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J. Biol. Chem., Vol. 279, Issue 26, 26885-26892, June 25, 2004

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Stimulatory Actions of Caffeic Acid Phenethyl Ester, a Known Inhibitor of NF-B Activation, on Ca²+-activated K⁺ Current in Pituitary GH₃ Cells^*

Ming-Wei Lin, Su-Rong Yang, Mei-Han Huang, and Sheng-Nan Wu

From the Institute of Basic Medical Sciences, National Cheng-Kung University Medical College, Tainan 701, Taiwan

Received for publication, January 13, 2004 , and in revised form, March 22, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Caffeic acid phenethyl ester (CAPE), a phenolic antioxidant derived from the propolis of honeybee hives, is known to be an inhibitor of activation of nuclear transcript factor NF-B. Its effects on ion currents have been investigated in pituitary GH₃ cells. This compound increased Ca²⁺-activated K⁺ current (I_K(Ca)) in a concentration-dependent manner with an EC₅₀ value of 14 ± 2 µM. However, the magnitude of CAPE-induced stimulation of I_K(Ca) was attenuated in GH₃ cells preincubated with 2,2'-azo-bis-(2-amidinopropane) hydrochloride (100 µM) or t-butyl hydroperoxide (1 mM). CAPE (50 µM) slightly suppressed voltage-dependent L-type Ca²⁺ current. In inside-out configuration, CAPE (20 µM) applied to the intracellular face of the detached patch enhanced the activity of large conductance Ca²⁺-activated K⁺ (BK_Ca) channels with no modification in single-channel conductance. After BK_Ca channel activity was increased by CAPE (20 µM), subsequent application of nordihydroguaiaretic acid (20 µM) did not further increase the channel activity. CAPE-stimulated channel activity was dependent on membrane potential. CAPE could also increase Ca²⁺ sensitivity of BK_Ca channels in these cells. Its increase in the open probability could primarily involve a decrease in the mean closed time. In current-clamp conditions, CAPE hyperpolarized the membrane potential and reduced the firing of action potentials. The stimulatory effects on these channels may partly contribute to the underlying mechanisms through which this compound influences the functional activities of neurons or neuroendocrine cells. Caution has to be used in attributing its response in the activation of NF-B.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Caffeic acid phenethyl ester (CAPE)¹ is a phenolic antioxidant that has been identified as one of the major components of honeybee propolis (1). It has been demonstrated to be a specific inhibitor of activation of nuclear transcript factor NF-B (2, 3). Previous studies have shown that this compound could protect the spinal cord and brain from ischemia reperfusion injury (4, 5) and prevent neurotoxic events caused by excessive inflammatory reaction in brain (6). Several lines of evidence also indicate that CAPE may modify the redox state in transformed fibroblast cells and in leukemic HL-60 cells (7–9). Furthermore, it has been reported that this compound inhibited the contractile response to phenylephrine or to high K⁺ solution in isolated rat thoracic aorta (10). However, to our knowledge, the effects of CAPE on ion currents have not been thoroughly studied.

Large conductance Ca²⁺-activated K⁺ (BK_Ca) channels play important roles in controlling the excitability of nerve, muscle, and other cells by stabilizing cell membrane at negative potentials (11). Their gating is known to be controlled by intracellular Ca²⁺ and/or membrane depolarization. The challenging of cells with oxidizing agents has been found to suppress the channel activity of these channels (12). Pituitary GH₃ cells have been demonstrated to exhibit the activity of these channels (13). Riluzole and ciglitazone, both of which were reported to prevent neuronal injuries, could enhance the activity of BK_Ca channels functionally expressed in these cells (14, 15). Importantly, the opener of these channels has been shown to counteract the deleterious effects of excitatory neurotransmitters following neurotoxic or ischemic injuries (16). Previous studies also revealed that the BK_Ca channel might be a relevant target of DNA synthesis in cultured Müller glial cells (17).

Therefore, the objective of this study was to (a) address the question of whether CAPE could affect Ca²⁺-activated K⁺ currents (I_K(Ca)) in GH₃ cells; (b) determine the effects of this compound on the activity of BK_Ca channels; and (c) examine whether it can influence the membrane potential. Interestingly, the present results indicate that in GH₃ lactotrophs, CAPE does not appear to affect the activation of NF-B exclusively, despite its ability to inhibit NF-B activation in these cells (18). The CAPE-induced increase in BK_Ca channel activity may account, at least in part, for its effects on cellular functions in neurons or neuroendocrine cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture—The clonal strain GH₃ cell line, originally derived from a rat anterior pituitary adenoma, was obtained from the Culture Collection and Research Center (CCRC-60015; Hsinchu, Taiwan). The detailed methods have been previously described (19). Briefly, the cells were cultured in Ham's F-12 medium (Invitrogen) supplemented with 15% heat-inactivated horse serum (v/v), 2.5% fetal calf serum (v/v), and 2 mM L-glutamine (Invitrogen) in a humidified environment of 5% CO₂/95% air. The experiments were generally performed 5 or 6 days after cells were subcultured (60–80% confluence).

Electrophysiological Measurements—Immediately before each experiment, the cells were dissociated, and an aliquot of cell suspension was transferred to a recording chamber positioned on the stage of an inverted microscope (DM IL; Leica, Wetzlar, Germany). The cells were bathed at room temperature (20–25 °C) in normal Tyrode's solution containing 1.8 mM CaCl₂. The recording pipettes were pulled from Kimax-51 capillaries (Kimble Glass, Vineland, NJ) using a two-stage microelectrode puller (PP-830; Narishige, Tokyo, Japan), and the tips were fire-polished with a microforge (MF-83, Narishige). When filled with pipette solution, their resistance ranged between 3 and 5 M. Ion currents were measured in the cell-attached, inside-out, and whole cell configurations of the patch-clamp technique, using an RK-400 patchclamp amplifier (Bio-Logic, Claix, France) (19).

Data Recording and Analysis—The signals were displayed on an analog/digital oscilloscope (HM 507; Hameg, East Meadow, NY) and on a liquid crystal projector (PJ550-2; ViewSonic, Walnut, CA). The data were stored in a Pentium III grade laptop computer (Slimnote VX₃; Lemel, Taipei, Taiwan) at 10 kHz through a Digidata 1322A interface (Axon Instruments, Union City, CA). This device was controlled by a commercially available software (pCLAMP 9.0; Axon Instruments). The currents were low pass filtered at 1 or 3 kHz. Ion currents obtained during whole cell experiments were stored without leakage correction and analyzed using the pCLAMP 9.0 software (Axon Instruments) or the Origin 6.0 software (Microcal, Northampton, MA).

To calculate the percentage of stimulation of CAPE on I_K(Ca), each cell was depolarized from 0 to +50 mV, and current amplitude during cell exposure to CAPE was measured and compared. The amplitude of I_K(Ca) in the presence of this compound at a concentration 200 µM was taken as 100%. The concentration of CAPE required to increase 50% of current amplitude was then determined using a Hill function, y = (E_max x [C]^nh)/(EC₅₀^nh +[C]^nh), where [C] is the CAPE concentration, EC₅₀ is the concentration required for a 50% increase; n_h is the Hill coefficient, and E_max is the CAPE-induced maximal increase in the amplitude of I_K(Ca).

The amplitudes of single BK_Ca channel currents were determined by fitting Gaussian distributions to the amplitude histograms of the closed and the open state. The channel open probability in a patch was expressed as N·P_o, which can be estimated using the following equation: N·P_o = (A₁ + 2A₂ + 3A₃ +... + nA_n)/(A₀ + A₁ + A₂ + A₃ +... + A_n), where N is the number of active channels in the patch, A₀ is the area under the curve of an all points histogram corresponding to the closed state, and A₁.... A_n represent the histogram areas reflecting the levels of distinct open state for 1 to n channels in the patch.

The relationships between the membrane potentials and the probability of channel openings obtained before and after the application of CAPE (20 µM) were fitted with a Boltzmann function of the form: N·P_o = n_P/{1 + exp[– (V – V)/k]}, where n_P = the maximal open probability, V = the membrane potential in mV, V is the voltage at which there is half-maximal activation, and k is the slope factor of the activation curve (i.e. the voltage dependence of the activation process in mV per e-fold change).

The averaged results are presented as the mean values ± S.E. The paired Student's t test was used for the statistical analyses. To further clarify the statistical difference among the two or four treatment groups, analyses of variance with Duncan's multiple range test for multiple comparisons were also performed. Differences between values were considered significant when p < 0.05.

Drugs and Solutions—CAPE (phenethyl caffeiate) was obtained from Cayman Chemical (Ann Arbor, MI). Curcumin, glibenclamide, nordihydroguaiaretic acid, and riluzole were obtained from Sigma/RBI (Natick, MA), and t-butyl hydroperoxide, dithiothreitol, and tetraethylammonium chloride from Sigma. 2,2'-Azo-bis(2-aminopropane) dihydrochloride (AAPH) was purchased from Wako Pure Industries, Ltd. (Osaka, Japan), paxilline from Biomol (Plymouth Meeting, PA), and cilostazol from Tocris Cookson Ltd. (Bristol, UK). Tetrodotoxin and apamin were obtained from Alomone Labs (Jerusalem, Israel), and fura-2 acetoxymethyl ester and fura-2 were from Molecular Probes (Eugene, OR). Squamocin was a gift from Dr. Yang-Chang Wu (Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung City, Taiwan).

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 the K⁺ currents or membrane potential, the recording pipette was backfilled with a solution consisting of 140 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. The free Ca²⁺ concentration of this solution was estimated to be 230 nM, assuming that the residual contaminating Ca²⁺ concentration was 70 µM, and the ratiometric fura-2 measurement with F-2500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) showed that this solution contained 205 ± 12 nM free Ca²⁺ for three different experiments. To measure voltage-dependent Ca²⁺ current, KCl inside the pipette solution was replaced with equimolar CsCl, and pH was adjusted to 7.2 with CsOH, whereas bathing solution contained 1 µM tetrodotoxin and 10 mM tetraethylammonium chloride.

For single-channel current recordings, the high K⁺ bathing solution contained 145 mM KCl, 0.53 MgCl₂ and 5 mM HEPES-KOH buffer, pH 7.4, and the pipette solution contained 145 mM KCl, 2 mM MgCl₂, and 5 mM HEPES-KOH buffer, pH 7.2. The value of free Ca²⁺ concentration was calculated assuming a dissociation constant for EGTA and Ca²⁺ (at pH 7.2) of 0.1 µM. To provide 0.1 µM free Ca²⁺ in bath solution, 0.5 mM CaCl₂ and 1 mM EGTA were added.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of CAPE on Ca²⁺-activated K⁺ Current (I_K(Ca)) in GH₃ Cells—In the first series of experiments, the whole cell configuration of the patch-clamp technique was used to investigate the effect of CAPE on ion currents in these cells. The cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl₂, and the pipette solution contained 0.1 mM EGTA and 3 mM ATP. ATP (3 mM) included in the pipette solution was effective at suppressing ATP-sensitive K⁺ channels (20). To inactivate other types of voltage-dependent K⁺ currents, each cell was held at the level of 0 mV. As illustrated in Fig. 1, when the cell was held at 0 mV and different potentials ranging from –10 to +60 mV with 10-mV increments were applied, a family of large, noisy, outward currents was elicited. These outward currents have been previously identified as I_K(Ca) (19). Interestingly, within 1 min of exposing the cells to CAPE (20 µM), the amplitude of outward currents was greatly increased throughout the entire range of voltage-clamp step. For example, when the cells were depolarized from 0 to +50 mV, current amplitudes measured at the end of the depolarizing pulses were increased to 552 ± 36 pA from a control of 228 ± 25 pA (p < 0.05; n = 8). This stimulatory effect was readily reversed after the removal of CAPE (Fig. 1). Fig. 1B illustrates the averaged I-V relationships for I_K(Ca) in control, during cell exposure to CAPE (20 µM) and washout of the compound.

View larger version (37K): [in this window] [in a new window]   FIG. 1. Stimulatory effect of CAPE on IK(Ca) in pituitary GH3 cells. The cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl2. A, superimposed current traces in control (top panel), during exposure to 20 µM CAPE (middle panel), and washout of CAPE (bottom panel). The cell was held at 0 mV, and the voltage pulses from –10 to +60 mV in 10-mV increments were applied. B, the averaged I-V relationships of outward currents measured at the end of voltage pulses in control (), after the application of 20 µM CAPE (), and after the washout of the compound (). Each point represents the mean ± S.E. (n = 6–9). C, concentration-response relationship for CAPE-induced stimulation of IK(Ca). Each cell was depolarized from 0 to +50 mV with a duration of 300 ms. The magnitude of IK(Ca) during cell exposure to CAPE (200 µM) was considered to be 100%. The smooth line represents the best fit to the Hill equation. The values for EC50 and the Hill coefficient were 14 ± 2 µM and 1.8, respectively. Each point represents the mean ± S.E. (n = 5–9).

Effect of CAPE on Voltage-dependent L-type Ca²⁺ Current (I_Ca,L) in GH₃ Cells—I_K(Ca) can be functionally coupled with Ca²⁺ influx through plasmalemmal voltage-dependent Ca²⁺ channels (21). A recent report also demonstrated that the action of CAPE on vasorelaxation in rat thoracic aorta could be due to the blockade of Ca²⁺ movement through the cell membrane (10). For these reasons, we further investigated whether it could exert any effect on I_Ca,L that was previously described in these cells (21, 22). These experiments were conducted with a Cs⁺-containing solution. The exposure to CAPE (20 µM) was found to be have little or no effect on I_Ca,L. However, this compound at a concentration of 50 µM slightly suppressed I_Ca,L, although it did not modify the I-V relationship of I_Ca,L (Fig. 2). For example, CAPE (50 µM) decreased the amplitude of I_Ca,L to 42 ± 3 pA from a control value of 51 ± 6 pA (p < 0.05; n = 7), when cells were depolarized from –50 to 0 mV. Therefore, this compound stimulated I_K(Ca) in a manner conceivably unlikely to be linked to an increase in the amplitude of I_Ca,L.

View larger version (24K): [in this window] [in a new window]   FIG. 2. Inhibitory effect of CAPE on voltage-dependent L-type Ca2+ current (ICa,L) in pituitary GH3 cells. In these experiments, the cells were bathed in normal Tyrode's solution that contained 1.8 mM CaCl2, 1 µM tetrodotoxin, and 10 mM tetraethylammonium chloride, and the recording pipettes were filled with a Cs+-containing solution. A, original currents showing the effect of CAPE (50 µM) on ICa,L. The cell was depolarized from –50 to 0 mV at a rate of 0.05 Hz. Trace a, control; trace b, CAPE (20 µM); trace c, CAPE (50 µM). B, the I-V relationships of ICa,L between the absence () and presence of 20 µM () and 50 µM () CAPE. Each point represents the mean ± S.E. (n = 5–7).

View larger version (25K): [in this window] [in a new window]   FIG. 3. Stimulatory effect of CAPE on the density of IK(Ca) in the absence (top panel) and presence of treatment with AAPH (middle panel) or t-butyl hydroperoxide (bottom panel). In these experiments, GH3 cells were incubated with AAPH (100 µM) or t-butyl hydroperoxide (1 mM) for 5 h. Each cell was held at 0 mV, and voltage pulses from –10 to +60 mV in 10-mV increments were applied. , control; , in the presence of 200 µM CAPE. Each point represents the mean ± S.E. (n = 4–8).

View larger version (33K): [in this window] [in a new window]   FIG. 4. Stimulatory effect of CAPE on the activity of BKCa channels in GH3 cells. The experiments were conducted with symmetrical K+ concentration (145 mM). Under inside-out configuration, the holding potential was set at +60 mV and the bath medium contained 0.1 µM Ca2+. A, the activity of BKCa channels recorded before (left panel) and during exposure (right panel) to 20 µM CAPE. The lower part in A shows current traces obtained in an expanded time scale corresponding to those labeled a and b in B and in the upper part of A. Upward deflection indicates the opening events of the channel. B, the time course of change in the channel open probability (0.5-s bin width) before and during the application of CAPE (20 µM) to the intracellular surface of the channel in an excised patch. C, amplitude histograms measured in the absence (left panel) and presence (right panel) of 20 µM CAPE.

View larger version (35K): [in this window] [in a new window]   FIG. 5. Stimulatory effects of CAPE, nordihydroguaiaretic acid, and riluzole on the activity of BKCa channels and the effects of CAPE on I-V relationship and Ca2+ sensitivity of these channels. The experiments were conducted in symmetrical K+ solution (145 mM), and the channels were recorded from inside-out patches of GH3 cells. A, original current traces recorded in control or in the presence of 20 µM nordihydroguaiaretic acid (NDGA), 20 µM nordihydroguaiaretic acid plus 20 µM CAPE, or 20 µM nordihydroguaiaretic acid plus 20 µM riluzole (Ril). The potential was held at +60 mV, and the compound tested was applied to the bath containing 0.1 µM Ca2+. The arrowheads indicate the zero current level. B, bar graph showing the effect of nordihydroguaiaretic acid, CAPE, nordihydroguaiaretic acid plus CAPE, CAPE plus riluzole, and nordihydroguaiaretic acid plus riluzole on the activity of BKCa channels. NDGA, 20 µM nordihydroguaiaretic acid; CAPE, 20 µM CAPE; Ril, 20 µM riluzole. In the experiments with nordihydroguaiaretic acid plus each compound (e.g. CAPE and riluzole), each compound was applied after the addition of nordihydroguaiaretic acid. In those with CAPE plus riluzole, riluzole was subsequently applied in continued presence of CAPE. Each point represents the mean ± S.E. (n = 6–9). *, significantly different from control group. **, significantly different from CAPE alone group. , significantly different from nordihydroguaiaretic acid alone group. C, examples of BKCa channels in the absence (left panel) and presence (right panel) of CAPE (20 µM) measured at different membrane potentials. The numbers shown to the left of the current traces mark voltage applied to the patch pipette. D, the I-V relationships of BKCa channels in the absence () and presence () of CAPE (20 µM). Each point represents the mean ± S.E. (n = 5–8). E, the relationship between the open probability of BKCa channels and membrane potential in the absence () and presence () of CAPE (20 µM). Each point represents the mean ± S.E (n = 4–8). F, stimulation of BKCa channels by CAPE at various concentrations of internal Ca2+. The potential was held at +60 mV, and various concentrations of Ca2+ in the bath before and during exposure to CAPE (20 µM) were applied (mean ± S.E.; n = 4–5 for each point).

Effect of CAPE on the Activation Curve of BK_Ca Channels— Fig. 5E shows the activation curve of BK_Ca channels in the absence and presence of CAPE (20 µM). The plot of open probability of BK_Ca channels as a function of membrane potential was fitted with a Boltzmann function as described under "Materials and Methods." In control, n_P = 0.35 ± 0.04, V = 75.4 ± 1.6 mV, and k = 10.7 ± 0.4 mV (n = 6), whereas in the presence of CAPE (20 µM), n_P = 0.71 ± 0.07, V = 61.2 ± 1.9 mV, and k = 10.9 ± 0.6 mV (n = 6). The data showed that the activation curve was shifted along the voltage axis to less positive potentials in the presence of CAPE. In contrast, no significant change in the slope (i.e. k value) of the activation curve was detected in the presence of this compound. Taken together, these results indicate that CAPE applied to the intracellular surface of the channel is capable of increasing the open probability in a voltage-dependent fashion.

Effect of Internal Ca²⁺ Concentration on CAPE-stimulated BK_Ca Channel Activity in GH₃ Cells—Whether the CAPE-induced increase in the activity of these channels is associated with internal Ca²⁺ concentration was also studied. In these experiments, when an excised membrane patch was formed, various concentrations of Ca²⁺ in the bath before and during exposure to CAPE (20 µM) were applied. As shown in Fig. 5F, the stimulatory effect of CAPE on BK_Ca channel activity was affected by changes in the level of intracellular Ca²⁺ concentration. For example, at the holding potential of +60 mV, the presence of CAPE (20 µM) caused a 2-fold increase in the open probability at internal Ca²⁺ concentration of 0.1 µM. However, at an internal Ca²⁺ concentration of 10 µM, CAPE at the same concentration stimulated channel activity by 4-fold.

Effect of CAPE on Kinetic Behavior of BK_Ca Channels in GH₃ Cells—The effect of CAPE on mean open and closed time of BK_Ca channels was examined and analyzed during recordings from patches showing only single-channel openings. As shown in Fig. 6, in control cells, the closed time histogram of BK_Ca channels at +60 mV can be fitted by a two-exponential curve with a mean closed time of 13.2 ± 2.8 and 55.8 ± 9.8 ms (n = 6). CAPE (20 µM) decreased the lifetime of the closed state to 10.9 ± 2.2 ms (n = 6). However, little or no change in mean open time was seen in the presence of CAPE (20 µM). Thus, the data demonstrate that its effect on BK_Ca channel activity in GH₃ cells is primarily due to a decrease in closed time.

View larger version (25K): [in this window] [in a new window]   FIG. 6. Effect of CAPE on mean closed time of BKCa channels in GH3 cells. Under symmetrical K+ condition, inside-out configuration was performed and potential was held at +60 mV. Closed time histogram in control (upper panel) was fitted by a two-exponential function with a mean closed time of 13.2 and 55.8 ms. The closed time histogram obtained after application of 20 µM CAPE to the bath (lower panel) was fitted by a single-exponential function with a mean closed time of 10.9 ms. The data were obtained from a measurement of 324 channel openings with a total recording time of 1 min in the control, and those obtained during the exposure to CAPE were measured from 421 channel openings with a total recording time of 30 s. The dashed lines shown in each lifetime distribution are placed at the value of the time constant in the closed state.

View larger version (30K): [in this window] [in a new window]   FIG. 7. Effects of CAPE on the firing of action potentials in GH3 cells and on the IK(Ca) underlying the ramp pulse with a duration similar to that in normal action potential waveforms of these cells. The cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl2. The patch pipettes were filled with a K+-containing solution. A, original potential trace showing the effect of CAPE on spontaneous action potentials. The horizontal bar indicates the application of CAPE (20 µM). B, bar graph showing the effect of CAPE in the absence and presence of paxilline on the firing of action potentials in these cells. CAPE, CAPE (20 µM); Pax, paxilline (1 µM). The parentheses shown with each bar indicates the number of cells examined. *, significantly different from control. **, significantly different from CAPE alone group. In C, the cell was held at the level of –50 mV, and the ramp pulses from +20 to –50 mV with a duration of 100 ms were applied to mimic the shape and duration of normal action potential waveforms present in GH3 cells. Trace A, control; trace b, in the presence of 20 µM CAPE. The upper panel in C indicates the voltage protocol used. D, summary of the data showing the effect of CAPE on the peak of IK(Ca) that is active in normal action potential waveforms. Each cell represents the mean ± S.E. (n = 6). CAPE, CAPE (20 µM); Pax, paxilline (1 µM). *, significantly different from control. **, significantly different from CAPE alone group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study shows that CAPE (a) increases the amplitude of I_K(Ca) in a concentration-dependent manner in pituitary GH₃ cells, (b) enhances the activity of BK_Ca channel in a voltage-dependent manner, and (c) reduces the repetitive firing of action potentials. This compound increased the probability of these channels in a mechanism unlikely to be linked to its inhibition of activation of NF-B. The stimulation by CAPE of I_K(Ca) conceivably could be one of the mechanisms underlying CAPE-induced actions, if similar results occur in neurons or neuroendocrine cells in vivo.

The EC₅₀ value of CAPE required for the stimulation of I_K(Ca) was 14 µM in this study. This value is similar to that required for the inhibition of NF-B activation (2). Therefore, there might be a link between the actions of CAPE on neurons or neuroendocrine cells and its observed effects on ion channels, although further experiments are required to find out whether CAPE can interact with the BK_Ca channel to influence I_K(Ca) in other types of cells. However, a lack of effect of glibenclamide excludes the involvement of activation of ATP-sensitive K⁺ channel in CAPE-stimulated I_K(Ca) in these cells.

A previous study showed that activation of NF-B could be accompanied by a decrease in the current density of smooth muscle L-type Ca²⁺ channel (29). However, the present results indicate that the CAPE-induced increase in I_K(Ca) does not depend on the increased availability of intracellular Ca²⁺ resulting from the enhanced Ca²⁺ influx through voltage-dependent Ca²⁺ channels, because it was not found to increase the amplitude of I_Ca,L. These observations are compatible with a recent report showing the inability of CAPE to alter the decrease in intracellular Ca²⁺ induced by low K⁺ solution in cerebellar granule cells (30). Our results demonstrating that this compound at a concentration of 50 µM produced a slight reduction in the amplitude of I_Ca,L can also account for its ability to inhibit high K⁺-induced vasoconstriction in isolated rat aorta (10).

It has been demonstrated that dithiothreitol could stimulate I_K(Ca) in GH₃ cells (31). In AAPH-treated cells, the stimulatory effect of CAPE on I_K(Ca) was attenuated, and a subsequent application of dithiothreitol effectively increased the amplitude of I_K(Ca). These results suggest that the sulfhydryl oxidizing and reducing agents can produce an effect on I_K(Ca) in GH₃ cells. It will be interesting to determine to what extent the decreased production of reactive oxygen species caused by CAPE affects the stimulatory effect of CAPE on I_K(Ca), because this compound is known to be a potent flavonoid antioxidant (1, 23). Moreover, it seems likely that a decrease in the production of reactive oxygen species caused by CAPE is upstream of its stimulation of I_K(Ca). Direct activation of BK_Ca channels and indirect inhibition of production of reactive oxygen species may synergistically contribute to the underlying cellular mechanisms through which this compound modifies the repetitive firing of these cells. In addition, like NS004 (32), CAPE was found to increase Ca²⁺ sensitivity of BK_Ca channels observed in GH₃ cells. Its ability to increase Ca²⁺ sensitivity of BK_Ca channels suggests that the CAPE molecule may modify the cysteine residues near the carboxyl-terminal, Ca²⁺ bowl domain of these channels (33).

Our study demonstrated that CAPE could not modify single-channel conductance of BK_Ca channels, but it did increase the channel open probability. The increase in the amplitude of I_K(Ca) caused by CAPE is primarily thought to be a result of a decrease in mean closed time. It was also seen that CAPE shifted the activation curve of BK_Ca channels to the left with no modification in the slope factor of this curve. This compound thus appears to produce the stimulation of BK_Ca channels by a direct effect on the channel or closely associated site, although the precise mechanisms of its action remain to be further elucidated. However, our data demonstrated that CAPE applied to the intracellular face of the excised patch produced a fraction of channel closings to shift to short-lived closings, resulting in one closed kinetic state.

It is worth mentioning that unlike the molecules of NS004, NS1619, or riluzole, the CAPE molecule has the juxtaposition of two aromatic rings, the unique structure of which is similar to those of some BK_Ca channel openers, such as nordihydroguaiaretic acid and resveratrol (Fig. 8) (34). The present results demonstrated that the stimulatory effect of CAPE and nordihydroguaiaretic acid on the BK_Ca channel was not additive. It is thus tempting to speculate that these two compounds, which are structurally related, may interact with the same binding site in the channel.

View larger version (14K): [in this window] [in a new window]   FIG. 8. Chemical structures of CAPE, nordihydroguaiaretic acid, and resveratrol.

Curcumin, another inhibitor of NF-B activation, was not found to have effects on BK_Ca channels, when it was applied to intracellular face of the excised patch. The results lead us to suggest that CAPE could induce the change in the activity of BK_Ca channels in GH₃ cells in a mechanism unlikely to be linked to its inhibition of NF-B activation. However, its change in membrane potential can be explained by the stimulatory effect on these channels. Such an effect may be responsible for its actions on neurons or neuroendocrine cells in vivo, despite the ability of this compound to inhibit activation of NF-B in GH₃ cells (18). Furthermore, CAPE and other structurally related compounds seem to be intriguing pharmacological tools used to characterize the properties of the BK_Ca channels. Elucidation of the structure of the binding site for CAPE or other structurally related compounds might provide a structural basis for the pharmacological modulation of BK_Ca channels.

	FOOTNOTES

 
^* This work was supported by National Science Council Grants NSC-91-2320B-006-106 and NSC-92-2320B-006-041. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Institute of Basic Medical Sciences, National Cheng-Kung University Medical College, No. 1, University Road, Tainan 701, Taiwan. Tel.: 886-6-2353535-5334; Fax: 886-6-2353660; e-mail: snwu{at}mail.ncku.edu.tw.

¹ The abbreviations used are: CAPE, caffeic acid phenethyl ester; BK_Ca, large conductance Ca²⁺-activated K⁺; AAPH, 2,2'-azo-bis(2-aminopropane) dihydrochloride.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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