Effect of Cilnidipine, a Novel Dihydropyridine Ca++-channel antagonist, on N-type Ca++ Channel in Rat Dorsal Root Ganglion Neurons

  1. Shigeo Fujii1,
  2. Kimiko Kameyama2,2,
  3. Masahiro Hosono1,
  4. Yutaka Hayashi1 and
  5. Kenji Kitamura
  1. 1Pharmaceuticals Research Laboratories, Fujirebio Inc., 51 Komiya-cho, Hachioji, Tokyo, 192 (S.F., M.H., Y.H.) and 2Department of Pharmacology, Fukuoka Dental College, Tamura, Fukuoka 814–01 (K.K., K.K.), Japan
     
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    Abstract

    We investigated the effects of cilnidipine, a dihydropyridine derivative, on neuronal Ca++ channels in rat dorsal root ganglion neurons. Voltage-dependent Ca++-channel currents were recorded, using 5 mM Ba++ as the charge carrier, by means of the whole-cell patch-clamp technique. The Ba++current was subdivided pharmacologically into calciseptine-sensitive (L-type), ω-conotoxin GVIA- (ωCgTx) sensitive (N-type), ω-agatoxin IVA- (ωAgTx) sensitive (P/Q-type) and toxin-resistant currents. Cilnidipine inhibited the L-type current with an IC50 of 100 nM in neurons pretreated with ωCgTx plus ωAgTx. In neurons pretreated with Cal plus ωAgTx, cilnidipine induced a potent inhibition of the N-type current, but was unable to block the residual Ba++ current. The IC50 for cilnidipine in respect of the N-type current was 200 nM. Cilnidipine (300–500 nM) modified neither the voltage-dependent inactivation curve nor the decay of the N-type current. Furthermore, elevation of the holding potential did not enhance the inhibitory action of cilnidipine (300 nM) on the N-type current. No effect was induced by 100 nM cilnidipine on the P/Q-type current. However, nicardipine (1 μM) barely inhibited the N-type current at a concentration that almost completely blocked the L-type current. In conclusion, cilnidipine has potent inhibitory actions on N-type as well as L-type voltage-dependent Ca++-channel in rat dorsal root ganglion neurons. The former action may bestow an additional clinical advantage for the treatment of hypertension, such as suppression of reflex tachycardia.

    The L-type VDCC is found in many excitable cells and contributes to physiological functions requiring [Ca++]i elevation. Although molecular cloning experiments have revealed different α1 subunits in different tissues (Hofmann et al., 1994), the L-type VDCC is still classed simply as a DHP-sensitive VDCC. Thus, DHP derivatives such as nicardipine and nifedipine have been used as pharmacological tools for the identification of L-type VDCC, and these drugs are particularly important for the treatment of cardiovascular disorders.

    Cilnidipine is a novel DHP derivative that has a slow-onset, long-lasting hypotensive effect in both hypertensive patients and animal models. As expected for a DHP derivative, cilnidipine concentration-dependently and voltage-dependently blocked L-type VDCC in the rabbit basilar artery (Oike et al., 1990). In the SHR, Hosono et al. (1995a) found that nicardipine and some other DHP derivatives reduced mean blood pressure, but had no effect on the pressor responses induced by acute cold stress. However, cilnidipine caused an inhibition of such pressor responses in addition to its hypotensive effect (Hosono et al., 1995a). As cilnidipine inhibited both elevations of plasma NE concentration and the release of [3H]NE in the rat mesenteric vasculature, the author speculated that cilnidipine might have inhibitory actions on sympathetic neurotransmission which were unique among DHP derivatives (Hosono et al., 1995a, b).

    With regard to the mechanisms underlying sympathetic neurotransmission, many papers have suggested that non-L-type VDCC, and particularly the N-type VDCC, might play a major role. This is because, whereas DHP derivatives have no effect, ωCgTx, a specific N-type VDCC blocker, inhibits nerve-mediated responses (Hirning et al., 1988;Clasbrummel et al., 1989; Pruneau and Angus, 1990;Rittenhouse and Zigmond, 1991; Fabi et al., 1993). Interestingly, cilnidipine has been found to partially displace in a concentration-dependent manner, the specific binding of [125I]ωCgTx in rat brain synaptosomes (Hosono et al., 1995b). This finding indicates a possible interaction between cilnidipine and the N-type VDCC.

    The aim of our experiments was to identify and clarify any inhibitory action of cilnidipine on the N-type VDCC. For this purpose, we used rat dorsal root ganglion neurons, which ‘have been shown to exhibit both N-type and other types of VDCC (Scroggs and Fox, 1992; Mintz and Bean, 1993).

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    Materials and Methods

    Drugs.

    The chemical structure of cilnidipine (MW 492.53) is shown in figure 1. Cilnidipine (Fuji-Rebio Inc., Tokyo, Japan) and nicardipine hydrochloride (Sigma Chemical Co., St. Louis, MO) were each dissolved in DMSO (Sigma) at 10 mM as stock solutions. The final concentration of DMSO was 0.1%. At this concentration, DMSO did not affect the depolarization-induced inward current in rat DRG neurons. Cal, ωCgTx and ωAgTx (all from Peptide Institute, Minoh, Japan) were dissolved in deionized water and diluted to their final concentration in the bathing solution. The concentrations of ωCgTx (≥3 μM) and ωAgTx (≥2 μM) used in our study have been reported selectively to block N- and P/Q-type VDCC, respectively (Aosaki and Kasai, 1989; Bleakman et al., 1995; Mintz and Bean, 1993; Wheeler et al., 1994; Sather et al., 1993). Cilnidipine and the other DHP derivatives were applied by superfusion in the bath. Biological toxins were delivered through a pressure-ejection pipette (experiment of fig.2) or superfused in the bath (other experiments).

    Figure 1
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    Figure 1

    Chemical structure of cilnidipine [2-methoxyethyl (E)-3-phenyl-2-propen-1-yl(±)-1,4-dihydro-2,6-dimethyl-4-(3-nitro-phenyl)pyridine-3,5-dicarboxylate].

    Figure 2
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    Figure 2

    Effect of type-specific Ca++ channel blockers in rat DRG neurons. Ba++ current was evoked by depolarizing pulses to 0 mV from a holding potential of -60 mV (30 msec duration; 0.18Hz). A, ωCgTx (3 μM), Cal (1 and 3 μM) and ωAgTx (2 μM) were sequentially applied through a pressure-ejection pipette. Inset shows current traces obtained at the times indicated by the arrows. B, Cal (100 nM alone, or 1 and 3 μM sequentially) applied through a pressure-ejection pipette to DRG neurons pretreated with 5 μM ωCgTx plus 2 μM ωAgTx. The Ba++ current before the application of Cal was normalized as 1.0.

    Cell culture.

    DRG were isolated from 1- to 5-day-old Wistar rats as follows. After chemical digestion in Ringer solution containing 0.1% collagenase (Wako Pure Chemicals, Osaka, Japan) and 0.05% trypsin (Sigma) at 37°C for 30 min, the DRG were rinsed twice with 2 ml enzyme-free Ringer solution. Single neuronal cell bodies were obtained by trituration through a fire-polished Pasteur pipette. The cell bodies so obtained were suspended in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 100 ng/ml 7S nerve growth factor, 50 μg/ml streptomycin sulfate and 50 U/ml penicillin G. They were then plated onto glass coverslips coated with poly-L-lysine (Sigma). Cultures were maintained at 37°C in a humidified air containing 5% CO2. Nonneuronal cell proliferation was reduced by the presence of 5 mM cytosine β-D-arabinofuranoside (free base; Sigma) for the first 24 hr. Current recordings were made using cells less than 5 days after plating. Small neuronal cell bodies (diameter < 30 mm) were selected for Ca++-channel current recording.

    Electrophysiology.

    Currents were recorded in a whole-cell configuration using a voltage-clamp amplifier (CEZ-2200, Nihon Khoden, Tokyo, Japan) from single cell bodies of DRG neurons. Currents were filtered at 3 kHz and stored in a computer (Apple Computer, 7100/80AV, Cupertino, CA) through an AD/DA interface (ITC-16, Instrutech Greatneck, New York, NY; sampling rate 10 kHz) using Axodata software (v1.2, Axon Instrum., Foster, CA). Electrode pipettes (2–3 MΩ in bathing solution) were made from borosilicated glass capillary tubes (Kimble Products, Owens, IL) using a multiple-step patch-electrode puller (P-97, Sutter Instrum., Novato, CA), and then were heat-polished (MF-83, Narishige Sci. Instr. Lab., Tokyo, Japan). Electrodes were manipulated with the aid of an electrically driven micromanipulator (Manipulator-E, Leitz, Wetzlar, FRG). The pipette solution consisted of (in mM) 135 CsCl, 5 MgCl2, 5 BAPTA (tetrapotassium salt; Dojin, Kumamoto, Japan), 10 HEPES, 5 ATP (disodium salt; Sigma) and 12 glucose (pH 7.0 after titration with CsOH). The bathing solution consisted of (in mM) 100 Tris hydroxymethyl aminomethane, 5 CsCl, 5 BaCl2, 1 MgCl2, 25 TEA-Cl, 5 HEPES and 20 glucose (pH 7.4 after titration with Tris HCl). Except where otherwise noted, currents were evoked by step depolarization to 0 mV from a holding potential of -60 mV (30 msec duration; 0.18Hz). This was done after allowing the cells to equilibrate for at least 5 min after the rupture of the patch membrane. All experiments were performed at room temperature (23–26°C).

    Data analysis.

    Capacitative and leak currents were subtracted using the P/4 method. The depolarization-induced Ba++ current was measured at peak, and the current recorded before drug application was normalized as 1.0. Drug action was assessed by calculating the difference between the peak current amplitudes before and 3 to 4 min after application of the relevant drug. Data are expressed as mean ± S.E.M.

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    Results

    Pharmacological classification of VDCC in rat DRG neurons.

    In the experiment shown in figure 2A, application of 5 μM ωCgTx reduced the Ba++ current by 43%. Sequential application of 1 μM Cal further attenuated the remaining Ba++ current. When the concentration of Cal was increased to 3 μM, the current was further decreased (to 22% of that recorded before the application of the toxins). Additional application of 2 μM ωAgTx reduced the remaining current even further. After sequential application of these three toxins, the Ba++ current in this neuron was almost abolished, but approximately 5% of the original current remained.

    Figure 2B shows the effect of 0.1 and 1 μM Cal on the Ba++ current in DRG neurons pretreated with 5 μM ωCgTx plus 2 μM ωAgTx for blockade of the N- and P/Q-type current. Cal inhibited the L-type current in a concentration-dependent manner. After application of 1 μM Cal, 3 μM Cal failed to affect the remaining Ba++ current. The attenuation of the Ba++current produced by 0.1 μM Cal was not reversed for at least 10 min after withdrawal of the toxin. Similarly, the reduced current remaining after application of 3 μM ωCgTx or 2 μM ωAgTx was unchanged by the removal of the relevant toxin from the superfusate, and a higher concentration of neither ωCgTx (5μM) nor ωAgTx (3 μM) inhibited the Ba++ current further (data not shown). The residual Ba++ current, observed after treatment with 3 μM Cal plus 3 μM ωCgTx plus 2 μM ωAgTx, was reversibly inhibited by superfusion with 100 μM CdCl2. At the holding potential used (-60 mV), application of a step-depolarization to -30 mV evoked no inward Ba++ current.

    Effect of cilnidipine on the L-type current.

    To examine the effect of cilnidipine on the neuronal L-type VDCC, DRG neurons were pretreated with a combination of 2 μM ωAgTx plus 5 μM ωCgTx to achieve blockade of the P/Q- and N-type currents. As shown in figure3A, the peak amplitude of the Ba++ current was then reduced by superfusion with as little as 1 nM cilnidipine and, moreover, cilnidipine reduced the Ba++ current in a concentration-dependent manner. In these experiments, cilnidipine simply reduced the amplitude of the Ba++ current without inducing a change in the current decay (fig. 3A). The mean size of the current reduction induced by 3 μM cilnidipine was 76 ± 7% in ωCgTx-, ωAgTx-pretreated neurons (n = 6). Nicardipine, at similar concentrations, also reduced the Ba++ current in ωCgTx-, ωAgTx-pretreated neurons. A high concentration of nicardipine (10 μM) reduced by 67 ± 4% the ωCgTx-, ωAgTx-resistant Ba++ current (n = 3). As shown in figure 2B, 1 μM Cal incompletely inhibited the ωCgTx-, ωAgTx-resistant Ba++ current; application of 3 μM Cal induced no additional inhibition. Therefore, to determine the concentration-response relationship for the L-type current, the total fall in the Ba++ current caused by either 3 μM cilnidipine or 10 μM nicardipine was taken to represent the L-type current component in ωCgTx-, ωAgTx-pretreated neurons. Figure 3B shows the effect of cilnidipine (n = 5–7) and nicardipine (n = 4) on this L-type current. Cilnidipine inhibited the L-type current with an IC50 of 100 nM. Nicardipine also inhibited the L-type current, and to much the same extent.

    Figure 3
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    Figure 3

    Effects of cilnidipine and nicardipine on the L-type current. DRG neurons were pretreated with 5 μM ωCgTx plus 2 μM ωAgTx for blockade of the N- and P/Q-type current. The Ba++ current was evoked by depolarizing pulses to 0 mV from a holding potential of -60 mV (30 msec duration; 0.18Hz). The current recorded before the application of the test drugs was normalized as 1.0. A, Time course of changes in the peak amplitude of the Ba++ current before and during application of cilnidipine. Cilnidipine (0.1 nM-3 μM), added to the superfusate, was cumulatively applied to a DRG neuron. Inset shows current traces obtained at the times indicated by the arrows. B, Relationship between the amplitude of the L-type current and the concentration of cilnidipine (m) and nicardipine (). The amplitude of the L-type current was calculated as the difference between the currents recorded in the absence or presence of the test drug (3 μM cilnidipine or 10 μM nicardipine). The line was fitted by a least-squares method. Each point represents mean ± S.E.M. from five to seven different neurons (for cilnidipine) or four different neurons (for nicardipine).

    Effect of cilnidipine on the non-L/N/P/Q-type current.

    When 3 μM ωCgTx was applied to a 3 μM Cal-, 2 μM ωAgTx- pretreated neuron, the peak amplitude of the Ba++ current was reduced by approximately 70% within 1 min. No additional inhibition was induced by the sequential application of 3 μM cilnidipine, but the residual Ba++ current was abolished by the application of 100 μM CdCl2 (fig. 4A). Overall, cilnidipine had no effect on the residual component of the Ba++ current (n = 3; fig. 4B).

    Figure 4
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    Figure 4

    Effect of cilnidipine on the non-L/N/P/Q-type current. A, DRG neurons were pretreated with 3 μM Cal plus 2 μM ωAgTx. Cilnidipine (3 μM) was superfused following treatment with 3 μM ωCgTx. Sequentially, CdCl2 (100 μM) was superfused. Inset shows current traces obtained at the times indicated by the arrows. B, Relative amplitude of the Ba++ current obtained before (open column) and after (filled column) application of cilnidipine to Cal-, ωCgTx-, ωAgTx-pretreated DRG neurons. Each column and vertical bar represents mean and S.E.M. from three different neurons. In A and B, the Ba++ current was evoked by depolarizing pulses to 0 mV from a holding potential of -60 mV (30 msec duration; 0.18Hz). The current recorded before application of ωCgTx was normalized as 1.0.

    Effect of cilnidipine on the N-type current.

    To investigate its effect on the N-type current, the inhibitory action of cilnidipine on the ωCgTx-sensitive current was studied in DRG neurons pretreated with 3 μM Cal plus 2 μM ωAgTx to achieve blockade of the L- and P/Q-type currents. When cilnidipine was cumulatively applied by superfusion before any application of ωCgTx to the neuron, the peak amplitude of the Ba++ current was reduced concentration-dependently (fig. 5A). On the other hand, superfusion with 1 μM nicardipine only slightly inhibited the Ba++ current (by 9% of control) in a Cal-, ωAgTx-pretreated neuron (fig. 5B). Superfusion with 3 μM ωCgTx then reduced the Ba++ current to 27% of the control current (recorded before application of nicardipine). The residual Ba++ current was abolished by application of 100 μM CdCl2 (fig. 5B). Figure 6 summarizes the effects of cilnidipine (n = 5–7) and nicardipine (n = 3) on the N-type current in DRG neurons. To obtain a concentration-response relationship for this action of cilnidipine, 3 μM ωCgTx was superfused at the end of the recording period in each Cal-, ωAgTx-pretreated neuron, and the difference between the current amplitudes before and after the application of ωCgTx was normalized as 1.0. The inhibitory action of cilnidipine occurred at concentrations similar to those that blocked the L-type current; in this case the IC50 was 200 nM. However, 1 μM nicardipine produced only an 11 ± 6% inhibition of the current (n = 3; fig. 6).When the holding potential was kept at -40 mV, 200 nM cilnidipine reduced the N-type current amplitude by 45 ± 11% (n = 4; fig. 6). As shown in figure 7, cilnidipine (500 nM) affected neither the voltage-dependent inactivation curve of the N-type current nor the membrane potential that reduced the amplitude of the N-type current to half (Vhalf; control, -44 ± 1 mV; cilnidipine, -45 ± 3 mV; n = 3). To investigate effect of cilnidipine on the decay of the N-type current, a long depolarizing pulse (300 msec duration) was applied in the absence or presence of 300 nM cilnidipine. Cilnidipine reduced the amplitude, but did not change the time-constant of the N-type current decay (control, 1.78 ± 0.33 sec; cilnidipine, 2.06 ± 0.24 sec; n = 4).

    Figure 5
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    Figure 5

    Effect of cilnidipine and nicardipine on the N-type current. DRG neurons were pretreated with 3 μM Cal plus 2 μM ωAgTx. The Ba++ current was evoked by depolarizing pulses to 0 mV from a holding potential of -60 mV (30 msec duration; 0.18 Hz). The current recorded before the application of cilnidipine was normalized as 1.0. Inset shows current traces obtained at the times indicated by the arrows. A, Changes induced by various concentrations of cilnidipine in the peak amplitude of the Cal-, ωAgTx-resistant Ba++ current. Cilnidipine (0.1 nM - 3 μM) was added to the superfusate. B, Changes induced by nicardipine in the peak amplitude of the Cal-, ωAgTx- resistant Ba++ current. Nicardipine (1 μM), ωCgTx (3 μM) and CdCl2 (100 μM) were superfused.

    Figure 6
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    Figure 6

    The relationship between the amplitude of the N-type current and the concentration of cilnidipine (•) or nicardipine (▴). The amplitude of the N-type current was taken as the difference between the Ba++ currents recorded before and after the application of 3 μM ωCgTx. In each experiment, 3 μM ωCgTx was superfused at the end of the recording period. The line was fitted by a least-squares method. Efect of 200 nM cilnidipine on the amplitude of the N-type current at the holding potential of -40 mV (○) was also plotted in the figure. Each point represents mean ± S.E.M. from four to seven (for cilnidipine) or three (for nicardipine) different neurons.

    Figure 7
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    Figure 7

    Voltage-dependent inactivation curves for the N-type current. Experimental protocol for voltage-dependent inactivation is also indicated in the figure. DRG neurons were pretreated with 3 μM Cal plus 2 μM ωAgTx. In each experiment, ωCgTx (3 μM) was superfused at the end of the recording period, and the amplitude of the N-type current was estimated from the difference between the Ba++ currents recorded before and after the application of ωCgTx. The N-type current amplitudes recorded without a conditioning potential were normalized as 1.0 (mean ± S.E.M.; control, ○; 500 nM cilnidipine, •; n = 3) and the N-type current amplitudes recorded after application of the drug are expressed relative to those recorded in the absence of drug (□; S.E.M. bars are not shown). The theoretical lines were drawn using the following equation: I=(Imax-C)/{1+exp[(V-Vhalf)/k]}+C, where I, Imax, V, Vhalf, k, and C are the relative amplitudes of the N- type current observed at various amplitudes of the conditioning pulse (I) or without a conditioning pulse (Imax), the amplitude of the conditioning pulse (V), and the amplitude of the conditioning pulse that reduced the amplitude of the N-type current to half (Vhalf), the slope factor (k), and the fraction of the noninactivating component of the N-type current (C). The curves in the absence or presence of cilnidipine were drawn using the following values: (control) Imax = 1.0, Vhalf45 mV, k = 5 mV, and C = .24; (cilnidipine) Imax = .6, Vhalf44 mV, k = 9 mV, and C = .10.

    Effect of cilnidipine on the P/Q-type current.

    To determine the effects of cilnidipine on the P/Q-type VDCC, DRG neurons were pretreated with a combination of 3 μM Cal and 5 μM ωCgTx to achieve blockade of the L- and N-type currents. A typical recording is shown in figure 8A. Superfusion with cilnidipine at concentrations up to 100 nM did not affect the Ba++ current (fig. 8Aa), but a higher concentration (1 μM) did inhibit it (fig.8Ab; by 62% of control in this example). Subsequent superfusion with 2 μM ωAgTx further reduced the Ba++ current in each neuron. To obtain a concentration-response relationship for this action of cilnidipine, 2 μM ωAgTx was superfused at the end of the recording period in each ωCgTx-, Cal-pretreated neuron and the difference between the current amplitudes before and after the application of ωAgTx was normalized as 1.0. Such a relationship, allowing an estimation of the amplitude of the P/Q-type current, is shown in figure 8B (n = 4–5). No inhibitory action was observed with 100 nM cilnidipine, whereas 1 μM cilnidipine inhibited the P/Q-type current by 45 ± 14%.

    Figure 8
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    Figure 8

    Effect of cilnidipine on the P/Q-type current. DRG neurons were pretreated with 3 μM ωCgTx plus 3 μM Cal. The Ba++ current was evoked by depolarizing pulses to 0 mV from a holding potential of -60 mV (30 msec duration; 0.18 Hz). The current recorded before application of cilnidipine was normalized as 1.0. A, Changes induced by cilnidipine in the peak amplitude of the Cal-, ωCgTx-resistant Ba++ current. Cilnidipine (Aa, 10 nM-100 nM; Ab, 1 μM) and ωAgTx (2 μM) were sequentially superfused. In the case of Ab, CdCl2 (100 μM) was superfused at the end of the recording period. B, Relationship between the amplitude of the P/Q-type current and cilnidipine concentration. The amplitude of the P/Q-type current was taken as the difference between the Ba++ currents recorded before and after application of 2 μM ωAgTx. In all experiments, 2 μM ωAgTx was superfused at the end of the recording period. Each point represents mean ± S.E.M. from four to five different neurons.

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    Discussion

    Our study showed that cilnidipine, a novel DHP derivative, has a potent inhibitory effect on the N-type VDCC, as well as on the L-type VDCC, in rat DRG neurons. The two inhibitory actions occurred at the similar concentration range. Furthermore, this drug exerted only a weak inhibitory action on the P/Q-type VDCC, and did not inhibit the residual component. Nicardipine had little inhibitory effect on the N-type VDCC. The partial blocking action of cilnidipine on the N-type VDCC may contribute to its therapeutic effects, possibly through a modulation of sympathetic neurotransmission (see below).

    Blockade of VDCC with specific blockers.

    The presence of multiple types of high-voltage-activated Ca++ channels in mammalian neurons is well known (L-, N-, P/Q-types etc.; Hofmannet al., 1994). Because of the difficulty of separating these channels by their kinetics or biophysical properties, we chose to separate them pharmacologically (Aosaki and Kasai, 1989; Bleakmanet al., 1995; Mintz and Bean, 1993; Wheeler et al., 1994; Sather et al., 1993). Cal causes a selective and irreversible block of the neuronal, cardiac and vascular L-type VDCC (De Weille et al., 1991; Teramoto et al., 1996). To study the actions of cilnidipine on non-L-type VDCC, we used 3 μM Cal, a concentration 3 times higher than that required for maximal blockade of the L-type current (data from our experiments), but which had no effect on the N-type VDCC in chick DRG neurons (De Weilleet al., 1991).

    We could record a Cd++-sensitive component of the Ba++ current even after application of Cal plus ωCgTx plus ωAgTx. Although we did not try to identify the current further, we suspect that this residual component might not be conducted through T-type VDCC because, in rat DRG neurons, T-type VDCC are almost inactivated at a holding potential of -60 mV (Fox et al., 1987; Scroggs and Fox, 1992). A possible contribution of R-type VDCC to the Cd++-sensitive component (Diochot et al., 1995) could not be excluded in the present experiments.

    Effect of cilnidipine on VDCC.

    DHP derivatives have a high-affinity for L-type VDCC in cardiac, skeletal and smooth muscle cells. In the present experiments, cilnidipine reduced the neuronal L-type current with an IC50 of 100 nM at a holding potential of -60 mV. Although we did not study in detail the action of nicardipine on the L-type current in our DRG neurons, this drug also clearly inhibited the L-type current. In fact, the same concentration-response relationship was obtained for cilnidipine and nicardipine (fig. 3B). This indicates that cilnidipine and nicardipine have an equipotent inhibitory action on the L-type current. Interestingly, Hosono et al. (1995a) reported that, in SHR, cilnidipine and nicardipine produced the same degree of hypotension.

    When L-type and P/Q-type currents were blocked with Cal plus ωAgTx, cilnidipine reduced the remaining Ba++ current. As this inhibition was not observed after ωCgTx-treatment, we concluded that cilnidipine acts on the N-type current in DRG neurons. In contrast, nicardipine (1 μM) induced only a slight inhibition of the Ba++ current in Cal-, ωAgTx-pretreated neurons. Actually, we think that this inhibition with 1 μM nicardipine did not result from a block of the N-type current, as nicardipine at 1 and 10 μM reduced the residual Ba++ current after blockade of L-, N- and P/Q-type currents (by 26 and 69%, respectively); in contrast 3 μM nifedipine, an other DHP derivative, did not attenuate the N-type current (unpublished observations).

    Micromolar and submillimolar concentrations of DHP derivatives have been reported to block 1) the N-type current (apparent IC50of nicardipine, 10 μM, Diochot et al., 1995), 2) the T-type current (K D of nicardipine, 3.5 μM; and of nifedipine, 5 μM, Akaike et al., 1989) and 3) the voltage-dependent Na+ (IC50 of nitrendipine, 3 μM, Yatani and Brown, 1985) and K+ currents (IC50 of nicardipine, 1 μM, Fagni et al., 1994). In frog sympathetic neurons, 10 μM of nifedipine is needed to inhibit 14% of the N-type current, 36% of the K+ current, and 12% of the Na+ current, but only 0.3 μM or less is needed to block 50% of the L-type current (Jones and Jacobs, 1990). These data show that the blocking action of nifedipine on the L-type current is more than 30 times stronger than its action on other ionic currents. In cardiac cells, Hume (1985) reported that, for nisoldipine, the K D for the L-type current was 1000 times lower than that for the K+ current. Therefore, inhibition of various ionic currents by high concentrations of DHP derivatives probably represents a nonspecific action. However, the inhibitory action of cilnidipine on the N-type current, as observed in our experiments, occurred at very low concentrations in comparison to those observed with other DHP derivatives. The size of the estimated IC50 value for the N-type current was close to that for the L-type current (N-type, 200 nM; L-type, 100 nM). However, these values might be underestimates, as the development of inhibition by cilnidipine was very slow, in fact, the inhibition induced by this drug had not reached the steady state level 3 to 4 min after its application, particularly at a low concentration. However, the P/Q-type current and the K+ outward current can be inhibited by only micromolar concentrations of cilnidipine (our experiments; Oikeet al., 1990). These results clearly indicate that the N-type-VDCC blocking action of cilnidipine is a specific action, and one which is not seen with other DHP derivatives.

    The reported Vhalf values for the N-type VDCC found in chick DRG neurons and for those expressed in oocytes and other cells are reported to be more negative than that found in our study (Fox et al., 1987; Ellinor et al., 1994; Bleakman et al., 1995). Although the reasons are not clear for such a discrepancy, it might be due to contamination of the measurements by other types of VDCC. However, our results clearly show that cilnidipine did not affect the voltage-dependent inactivation curve of the ωCgTx-sensitive component. Cilnidipine did not change the decay of the N-type current, not did elevation of the holding potential enhance the inhibitory action of cilnidipine on the N-type current. These results indicate that cilnidipine inhibits the N-type current in a voltage-independent manner. As Oike et al. (1990) reported that cilnidipine induced a voltage-dependent inhibition of the L-type current, the mechanisms underlying its inhibition of the N-type and L-type currents might differ.

    Concerning the action of cilnidipine on ωCgTx binding, Hosonoet al. (1995b) reported that cilnidipine (10 nM - 10 μM) inhibited [125I]ωCgTx binding at most by 25%, whereas nicardipine failed to displace the binding at all in rat brain synaptosomes. Our results were qualitatively in accord with those binding experiments. However, it is worth noting the small quantitative difference between the inhibition of the N-type current in our DRG neurons and the displacement of ωCgTx binding in synaptosomes. This difference might be due to the difficulty experienced by cilnidipine in displacing tightly bound [125I]ωCgTx from the N-type VDCC, as ωCgTx has a very high affinity for rat brain synaptosomes (Abe et al., 1986; Barhanin et al., 1988;Feigenbaum et al., 1988), and as this toxin induces an irreversible block of the N-type VDCC (Aosaki and Kasai, 1989; Bleakmanet al., 1995). Another possible interpretation is that cilnidipine only recognizes some of the ωCgTx binding sites. Lampeet al. (1993) reported that, as with ωCgTx, ω-gramotoxin SIA, a polypeptide toxin, inhibited chick synaptosomal45Ca++ influx, but it did not displace [125I]ωCgTx binding to rat brain membrane fragments. Further experiments will be required to clarify compare and contrast the cilnidipine-, ωCgTx-binding sites on the N-type VDCC, such as experiments using chimera channels.

    Contribution of N-type VDCC blockade to antihypertensive effects.

    Evidence has been accumulating about the contribution of the sympathetic nervous system to the elevation of blood pressure in hypertensive patients and animals (Lee et al., 1987;Anderson et al., 1989), and a number of authors have shown that the N-type VDCC is closely related to sympathetic neurotransmission (Hirning et al., 1988; Clasbrummelet al., 1989; Pruneau and Angus, 1990; Rittenhouse and Zigmond, 1991; Fabi et al., 1993). In addition, Rittenhouse and Zigmond (1991) noted that N-type VDCC are involved in the activation of tyrosine hydroxylase, a rate-limiting enzyme in the biosynthesis of catecholamines, including NE. Indeed, i.v. administration of ωCgTx causes potent hypotension in conscious SHR (Pruneau and Bélichard, 1992), even though this peptide does not inhibit arterial high-voltage-activated Ca++ channels (McCleskey et al., 1987). Hosono et al. (1995a)reported that cilnidipine, but not other DHP antagonists, reduced both the plasma NE concentration and the pressor response induced by acute cold stress in SHR. Furthermore, cilnidipine, but not nicardipine, inhibited the release of [3H]NE from the rat mesenteric vasculature (Hosono et al., 1995b). These inhibitory actions of cilnidipine can presumably be explained, at least in part, by its blocking action on N-type VDCC in the nerve terminals. Significantly, ωCgTx causes a marked suppression of [3H]NE release evoked by periarterial nerve stimulation (Hosono et al., 1995b).

    As neuronal L-type and N-type VDCC participate in cell excitation and sympathetic neurotransmission (Hirning et al., 1988;Clasbrummel et al., 1989; Pruneau and Angus, 1990;Rittenhouse and Zigmond, 1991; Fabi et al., 1993; Hellet al., 1993), the inhibitory action of cilnidipine on N-type VDCC may make a synergistic contribution to its therapeutic effects. If so, “N-type VDCC blockers” such as cilnidipine could lead to a new therapeutic strategy in the battle against hypertension. However, as cilnidipine had a weak action on the P/Q-type VDCC, this particular drug may slightly affect the neurotransmission regulated by P/Q-type VDCC both in the central nervous system (Mintz et al., 1992; Mintz and Bean, 1993; Takahashi and Momiyama, 1993;Wheeler et al., 1994) and at the neuromuscular junction (Uchitel et al., 1992; Sugiura et al., 1995).

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    Acknowledgments

    The authors thank Dr. R. J. Timms for editing the English and Ms. Yukiko Okazaki for typing the manuscript.

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    Footnotes

    • Send reprint requests to: Dr. S. Fujii, Pharmaceuticals Research Laboratories, Fujirebio Inc., 51 Komiya-cho, Hachioji, Tokyo, 192, Japan.

    • Abbreviations:
      DRG
      dorsal root ganglion
      VDCC
      voltage-dependent Ca++ channel
      DHP
      dihydropyridine
      Cal
      calciseptine
      ωCgTx
      ω-conotoxin GVIA
      ωAgTx
      ω-agatoxin IVA
      NE
      norepinephrine
      DMSO
      dimethylsulfoxide
      SHR
      spontaneously hypertensive rat
      • Received August 5, 1996.
      • Accepted November 26, 1996.
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