Rho kinase (ROCK) and nitric oxide (NO) are important targets in cardiovascular diseases. Therefore, we investigated the possible influence of NO on Rho kinase (ROCK-2 isoform) expressions in cultured rat coronary microvascular endothelial cells. The cells were isolated from Wistar rats on a Langendorff system, and were incubated overnight (similar to 16 h) with an NO generator, A-23187 (10(-7) to 10(-6) M) NO donors, such as sodium nitroprusside (10(-7) to 10-6 M) glyceryl trinitrate (10(-7) to 10-6 M), 2,2'-(hydroxynitro- sohydrazono)bis-ethanimine (10(-7) to 10(-6) M), and NaNO2 (10(-4) to 10(-3) M) or a nitric oxide synthase (NOS) inhibitor, N-G-nitro-L-arginme methylester (2x10(-4) M), or two ROCK inhibitors, (-+)(R)-trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10(-5) M) and fasudil (10(-5) M) in the absence or presence of thrombin (4 U/mL). ROCK-2 and endothelial NOS (eNOS) expressions were detected by Western blotting. Moreover, nitrite/nitrate levels were detected by Griess method in the presence of the ROCK inhibitors. The NO donors and the NO generator had no significant effects on ROCK-2 expression. Y-27632 and fasudil did not alter eNOS expression and NO production. Nitrite/nitrate levels were 4.4 +/- 0.32 mu M in control and 4.0 +/- 0.93 mu M and in Y-27632 group. These results demonstrate that prolong NO donation could not suppress the expression of ROCK-2 protein, and the ROCK inhibitor did not change e-NOS expression and NO production in the cultured rat coronary microvascular endothelial cells.