Selectivities of Dihydropyridine Derivatives in Blocking Ca Channel Subtypes Expressed in Xenopus Oocytes

Some dihydropyridines (DHPs), such as amlodipine and cilnidipine, have been shown to block not only L-type but also N-type Ca channels; therefore, DHPs are no longer considered as L-type-specific Ca channel blockers. However, selectivity of DHPs for Ca channel subtypes including N-, P/Q-, and R-types are poorly understood. To address this issue at the molecular level, blocking effects of 10 DHPs (nifedipine, nilvadipine, barnidipine, nimodipine, nitrendipine, amlodipine, nicardipine, benidipine, felodipine, and cilnidipine) on four subtypes of Ca channels (L-, N-, P/Q-, and R-types) were investigated in the Xenopus oocyte expression system with the use of the two-microelectrode voltage-clamp technique. L-type Ca channels expressed as a1Ca2b1a combination were profoundly blocked by all DHPs examined, whereas blocking actions of these DHPs on R-type (a1Ea2b1a) channels were equally weak. In contrast, 5 of the 10 DHPs (amlodipine, benidipine, cilnidipine, nicardipine, and barnidipine) significantly blocked N-type (a1Ba2b1a) and P/Q-type (a1Aa2b1a) Ca 21 channels. These selectivities of DHPs in blocking Ca channel subtypes would provide useful pharmacological and clinical information on the mode of action of the drugs including side effects and adverse effects. High voltage-activated Ca channels in excitable cells such as myocytes, smooth muscle cells, and neurons play important roles, including contraction of myocytes, electrical excitement in neurons, and modulation of hormone and neurotransmitter release (Tsien et al., 1991). High voltage-activated Ca channels are pharmacologically classified into at least five different subclasses (L-, N-, P-, Q-, and R-type), the characteristics of which are determined by the pore-forming a1 subunit. The subunits a1C, a1D, and a1S form L-type Ca 21 channels and bind dihydropyridines (DHPs), phenylalkylamines, and benzothiazepines with high affinity, whereas the subunits a1B, a1A, and a1E form N-, P/Q-, and R-type Ca channels, respectively, which show low affinities for these drugs (Hockerman et al., 1997; Hering et al., 1998; Striessnig et al., 1998). Because nifedipine, the prototype of the DHPs, exclusively blocked muscular L-type Ca channels (Fleckenstein, 1983), DHPs had been considered as selective blockers for L-type channels. Recent studies have shown, however, that two DHPs, amlodipine (Furukawa et al., 1997) and cilnidipine (Fujii et al., 1997; Uneyama et al., 1997), blocked N-type Ca channels as well. These findings indicate that DHPs are no longer considered L-type specific blockers, and suggest that some DHPs may block other subtypes of Ca channels, such as P/Q-, and R-types. DHPs are widely used clinically in the treatment of hypertension, angina pectoris, and cerebrovascular diseases. However, pharmacological profiles of the effects of DHP on each Ca channel subtype are not understood well enough for them to be used with confidence with these drugs. Non-L-type Ca channels are diversely distributed in peripheral and central nervous cells (Tsien et al., 1991). However, native neuronal cells and cell lines possess multiple subtypes of Ca channels in a single cell, which hampers quantitative comparison of effects of a given drug on a single subtype of Ca channel. To address these issues at the molecular level, a single subclass of the Ca channel a1 subunit (a1A, a1B, a1C, or a1E) was coexpressed with the same auxiliary a2 and b subunits in Xenopus oocytes, and 10 DHP derivatives used clinically were examined for their channelblocking effects. Materials and Methods Methods for in vitro transcription of cRNAs specific to the Ca channel a1, a2, and b1a subunits and procedures for functional exReceived for publication March 1, 1999. 1 This study was supported in part by research grants from the Ministry of Education, Science and Culture of Japan to T.F. (Grant 10670676) and to T.N. (Grant 08680855). ABBREVIATIONS: DHP, dihydropyridine; DMSO, dimethyl sulfoxide; I-V, current-voltage; IC50, concentration at half-blockade. 0022-3565/99/2912-0464$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 291, No. 2 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 291:464–473, 1999 464 at A PE T Jornals on July 9, 2017 jpet.asjournals.org D ow nladed from pression of Ca channels in Xenopus oocytes were described previously (Furukawa et al., 1998). After removal of the follicular cell layer, Xenopus oocytes were injected with 0.3 mg/ml a1 [a1C (Mikami et al., 1989), a1B (Fujita et al., 1993), a1A (Mori et al., 1991), or a1E (Niidome et al., 1992)] cRNA in combination with 0.2 mg/ml a2 (Mikami et al., 1989) cRNA and 0.1 mg/ml b1a (Mori et al., 1991) cRNA. In some experiments, the cRNA for the b2b or b3 subunit (Hullin et al., 1992) was used instead of that for b1a subunit. The oocytes were cultured for 2 to 4 days and then subjected to electrophysiological measurement. The oocytes were placed in a small chamber perfused with extracellular solution (10 mM Ba, 90 mM Na, 2 mM K, 5 mM HEPES, and 0.3 mM niflumic acid, pH 7.5, with methanesulfonic acid), and Ba currents through expressed channels were measured by the two-microelectrode voltageclamp method with a GeneClamp 500 amplifier (Axon Instruments, Foster City, CA). The experimental chamber was 0.5 ml in volume, and it was perfused continuously (2.0–3.0 ml/min) with the extracellular solution. Commercial software (pClamp version 6.0; Axon Instruments) was used for generating voltage pulses, acquiring data, and analyzing the currents. Typically, oocytes were clamped at a holding potential of 2100, 280, or 260 mV and depolarized to 110 mV for 200 ms every 15 s. Microelectrodes were filled with 3 M KCl, and those showing a resistance of 0.5 to 1.2 VM were used. The drug effects were evaluated after a 5-min perfusion of bath solution containing a DHP derivative. In experiments to obtain concentration-response relationships, the concentrations of the DHPs were changed successively. Each experiment was finished within 20 min to avoid possible run down of Ba currents. Because no detectable current change was observed during exposure to the vehicle for DHP [0.2% dimethyl sulfoxide (DMSO)] as reported previously (Furukawa et al., 1997), the concentration of DMSO in the bath solution was maintained at 0.2% throughout the experiments. DHPs were dissolved into DMSO just before each experiment and added to the bath solution to make the final concentration. The following DHPs were generous gifts from pharmaceutical companies: amlodipine from Sumitomo Pharmaceuticals Co., Ltd. (Tokyo, Japan); benidipine from Kyowa-Hakko Pharmaceuticals Co., Ltd. (Tokyo, Japan); barnidipine from Yamanouchi Seiyaku Co., Ltd. (Tokyo, Japan); cilnidipine from Ajinomoto Co., Ltd. (Tokyo, Japan); felodipine from Hoechst-Marion-Roussel (Tokyo, Japan); and nilvadipine from Fujisawa Pharmaceuticals Co., Ltd. (Tokyo, Japan). Other drugs were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Statistical data were represented by the mean 6 S.E.