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Home , Antimigraine drug, Dimetotiazine, Lomerizine

Inhibitory Effect of , a Diphenylpiperazine Ca2+ -, on Ba2+ Current through Voltage-Gated Ca2+ Channels in PC 12 Cells

Tomokazu Watanol, Hideaki Hara2,* and Takayuki Sukamoto2

1 Department of , New 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- 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 were > 4 times more potent than , , and . 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) dihydrochloride has been fate (Wako) at 37C in an atmosphere of 5% CO2. They reported to inhibit [3H] 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 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- 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). The 1,4 channels, recently they have been reported to possess an dihydropyridine receptor is located on the surface of the N-type and c)-agatoxin-IVA-sensitive (P/Q-type) compo channel; on the other hand, the diltiazem and verapamil nent, but not the T-type (12, 13). However, the ex binding sites are located internally, deep within the chan perimental condition (-60 mV holding potential) in the nel. Lomerizine has been supposed to bind to the 1,4-di present study is suitable for detecting L-type, but not N hydropyridine binding site from the results obtained by a or T-type (11), Ca 21 channel activity. Lomerizine inhibits [3H]nitrendipine binding study using synaptosome mem branes in guinea pig cortex (2). These different locations of the binding sites of the drugs may explain the different time course of the inhibition of Ba2+ current among the drugs tested in the present experiment. The potencies of lomerizine and other calcium channel blockers tested in inhibiting the Ba2+ currents through the high voltage-activated Ca 21 channels were in the follow ing order: lomerizine = nicardipine > flunarizine > verap amil > diltiazem > dimetotiazine. Although the order obtained with lomerizine and flunarizine are well-consis tent with that obtained by a [3H]nitrendipine binding study (2), the inhibitory effect of nicardipine in this ex periment was somewhat weaker than that expected from the binding study. Nicardipine was about 30 times more potent than lomerizine in inhibiting [3H]nitrendipine binding to synaptosome membranes in guinea pig cortex. Nicardipine has been reported to suppress the L-type Ca2+ channel current found in PC12 cells with an IC50 value of 2.7 pM (12). The IC50 value for nicardipine in Fig. 3. Concentration-response curves for inhibitory effects of PC12 cells is consistent with that reported here (2 pM). lomerizine (0), nicardipine (0), flunarizine (A), diltiazem (A), The inhibitory effect of 1,4-dihydropyridine derivatives verapamil ([:]) and dimetotiazine (/) on Ba2+ current in PC 12 cells. such as nicardipine on the Ba2+ currents through the The peak Ba2+ currents were obtained from Fig. 2. The data recorded in the presence of each concentration of drug was normal high-voltage activated Ca2+ channels has been reported to ized with respect to that recorded before the application. Data are be weaker in neuronal cells than in other cells such as presented as means ±S.E. (n=8). cardiac cells and smooth muscle cells (15, 16). The effect of lomerizine on the high-voltage activated Ca" channels diphenylpiperazines on [3H]nitrendipine binding. Jpn J has not yet been determined in cells other than neuronal Pharmacol 48, 241-247 (1988) 3 Iwamoto T, Morita T and Sukamoto T: Calcium antagonism by cells. Therefore, further studies are needed to address KB-2796, a new diphenylpiperazine analogue, in dog vascular whether the inhibitory effect of lomerizine on the Ca" smooth muscle. J Pharm Pharmacol 43, 535-539 (1991) channels found in neuronal cells is different from those 4 Akaike N, Ishibashi H, Hara H, Oyama Y and Ueha T: Effect found in peripheral cells. of KB-2796, a new diphenylpiperazine Ca2+ antagonist, on Lomerizine has been reported to preferentially inhibit voltage-dependent Ca2+ currents and oxidative in T-type Ca2+ channels rather than L-type Ca2+ channels in dissociated mammalian CNS neurons. Brain Res 619, 263-270 rat pyramidal cells (4). The profile of lomerizine resem (1993) bled that of flunarizine (4). Furthermore, the effect of 5 Gotoh F, Fukuuchi Y, Tashiro K, Katsuzawa N, Katayama S, Hirai S, Otomo E, Kito S, Terashi A, Tazaki Y, Sakai F, Ito E, lomerizine on the Ba2+ currents in the present study was Takahashi A, Sawada T, Takayanagi T, Takahashi K, Fujishima also similar to that of flunarizine. The similarity be M and Goto I: Clinical evaluation of KB-2796 on migraine tween lomerizine and flunarizine may be due to the com -double-blind study in comparison with dimetotiazine-. Clin mon diphenylpiperazine structure. Eval 23, 183-214 (1995) (Abstr in English) Lomerizine has shown to reduce the number of 6 Hara H, Morita T, Sukamoto T and Cutrer FM: Lomerizine migraine attacks in patients with migraine (5). Flunarizine (KB-2796), a new . CNS Drug Rev 1, 204-226 and nicardipine have been reported to show better thera (1995) 7 Inoue K and Kenimer JG: Muscarinic stimulation of calcium peutic effects than the placebo in double-blind and influx and norepinephrine release in PC12 cells. J Biol Chem placebo-controlled clinical trials in migraine patients (17, 263, 8157-8161 (1988) 18). The beneficial effects of lomerizine in migraine 8 Hamill OP, Marty A, Neher E, Sakmann B and Sigworth FJ: patients are supposed to mainly result from the anti Improved patch-clamp techniques for high-resolution current vasoconstrictor effect and inhibitory effect on spreading recording from cells and cell-free membrane patches. Pflugers by suppression of L-type Ca2+ channels in Arch 391, 85-100 (1981) smooth muscle and neuronal cells, respectively (6). Taken 9 Nakazawa K, Inoue K, Ohara-Imaizumi M, Fujimori K and together, the data in the present study and the above Takanaka A: Inhibition of Ca-channels by diazepam compared with that by nicardipine in pheochromocytoma PC12 cells. findings indicate that the mechanism of the anti-migraine Brain Res 553, 44-50 (1991) effect of lomerizine may be similar to those of flunarizine 10 Ito K, Nakazawa K, Koizumi S, Liu M, Takeuchi K, Hashimoto and nicardipine from the viewpoint of the inhibitory T, Ohno Y and Inoue K: Inhibition by drugs of effects on L-type Ca2+ channels. L-type Ca2+ channel current in PC12 cells. Eur J Pharmacol Dimetotiazine, a classical anti-migraine drug with 314, 143 150 (1996) H1 and 5-HT2-receptor blocking 11 Tsien RW, Lipscombe D, Madison DV, Bley KR and Fox AP: effects, showed a weak inhibitory effect on the Ba2+ cur Multiple types of neuronal calcium channels and their selective modulation. Trends Neurosci 11, 431-438 (1988) rents, like diltiazem and verapamil did, although the 12 Liu H, Felix R, Gurnett CA, Waard MD, Witcher DR and potency of dimetotiazine was approximately 10 times less Campbell KP: Expression and subunit interaction of voltage than that of lomerizine. Therefore, we can not rule out dependent Ca2+ channels in PC12 cells. J Neurosci 16, the possibility that the mechanism of action of dimetotia 7557-7565 (1996) zine on migraine is partly due to the suppression of L-type 13 Usowicz MM, Porzig H, Becker C and Reuter H: Differential Ca2+ channels in smooth muscle and/or neuronal cell. expression by nerve growth factor of two types of Ca2+ chan Further studies are needed to clarify the mechanism. nels in rat phaeochromocytoma cell lines. J Physiol (Lond) 426, 95 -116 (1990)

Acknowledgments 14 Triggle DJ: Calcium-channel drugs: structure-function relation The authors would like to thank Dr. Ken-ichi Nakazawa, Division ships and selectivity of action. J Cardiovasc Pharmacol 18, of Pharmacology, National Institute of Health Sciences, for helpful S1-S6 (1991) discussions and Dr. Tomoyuki Ono, Department of Pharmacology, 15 Hess P, Lansman JB and Tsien RW: Different modes of Ca Fukushima Medical College, for helpful advice on the culture channel gating behaviour favoured by dihydropyridine Ca and serum. Furthermore, we are grateful to Dr. Koji Yamamoto, agonists and antagonists. Nature 311, 538-544 (1984) New Drug Discovery Research Lab., Kanebo Ltd., for his advice on 16 Friedman ME, Suarez Kurtz G, Kaczorowski GJ, Katz GM and the culture method. Reuben JP: Two calcium currents in a smooth muscle cell line. Am J Physiol 250, H699-H703 (1986)

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