Role of RhoA in Regulating the Pump Function of Isolated Lymphatics From Hemorrhagic Shock Rats

Abstract

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.

Reduction of contractility and reactivity in isolated lymphatics from hemorrhagic shock rats with resuscitation

“…These results of lymphatic contractility and reactivity following hemorrhage and resuscitation were similar with that of lymphatics from hemorrhagic shock without resuscitation in previous report 6 . Because lymphatic pumping plays a vital role in the regulation of interstitial fluid volume 12 , therefore, the treatment of enhancing lymphatic pump function may be beneficial to hemorrhagic shock.…”

“…Previous studies demonstrated that lymphatic reactivity to vasoactive substances is associated with lymphatic contractile activity 5,6,10,11 . Therefore, in this study, furthermore, we tested the isolated lymphatic contractile response to gradient concentration of SP, and found that the difference value of indexes of CF, TI and FPF decreased at certain concentrations of SP, significantly.…”

“…After anesthetization with 1% pentobarbital sodium (50 mg/kg, Merck, Germany), all of the rats received femoral operations to separate the right femoral vein for anticoagulation and bilateral femoral arteries for hemorrhage, and monitoring of mean arterial pressure (MAP) as previously reported 6 . After 30 min-equilibrium period, the rats in the shock group, establishment of hemorrhagic shock model was performed with the method of hemorrhage through the left femoral artery, and the MAP was maintained at a level of 40 mmHg for 90 min by withdrawing or reinfusing shed blood.…”

“…At 30 min after surgical operation in the sham group or at 3 h after resuscitation finished in the shock group, the lymphatic vessel was obtained from thoracic duct under a SZ2-ILST stereomicroscope (Olympus, Tokyo, Japan) as previously described 6 .…”

“…After acute hemorrhage, in vivo, lymphatic pumping function was enhanced in the early stage, which plays an important role in blood volume and protein restitution 2,3 ; however, the contractility of lymphatic vessels was decreased in the late stage, which is the one of reasons leaded to severe shock and death 4 . In addition, there were biphasic changes in contractility and reactivity to norepinephrine (NE) and substance P (SP) of isolated lymphatics obtained from thoracic ducts of hemorrhagic shocked rats without resuscitation 5,6 . Hence, lymphatic circulation plays an important role during hemorrhagic shock.…”