Ultrasound at 27 kHz increases tissue expression and activity of nitric oxide synthases in acute limb ischemia in rabbits

Abstract

Transcutaneous low-frequency ultrasound (US) preserves myocardial and skeletal muscle viability by increasing tissue perfusion through an undefined nitric oxide (NO)-dependent mechanism. We have examined whether US increases tissue expression and activity of the three nitric oxide synthase (NOS) isoforms: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS). The two femoral arteries of four New Zealand rabbits were ligated for a total of 120 min. After 60 min of ligation, transcutaneous low-frequency US (27 kHz, 0.13 W/cm2) was applied for 60 min to one thigh, while the contra-lateral artery served as a control (total ischemia time=120 min). Calcium-dependent (cNOS) and -independent (ciNOS) NOS activity, and concentration of total eNOS, ser-1177 phosphorylated eNOS (P-eNOS), nNOS and iNOS were then determined in the gracilis muscle. Compared with the control, US application significantly increased cNOS activity [3.34+/-0.28 versus 3.87+/-0.10x1000 counts per minute (cpm), respectively, p=0.031] and ciNOS activity (1.99+/-0.09 versus 3.26+/-0.68 cpm, respectively, p<0.001). Western immunoblotting revealed a significant increase in protein content of both iNOS (184.5+/-1.08%; p<0.0001) and P-eNOS (381.5+/-2.47%; p<0.001), with only a small increase in total eNOS and nNOS expression. In conclusion, application of transcutaneous low-frequency US to ischemic muscular tissue significantly increases both cNOS and ciNOS activity by increasing eNOS phosphorylation and iNOS expression, respectively.