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Calcium channels are common therapeutic targets for the treatment of cardiovascular disorders such as hypertension (Ishikawa et al., 1997), however they have not always been as successful as might be expected against vasospasm and stroke. Recently a novel high voltage activated, nifedipine-insensitive, mibefradil-sensitive calcium channel was described in small mesenteric arteries of guinea pigs and rats (Morita et al., 1999, 2002), and called the “M-type” voltage dependent calcium channel (mVDCC). This channel has also been found in rabbit mesenteric arteries, where it is suggested to play a role in diameter regulation (Itonaga et al., 2002). The aim of the present study was to determine if cerebral arteries possess similar nifedipine-insensitive VDCCs which could be used as targets for cerebrovascular disorders.
Juvenile (14-17 day old) male Wistar rats were anaesthetized with ether and decapitated. The basilar artery was removed from the brain and superfused with physiological Krebs solution at 33-37°C. Diameter was monitored as a measure of vascular tone using an edge-tracking computer program. Membrane potential was measured with sharp intracellular microelectrodes (100-180 MΩ), and current pulses (1-2 min) were applied to short segments of artery (less than 800µm) using discontinuous current clamp mode (Axoclamp 2B). Change in intracellular calcium concentration ([Ca]i) was measured with the ratiometric calcium indicator Fura-2 AM and a photometry system. After 30 minutes the arteries developed spontaneous rhythmical oscillations in diameter (vasomotion) and membrane potential with the most negative potential around -45mV. Application of the L-type VDCC blocker, nifedipine, abolished vasomotion but did not alter tone, membrane potential or [Ca]i in basilar arteries, while inhibition of the IP3 pathway with U73122 also abolished vasomotion but caused hyperpolarization, relaxation and a decrease in [Ca]i. Small hyperpolarizing current steps which took the membrane potential to -50mV caused immediate abolition of vasomotion and relaxation. Relaxation occurred in the presence or absence of nifedipine. Application of the T- and M-type VDCC blocker, mibefradil, hyperpolarized and relaxed the artery, decreasing [Ca]i, while the T-type VDCC blocker, nickel chloride, only relaxed the artery at a high non-specific concentration (1mM). After the artery was hyperpolarized and relaxed with U73122, application of 40mM KCl caused depolarization and constriction in the presence of nifedipine. A similar result was obtained when 2-APB, an IP3 inhibitor was present together with nifedipine, suggesting that the effect of voltage was not on calcium release from intracellular stores. Taken together the results suggest that nifedipine-insensitive, mibefradil-sensitive VDCCs play a role in vascular tone in the rat basilar artery. These channels are activated at depolarized potentials and rapidly closed by small hyperpolarizations. They are thus unlikely to be T-type VDCCs, which are activated at more negative potentials and rapidly inactivated during prolonged depolarization. We suggest that these nifedipine-insensitive high voltage-activated calcium channels may provide a novel therapeutic target for cerebrovascular disorders.
Ishikawa, K., Nakai, S., Takenaka, T., Kanamasa, K., Hama, J., Ogawa, I., Yamamoto, T., Oyaizu, M., Kimura, A., Yamamoto, K., Yabushita, H. & Katori, R. (1997) Circulation, 95, 2368-2373.
Itonaga, Y., Nakajima, T., Morita, H., Hanano, T., Miyauchi, Y., Ito, Y. & Inoue, R. (2002) Life Sciences, 72, 487-500.
Morita, H., Cousins, H., Onoue, H., Ito, Y. & Inoue, R. (1999) Circulation Research, 85, 596-605.
Morita, H., Shi, J., Ito, Y. & Inoue, R. (2002) British Journal of Pharmacology, 137, 467-476.