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Cilostazol, an Inhibitor of Type 3 Phosphodiesterase, Stimulates Large-Conductance, Calcium-Activated Potassium Channels in Pituitary GH₃ Cells and Pheochromocytoma PC12 Cells

Institute of Basic Medical Sciences (S.-N.W., M.-H.H.), National Cheng-Kung University Medical College, 701 Tainan, Taiwan; and Department of Surgery (S.-.I.L.), Kaohsiung Veterans General Hospital, 813 Kaohsiung City, Taiwan

Address all correspondence and requests for reprints to: Dr. Sheng-Nan Wu, Institute of Basic Medical Sciences, National Cheng-Kung University Medical College, No. 1, University Road, 701 Tainan, Taiwan. E-mail: snwu{at}mail.ncku.edu.tw.

	Abstract

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The effects of cilostazol, a dual inhibitor of type 3 phosphodiesterase and adenosine uptake, on ion currents were investigated in pituitary GH₃ cells and pheochromocytoma PC12 cells. In whole-cell configuration, cilostazol (10 µM) reversibly increased the amplitude of Ca²⁺-activated K⁺ current [I_K(Ca)]. Cilostazol-induced increase in I_K(Ca) was suppressed by paxilline (1 µM) but not glibenclamide (10 µM), dequalinium dichloride (10 µM), or ß-bungarotoxin (200 nM). Pretreatment of adenosine deaminase (1 U/ml) or ,ß-methylene-ADP (100 µM) for 5 h did not alter the magnitude of cilostazol-stimulated I_K(Ca). Cilostazol (30 µM) slightly suppressed voltage-dependent L-type Ca²⁺ current. In inside-out configuration, bath application of cilostazol (10 µM) into intracellular surface caused no change in single-channel conductance; however, it did increase the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels. Cilostazol enhanced the channel activity in a concentration-dependent manner with an EC₅₀ value of 3.5 µM. Cilostazol (10 µM) shifted the activation curve of BK_Ca channels to less positive membrane potentials. Changes in the kinetic behavior of BK_Ca channels caused by cilostazol were related to an increase in mean open time and a decrease in mean closed time. Under current-clamp configuration, cilostazol decreased the firing frequency of action potentials. In pheochromocytoma PC12 cells, cilostazol (10 µM) also increased BK_Ca channel activity. Cilostazol-mediated stimulation of I_K(Ca) appeared to be not linked to its inhibition of adenosine uptake or phosphodiesterase. The channel-stimulating properties of cilostazol may, at least in part, contribute to the underlying mechanisms by which it affects neuroendocrine function.

	Introduction

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CILOSTAZOL, A THROMBOLYTIC AND vasodilator drug, has been developed as a selective inhibitor of cyclic nucleotide phosphodiesterase type 3A (1, 2). It has been regarded as a useful tool to investigate the adenylyl cyclase-cAMP pathway (3, 4, 5). However, cilostazol appears to possess an additional effect that does not require the inhibition of phosphodiesterases (2, 5). It was reported to be an inhibitor of adenosine uptake in a variety of cells (5, 6). For example, this drug could enhance the adenosine-induced increase in cAMP in Chinese hamster ovary cells in which adenosine receptors were overexpressed (6). In Calu-3 cell monolayers, cilostazol has been shown to produce a short circuit current that was sensitive to inhibition by glibenclamide (5). It could potentiate the increase of this current caused by the stimulation of adenosine receptors with adenosine (5). In addition, it has been reported to suppress acetylcholine-induced catecholamine secretion in bovine adrenal chromaffin cells (7). The latter findings appeared not to be related to an increase in intracellular cAMP. On the other hand, this drug has been reported to improve blood flow, facilitate axonal regeneration, and prevent impairment of slow axonal transport in streptozotocin-induced diabetic rats (8, 9, 10, 11, 12). It has been found to have a neuroprotective effect against cerebral infarct (13, 14). However, the effect of cilostazol on ion currents in neurons or neuroendocrine cells has not been studied extensively, although a recent report (15) showed the ability to stimulate Ca²⁺-activated K⁺ currents [I_K(Ca)] in human neuroblastoma SK-N-SH cells.

The large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel, the gating of which is controlled by intracellular Ca²⁺ levels and/or membrane depolarization, plays a role in the regulation of neuronal excitability and the coupling of excitation-contraction and stimulus-secretion (16). Cloning and expression of - and ß-subunits, which these channels comprise, will enable us to elucidate the structural components that control their gating (16, 17). We have demonstrated that riluzole and ciglitazone, both of which were reported to prevent neuronal injuries, could enhance the activity of BK_Ca channels found in GH₃ cells (18, 19). Importantly, previous studies have demonstrated that the opener of BK_Ca channels could counteract the deleterious effects of excitatory neurotransmitters after neurotoxic injuries (20).

Therefore, the goal of this study was to examine the effect of cilostazol on I_K(Ca) in pituitary GH₃ cells, address the issue whether cilostazol affects the activity and kinetic properties of BK_Ca channels, and determine whether cilostazol can affect the activity of BK_Ca channels in pheochromocytoma PC12 cells. Interestingly, these results indicate that cilostazol can enhance the activity of BK_Ca channels in a mechanism unlikely to be linked to the inhibition of phosphodiesterase or adenosine uptake.

	Materials and Methods

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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 (21). Briefly, cells were cultured in Ham’s F-12 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 15% heat-inactivated horse serum (vol/vol), 2.5% fetal calf serum (vol/vol), and 2 mM L-glutamine (Life Technologies) in a humidified environment of 5% CO₂/95% air. The experiments were performed 5 or 6 d after cells were subcultured (60–80% confluence). In a separate series of experiments, GH₃ cells were incubated with adenosine deaminase (1 U/ml) or ,ß-methylene-ADP (AOPCP, 100 µM) at 37 C for 5 h.

Stock cultures of rat pheochromocytoma PC12 cells were also obtained from the Culture Collection and Research Center (CCRC-60048). PC12 cells were maintained in RPMI 1640 medium (Life Technologies) supplemented with 10% heat-inactivated horse serum (vol/vol) and 5% fetal bovine serum (vol/vol) in a 5% carbon dioxide atmosphere at 37 C.

Electrophysiological measurements
Immediately before each experiment, 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 Microsystems, Wetzlar, Germany). 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 thin-walled borosilicate glass capillaries (Kimax-51, 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 recorded in the cell-attached, inside-out, and whole-cell configurations of the patch-clamp technique, using an RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) (21). All potentials were corrected for liquid junction potential, a value that would always develop at the tip of the pipette when the composition of the pipette solution was different from that in the bath.

Data recording and analysis
The signals were displayed on an analog/digital oscilloscope (HM 507, Hameg Inc., East Meadow, NY) and a liquid crystal projector (PJ550-2, ViewSonic Corp., Walnut, CA). The data were on-line stored in a Pentium III-grade laptop computer (Slimnote VX₃, Lemel, Taipei, Taiwan) via a universal serial bus port at 10 kHz through a high-speed/low-noise analog/digital interface (Digidata 1322A, Axon Instruments, Union City, CA). This device was controlled by a commercially available software (pCLAMP 9.0, Axon Instruments). Currents were low-pass filtered at 1 or 3 kHz. Ion currents recorded during whole-cell experiments were stored without leakage correction and analyzed subsequently using the pCLAMP 9.0 software (Axon Instruments), the Origin 6.0 software (Microcal Software, Inc., Northampton, MA), SigmaPlot 7.0 software (SPSS, Inc., Apex, NC), or custom-made macros in Excel (Microsoft, Redmont, WA). The pCLAMP-generated voltage-step protocols were used to examine the current-voltage (I-V) relations for ion currents.

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, respectively. Channel dwell times were determined by applying a standard half-amplitude crossing protocol using the pCLAMP 9.0 software (Axon Instruments). The activity of the channel 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 single-channel conductance was calculated by linear regression using mean current amplitudes measured at different voltages.

To calculate the concentration-response relationship for cilostazol-stimulated BK_Ca channel activity, the potential was held at the level of +60 mV and the bath medium contained 0.1 µM Ca²⁺. The probability of channel openings measured during the exposure to cilostazol (100 µM) was taken as 100%. The concentration-dependent relationship of cilostazol on the channel open probability was fitted to a Hill equation: y = (E_max x [C]ⁿ_h)/(EC₅₀ⁿ_h +[C]ⁿ_h), where [C] is the concentration of cilostazol; EC₅₀ and n_h are the concentration required for a 50% inhibition and the Hill coefficient, respectively; and E_max is the cilostazol-induced maximal increase in channel activity. The Solver subroutine in Microsoft Excel was used to fit data by a least-squares minimization procedure.

To determine the effect of cilostazol on the activation curve of BK_Ca channels, the ramp pulses from 0 to +80 mV with a duration of 1 sec were delivered from pCLAMP 9.0 software. The activation curve was calculated by averaging current traces in response to 20 voltage ramps and dividing each point of the mean current by the unitary amplitude of each potential after the leakage component was corrected. The activation curves obtained before and after the addition of cilostazol were fitted with Boltzmann function of the form: relative N P_o = n_P/{1 + exp[-(V - a)/b]}, where n_P is the maximal relative N P_o, b is the slope factor of the voltage-dependent activation (i.e. the change in the potential required to result in an e-fold increase in the activation), and a is the voltage at which there is half-maximal activation.

The averaged results are presented as the mean values ± SEM. The paired or unpaired t test and one-way ANOVA with the least significance difference method for multiple comparisons were used for the statistical evaluation of differences among the mean values. Differences between values were considered significant when P < 0.05.

Drugs and solutions
Cilostazol [Pletal, 6-(4-[1-cyclohexyl-1H-tetrazol-5-yl]butoxy)-3,4-dihydro-2-(1H)-quinolinone] and dequalinium dichloride were obtained from Tocris Cookson Ltd. (Bristol, UK), and sp-adenosine-3',5'-cyclic monophosphorothioate (sp-cAMPS) was from Biomol (Plymouth Meeting, PA). Adenosine, OPCP, glibenclamide, and tetraethylammonium chloride were purchased from Sigma (St. Louis, MO). Tetrodotoxin was obtained from Alomone Labs (Jerusalem, Israel), adenosine deaminase was from Roche Molecular Biochemicals (Indianapolis, IN), and tetrandrine was from Aldrich Chemical (Milwaukee, WI). ß-Bungarotoxin was a kind gift of Dr. Long-Sen Chang (Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung City, Taiwan). Magnolol was kindly provided by Dr. Chien-Chich Chen (National Institute of Chinese Medicine, Taipei City, Taiwan). All other chemicals were commercially available and of reagent grade. The twice-distilled water that had been deionized through a Millipore-Q system (Millipore, Bedford, MA) was used in all experiments.

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 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). To measure Ca²⁺ current, KCl inside the pipette solution was replaced with equimolar CsCl, and pH was adjusted to 7.2 with CsOH.

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. The pipette solution was filtered on the day of use with a 0.22-µm pore size syringe filter (Millipore).

	Results

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Effect of cilostazol on 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 cilostazol on ion currents in these cells. 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. ATP (3 mM) contained in the pipette solution could be effective in suppressing ATP-sensitive K⁺ channels (22). 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 +70 mV with 20-mV increments were applied, a family of large, noisy, outward currents were elicited. These outward currents have been previously identified as Ca²⁺-activated K⁺ currents [I_K(Ca)]. Interestingly, within 1 min of exposing the cells to cilostazol (10 µM), the amplitude of outward currents was greatly increased throughout the entire range of voltage-clamp step. This stimulatory effect was readily reversed after the removal of cilostazol (Fig. 1). Figure 1B illustrates the averaged I-V relations for I_K(Ca) in the absence and presence of cilostazol (10 µM).

View larger version (45K): [in this window] [in a new window]   FIG. 1. Stimulatory effect of cilostazol on IK(Ca) in GH3 cells. A, Superimposed current traces obtained in the control, during cell exposure to cilostazol and washout of the drug. Cells were bathed in normal Tyrode’s solution containing 1.8 mM CaCl2. The cell was held at 0 mV, and different voltage steps ranging from +10 to +70 mV in 20-mV increments were applied. The uppermost part in A indicates the voltage protocol used. Arrowheads indicate the zero current level. CLZ, Cilostazol (10 µM). B, Averaged I-V relationships of IK(Ca) measured at the end of each voltage pulse in control () and during exposure to 10 µM cilostazol () and washout of cilostazol (). Each point represents the mean ± SEM (n = 6–11). C, Effect of glibenclamide, dequalinium dichloride, ß-bungarotoxin, paxilline, and tetrandrine on cilostazol-stimulated IK(Ca). Each cell was depolarized from 0 to +50 mV. The amplitude of IK(Ca) in the presence of 10 µM cilostazol was considered to be 1.0 and the effects of various compounds on cilostazol-increased IK(Ca) were compared. The parentheses above each bar denote the number of cells from which the data were taken. Mean ± SEM. *, Significantly different from cilostazol alone group. CLZ, Cilostazol (10 µM); Glib, glibenclamide (10 µM); Deq, dequalinium dichloride (10 µM); ßBT, ß-bungarotoxin (200 nM); Pax, paxilline (1 µM); Tet, tetrandrine (10 µM).

Effect of cilostazol on I_K(Ca) in cells preincubated with AOPCP or adenosine deaminase
It has been shown that the effect of cilostazol was associated with its inhibition of adenosine uptake (1, 6, 27, 28). The binding of adenosine receptor by adenosine analogs was also found to inhibit prolactin release from GH₃ cells (29). The effect of cilostazol on I_K(Ca) was thus assessed in cells treated with AOPCP (100 µM) or adenosine deaminase (1 U/ml) for 5 h. AOPCP is an inhibitor of ecto-5'-nucleotidase, whereas adenosine deaminase can degrade adenosine to inosine. However, unexpectedly, in these GH₃ cells preincubated with adenosine deaminase or AOPCP for 5 h, the stimulatory effect of cilostazol on the I-V relationship of I_K(Ca) was little altered (Fig. 2). For example, in cells pretreated with adenosine deaminase (1 U/ml), cilostazol (10 µM) could increase the amplitude of I_K(Ca) at the level of +50 mV from 281 ± 37 to 560 ± 71 pA (n = 8). There was no significant difference in the magnitude of cilostazol-stimulated effect on I_K(Ca) between control cells and cells treated with AOPCP or adenosine deaminase. Therefore, these results suggest that the stimulatory effect of cilostazol on I_K(Ca) is not attributed to its inhibition of adenosine uptake.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Effect of cilostazol on the averaged I-V relations of IK(Ca) in GH3 cells treated with adenosine deaminase (upper) or AOPCP (lower). GH3 cells were preincubated with adenosine deaminase (1 U/ml) or AOPCP (100 µM) for 5 h. In each cell examined, IK(Ca) was elicited from 0 mV to different potentials ranging from +10 to +70 mV with 20-mV increments. Each point represents the mean ± SEM (n = 6–8). , Control; , in the presence of cilostazol (10 µM).

View larger version (31K): [in this window] [in a new window]   FIG. 3. Stimulatory effect of cilostazol 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) and during exposure (right) to 10 µM cilostazol (CLZ). Upward deflection indicates the opening events of the channel. Note that the channel exhibits only a few, brief openings in the control and the exposure to cilostazol produces an increase in channel activity. B, Amplitude histograms measured in the absence (left) and presence (right) of 10 µM cilostazol (CLZ). The closed state corresponds to the peak at 0 pA. C, Bar graph showing the effect of cilostazol, magnolol, paxilline, and sp-cAMPS on the open probability of BKCa channels. Parentheses above each bar indicate the number of cells from which the data were taken. *, Significant from control. CLZ, Cilostazol (10 µM); Mag, magnolol (10 µM); Pax, paxilline (1 µM); sp-cAMPS (100 µM).

The relationship between the concentration of cilostazol and the channel open probability is shown in Fig. 4A. This drug increased the channel open probability in a concentration-dependent manner with an EC₅₀ value of 3.5 µM. At a concentration of 100 µM cilostazol fully increased the channel activity. The Hill coefficient was found to be 2.3, suggesting that there was a positive cooperativity for the stimulation of BK_Ca channels.

View larger version (24K): [in this window] [in a new window]   FIG. 4. Concentration- and voltage-dependent effects of cilostazol on BKCa channels in GH3 cells. The experiments were conducted with symmetrical K+ concentration (145 mM). Under inside-out configuration, the holding potential was +60 mV and bath medium contained 0.1 µM Ca2+. Cilostazole was applied to the intracellular surface of excised membrane patches. A, Concentration-response curve for the cilostazol-induced activation of BKCa channels. The channel open probability in the presence of cilostazol (100 µM) was considered to be 100%. The curve was fitted with the Hill equation as described in Materials and Methods. The EC50 value was 3.5 µM and the Hill coefficient was 2.3. Each point represents the mean ± SEM (n = 5–9). B, The effect of cilostazol on the activation curve of BKCa channels. The activation curves were obtained when the ramp pulses were applied from +20 to +120 mV with a duration of 1 sec. The curves were fitted with the Boltzmann equation as described in Materials and Methods. CLZ, Cilostazol (10 µM). C, Lack of effect of cilostazol on single-channel conductance of BKCa channels. Under symmetrical K+ condition, the holding potential was 0 mV in inside-out configuration and bath medium contained 0.1 µM Ca2+. The voltage ramp pulses from 0 to +50 mV were used to measure single-channel conductance. The straight lines with a reversal potential of 0 mV represent the I-V relationships of BKCa channels in the absence and presence of cilostazol (3 and 10 µM). Note that no modification in single-channel conductance was found in the presence of cilostazol, although it could increase the channel open probability.

Lack of effect of cilostazol on single-channel conductance of BK_Ca channels
Whether cilostazol affects single-channel conductance was also examined. To construct the plots of current amplitude as a function of membrane potential, the voltage ramp pulses from 0 to +50 mV with a duration of 1 sec were applied at a rate of 0.05 Hz. Figure 4C illustrates the I-V relationships of BK_Ca channels in the absence and presence of cilostazol (3 and 10 µM). The single-channel conductance calculated from the linear I-V relationship in control was 185 ± 7 pS (n = 8) with a reversal potential of 0 ± 2 mV (n = 8). The value for these channels in the control did not differ significantly from that obtained in the presence of 10 µM cilostazol (187 ± 7 pS, n = 8).

Effect of cilostazol on kinetic behavior of BK_Ca channels
The effect of cilostazol on the gating of these channels was analyzed because of its inability to modify single-channel conductance. In an excised patch of control cell, open and closed time histograms at the level of +30 mV can be fitted by a one or two exponential curve (Fig. 5). The time constant for the open-time histogram was 2.3 ± 0.5 msec, whereas those for the fast and slow components of the closed time histogram were 6.9 ± 1.2 and 85 ± 4 msec, respectively (n = 7). In inside-out patches, the application of cilostazol (10 µM) to the bath increased the time constant of the open state to 4.8 ± 0.6 msec and decreased the mean closed times to 3.2 ± 0.2 and 24 ± 2 msec (n = 7). Thus, the results showed that this drug produced an increase in channel open time and a decease in channel closed time. These changes may primarily explain its stimulatory effect on the channel activity.

View larger version (31K): [in this window] [in a new window]   FIG. 5. Effect of cilostazol on mean open (A and B) and closed times (C and D) of BKCa channels in GH3 cells. Inside-out configuration was performed in these experiments and the potential was held at +30 mV to reduce the number of channel openings. Cells were bathed in symmetrical K+ solution (145 mM) and bath medium contained 0.1 µM Ca2+. The open (A and B) and closed time (C and D) histograms in the control (A and C) and after application (B and D) of cilostazol (10 µM) are shown on the right. Of note, the abscissa and ordinate show the logarithm of apparent open or closed times (msec) and the square root of the number of events (n1/2), respectively. Control data were obtained from measuring 403 channel openings with a total record time of 3 min, whereas those obtained in the presence of cilostazol were measured from 503 channel openings with a total record time of 2 min. The dashed lines shown in each lifetime distribution are placed at the value of the time constant in open or closed state.

View larger version (25K): [in this window] [in a new window]   FIG. 6. Inhibitory effect of cilostazol on voltage-dependent L-type Ca2+ current (ICa,L) in pituitary GH3 cells. In these experiments, 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 cilostazol (30 µM) on ICa,L. The cell was depolarized from -50 to 0 mV at a rate of 0.05 Hz. a, Control; b, cilostazol (10 µM). B, The I-V relationships of ICa,L between the absence () and presence () of cilostazol (10 µM). Each point represents the mean ± SEM (n = 6–9).

View larger version (61K): [in this window] [in a new window]   FIG. 7. Effect of cilostazol and other related compounds on the firing of action potentials in GH3 cells. Cells were bathed in normal Tyrode’s solution containing 1.8 mM CaCl2. Patch pipettes were filled with a K+-containing solution. Change in membrane potential was measured under current-clamp configuration. A, Original potential trace showing the effect of cilostazol on spontaneous action potentials. Horizontal bar indicates the application of cilostazol (10 µM). B, Comparison between the effect of cilostazol and those of adenosine, magnolol, and paxilline on the firing of action potentials. The parentheses above each bar indicate the number of cells from which the data were taken. Ctrl, Control; Ado, adenosine (100 µM); CLZ, cilostazol (10 µM); Mag, magnolol (10 µM); Pax, paxilline (1 µM). *, Significantly different from control.

View larger version (29K): [in this window] [in a new window]   FIG. 8. Effect of cilostazol on the I-V relationship of BKCa channels in pheochromocytoma PC12 cells. A, Examples of BKCa channels in the absence (left) and presence (right) of cilostazol (10 µM) measured from an inside-out patch at various potentials. Cilostazol was applied to the bath medium. CLZ, Cilostazol (10 µM). The number shown at the beginning of each current trace indicates the voltage applied to the patch pipette. B, The I-V relationships of BKCa channels in the absence and presence of cilostazol (10 µM). Values are mean ± SEM (n = 6–9). C, Bar graph showing the effect of cilostazol and magnolol on the probability of these channels in the absence and presence of paxilline. Parentheses above each bar indicate the number of cells examined. Ctrl, Control; CLZ, cilostazol (10 µM); Mag, magnolol (10 µM); Pax, paxilline (1 µM). *, Significantly different from control. **, Significantly different from magnolol or cilostazol alone group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Several lines of evidence have been presented to indicate that cilostazol may block the uptake of adenosine into the cells (1, 6, 27, 28). The activation of adenosine receptors caused by adenosine agonists has been previously reported to stimulate prolactin secretion in GH₃ cells (29). Therefore, the question arises as of whether the stimulatory effect of cilostazol on I_K(Ca) observed in GH₃ cells could be due to an increase in the level of endogenous adenosine when adenosine uptake was blocked by cilostazol. However, this idea is difficult to reconcile with the present results showing that in GH₃ cells preincubated with adenosine deaminase or AOPCP, the stimulatory effect of cilostazol on I_K(Ca) remained unaltered. Adenosine (100 µM) was found to have no effect on the firing of action potentials. Therefore, under our experimental conditions, the stimulation of I_K(Ca) by cilostazol in GH₃ cells was not associated with changes in the level of endogenous adenosine. It is unlikely that cilostazol stimulated I_K(Ca) in a manner independent on its inhibition of adenosine uptake. Indeed, single-channel experiments with inside out configuration showing that cilostazol applied to the intracellular surface was found to enhance BK_Ca channel activity also exclude this possibility.

The present results also provide the evidence that the cilostazol-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 cilostazol was not found to increase the amplitude of I_Ca,L. These observations are compatible with a previous report showing the ability of this drug to suppress the tonic contraction induced by high K⁺ solution in vascular smooth muscle (34). In addition, because the increased level of intracellular cAMP was found to increase Ca²⁺ influx through L-type Ca²⁺ channels in pituitary tumor (GH₄C₁) cells (33), the inhibition of I_Ca,L caused by cilostazol at a concentration greater than 30 µM appears to be direct and unlikely to be linked to its inhibition of phosphodiesterase.

The results from single-channel experiments with excised patches led to the interpretation that the stimulatory effect of cilostazol on BK_Ca channel activity does not require cytosolic diffusible substances (e.g. cAMP) inside the cell and is primarily due to the result of direct binding to the inner surface of the channel. Inability of sp-cAMPS, an analog of cAMP, to stimulate BK_Ca channels also supports this notion. However, the binding site for cilostazol could not be located intracellular to the central pore cavity because of no modification of single-channel conductance in the presence of cilostazol. This drug may thus bind to the BK_Ca channel at the sites that are important for channel gating, given that there was an increase in mean open time and a decrease in mean closed time in the presence of cilostazol.

Cilostazol could increase the activity of BK_Ca channels in a voltage-dependent fashion possibly by acting at a site that is accessible from the intracellular side of the channel. Therefore, it seems reasonable to assume that the sensitivity of neurons or neuroendocrine cells to this drug would depend on the level of resting membrane potential, the firing of action potentials, and the concentration of cilostazol used, if the cilostazol action in vivo is the same as those in GH₃ cells shown herein. Indeed, we also found that, like magnolol (31), it was effective in suppressing the repetitive firing of action potentials in GH₃ cells.

The EC₅₀ value of cilostazol required for the stimulation of BK_Ca channels was 3.5 µM, a value that is similar to that required for the inhibition of adenosine uptake into the cells (6, 27) but is lower than that used to inhibit platelet aggregation (35). Plasma concentrations of cilostazol in humans are 1–5 µM (36). Because its effect shown here may occur at a concentration achievable in humans (36), the present findings suggest that the BK_Ca channel may be a target for the action of cilostazol. Regardless of the mechanism of its actions, the present results should also be noted with caution in relation to its increasing use as an inhibitor of type 3 phosphodiesterase or adenosine uptake (3, 6, 27, 28, 37, 38, 39).

The concentration-response curve of channel activation by cilostazol had a Hill coefficient of 2.3, suggesting that more than one cilostazol molecule bind to the channel, although the stoichiometry of cilostazol binding to the channel is currently unknown. It is possible that cilostazol has a unique structure that interacts with the BK_Ca channel to increase the amplitude of I_K(Ca) in GH₃ cells. Interestingly, cilostazol is noted to have a structure that bears resemblance to chlorzoxazone, riluzole, and the benzimidazolones, the structurally related compounds of which have been known to be BK_Ca channel openers (40). A recent report showed that hemin could bind to the heme-binding amino acid sequence motif found in Slo BK and native BK_Ca channels with a high affinity, thus leading to a decrease in channel activity (41). It will be of interest to explore whether cilostazol can interact with this similar motif to affect the activity of BK_Ca channels.

The results of this study show a significant increase of BK_Ca channel activity by cilostazol in GH₃ and PC12 cells. This effect is presumably not mediated by its inhibitory effects on the uptake of adenosine or on the activity of phosphodiesterases. It is possible that cilostazol-induced effects on neurons or neuroendocrine cells are partly, if not entirely, attributed to a direct simulation of the BK_Ca channels. Furthermore, these results suggest that in addition to the increase in intracellular cAMP, the direct effect of cilostazol on the stimulation of BK_Ca channels may have important implications in understanding its relaxation of vascular smooth muscle (42, 43). In fact, this drug has been used for the management of chronic peripheral arterial occlusive disease (14, 27, 42, 43, 44, 45).

Recent observations showed that the BK_Ca channel activity might play a role in time- and cell-dependent modulation of physiological outputs in anterior pituitary cells (46). The increase of these channel caused by cilostazol might reduce hormonal secretion. The activity of these channels was found to be activated in the course of steroidogenesis induced by human chorionic gonadotropin (47). Therefore, it will be of interest to determine to what extent cilostazol affects the steroidogenesis in the ovary.

	Acknowledgments

 
The authors would like to thank Su-Rong Yang for technical assistance in the preparation of cultured cells.

	Footnotes

 
This work was supported by grants from the National Science Council (NSC-91-2320B-006-106 and NSC-92-2320B-006-041), Taiwan.

Abbreviations: AOPCP, ,ß-Methylene-ADP; BK_Ca, large-conductance Ca²⁺-activated K⁺; I_Ca,L, voltage-dependent L-type Ca²⁺ current; I_K(Ca), Ca²⁺-activated K⁺ current; I-V, current-voltage; sp-cAMPS, sp-adenosine-3',5'-cyclic monophosphorothioate.

Received October 23, 2003.

Accepted for publication November 18, 2003.

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