Changes of Rho kinase activity after hemorrhagic shock and its role in shock-induced biphasic response of vascular reactivity and calcium sensitivity.
ABSTRACT The purpose of the present study is to investigate the changes of Rho kinase activity and its role in biphasic response of vascular reactivity and calcium sensitivity after hemorrhagic shock. The vascular reactivity and calcium sensitivity of superior mesenteric artery (SMA) from hemorrhagic shock rats were determined via observing the contraction initiated by norepinephrine (NE) and Ca under depolarizing conditions (120 mmol/L K) with isolated organ perfusion system. At same time, Rho kinase activity in mesenteric artery was measured, and the effects of Rho kinase activity-regulating agents, angiotensin II (Ang-II), insulin, and Y-27632, on vascular reactivity and calcium sensitivity were also observed. The results indicated that the vascular reactivity and calcium sensitivity were increased at early shock (immediate and 30 min after shock) and decreased at late shock (1 and 2 h after shock). The maximal contractions of NE and Ca were significantly increased (P < 0.05 or P < 0.01) at early shock. But they were significantly decreased at late shock (P < 0.05 or P < 0.01). Rho kinase activity was significantly increased at early shock (immediate after shock) (P < 0.05) but significantly decreased at 1 and 2 h after shock (P < 0.05 or P < 0.01). It was positively correlated with the changes of vascular reactivity and calcium sensitivity. Insulin decreased the increased contractile response of SMA to NE and Caat early shock (P < 0.05 or P < 0.01). Angiotensin II increased the decreased contractile response of SMA to NE and Ca at 2-h shock (P < 0.05 or P < 0.01); Y-27632, Rho kinase-specific antagonist, decreased the contractile response of SMA to NE and Ca at 2-h shock, and abolished Ang-II induced the increase of vascular reactivity and calcium sensitivity. The results suggest that Rho kinase may be involved in the biphasic change of vascular reactivity and calcium sensitivity after hemorrhagic shock. Rho kinase may regulate vascular reactivity through the regulation of calcium sensitivity. Rho kinase-regulating agents may have some beneficial effects on shock-induced vascular hyporeactivity.
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ABSTRACT: It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V(max). Activity of MP with different substrates also was inhibited by phosphorylation. Two major sites of phosphorylation on MYPT1 were Thr(695) and Thr(850). Various point mutations were designed for these phosphorylation sites. Following thiophosphorylation by Rho-kinase and assays of phosphatase activity it was determined that Thr(695) was responsible for inhibition. A site- and phosphorylation-specific antibody was developed for the sequence flanking Thr(695) and this recognized only phosphorylated Thr(695) in both native and recombinant MYPT1. Using this antibody it was shown that stimulation of serum-starved Swiss 3T3 cells by lysophosphatidic acid, thought to activate RhoA pathways, induced an increase in Thr(695) phosphorylation on MYPT1 and this effect was blocked by a Rho-kinase inhibitor, Y-27632. In summary, these results offer strong support for a physiological role of Rho-kinase in regulation of MP activity.Journal of Biological Chemistry 01/2000; 274(52):37385-90. · 4.65 Impact Factor
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ABSTRACT: Hemorrhage-induced vascular hyporeactivity to norepinephrine (NE) and the possible effector roles of nitric oxide (NO) and endothelin (ET) were investigated in different vascular beds of rats. Under urethane anesthesia, rats (n = 7 per group) were hemorrhaged to a mean arterial pressure (MAP) of 50 mm Hg for 60 min. A group of rats was pretreated with either NG-nitro-L-arginine methyl ester (10 mg/kg), an NO synthase inhibitor, or PD142893 (0.1 mg/kg), an ET receptor antagonist 15 min before the end of the hypotensive period. Operated, euvolemic rats served as controls. The responses of MAP and the blood flow of the superior mesenteric (SMA), celiac (CA), left renal (LRA), and left femoral arteries (LFA) to NE (3 microg/kg, i.v.) were measured at baseline (prehemorrhage), at the end of the hypotensive period (0 h), and at 1, 2, and 4 h after the end of the hypotensive period. The pressor responses to NE on MAP at 0, 1, 2, and 4 h in the 60-min hemorrhage groups were reduced to 45.9%, 37.8%, 29.2%, 18.4% of baseline pressor response, respectively. At these same times, the fall in blood flow in response to NE in SMA, CA, LRA, and LFA was significantly blunted (P < 0.01). This loss of responsiveness in CA and LFA was more severe than in SMA and LRA (P < 0.05-P < 0.01). Pretreatment with L-NAME or PD142893 significantly improved the pressor response of MAP and the blood flow responses of the four arteries to NE (P < 0.01). Hypotension at 50 mm Hg for 60 min resulted in an apparent loss of vascular reactivity to NE, and the four vasculatures studies were not affected to the same extent. In addition, NO and ET appear to contribute to the loss of vascular reactivity in different vasculatures in hemorrhagic shock.Shock 03/2003; 19(3):208-14. · 2.61 Impact Factor
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ABSTRACT: Our previous work observed that vascular hyporeactivity to norepinephrine (NE) developed after hemorrhage and the response was not the same in the 4 arteries examined. To evaluate possible mechanisms involved, the present study investigated the gene expression of iNOS, eNOS, IL-1beta, IL-6, TNF-alpha, and endothelin-1 in the corresponding organs, and the roles of nitric oxide (NO) and endothelin (ET). LAnesthetized rats (n=7/time point/group) were hemorrhaged to a mean arterial pressure of 50 mmHg for 60 min. The vascular reactivity of the superior mesenteric (SMA), celiac (CA), left renal (LRA), and left femoral arteries (LFA) to NE was measured at baseline, at the end of the hypotensive period (E), and at 1, 2, and 4 h later in the three groups (hemorrhage, hemorrhage+NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, or hemorrhage+PD142893, an ET receptor antagonist). Gene expression in ileum, left kidney, liver, and skeletal muscle was determined by quantitative RT-PCR at these times. Vascular reactivity of SMA, CA, LRA, and LFA to NE decreased as much as 98% over 4 h compared with baseline. This loss of responsiveness in CA and LFA was more severe than in SMA and LRA. Gene expression of iNOS, eNOS, IL-1beta, IL-6, TNF-alpha, and endothelin-1 in the corresponding organs of select vasculatures increased markedly over baseline levels and the fold increase in mRNA levels of these enzymes and mediators in liver and skeletal muscle was higher than in ileum and left kidney. For example, at 4 h, iNOS expression was over 16-fold higher than baseline in liver and skeletal muscle, but 5- and 7-fold higher in ileum and kidney, respectively. L-NAME or PD142893 partially attenuated the decreased vascular reactivity induced by hemorrhagic shock and attenuated the changes in gene expression observed. These findings suggest that the differential expression of NOS, cytokines, and endothelin-1 in different organs are associated with the development of vascular hyporeactivity after hemorrhagic shock and may account, at least in part, for the vascular bed diversity observed.Journal of Surgical Research 06/2005; 125(2):128-36. · 2.02 Impact Factor