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The pharmacological properties of YM-254890, a specific G(alpha)q/11 inhibitor, on acute thrombosis and chronic neointima formation after vascular injury have been investigated. FeCl3 was used to induce vascular injury in the carotid artery of mice. For the thrombosis studies, the test drug was either intravenously or orally administered before vascular injury. For the neointima studies, the test drug was orally administered 1 h before and twice daily for 1 week after vascular injury. Histological analysis was then performed 3 weeks later. YM-254890 significantly inhibited ex vivo platelet aggregation 5 min after intravenous bolus injection at 0.03 mg/kg or more, and 1 h after oral administration at 1 mg/kg. YM-254890 significantly inhibited thrombus formation after intravenous bolus injection at 0.03 mg/kg as well as after oral administration at 1 mg/kg, but tail transection bleeding time was significantly prolonged at 0.1 mg/kg for intravenous injection and 3 mg/kg for oral administration. Furthermore, oral administration of YM-254890 dose-dependently inhibited neointima formation 3 weeks after vascular injury with significant effects at 1 mg/kg twice daily for 1 week. Clopidogrel also significantly inhibited neointima formation at its antithrombotic dose, but its inhibitory potency was less than that of YM-254890. However, YM-254890 significantly reduced systemic blood pressure at doses 3 times higher than those that produced significant inhibitory effects on thrombosis and neointima formation. Though the systemic use of YM-254890 may be limited, owing to its narrow therapeutic window, this unique compound is a useful research tool for investigating the physiological roles of G(alpha)q/11 .

In cardiac excitation-contraction coupling, Ca2+-induced Ca2+ release (CICR) from ryanodine receptors (RyRs), triggered by Ca2+ entry through the nearby L-type Ca2+ channel, induces Ca2+-dependent inactivation (CDI) of the Ca2+ channel. Aiming at elucidating the physiological role of CDI produced by CICR (CICR-dependent CDI), we investigated the contribution of the CICR-dependent CDI to action potential (AP) waveform and the amount of Ca2+-influx through Ca2+ channels during AP in rat ventricular myocytes. The elimination of the CICR-dependent CDI, by depletion of the SR Ca2+ with thapsigargin, significantly prolonged AP duration (APD). APD changed in parallel with the magnitude of CICR during the recovery of the SR Ca2+ content after transient depletion by caffeine. Such CICR-dependent change of APD persisted under the highly Ca2+ buffered condition where the Ca2+ signalling was restricted to nanoscale domains. Blockers of the Ca2+-dependent Cl- channel or the BK channel did not affect AP waveform. The amount of Ca2+-influx through Ca2+ channels during the SR-depleted type AP waveform, measured in the SR-depleted myocyte, was increased by 40 % over that during the SR-intact type AP waveform measured in the SR-intact myocyte. The protein kinase A stimulation further enhanced the Ca2+-influx during AP under the SR-depleted condition to 70 % of that under the SR-intact condition. These results indicate that the CICR-dependent CDI of L-type Ca2+ channels, under control of the privileged cross-signalling between L-type Ca2+ channels and RyRs, play important roles for monitoring and tuning the SR Ca2+ content via changes of AP waveform and the amount of Ca2+-influx during AP in ventricular myocytes.

In cardiac excitation_contraction coupling (E-C coupling), gating kinetics of L-type Ca 2+ channels and ryanodine receptors (RyRs) are mutually regulated via advantageous cross-signalling between the two channels in the confined space of the dyad junction that is largely inaccessible to exogenous Ca 2+ buffers (Sham et al. 1995;Adachi-Akahane et al. 1996).It is well known that the inactivation of L-type Ca 2+ channels depends on voltage and cytosolic Ca 2+ ([Ca 2+ ] i ) (Bers, 2001). Recent studies proposed that, in response to Ca 2+ influx through the L-type Ca 2+ channel, the Ca 2+bound calmodulin (CaM) interacts with the IQ motif located in the carboxyl tail of the pore-forming a 1C subunit of L-type Ca 2+ channels to cause a conformational change of the Ca 2+ channel leading to Ca 2+ -dependent inactivation (CDI) (Pitt et al. 2001;Erickson et al. 2001). In contrast to recent advances in the clarification of the molecular mechanism underlying CDI of the Ca 2+ channel, however, its physiological role in cardiac E-C coupling, with respect to its contribution to action potential (AP) waveform still remains to be elucidated. In rat ventricular myocytes, the fast Ca 2+ -dependent inactivation of the L-type Ca 2+ channel produced by the Ca 2+ -induced Ca 2+ release (CICR-dependent CDI) occurs during AP. In adult ventricular myocytes especially, the CICR trigged by the nanoscale cross communication between the Ca 2+ channel and RyR, further accelerates the inactivation rate of Ca 2+ channels so its time constant becomes less than 10 ms (Hadley & Hume, 1987). The role of CDI on AP waveform or on the amount of Ca 2+ influx during the fixed AP waveform has been discussed based on experimental and simulation studies (Linz & Meyer, 1998;Winslow et al. 1999; Fanconnier et al. 2003). However, the contribution of the CICR-dependent CDI of the L-type Ca 2+ channel to AP waveform and the consequent change of the total amount of Ca 2+ influx during AP has never been directly addressed. In this study, we aimed at elucidating the physiological role of the CICR-dependent CDI of Ca 2+ channels in the regulation of AP waveform and also the total amount of Ca 2+ influx during AP in ventricular

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