The vasodilatation elicited by Rho kinase inhibition in diabetic rat aorta is potentiated in the presence of endothelium

SS Yildirim, FA Cicek, HB Kandilci, B Turan. Ankara University Faculty of Medicine, Department of Biophysics, 06100, Turkey

Diabetes-induced endothelial dysfunction is characterized by a decrease in production and bioavailability of nitric oxide (NO) in blood vessel wall 1 . This phenomenon may in part be due to alterations in Rho-kinase (ROCK) signaling 2 . It has been shown that the activity of endothelial nitric oxide synthase (eNOS) is regulated by ROCK pathway while NO inhibits ROCK activity contemporaneously 3,4 . The aim of the present study is to investigate the role of endothelium in Rho-kinase signalling in diabetic rat aorta. Thoracic aorta excised from streptozotocin-treated (50 mg/kg) rats were cut into rings and then immersed in Krebs-Heinseleit solution for isometric tension recordings. Endothelium was removed mechanically in some of the preparations. For immunoblotting, endothelium denuded or intact aortas were cleaned and stored at -80˚C until protein extraction. Data are expressed as mean ± S.E.M. EC50 values were derived using Graph-Pad Prism. Mean differences among experimental groups were evaluated by unpaired t-test. P < 0.05 was accepted as significant. ROCK2 isoform protein level was increased (2.455 ± 0.12 fold increase vs. control, n=5, P<0.05) in endothelium intact diabetic samples. Endothelium removal abolished this increase (0.79 ± 0.08 fold decrease vs. control, n=4, P>0.05). Total eNOS was upregulated (2.0 ± 0.49 fold increase vs. control, n=5, P<0.05) where its ROCK dependent phosphorylated inactive form (Ser1177) was downregulated (0.47± 0.25 fold decrease vs. control, n=3, P<0.05) in diabetic rats. Y27632 (1 µM, 30 min), a selective inhibitor of ROCK, attenuated phenylephrine (10-7 M) induced contractions (0.40.5 ± 0.16 fold decrease vs control, n=5, P<0.05) in endothelium intact but not in endothelium denuded diabetic rat aortas. Moreover, endothelium removal shifted Y27632 (10-8-10-5 M) induced vasodilatory concentration response curve to the left (e(-): pEC50=4.26 ± 0.31, e(+): pEC50=5.75 ± 0.27, n=3, P<0.05) in diabetic aortas. In conclusion, these results suggest that an endogenous factor released from endothelium, possibly NO, may tonically inhibit ROCK activity. Furthermore, ROCK signalling pathway induced by diabetes can inactivate eNOS and decrease NO production. Therefore, in diabetes, there might be a vicious cycle between impaired NO production and decreased ROCK inhibition.

1 Serizawa et al. Cardiovascular Diabetology 2011, 10:105

2 Snah et al. Molecular and Cellular Biochemistry 2006, 283:191

3 Sugimoto et al. Biochemical and Biophysical Research Communications 2007, 361:462

4 Chitaley et al. Hypertension 2002, 39:438