Objective: We have demonstrated that, besides his role in the calcium-sensitization of the contractile process in smooth muscle, Rho-dependent kinase (ROK) is involved in noradrenaline-activated calcium entry in rat arteries (Ghisdal et al. 2003). The objective of the present study was to investigate the involvement of the ROK pathway in the calcium signal in cultured vascular smooth muscle cells, by comparing the effect of the ROK inhibitor, Y-27632, in whole artery and in cultured vascular smooth muscle cells. Methods: Endothelium-denuded rat aorta segments, primary cultured rat aortic smooth muscle cells, and the aortic smooth muscle cell line A7r5 were used. Cytosolic calcium concentration was measured in fura-2-loaded artery or cells. Vasopressin (10 nM) was first applied in Ca²⁺ - free solution to release intracellular Ca²⁺ and Ca²⁺ entry was activated by the addition of Ca²⁺ in the bath solution. Involvement of the actin-cytoskeleton was tested by pre-incubation with cytochalasin-D (20 mM). ROK expression was measured by western blot and its activity was determined by measuring its downstream target, phospho-ERM. Results: In isolated rat aorta, the ROK inhibitor Y-27632 (10 mM) completely inhibited vasopressin-evoked contraction and depressed calcium entry signal by 57 ± 14 % (n=4). The inhibition of the calcium signal by Y-27632 was unchanged in cytochalasin-D-treated artery, which did not develop any contractile response. In cultured aortic smooth muscle cells as in A7r5 cells, Y-27632 did not affect the calcium entry signal evoked by vasopressin, although ROK expression and its activation by vasopressin were confirmed in aorta, cultured smooth muscle cells and A7r5. Conclusion: These results suggest that the mechanism of agonist-evoked Ca²⁺ entry is different in the whole artery compared to cultured cells, which probably lack an essential effector in the ROK pathway. Reference: Ghisdal, P., Vandenberg, G. & Morel, N. 2003. J Physiol 551, 855-867.