Different potencies of dihydropyridine derivatives in blocking T-type but not L-type Ca2+ channels in neuroblastoma-glioma hybrid cells
Introduction
T-type Ca2+ channels are present in a variety of excitable cells including skeletal (Cognard et al., 1986) and smooth muscle fibers (Loirand et al., 1986), cardiomyocytes (Mitra and Morad, 1986), peripheral (Carbone and Lux, 1984) and central neurons (Akaike, 1991). In neurons of certain brain regions, e.g., in the thalamus, T-type Ca2+ channels are responsible for Ca2+ spikes (Coulter et al., 1989) and are involved in the transition to phasic bursting behavior (Llinás and Jahnsen, 1982). In combination with Ca2+-activated K+ currents, membrane potential oscillations are generated which dominate thalamocortical interactions in sleep and under pathophysiological conditions. Thus T-type Ca2+ channels are thought to be involved in generalized absence seizures (Steriade and Llinás, 1988). Moreover, T-type Ca2+ conductance may also contribute to abnormal cortical synchronous rhythms (Connors and Amitai, 1993). Electrophysiological features of T-type Ca2+ currents are variable. In addition, sensitivities to inorganic and organic Ca2+ channel blockers differ considerably (Akaike, 1991). Furthermore, the classical L-type Ca2+ channel blockers with the dihydropyridine structure also inhibit T-type Ca2+ current but with varying potencies. In a study on freshly isolated hypothalamic neurons, T-type Ca2+ current was reduced in the order of potency nicardipine>nifedipine>nimodipine with a Kd between 3.5 and 7 μM (Akaike et al., 1989). In hippocampal neurons, nicardipine blocked T-type Ca2+ current with an even higher potency than L-type Ca2+ current (Takahashi and Akaike, 1990). Since in most of the concentration–response studies on reduction of T-type Ca2+ current by dihydropyridines enzymatically dispersed cells were used, it was intended to compare T- and L-type Ca2+ channel block in cultured cells of the neuroblastoma-glioma hybrid cell line NG108-15. Differently substituted derivatives including nimodipine and nicardipine were studied. The results are discussed on the background of known therapeutical properties of dihydropyridine derivatives, in particular in neuropsychiatric disorders or corresponding animal models. Some of the results have been communicated in preliminary form (Stengel et al., 1996).
Section snippets
Cell culture
NG108-15 mouse neuroblastoma×rat glioma hybrid cells (kindly provided by Professor Wellhöner, Hannover) were grown in a culture medium composed of 90% Dulbecco's modified Eagle's medium (DMEM), 10% fetal calf serum (FCS), hypoxanthine–aminopterin–thymidine supplement (HAT), 100 IU/ml penicillin and 100 μg/ml streptomycin. After transferring 3.5×104 cells/ml (passage number 20–40) to multiwell-plates (Nunc, Roskilde) containing 10 mm diameter-glass coverslips which had been coated with poly-l
Separation of voltage-activated Ca2+ channel currents in differentiated NG108-15 neuroblastoma-glioma hybrid cells
Low voltage- and high voltage-activated Ca2+ channel currents were distinguished due to their different potential range of activation, potential-dependent inactivation and inactivation kinetics. High voltage-activated current components were pharmacologically separated, since potential range of activation, potential-dependent inactivation and kinetic properties were similar. High voltage-activated currents were characterized by an activation threshold at −20 mV, maximum activation at 0 mV and a
Discussion
The present results indicate that several dihydropyridine derivatives exhibit widely varying potencies for T-type but similar potencies for L-type Ca2+ channel block. In contrast, T-type Ca2+ channel block by nicardipine, nimodipine and nifedipine was less variable in a study on freshly isolated hypothalamic neurons (Akaike et al., 1989). It is suggested that structural differences of T-type Ca2+ channels account for a lower affinity and potency of nimodipine and nifedipine in the present
Acknowledgements
The authors wish to thank R. Weitschat for excellent technical assistance, Dr. Bräter and Dr. Matthes for providing cultured cells. Instrumentation grants by Dr. Wulf Vater, Leverkusen, and drug gifts by Dr. Seuter, Tropon-Werke Köln, are gratefully acknowledged. We thank Professor Dr. U. Ravens for helpful comments on the manuscript.
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