Wang and Lemos, 1992); that is, the channel activity present in these cells is sensitive to charybdotoxin or iberiotoxin but is not suppressed by apamin. Previous studies also showed that the cells with a human BK_Ca channel (hSlo) phenotype were less sensitive to tetrandrine than those in which hSlo and hSlo were coexpressed (Dworetzky et al., 1996; Gribkoff et al., 1996). Taken together, these results reflect that the existence of different splice variants of the channel or association of the channel with additional subunits may underline the observed difference. Previous reports showed that most BK_Ca channels present in smooth muscle cells are composed to - and -subunits (Vogalis et al., 1996; Tanaka et al., 1997). Therefore, BK_Ca channels found in this cell line appear to contain additional subunits that can modulate the sensitivity to tetrandrine. Because a cell line used in the present study might diverge from its original properties, further investigations are required before any conclusion can be reached as to the inhibitory effects of tetrandrine on BK_Ca channels in a variety of endothelial cells. Nevertheless, the tetrandrine-induced reduction in the amplitude of whole-cell outward current may arise from different types of single-channel kinetic behavior. Our study reported here clearly shows that a tetrandrine-mediated decrease in outward currents in these cells is not due to a decrease in single-channel conductance but rather to a decrease in mean open time and an increase in mean closed time. The effect of tetrandrine on the kinetic behavior of BK_Ca channels appears to be different from that of iberiotoxin, because iberiotoxin usually causes long periods of closure (Candia et al., 1992).

It has been reported that tetrandrine can block Ca²⁺ entry induced by the depletion of Ca²⁺ stores in various type of cells, including endothelial cells (Leung et al., 1994; Liu et al., 1995; Low et al., 1996; Takemura et al., 1996). The nonselective cation channels present in endothelial cells were found to be involved in the regulation of intracellular Ca²⁺ concentration and in the control of the activity of BK_Ca channels (Fichtner et al., 1987; Ling and O'Neill, 1992; Nilius et al., 1997). Tetrandrine may block these channels, thus leading to a decrease in intracellular Ca²⁺ concentration. However, the concentration required for the inhibition of thapsigargin-induced Ca²⁺ entry (IC₅₀ = ~20 µM) is relatively higher than that used for the blockade of BK_Ca channels (Wang and Lemos, 1992; Leung et al., 1994; Liu et al., 1995). Therefore, it requires further study to determine the extent to which tetrandrine-mediated blockade of these cation channels contributes to its inhibitory effect on the activity of BK_Ca channels.

In summary, the present study clearly demonstrates that in addition to the blockade of Ca²⁺ channels, tetrandrine at therapeutically relevant concentrations can directly inhibit the activity of BK_Ca channels in cultured endothelial cells of human umbilical veins. Because lining endothelial cells are functionally coupled to vascular smooth myocytes, the change in membrane potential is likely to be electrotonically transmitted to smooth muscle cells (Bény, 1999). The blockade of BK_Ca channels has been demonstrated to cause membrane depolarization and vasoconstriction (Cabell et al., 1994; Wu et al., 1995; Koichi et al., 1997; Edwards et al., 1998). Thus, assuming that similar results were found in endothelial cells in vivo to those occurring in these cells, the inhibitory effect of tetrandrine on BK_Ca channels might significantly contribute to its change in functional activities of endothelial cells lining microvascular wall and to its vasoconstrictor effects.