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Vol. 292, Issue 1, 96-103, January 2000


Effect of Mibefradil on Voltage-Dependent Gating and Kinetics of T-Type Ca2+ Channels in Cortisol-Secreting Cells

Juan Carlos Gomora, Lin Xu, Judith A. Enyeart and John J. Enyeart1

Department of Pharmacology (J.C.G., L.X., J.A.E., J.J.E.) and The Neuroscience Program (J.J.E.), The Ohio State University, College of Medicine and Public Health, Columbus, Ohio

    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We have studied the effect of the Ca2+ antagonist mibefradil on low voltage-activated T-type Ca2+ channels in whole-cell patch clamp recordings from bovine adrenal zona fasciculata (AZF) cells. AZF cells are distinctive in expressing only T-type Ca2+ channels, allowing the mechanism of pharmacological agents to be explored without interference from other Ca2+ channels. The inhibition of T-type Ca2+ channels by mibefradil was voltage- and use-dependent. When Ca2+ currents were activated from holding potentials of -80 and -60 mV, mibefradil inhibited currents with IC50 values of 1.0 and 0.17 µM, respectively. When T-type Ca2+ current (IT) was activated from a holding potential of -90 mV in the presence of 2 µM mibefradil, a single voltage step to -10 mV inhibited IT by 16.2% ± 2.9% (n = 10). With subsequent voltage steps, applied at 10-s intervals, block reached a steady-state value of 51.9% ± 5.0% (n = 5). Mibefradil (1 µM) produced a leftward shift of 5.7 mV (n = 4) in the voltage-dependent steady-state availability curve such that T-type Ca2+ channels inactivated at more negative potentials, but this drug did not change the voltage-dependence of T channel opening. Mibefradil failed to alter the kinetics of T channel activation, inactivation, or deactivation, but markedly slowed T channel recovery following an inactivating prepulse. Mibefradil inhibited adrenocorticotropin-stimulated cortisol secretion from AZF cells with an IC50 value of 3.5 µM. These results show that mibefradil is a relatively potent antagonist of T-type Ca2+ channels in cortisol-secreting cells. The enhanced potency of mibefradil with sustained or repetitive depolarizations, its shifting of the steady-state inactivation curve, and its slowing of recovery all indicate that this drug preferentially interacts with Ca2+ channels in the open or inactivated state. The inhibition of cortisol secretion by mibefradil at concentrations similar to those that block IT is consistent with a requirement for these channels in corticosteroidogenesis.

    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Mibefradil is a Ca2+ channel antagonist that has been used clinically as an antihypertensive and antianginal agent (Noll and Lusher, 1998). Among organic Ca2+ antagonists, mibefradil is distinctive because it preferentially blocks low voltage-activated T- rather than L-type Ca2+ channels (Mishra and Hermsmeyer, 1994b; Mehrke et al., 1994; Ertel and Ertel, 1997).

The study of the mechanism of T channel block by mibefradil has been hampered by the presence of multiple Ca2+ channel subtypes in most excitable cells, including those of the heart, vascular smooth muscle, and neurons. The presence of both low and high voltage-activated Ca2+ channels makes it difficult to isolate T-type currents in whole-cell recordings, and to measure voltage-dependent gating and kinetic parameters of these channels. Consequently, it is also difficult to accurately measure the effects of pharmacological agents on these parameters.

Studies aimed at determining the mechanism of T channel inhibition by mibefradil have produced conflicting results. Block of T-type Ca2+ channels in the h-MTC human thyroid C cell line was reported to show no voltage- or use-dependence (Mehrke et al., 1994). No increased potency of the drug was observed with membrane depolarization or increased frequency of stimulation. In contrast, block of T-type Ca2+ channels by mibefradil in mouse spermatozoa, rat cerebellar Purkinje neurons, and rat sensory neurons displays marked voltage- and use-dependence (Arnoult et al., 1998; Todorovic and Lingle, 1998; McDonough and Bean, 1998).

Bovine adrenal zona fasciculata (AZF) cells are an excellent choice for studying the properties of T-type Ca2+ channels and their modulation by pharmacological agents. The majority of AZF cells (>95%) express only T-type Ca2+ channels, which have been extensively characterized with respect to voltage-dependent gating and kinetics (Mlinar et al., 1993). Freshly plated AZF cells are small (diameter <20 µM) and spherical allowing for rapid and accurate voltage control and current recording in whole-cell patch clamp experiments (Mlinar and Enyeart, 1993; Mlinar et al., 1993). Ca2+ influx through T-type channels may mediate depolarization-dependent cortisol secretion (Enyeart et al., 1993).

We have characterized the action of mibefradil on T-type Ca2+ current (IT) in bovine AZF cells, including its potency, voltage, and use dependence, as well as its action on voltage-dependent gating and kinetic parameters. The effect of mibefradil as an antagonist of adrenocorticotropin (ACTH)-stimulated cortisol secretion also was determined.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Tissue culture media, antibiotics, fibronectin, and fetal bovine serum (FBS) were obtained from Gibco Laboratories (Grand Island, NY). Coverslips were from Bellco Glass, Inc. (Vineland, NJ). Enzymes, MgATP, ACTH(1-24), and 1,2,-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid were obtained from Sigma Chemical Co. (St. Louis, MO). Mibefradil was obtained from Hoffman-La Roche Inc. (Basel, Switzerland).

Isolation and Culture of Adrenocortical Cells. Bovine adrenal glands were obtained from steers (age range 1 to 3 years) within 60 min of slaughter at a local slaughterhouse. Fatty tissue was removed immediately and the glands were transported to the laboratory in ice-cold PBS containing 0.2% dextrose. Isolated AZF cells were prepared as previously described (Gospodarowicz et al., 1977) with some modifications. In a sterile tissue culture hood, the adrenals were cut in half lengthwise and the lighter medulla tissue trimmed away from the cortex and discarded. The capsule with attached glomerulosa and thicker fasciculata layer were then dissected into large pieces (~1.0 × 1.0 × 0.5 cm). A Stadie-Riggs tissue slicer (Thomas Scientific, Philadelphia, PA) was used to separate fasciculata tissue from the glomerulosa layers by slicing 0.3- to 0.5-mm slices from the larger pieces. The first medulla/fasciculata slices were discarded. One to two subsequent fasciculata slices were saved in cold sterile PBS/0.2% dextrose. The fasciculata/glomerulosa margin (~0.5 mm) and capsule with attached glomerulosa were discarded. Fasciculata tissue slices were then diced into 0.5-mm3 pieces and dissociated with 2 mg/ml (~200-300 U/ml) of Type I collagenase (neutral protease activity not exceeding 100 U/mg of solid), 0.2 mg/ml deoxyribonuclease in Dulbecco's modified Eagle's medium (DMEM)/F12 for ~1 h at 37°C, triturating after 30 and 45 min with a sterile, plastic transfer pipette. The tissue/cell suspension was filtered through two layers of sterile cheesecloth, then centrifuged to pellet cells at 100g for 5 min. Undigested tissue remaining in the cheesecloth was collagenase-treated for an additional hour. Pelleted cells were washed with DMEM/0.2% BSA, centrifuging as before. After resuspension in DMEM, cells were filtered through 200-µm stainless steel mesh to remove clumps. Dispersed cells were again centrifuged and either resuspended in DMEM/F12 (1:1) with 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin and plated for immediate use, or resuspended in FBS/5% dimethyl sulfoxide, divided into 1-ml aliquots each containing ~4 × 106 cells, and stored in liquid nitrogen for future use. Cells were plated in 35-mm dishes containing 9-mm2 glass coverslips that had been treated with fibronectin (10 µg/ml) at 37°C for 30 min then rinsed with warm, sterile PBS immediately before adding cells. Dishes were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Patch Clamp Experiments. Patch clamp recordings of IT were made in the whole-cell configuration. The standard pipette solution was 120 mM CsCl, 1 mM CaCl2, 2 mM MgCl2, 11 mM 1,2,-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 10 mM HEPES, and 1 mM MgATP with pH titrated to 7.2 with CsOH. The external solution consisted of 117 mM tetraethylammonium chloride, 5 mM CsCl, 10 mM CaCl2, 2 mM MgCl2, 5 mM HEPES, and 5 mM glucose, with pH adjusted to 7.4 with tetraethylammonium hydroxide. All solutions were filtered through 0.22 µM cellulose acetate filters.

AZF cells were used for patch clamp experiments 2 to 12 h after plating. Typically, cells with diameters < 15 µm and capacitances of 8 to 15 picofarads were selected. Coverslips were transferred from 35-mm culture dishes to the recording chamber (volume, 1.5 ml) that was continuously perfused by gravity at a rate of 3 to 5 ml/min. Patch electrodes with resistances of 1.0 to 2.0 MOmega were fabricated from Corning 0100 glass (World Precision Instruments, Sarasota, FL) with a Brown-Flaming model P-80 microelectrode puller (Sutter Instrument Company, Novato, CA). Ca2+ currents were recorded at room temperature (22-25°C) following the procedure of Hamill et al. (1981) with an Axopatch ID patch clamp amplifier (Axon Instruments, Inc., Burlingame, CA). Access resistance during recording, estimated from the transient cancellation controls of the patch clamp amplifier was 2 to 4 MOmega . The combination of access resistance and cell capacitance yielded voltage clamp time constants of <100 µs.

Pulse generation and data acquisition were done with a personal computer and PCLAMP software with TL-1 interface (Axon Instruments, Inc.). Currents were digitized at 2 to 20 kHz after filtering with an eight-pole Bessel filter (Frequency Devices Inc., Haverhill, MA). Linear leak and capacity currents were subtracted from current records with scaled hyperpolarizing steps of one-third to one-fourth amplitude. Data were analyzed and plotted with PCLAMP 5.5 and 6.02 (Clampan and Clampfit) and SigmaPlot (version 4.0; SPSS, Inc., Chicago, IL). Series resistance compensation was not used because the amplitude of IT current seldom exceeded 500 pA. A current of this size in combination with a 4 MOmega access resistance produced a voltage error of only 2 mV, which was not corrected.

Secretion Experiments. AZF cells were cultured on fibronectin-treated 35-mm plates at a density of 4.4 × 105 cells/dish in DMEM/F12 containing 10% FBS, 100 U/ml penicillin, 0.1 mg streptomycin, and the antioxidants 1 µM tocopherol, 20 nM selenite, and 100 µM ascorbic acid. After 24 h, the media was aspirated and changed to defined media consisting of DMEM/F12 (1:1), 50 µg/ml BSA, 100 µM ascorbic acid, 1 µM tocopherol, 10 nM insulin, and 10 µg/ml transferrin. Drugs were added directly to media from concentrated stocks. We collected 200-µl samples of media at selected times and they were frozen at -20°C and later assayed for cortisol with a solid-phase radioimmunoassay kit (Diagnostic Products Corp., Los Angeles, CA). Experiments were performed on triplicate 35-mm dishes, and hormone assays were performed in duplicate at several dilutions.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In whole-cell patch clamp experiments on bovine AZF cells, voltage steps to -10 mV, applied from a holding potential of -80 mV, activate a rapidly inactivating IT, the only Ca2+ current detectable in the majority of these cells (Fig. 1A). T-type Ca2+ channels are distinguished by their slow rate of closing, which is observed in whole-cell recordings as a decaying tail current upon repolarization after a brief activating voltage step (Fig. 1B). The inhibition of these tail currents by mibefradil was used to determine the potency of this drug as an antagonist of T-type Ca2+ channels. When T-type Ca2+ currents were activated by 10-ms voltage steps to -10 mV applied at 30-s intervals from a holding potential of -80 mV, mibefradil blocked this current with an IC50 value of 1.0 µM (Fig. 1C). Mibefradil was even more potent when the Ca2+ currents were activated by longer voltage steps of 300-ms duration (Fig. 1A). Inhibition of IT by 10 µM mibefradil was reversed by 46.3% ± 3.1% (n = 6) after washing in control saline for periods of 15 to 20 min.


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  		Fig. 1.   Concentration-dependent inhibition of IT by mibefradil. Ca2+ currents was activated by short (10-ms) or long (300-ms) voltage steps to -10 mV applied at 30-s intervals from a holding potential of -80 mV. Mibefradil was superfused at increasing concentrations. A and B, current records showing steady-state block of IT by 0.1, 1, and 10 µM mibefradil with short (B) or long (A) depolarizing steps. C, inhibition curve obtained from experiments as described in (B). Normalized current is plotted against mibefradil concentration. Results are means ± S.E. of 6 to 10 measurements at each concentration. Inhibition curve was generated by fitting data with an equation of the form: I/Imax = 1/[1 + (B/IC50)X], where B is the mibefradil concentration, IC50 is the concentration that reduces IT by 50%, and X is the Hill coefficient.

Although mibefradil effectively blocked IT over a wide range of test potentials, it was slightly more potent at more positive voltages. In the experiments illustrated in Fig. 2, current-voltage relationships for IT were obtained by applying voltage steps from -80 mV to a range of test potentials between -60 and +40 mV, before and after superfusing cells with 1 µM mibefradil. Figure 2B shows an averaged current-voltage relationship obtained by plotting the mean peak current derived from three cells at each potential before and after superfusing the cell with mibefradil. At five test potentials between -35 and -15 mV, where Ca2+ current could be accurately measured, mibefradil (1 µM) inhibited IT by an average of 51.7% ± 2.8%. By comparison, at potentials between +10 and +30 mV, IT was inhibited by 62.3% ± 1.1%.


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  		Fig. 2.   Effect of mibefradil on current-voltage relationship. T-type Ca2+ currents were activated from -80 mV by voltage steps applied at 0.1 Hz to various test potentials between -60 and +40 mV before and after superfusing 1 µM mibefradil. A, current traces recorded at the indicated test potential before and after superfusion of 1 µM mibefradil. B, current-voltage relationship: peak current amplitudes from three cells in the absence and presence of mibefradil were averaged and plotted against test potential. Values are means ± S.E.

Voltage- and Use-Dependent Block of IT by Mibefradil. Many L-type Ca2+ channel antagonists preferentially bind to or occlude Ca2+ channels that are in the open or inactivated conformation. Blockers of this type display voltage- and use-dependence marked by enhanced potency in rapidly firing or depolarized cells (Lee and Tsien, 1983; Bean, 1984; Sanguinetti and Kass, 1984). Block of T-type Ca2+ channels in AZF cells by mibefradil was both voltage- and use-dependent.

Figure 3 shows that the potency of mibefradil was enhanced when T-type Ca2+ currents were activated from -60 rather than -80 or -90 mV. In Fig. 3A, Ca2+ currents were activated by identical voltage steps to -10 mV from a holding potential of -60 mV (right, c) and then -90 mV (left, c). In each case, tail currents were measured after returning to -90 mV. The cell was then superfused with 2 µM mibefradil while recording Ca2+ currents activated from a holding potential of -90 mV (left, m). After steady-state block was achieved, Ca2+ currents were then activated from a holding potential of -60 mV in the presence of mibefradil (right, m).


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  		Fig. 3.   Voltage-dependent block of IT by mibefradil. A, effect of varying holding potential on extent of block. IT was first activated at 15-s intervals from a holding potential of -60 mV by 10-ms voltage steps to -10 mV, and tail currents were measured upon returning to the holding potential (right, c). The holding potential was switched to -90 mV and currents were recorded in response to identical voltage steps in control saline (left, c), and after superfusing 2 µM mibefradil (left, m). The holding potential was switched to -60 mV, and additional currents recorded until inhibition reached a steady-state value (right, m). Block of IT increased from 45 to 83% when the holding potential was switched from -90 to -60 mV. B, effect of holding potential on concentration-dependent inhibition by mibefradil. Concentration-dependent inhibition of IT was measured for currents activated from -60 mV. Inhibition curves were generated as described in the legend of Fig. 1. Results are means ± S.E. of three to four cells for each concentration.

In this cell, mibefradil inhibited IT by 45%, when currents were activated from a holding potential of -90 mV (Fig. 3A, left), and steady-state block increased to 83% when currents were activated from -60 mV (right). Overall, in four similar experiments mibefradil (2 µM) inhibited IT by 47.9% ± 6.2% at a holding potential of -90 mV, and by 83.7% ± 5.5% when the holding potential was reduced to -60 mV.

Comparison of the inhibition curves for mibefradil measured from a holding potential of -60 mV to that obtained at -80 mV also indicated a significant increase in potency at more depolarized holding potentials (Fig. 3B). At a holding potential of -60 mV, mibefradil inhibited IT with an IC50 value of 0.17 µM compared with a value of 1.0 µM obtained at a holding potential of -80 mV (Fig. 3B). Overall, block of T-type Ca2+ channels in AZF cells by mibefradil was clearly voltage-dependent and enhanced at more depolarized holding potentials.

Inhibition of T-type Ca2+ channels by mibefradil also displayed a marked use-dependence. In the experiment illustrated in Fig. 4, Ca2+ currents were first recorded at 10-s intervals in control saline before superfusing the cell with 2 µM mibefradil for 5 min in the absence of depolarizing steps (A and B, 1). When voltage steps were resumed, IT was initially inhibited by 14.5% (A and B, 2). The extent of inhibition increased with subsequent test pulses, reaching a maximum of 54% at 3.5 min (A and B, 3).


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  		Fig. 4.   Use-dependent block of IT by mibefradil. T-type Ca2+ currents were activated at 10-s intervals by 10-ms voltage steps to -10 mV applied from a holding potential of -80 mV. Tail currents were recorded upon repolarization to -80 mV. A, after recording currents in control saline (1) the cell was superfused with 2.5 µM mibefradil for 5 min in the absence of voltage steps. Depolarizing steps were then applied at 10-s intervals for 2.5 min in the continued presence of mibefradil (2, 3). After a second 5-min pulse-free period in the presence of mibefradil, voltage steps were again applied at 10-s intervals for 2.5 min (4, 5). IT amplitudes are expressed as a fraction of the maximum IT and plotted against time. B, current traces recorded at times as indicated by numbers in (A).

This use-dependent block was partially relieved upon cessation of test pulses. In the experiment illustrated, a 5-min pulse-free period resulted in a 50% reversal of the use-dependent component of inhibited IT (Fig. 4, A and B, 4). When test pulses were resumed, block again increased to its maximum value (A and B, 5). Overall, at a holding potential of -90 mV, in the absence of stimulation, 2 µM mibefradil inhibited IT by 16.2% ± 2.9% (n = 10). When 10-ms voltage steps to -10 mV were applied at 10-s intervals in the presence of the drug, block increased to 51.9% ± 5.0% (n = 5) within 2.8 ± 0.2 min.

Effect of Mibefradil on Voltage-Dependent Gating. Inhibition of IT by mibefradil could occur through a direct occlusion of the pore by the drug, or by an allosteric mechanism wherein binding produces a shift in the voltage-dependence of IT activation or steady-state inactivation. In this regard, mibefradil might reduce IT amplitude measured at selected test voltages by shifting the channel activation or fraction open curve to the right along the voltage axis, such that stronger depolarizations are required to open these channels.

We have explored the effect of mibefradil on voltage-dependence of IT activation. The voltage-dependence of IT opening in the absence and presence of 1 µM mibefradil was determined by applying activating voltage-steps of varying size from a holding potential of -80 mV and then stepping the voltage back to the constant holding potential where the peak tail current was measured (Fig. 5A). Under these conditions, the tail current amplitude is directly proportional to the number of channels that are open at the end of the activating pulse.


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  		Fig. 5.   Mibefradil and voltage-dependent activation. The effect of mibefradil on voltage-dependence of IT activation was measured by recording tail currents at a constant potential (-80 mV) after applying activating voltage steps of 10-ms duration to potentials between -60 and +30 mV before and after superfusing cells with 1 µM mibefradil. A, current traces at indicated test potentials before and after superfusing 1 µM mibefradil. B, activation curves: tail current amplitudes were normalized to the maximum value and plotted as the fraction of open channels against test potential. Values are means ± S.E. from seven different cells. Data points were fit with a Boltzmann expression of the form Fraction open = 1/[1 + exp(v1/2- v)/K], where v1/2 is the voltage where half of the channels are in the open configuration and K is the slope factor. Control, ; mibefradil, open circle .

Activation curves were obtained by plotting the initial amplitude of the tail current as a function of the activating voltage. Tail current amplitudes were normalized and expressed as the fraction of open channels. Data points were fit by a Boltzmann function of the form: fraction open = 1/[1 + exp (v1/2 - v)/K], where v1/2 is the voltage where half of the channels are in the open configuration and K is the slope factor. Curves were fit to data points acquired at test potentials between -60 and +30 mV.

Figure 5B shows that mibefradil (1 µM) had no significant effect on the voltage-dependence of channel opening derived from measurements on seven separate cells. The midpoint of the fraction open curve and slope factor were -23.8 and 6.4 mV, respectively, in control saline, compared with -22.7 and 6.1 mV in the presence of mibefradil.

The increased potency of mibefradil observed at depolarized holding potentials suggests that this drug might alter the voltage-dependence of T channel inactivation. Mibefradil produced a leftward shift in the voltage-dependent steady-state availability of T-type channels such that channels inactivate at more negative potentials.

The voltage-dependent steady-state inactivation of the low voltage-activated Ca2+ current was assessed in the absence and presence of mibefradil by applying 5-s conditioning pulses to various potentials between -90 and -30 mV, followed by activating steps to -10 mV. Normalized Ca2+ currents from four separate cells were averaged and the mean values were fit with a smooth curve according to the Boltzmann relationship: I/IMAX = 1/[1 + exp(v - v1/2)/K], where Imax is the current activated from a holding potential of -85 mV, v1/2 is the voltage where one-half of the channels are in the open configuration, and K is the slope factor (Fig. 6). Mibefradil (1 µM) shifted the v1/2 by 5.7 mV from -49.9 ± 0.30 to -55.6 ± 0.37 mV (n = 4). Thus, mibefradil altered the voltage dependence of T channel inactivation but not activation.


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  		Fig. 6.   Mibefradil and voltage-dependent steady-state inactivation. IT was activated by voltage steps to -10 mV after 10-s conditioning pulses to potentials between -90 and -30 mV in 7.5-mV increments, before and after superfusing cell with 1 µM mibefradil. Steady-state inactivation curves (or availability curves) were obtained by fitting averaged data from four separate cells to an expression of the form I = Imax/[1 + exp(v - v1/2)/K], where I is the measured current amplitude, Imax is the maximal current amplitude, v is the conditioning potential, v1/2 is the potential at which half of the channels are available for activation, and K is the slope factor. Values are means ± S.E. for control () and mibefradil (open circle ).

Effect of Mibefradil on T Channel Gating Kinetics. Rather than occluding T-type Ca2+ channels or allosterically altering their steady-state availability, mibefradil might reduce T current amplitude through an action on channel gating kinetics, including the rates of activation, inactivation, or recovery from inactivation. For example, by slowing activation, mibefradil could reduce the maximum number of channels that are simultaneously open during a voltage step, thereby reducing peak current. Alternatively, acceleration of inactivation kinetics could produce a similar reduction in the measured peak current as T channels are rapidly swept into the inactivated state soon after opening.

It was found that mibefradil failed to alter the kinetics of T channel activation or inactivation, as well as deactivation, but markedly slowed T channel recovery following an inactivating prepulse. Activation kinetics of IT in AZF cells is described by an equation of the form IT = IMAX[1 - exp(-t/tau a)]4 (Mlinar et al., 1993). Mibefradil did not significantly change the activation time constant (tau a) measured in response to voltage steps to -10 mV, applied from a holding potential of -80 mV (Table 1).

                              
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  		TABLE 1
Effect of mibefradil on IT gating kinetics

IT in AZF cells (activated by 300-ms voltage steps to -10 mV from a holding potential of -80 mV) inactivates with a single time constant that did not significantly change in the presence of mibefradil. In the experiment illustrated in Fig. 7, the control current inactivated with a tau i of 25.9 ms compared with a value of 26.3 ms after steady-state block with 1 µM mibefradil. Overall, in a total of 19 cells, T-type current inactivated with a time constant of 27.5 ± 1.8 ms in control saline compared with 25.1 ± 1.1 ms after exposure to 1 µM mibefradil (Table 1).


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  		Fig. 7.   Mibefradil does not alter T current inactivation kinetics. T-type Ca2+ current was activated by voltage steps to -10 mV from a holding potential of -80 mV before (1) and after (2) superfusing the cell with 1 µM mibefradil. The inactivating components of the currents were fit with single exponentials of the form I = IMAXe-T/tau i, where tau i is the inactivation time constant.

T channels opened by membrane depolarization relax to the closed state upon repolarization (to -80 mV) with a single time constant (tau d) that was not altered by mibefradil (Table 1).

In contrast to its lack of effect on other kinetic parameters, mibefradil markedly slowed the kinetics of K+ channel repriming after membrane depolarization. Recovery kinetics were studied by a two pulse protocol in which cells were first voltage clamped at -10 mV for 500 ms to completely inactivate T channels, then stepping the membrane potential to -80 mV for various periods up to 80 s, before applying an activating test pulse to -10 mV.

In control saline, channels returned from the inactive to the closed state by a process described by two exponential time constants that differ by approximately one order of magnitude. In the presence of 1 µM mibefradil, recovery could still be described by two time constants, but the slow time constant (tau RS) had increased ~10-fold from 2.37 ± 0.35 to 21.6 ± 11.5 s (n = 4), whereas the fast time constant (tau RF) was not increased (Fig. 8 and Table 1).


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  		Fig. 8.   Effect of mibefradil on T channel recovery. A two-pulse protocol was used to determine the effect of mibefradil on the kinetics of T channel recovery following an inactivating prepulse. T channels were inactivated by a 500 ms to -10 mV after which the membrane potential was stepped to -80 mV for periods ranging from 100 ms to 20 s for control cells (A) or 100 ms to 80 s for mibefradil-treated cells (B) at which time an activating voltage step to -10 mV was applied and the tail current amplitude used as a measure of T channel recovery. Normalized tail currents were plotted as a function of time and fit with a function of the form I/IMAX = a[1 - exp(-t/tau RF)] + b[1 - exp(-t/tau RS)], where tau RF and tau RS are fast and slow time constants. Values are means ± S.E. of four separate cells for control saline (A) and in 1 µM mibefradil (B).

Effect of Mibefradil on Cortisol Secretion. Studies with less selective Ca2+ channel blockers indicated that T-type Ca2+ channels were required for ACTH-stimulated cortisol secretion (Enyeart et al., 1993). We have found that mibefradil inhibits ACTH-stimulated cortisol secretion from cultured AZF cells with an IC50 value of 3.5 µM, a concentration only slightly higher than that which blocks T-type Ca2+ current (Fig. 9).


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  		Fig. 9.   Inhibition of cortisol secretion by mibefradil. Cultured AZF cells were incubated for 24 h in serum-containing media as described in Materials and Methods before switching to test media containing ACTH (200 pM) and mibefradil at several different concentrations. After 5 h, media samples were collected and assayed for cortisol. Results are means ± S.E. of triplicate determinations. Curves were fit with an equation of the form y = 1/[1 + B/IC50], where y is the quantity of cortisol secreted, B is the mibefradil concentration, and IC50 is the concentration of mibefradil that inhibits cortisol secretion half-maximally.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

It was discovered that mibefradil effectively blocks T-type Ca2+ channels in bovine AZF cells and ACTH-stimulated cortisol secretion with a potency similar to that reported for inhibition of T channels in heart, brain, and vascular smooth muscle. Inhibition of IT was promoted by sustained or repetitive depolarizations, and was accompanied by a hyperpolarizing shift in the steady-state availability curve and slowing of recovery after inactivation. These results are consistent with a model wherein mibefradil preferentially interacts with T channels in AZF cells that have been opened or inactivated by membrane depolarization.

Relative Potency of Mibefradil. Mibefradil inhibited T-type Ca2+ channels in AZF cells at concentrations similar to those that inhibit T-type Ca2+ channels in cells, including cerebellar Purkinje neurons (McDonough and Bean, 1998) and vascular smooth muscle cells (Mishra and Hermsmeyer, 1994a). In several other cell types, including mouse spermatozoa, thyroid C cells, and rat sensory neurons, slightly higher IC50 values have been reported for T channel inhibition (Mishra and Hermsmeyer, 1994b; Arnoult et al., 1998; Todorovic and Lingle, 1998). However, given the marked dependence of mibefradil potency on membrane potential and frequency of depolarization, some of this variability could certainly be due to differing experimental conditions.

Furthermore, in some cases, the measurement of mibefradil potency may have been compromised by the inability to effectively isolate T current from that carried by other Ca2+ channel subtypes. In this regard, it is clear that at least 10-fold higher concentrations of mibefradil are required to block current through L-, N-, Q-, and R-type Ca2+ channels (Mehrke et al., 1994; Bezprozvanny and Tsien, 1995).

Use and Voltage Dependence. The clear use- and voltage-dependent block of T channels in AZF cells by mibefradil is consistent with results reported for T channels in several other cell types (Arnoult et al., 1998; McDonough and Bean, 1998; Todorovic and Lingle, 1998). However, the inhibition of T-type Ca2+ channels by mibefradil in the human h-MTC thyroid C cell line has been reported to be completely independent of voltage and stimulation frequency (Mehrke et al., 1994). It is not known whether these discordant results stem from authentic differences in the Ca2+ channels or variations in experimental conditions.

In this regard, major differences also exist in the reported mechanisms of mibefradil block of high voltage-activated Ca2+ channels. Inhibition of cloned L, N, Q, and R Ca2+ channels by mibefradil showed prominent use- and voltage-dependence (Mehrke et al., 1994; Bezprozvanny and Tsien, 1995). In contrast, Mishra and Hermsmeyer (1994a) reported that block of L channels in a vascular smooth muscle cell line was independent of stimulation frequency and membrane potential. Currently, no satisfactory explanation for these apparently contradictory results exists.

Effect of Mibefradil on Voltage-Dependent Gating. The voltage- and use-dependent block of T-type Ca2+ channels by mibefradil in AZF cells resembles block of Na+ channels by lidocaine and L-type Ca2+ channels by many organic Ca2+ channel blockers (Bean et al., 1983; Lee and Tsien, 1983; Bean, 1984). According to the modulated receptor hypothesis of Hille (1977) and Hondeghem and Katzung (1977), voltage- and use-dependent block occur when drugs preferentially bind to channel conformations prevalent at depolarized membrane potentials. This model predicts that preferential binding and stabilization of the inactivated state would shift the steady-state inactivation curve in the hyperpolarizing direction. The leftward shift in the steady-state availability curve that we observed is consistent with the hypothesis that mibefradil binds to and stabilizes the inactivated state of T-type Ca2+ channels in AZF cells. In contrast to its effect on inactivation, mibefradil had absolutely no effect on the voltage-dependence of T channel activation, indicating a relatively specific action of the drug on T channel gating machinery.

Effect of Mibefradil on Gating Kinetics. The results of experiments measuring the action of mibefradil on gating kinetics provide information about its molecular mechanism. Rather than occluding the channel pore, some agents reduce peak currents by slowing or accelerating gating transitions. Lanthanides reduce voltage-gated K+ currents by markedly slowing the rate of channel activation (Enyeart et al., 1998). In the simplified gating scheme shown, the lack of effect of mibefradil on K1 (or 1/tau a) indicates that an effect on activation kinetics does not contribute to mibefradil inhibition of T channels (see scheme).


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  		Scheme. Gating scheme.

Mibefradil also did not accelerate the rate of T channel inactivation (K3). Besides demonstrating that mibefradil does not alter the transition of open channels moving to the inactivated state, this result suggests that the drug does not function by blocking open channels. Besides reducing the amplitude of the peak current, agents that act by blocking open channels frequently accelerate the apparent rate of inactivation as recently opened channels are rapidly blocked (Lee and Tsien, 1983; Hille, 1992). Mibefradil has been shown to speed the decay of high voltage-activated Ca2+ channels, a result consistent with open channel block (Bezprozvanny and Tsien, 1995). The lack of effect of mibefradil on the kinetics of T current decay suggests that this agent does not preferentially bind to and block open T-type Ca2+ channels in AZF cells. Furthermore, the lack of effect of mibefradil on the kinetics of open channels returning to the closed state with repolarization (tau d) also argues against a preferential interaction of mibefradil with open T channels. However, our results do not exclude the possibility that mibefradil preferentially binds to and blocks open T channels with extremely rapid kinetics.

Of the kinetic parameters studied, mibefradil altered only the kinetics of T channel recovery following a depolarization. This result is consistent with preferential binding to and stabilization of the inactivated state (Bean et al., 1983). Along with the hyperpolarizing shift in the steady-state inactivation curve, the slowing of recovery upon repolarization, serve as hallmarks of local anesthetics and Ca2+ channel blockers that display use- and voltage-dependence (Bean et al., 1983; Bean, 1984; Sanguinetti and Kass, 1984).

According to the modulated receptor hypothesis, the slowed recovery produced by local anesthetics and Ca2+ channel blockers results from preferential binding and stabilization of the inactivated state (Hille, 1977; Hondeghem and Katzung, 1977). However, this interpretation will have to be reevaluated in view of the recent finding that lidocaine does not slow the kinetics of reopening of the fast inactivation gate of Na+ channels, although current recovers extremely slowly (Vedantham and Cannon, 1999). In AZF cells where T channels recover from inactivation by a process described by two time constants, mibefradil markedly increased only the slow recovery time constant (Mlinar et al., 1993). Whether this represents a slowing of inactivated channels returning to the closed state, or the time course of mibefradil dissociation from and unblocking of closed T channels is unknown. Regardless, mibefradil does not alter the fast recovery time constant in AZF cells, suggesting that the drug does not alter the kinetics of T channels that recover from inactivation along this rapid pathway.

The inhibition of ACTH-stimulated cortisol secretion by mibefradil at concentrations that blocked IT are consistent with a model for secretion that requires depolarization-dependent Ca2+ entry through T channels (Enyeart et al., 1993, 1996). The selectivity of mibefradil as a T channel blocker is important in this respect.

In summary, mibefradil is an effective new antagonist of T-type Ca2+ channels in AZF cells. Although it is less potent than diphenylbutylpiperidine antipsychotics as an antagonist of T-type Ca2+ channels in these cells (Enyeart et al., 1993), its relative selectivity for these low voltage-activated channels makes it a useful tool for the study of T channel function in this and other secretory cells. However, the apparent uniform block by mibefradil of T-type Ca2+ channels in neurons, muscle, and endocrine cells could limit its utility as a therapeutic agent.

    Footnotes

Accepted for publication August 2, 1999.

Received for publication June 10, 1999.

1 J.J.E. was supported by National Institute of Diabetes and Digestive and Kidney Grant DK-47875 and by National American Heart Association Grant-in-Aid 94011740.

Send reprint requests to: Dr. John J. Enyeart, Department of Pharmacology, The Ohio State University, College of Medicine, 5188 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210-1239. E-mail: enyeart.1{at}osu.edu

    Abbreviations

AZF, bovine adrenal fasciculata; IT, T-type Ca2+ current; ACTH, adrenocorticotropin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.

    References
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

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