and purpose: Myosin light chain kinase (MLCK) plays a pivotal role in regulation of cellular functions the evidence often relying on the effects of extracelluarly administered drugs such as ML-9. at which the effects of drugs reached the maximum. We therefore empirically chose the time point of 5?s after the onset of drug application. Although this would introduce some errors with the same method we could get similar results with 1-oleoyl-2-acetyl-relationship constructed by a 2?s rising ramp voltage was different at ?100 or 100?mV ((3.8?μM; Saitoh et al. 1987 More importantly the cooperativity factor (nH) for α1-adrenoceptor NSCC (2.5) is more than twice that of overexpressed TRPC6 channel which is equal to unity (1.0) (Aromolaran et al. 2000 present study). These stoichiometric data suggest that ML-9 may exert at least two distinct effects that is inhibition of MLCK activity and more direct inhibition of NSCC. Both effects seem to be present in the α1-adrenoceptor NSCC whereas only the latter may be operative in TRPC6 channels expressed in HEK293 cells where as compared with calmodulin-dependent kinase II Brefeldin A the physiological impact of MLCK activity on NSCC activation would be much weaker than in muscle tissues. Indeed this may have partly been manifested as reduced availability of TRPC6 channel with overexpresion of mutant MLCK (Figure 4d) but we did not further pursue this issue in the present study. On the other hand ML-7 another naphthalene sulphonamide derivative is more than 30-fold more potent in inhibiting MLCK (IC50=300?nM; Saitoh et al. 1987 than the TRPC6 channel (IC50 appears slightly larger than 10?μM; see Figure 5c). This Brefeldin A means that even though this compound’s MLCK-independent effect on TRPC6 channel would be operative in the micromolar range this would already be masked by its submicromolar inhibitory effect on MLCK thus the former effect would not be clearly observable. This actually seems to occur in rabbit portal vein α1-adrenoceptor NSCC which ML-7 (IC50=0.8?μM) inhibits more effectively than ML-9 but the value of nH is nearly equal to unity Brefeldin A (Aromolaran et al. 2000 In this regard to separate the effects on MLCK and TRPC6 channel ML-7 may be more advantageous than ML-9 when it is used at low micromolar concentrations. The non-specific inhibitory effects of ML-9 on Ca2+ transporting activities have been documented in the literature. For instance in porcine aortic endothelial cells one study reported that bradykinin or Brefeldin A thapsigargin-induced Ca2+ entry was suppressed by both ML-9 and wortmannin with a parallel inhibition of MLC phosphorylation (Watanabe et al. 1998 However in the same preparation another study provided a conflicting evidence that only ML-9 but not wortmannin-inhibited thapsigargin-induced Ca2+ entry (Kuroiwa-Matsumoto Brefeldin A et al. 2000 In guinea pig tracheal smooth muscle while ML-9 was found to be able to reduce Ca2+ entry and concomitant contraction induced by high K+ methacholine or thapsigargin wortmannin only inhibited the contraction Brefeldin A without affecting [Ca2+]i (Ito et al. 2004 All these findings appear to be compatible with the idea that ML-9 may act as a direct inhibitor of several different types of Ca2+ entry channels rather than via its effects on MLCK activity. TRPC6 is known to be a major TRPC isoform expressed in S1PR3 vascular smooth muscle cells (Inoue et al. 2006 but its relatively abundant expression has also been detected in airway smooth muscle cells and endothelial cells (Ong et al. 2003 Yip et al. 2004 It is thus possible that part of the inhibitory actions of ML-9 observed in the above studies may also reflect the nonspecific effects of ML-9 on Ca2+ entry channels per se as observed in the present study. In summary the present results clearly show that ML-9 effectively..