In this respect, our present results have highlighted a new important target for the vasomotor control via hitherto-unidentified P2Y receptor subtypes in vascular smooth muscle. molecular level. Detailed electrophysiological analysis of mVDCC has revealed that, despite its rapidly inactivating nature, there is a range of membrane potential in which constant or non-inactivating Ca2+ influx occurs. The physiological significance of non-inactivating Ca2+ influx has been emphasized for L-type VDCC, as the crucial determinant of free Ca2+ concentration ([Ca2+]i) in arterial easy muscle cells and thus of the arterial diameter or tone under pressurized conditions (for review see Nelson 1990). Furthermore, this Ca2+ influx has been thought to be effectively regulated by the modulatory actions of various vasoactive substances such as neurotransmitters (e.g. noradrenaline, neuropeptide Y, acetylcholine, vasointestinal peptide and calcitonin gene-related peptide), vasoactive autacoids which are released from the vascular endothelium or produced during local inflammatory processes (e.g. nitric oxide, endothelium-derived hyperpolarizing factor, endothelin, histamine and bradykinin) and circulating hormones released from distant endocrine organs (e.g. angiotensin II and vasopressin) (Beech, 1998; Kuriyama 1998). In the present study, we have therefore resolved the question Shikimic acid (Shikimate) of whether receptor-mediated regulation has a comparable physiological significance in modifying the mVDCC activity. To GREM1 this end, we screened the effects on mVDCC of vasoactive substances known to affect the electrical and contractile properties of vascular easy muscle. We have found that ATP, a well established fast neurotransmitter of the vascular sympathetic nerves (Burnstock, 1990), exerts the most pronounced dose-dependent modulatory effects on mVDCCs through three distinct mechanisms. The preliminary account of this work has been presented in the 73rd annual getting together with of the Japanese Pharmacological Society (Morita 2000). METHODS Cell dispersion and electrophysiological measurements Procedures used Shikimic acid (Shikimate) for cell dispersion and the system for patch clamp experiments were the same as described previously (Morita 1999) and performed according to the guidelines approved by a local animal ethics committee of Kyushu University. In brief, guinea-pigs of either sex weighing 200C500 g were killed by decapitation after stunning under light anaesthesia with inhalation of diethyl ether. Short segments from the distal half of terminal branches of mesenteric artery measuring 70C100 m in diameter were mechanically dissected with fine scissors and forceps, and incubated successively in nominally Ca2+-free Krebs solutions without and with 2 mg ml?1 collagenase (Sigma type I) at 35C for 30 and 60 min, respectively. Single cells, yielded by gently triturating these digested segments using a blunt tipped pipette 20 to 30 occasions, were stored in 0.5 mm Ca2+-made up of Krebs solution at 10C until use. A commercial amplifier (Axopatch 1D, Axon Devices) in conjunction with an A/D, D/A converter was used to generate voltages and sample current signals after low-pass filtering at 1 kHz (digitized at 2 kHz), under the control of an IBM computer (Aptiva) which was driven by a commercial software Clampex v.6.02 (Axon Devices). The P/4 or P/2 method was used to subtract leak currents, and 50 to 70 %70 % of series resistance (10C15 M) was electronically compensated. Data analyses and illustration were performed using Clampfit v.6.02 (Axon Devices). All experiments were performed at room heat (22C25C). Solutions Solutions of the following composition were used (mm): 5 Ba2+-external answer: Na+ 140, K+ 6, Ba2+ 5, Mg2+ 1.2, Cl? 158.4, glucose 10, Hepes 10 (pH 7.4; adjusted by Tris base); divalent cation-free external answer: Na+ 140, K+ 6, Cl? 146, EDTA 0.2, glucose 10, Hepes 10 (pH 7.4; adjusted by Tris base). All external solutions were supplemented Shikimic acid (Shikimate) with nifedipine 10 m and were superfused at a rate of 1C2 ml min?1 into the recording chamber (volume 0.2 ml), via a gravity-fed perfusion system (time of complete solution change 30 s); Cs+-internal answer: Cs+ 140, Mg2+ 2, Cl? 144, phosphocreatine 5, Na2ATP 1, GTP 0.2, EGTA 10, Hepes 10 (pH 7.2; adjusted by Tris base). Free ATP and Ba2+ concentrations (Fig. 3) were calculated using Fabiato.