Inhibitory Effect of Lomerizine, a Diphenylpiperazine Ca2+ -Channel Blocker, on Ba2+ Current Through Voltage-Gated Ca2+ Channels in PC 12 Cells
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Inhibitory Effect of Lomerizine, a Diphenylpiperazine Ca2+ -Channel Blocker, on Ba2+ Current through Voltage-Gated Ca2+ Channels in PC 12 Cells Tomokazu Watanol, Hideaki Hara2,* and Takayuki Sukamoto2 1 Department of Pharmacology, New Drug Discovery Research Laboratory, Kanebo Ltd., 1-5-90 Tomobuchi-cho,of Pharmacology, Miyakojima-ku, New Drug Osaka R & 534,D Laboratory, Japan2Department Kanebo Ltd., 1-5-90 Tomobuchi-cho, Miyakojima-ku, Osaka 534, Japan Received July 8, 1997 Accepted August 22, 1997 ABSTRACT-We investigated the effect of lomerizine, an anti-migraine drug, on the Ba2+ current through voltage-gated Ca2+ channels in rat pheochromocytoma (PC12) cells using a whole-cell voltage-clamp tech nique. Lomerizine inhibited the Ba2+ current with an IC50 value of 1.9 ƒÊM. Lomerizine and nicardipine were > 4 times more potent than flunarizine, diltiazem, verapamil and dimetotiazine. The time course of inactivation induced by lomerizine was similar to that induced by nicardipine and flunarizine. These data indicate that lomerizine may inhibit the Ca2+ channel in a similar manner to nicardipine and flunarizine, and its potency is almost equal to that of nicardipine. Keywords: Lomerizine , Voltage-gated Ca2+ channel, PCl2 cell Calcium channel blockers are mainly divided into three Bio Medical, Kyoto) containing 7° fetal bovine serum types, namely, 1,4-dihydropyridine, phenylalkylamine (Moregate, Melbourne, Australia), 7% heat-inactivated and benzothiazepine types, from their structure (1). horse serum (Gibco, Grand Island, NY, USA), 2 MM L Lomerizine, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4 glutamine (Wako, Osaka) and 50 pg/ml gentamicin sul trimethoxybenzyl)piperazine dihydrochloride has been fate (Wako) at 37C in an atmosphere of 5% CO2. They reported to inhibit [3H]nitrendipine binding to synapto were then plated onto collagen-coated glass slides. After some membranes in guinea pig cerebral cortex (2) and an additional 2 to 3 days in culture, the cells were used for canine aorta (3), to inhibit the voltage-gated Ca 2+ current the following experiments. in rat hippocampal pyramidal neurons (4) and to reduce Membrane potentials and currents were measured the number of migraine attacks in patients with migraine using whole-cell recordings and improved patch-clamp (5, 6). Thus, lomerizine is regarded as a new class of cal techniques (8). The cells were continuously superfused cium channel blocker with different structure from the with an extracellular solution containing 140 mM NaCI, representative calcium channel blockers as described the 5.4 mM KCI, 1.8 mM CaC12and 10 mM 2-[4-(2-hydroxy above. However, the pharmacological profile of lomeri ethyl)-1-piperazinyl]ethanesulphonic acid (HEPES, zine has not yet been fully clarified in comparison with Wako), pH adjusted to 7.4 with NaOH. The heat that of existing calcium channel blockers. Therefore, we polished patch pipettes each had a tip resistance of about investigated the effect of lomerizine on the Ba2+ current 5 MQ when filled with an intracellular solution containing through the voltage-gated Ca 2+ channel in rat 150 mM CsCl, 10 mM HEPES and 5 mM O,O'-bis(2 pheochromocytoma (PC12) cells and compared it with aminoethyl)ethyleneglycol-N,N, N, N'tetraacetic acid those of various other types of calcium channel blockers (EGTA, Wako), pH adjusted to 7.3 with CsOH. Mem and that of dimetotiazine, an anti-migraine drug. brane potentials were controlled by a voltage-clamp am PC 12 cells (Riken Cell Bank, Ibaraki) were prepared as plifier (Axopatch 200A; Axon, Foster City, CA, USA). previously described (7). In brief, PC12 cells were cul Membrane currents were acquired on-line and analyzed tured in Dulbecco's Modified Eagle's Medium (Nikken later by a computer (PC-5/120; Inter Medical, Nagoya) using pCLAMP6 software (Axon). As Ca2+ channels are * To whom correspondence should be addressed . more permeable to Ba2+ than to Ca2+ (9), extracellular CaC12 was replaced with BaC12 (10.8 mM) to obtain a steady-state blockade was obtained by 60 sec after the large inward Ba2+ current permeating through voltage start of the superfusion (Fig. 1A) at concentrations of gated Ca2+ channels. Compensation was made for series 0.1-10 ,uM. This effect was similar to those produced by resistance, and electrical signals were filtered at 5 kHz. nicardipine, flunarizine and dimetotiazine (data not The inward current was activated by a depolarizing step shown). On the other hand, the steady-state blocking to +10 mV from a holding potential of 60 mV. The effect of diltiazem reached a steady state level at 120 sec experiments were performed at approximately 361C using after the start of the superfusion at concentrations of a TC2b;p bipolar temperature controller (Bioptechs, Inc., 1-100 ,uM in most of the cells examined (Fig. 1B). The Butler, PA, USA). The IC50 value for each drug in in profile of the inhibition produced by verapamil was simi hibiting Ba2+ current was calculated by Probit analysis. lar to that seen with diltiazem (data not shown). There Lomerizine 2HC1 (Kanebo, Osaka); nicardipine HC1 fore, for routine assessment of the typical inhibition flunarizine 2HCl, (±)-verapamil, diltiazem HCl (Sigma, produced by each drug and to derive the concentration St. Louis, MO, USA); and dimetotiazine mesylate inhibition curves, the effects on the Ba2+ current of (Shionogi, Osaka) were first dissolved in dimethyl sulf lomerizine, nicardipine, flunarizine and dimetotiazine in oxide (DMSO, Wako) and added to extracellular solu concentrations of 0.1-10 ,uM were tested at 60 sec after tions so that the final concentration of DMSO was the beginning of their superfusion, whereas the effects of <<0.1%. diltiazem and verapamil in concentrations of 1-100 ,uM Figure 1 shows typical records of the blocking effect of were tested at 120 sec. lomerizine and diltiazem on the Ba2+ current through Figure 2 shows typical examples of the inhibitory voltage-gated Ca 21 channels in PC 12 cells. The inhibition effects of lomerizine, nicardipine, flunarizine, diltiazem, produced by lomerizine was time-dependent: it was first verapamil and dimetotiazine on the Ba2+ current. Cur observed at 20 sec after the start of the superfusion, and a rent traces obtained just before (Control) and after the Fig. 1. Time-dependency of inhibition by lomerizine and diltiazem of Ba2+ current in PC12 cells. The Ba2+ current was acti vated by a 200 msec depolarizing step pulse from -60 mV to + 10 mV every 10 sec. A: Current traces recorded 60 sec before (-60), just before (0) and 10-70 sec after the application of lomerizine (10 PM). B: Current traces recorded 60 sec before (-60), just before (0) and 10-130 sec after the application of diltiazem (10 pM). Fig. 2. Effects of lomerizine, nicardipine, flunarizine, diltiazem, verapamil and dimetotiazine on Ba2+ current in PC12 cells. The Ba2+ current was activated by a 200-msec depolarizing step pulse from -60 mV to +10 mV every 10 sec. Panels for lomerizine (A), nicardipine (B), flunarizine (C) and dimetotiazine (F) show superimposed the current traces from just before (control) and 60 sec after the application of the doses shown. Those for diltiazem (D) and verapamil (E) show superimposed current traces from just before (control) and 120 sec after the application of the doses shown. superfusion of a given drug at various concentrations rent. In a preliminary experiment, the current-voltage (between 0.1 and 100 pM) are superimposed. Each of the (I-V) relationship exhibited a bell-shape, peaking at + 10 drugs concentration-dependently inhibited the Ba2+ cur mV, as has been previously reported (9, 10); and lomeri zine did not affect the I-V relationship. Therefore, we both L-type and T-type Ca 2+ currents in a hippocampal investigated the time-dependency and concentration CA1 pyramidal single neuron (4) and does not have any response relationship with a depolarizing step to +10 affinity for N-type Ca2+ channels in a [1211]w-conotoxin mV. binding study using rat cortical membranes (unpublished Figure 3 shows the concentration-response relationship observation, H. Hara et al.). for these drugs. The IC50 values (with 95010 confidence In this experiment, lomerizine concentration-depend limits) for lomerizine, nicardipine, flunarizine, diltiazem ently inhibited high-voltage activated inward Ba2+ cur and verapamil were 1.9 (1.1-3.9), 2.0 (0.86-6.2), 7.9 rents. The time course (inhibition pattern) of the effect of (3.1 52), 13.0 (6.7 27) and 10.4 (5.1 21) pM, respec lomerizine on Ba2+ current was similar to those of tively. Dimetotiazine produced only an approximately nicardipine and flunarizine, but different from those of 40% inhibition at a concentration of 10 pM (the highest diltiazem and verapamil. Maximal steady state inhibition concentration used). Lomerizine and nicardipine were induced by lomerizine, nicardipine or flunarizine was ob both > 4 times more potent than the other three calcium served faster than that induced by diltiazem or verapamil. channel blockers and dimetotiazine. These data indicate that the mode of action of lomerizine We examined the effect of lomerizine on Ca2+ channels in inhibiting L-type Ca 2+ channels may be similar to that in PC12 cells and compared the effect with those of four of nicardipine and flunarizine and different from that of other calcium channel blockers and with that of diltiazem and verapamil. dimetotiazine. PC12 cells have been used widely in studies Each of the 1,4-dihydropyridines studied here, diltia requiring a neuron-like clone cell-line. The Ba2+ current zem and verapamil, interacts with a specific receptor in PC12 cells was classified as an example of L-type domain found on a large (about 165 kD) membrane Ca 21 channel activity by Tsien et al. (11). Although un spanning protein that constitutes a substantial portion differentiated PC 12 cells express predominantly L-type of the L-type, voltage-gated Ca 2+ channel (14).