Role of RhoA in regulating the pump function of isolated lymphatics from hemorrhagic shock rats.

The aim of this present study was to examine changes in RhoA protein levels and the role in RhoA in lymphatic contractility and reactivity after hemorrhagic shock. Levels of RhoA and phospho-RhoA in lymphatic tissue isolated from hemorrhagic shock rats were measured, and the contractility and reactivity to substance P of lymphatics isolated from control rats and rats subjected to shock 0.5 and 2 h were determined with an isolated lymphatic perfusion system at a transmural pressure of 3 cmH2O. At the same time, lymphatics isolated from rats subjected to shock 0.5 and 2 h were incubated with agonists and antagonists of RhoA/Rho kinase signaling. Contractile frequency, end-diastolic and end-systolic diameter, and passive diameter were recorded and used to calculate lymphatic tonic index, contractile amplitude, and fractional pump flow. After stimulation with a gradient of substance P, the differences between the preadministration and postadministration values of contractile frequency, contractile amplitude, tonic index, and fractional pump flow were calculated to further assess lymphatic reactivity. RhoA protein levels were significantly increased at 0.5 h after shock but decreased at 2 and 3 h after shock; p-Rho levels were initially increased after shock and subsequently decreased. The contractility and reactivity of 0.5-h-shocked lymphatics were significantly reduced by the RhoA antagonist C3 transferase and the Rho kinase antagonist Y-27632. The RhoA agonist U-46619 increased the contractility and reactivity of 2-h-shocked lymphatics, whereas Y-27632 suppressed the effect of U-46619. Okadaic acid, an inhibitor of myosin light-chain phosphatase, had no effect on the contractility of 2-h-shocked lymphatics, but improved lymphatic reactivity. These results suggest that RhoA is involved in the modulation of lymphatic pump function during hemorrhagic shock and that its effects may be mediated by Rho kinase and MLCP.