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CHANGES OF RHO KINASE ACTIVITY AFTER HEMORRHAGIC SHOCK AND ITS ROLE IN SHOCK-INDUCED BIPHASIC RESPONSE OF VASCULAR REACTIVITY AND CALCIUM SENSITIVITY

Li, Tao; Liu, Liangming; Xu, Jing; Yang, Guangming; Ming, Jia

doi: 10.1097/01.shk.0000228796.41044.41
Basic Science Aspects
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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 Ca2+ 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 Ca2+ 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 Ca2+at early shock (P < 0.05 or P < 0.01). Angiotensin II increased the decreased contractile response of SMA to NE and Ca2+ 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 Ca2+ 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.

State Key Laboratory of Trauma, Burns and Combined Injury, The 2nd Department of Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China

Received 13 Mar 2006; first review completed 27 Mar 2006; accepted in final form 1 May 2006

Address reprint requests to Liangming Liu, MD, PhD, The 2nd Department of Research Institute of Surgery, Daping Hospital, Third Military Medical University, Daping, Chongqing 400042, People's Republic of China. E-mail: liulmh@online.cq.cn.

This work was supported by the National Natural Science Foundation of China (No. 30271266 and 30370563), the Scientific Research Foundation for the Returned overseas Chinese Scholars, State Education Ministry, and the National Basic Research Program of China (2005CB522601).

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INTRODUCTION

After severe trauma or shock, including hemorrhagic, endotoxic, and septic shock, vascular reactivity to vasoconstrictors and vasodilators is greatly reduced (1, 2). Previous studies showed that it may be related to the functional disorder of the K+ and Ca2+ channels in vascular smooth muscle cell (VSMC) or the hyperpolarization of cell membrane (3, 4). However, recovering the function of the K+ and Ca2+ channels and polarization state of the cell membrane cannot restore the vascular reactivity completely. Our previous study showed that calcium overload and calcium desensitization (the decrease of force/Ca2+ ratio) coexisted in myocardium cell after severe trauma or shock, which resulted in the less reactivity of the myocardium cell to the traditional cardiotonics (5). In addition, calcium overload was also found in VSMC after shock, calcium desensitization existed in VSMC after hemorrhagic shock, and calcium desensitization played an important role in vascular hyporeactivity in our previous study (6). Vascular reactivity and calcium sensitivity appeared biphasic change during shock.

Rho kinase is a Ser/Thr protein kinase and was identified as a guanosine triphosphate (GTP)-Rho binding protein. Rho is a small GTPase, and it was thought to be a molecular switch to mediate signals to various molecules (7). When cells are stimulated by certain extracellular signals such as lysophosphatidic acid, guanosine diphosphate (GDP)-Rho, the inactive form of Rho, is converted to GTP-Rho, the active form of Rho (8). Research showed that Rho was involved in the regulation of many biological cellular functions, such as stress-fiber and focal-adhesion formation, smooth muscle contraction, and so on (9). Several proteins including protein kinase Nitrogen, Rho kinase, and the myosin-binding subunit of myosin phosphatase (10, 11) have been identified as the effectors of Rho. Among these effectors, Rho kinase implicated its important downstream molecule.

Many studies found that Rho/Rho kinase took part in the regulation of VSMC calcium sensitivity. Rho/Rho kinase can be activated by several pathological mediators, including endothelin-1 (ET-1), 5-hydroxytryptamine, and oxyhemoglobin. The activation of Rho/Rho kinase leads to the inhibition of myosin light chain phosphatase (MLCP) and consequently results in an increase of vascular tone, which might contribute to the development of hypertension and coronary or cerebral vasospasm (12). At same time, our pervious studies showed that calcium desensitization existed in VSMC after hemorrhagic shock, and Rho kinase took part in the regulation of calcium sensitivity. Calcium desensitization played an important role in the occurrence of vascular hyporeactivity after hemorrhagic shock, vascular reactivity and calcium sensitivity appeared biphasic change after shock (6). But how the activity of Rho kinase is changed after hemorrhagic shock and whether it is involved in the biphasic response of vascular reactivity and calcium sensitivity are unclear.

To elucidate this problem and get more understanding about the role of Rho kinase in the occurrence of vascular hyporeactivity, we observed the changes of vascular reactivity and calcium sensitivity of superior mesenteric artery (SMA) and the changes of Rho kinase activity in mesenteric artery at different time after shock and analyzed their relationship first; secondly, we used insulin and angiotensin II (Ang-II) as tool agents to inhibit or increase the activity of Rho kinase to observe their effects on vascular reactivity and calcium sensitivity (13); thirdly, inasmuch as Ang-II is not a specific Rho kinase agonist, we used Rho kinase-specific inhibitor, Y-27632, to testify if Ang-II increases the vascular reactivity and calcium sensitivity through Rho kinase.

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MATERIALS AND METHODS

Instrumentation

Seventy-two Wistar rats weighing 200 to 250 g were fasted for 12 h but allowed water ad libitum before the experiment. On the day of the experiment, eight rats were randomized to the sham-operated group, and the rest were subjected to hemorrhagic shock. Rats were first anesthetized with sodium pentobarbital. The right femoral arteries were then catheterized with polyethylene tubing (outer diameter, 0.965 mm; inner diameter, 0.58 mm) for monitoring the mean arterial pressure (MAP) and bleeding. To prevent clot formation, the tubing was filled with normal saline containing 30 U/mL of heparin. After the completion of the surgical procedure, the rats were allowed to stabilize for 10 min, then rapidly hemorrhaged (within 10 min) from the right femoral arterial catheter until the MAP dropped to 40 mmHg and maintained for a necessary time according to experimental design. The average bleeding rate was 0.45 ± 0.08 mL/min, and the total bleeding volume was 21.5 ± 3.41 mL/kg.

This study was approved by the Research Council and Animal Care and Use Committee of Research Institute of Surgery, Daping Hospital, Third Military Medical University. All experiments were conformed to the guidelines of ethical use of animals, and all efforts were made to minimize animal suffering and to reduce the number of animals used.

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Experimental design

Seventy-two Wistar rats were randomly divided into five groups: sham-operated (n = 8), 0-h shock (immediate) (n = 16), 30-min shock (n = 8), 1-h shock (n = 8), and 2-h shock (n = 32) groups. Among them, 0-h shock group was further divided into 0-h control group and insulin group (n = 8/group); 2-h shock group was further divided into 2-h control, Ang-II, Y-27632, and Y-27632 + Ang-II groups (n = 8/group). After the completion of surgical procedure as described above, the rats were hemorrhaged to a MAP at 40 mmHg and maintained at this level for 0 h, 30 min, 1 h, and 2 h, respectively. Rats in sham-operated group received identical operations as shock group, but without hemorrhage. Finally, a laparotomy was performed, and mesenteric arteries were obtained from the sham-operated or shocked rats. After cleaning off the adhering tissues carefully, each SMA was cut into two rings of 2- to 3-mm long for the experiments. One ring was used for the measurement of vascular reactivity, and the other was used for the measurement of calcium sensitivity. The rest of mesenteric artery was used for the measurement of Rho kinase activity.

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Measurement of the vascular reactivity of SMA ring

Artery rings were mounted on wire and suspended between a force transducer and a post attached to a micrometer, then immersed into a 10-mL isolated organ chamber (Scientific Instruments, Barcelona, Spain) containing Krebs-Henseleit (K-H) solution (in mmol/L): 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.03 KH2PO4, 0.45 MgSO4 · 7H2O, 2.5 CaCl2, and 11.1 Glucose at pH 7.4, which was continuously bubbled with 95%O2/5%CO2 and the temperature was maintained at 25°C. Preload was given 0.5 g, and the K-H solution was replaced every 20 min. The tension of the artery rings was determined by a Power Lab System via a force transducer (AD Instruments, Castle Hill, Australia). After 2-h equilibration, the contractile responses of artery rings to norepinephrine (NE) (1 × 10−9, 1 × 10−8, 1 × 10−7, 1 × 10−6, 1 × 10−5, 1 × 10−4 mol/L) in sham-operated and shock groups were determined. Artery rings in insulin, Ang-II, and Y-27632 groups were incubated with insulin (100 nmol/L), Ang-II (1 nmol/L), and Y-27632 (10 μmol/L), respectively, for 10 min first, then the vascular reactivity of SMA to NE were determined. Artery rings in Y-27632 + Ang-II group were incubated with Y-27632 (10 μmol/L) for 10 min, followed by Ang-II (1 nmol/L) for 10 min, and then the vascular reactivity of SMA to NE were determined.

The dosage of insulin, Ang-II, and Y-27632 selected in the present study was based on the previous reports and our own study (6, 13, 14).

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Measurement of the calcium sensitivity of SMA ring

Artery rings were incubated and equilibrated in the K-H solution for 2 h as described above, then the solution was replaced with depolarizing solution containing (in mmol/L): 2.7 NaCl, 120 KCl, 25 NaHCO3, 1.03 KH2PO4, 0.45 MgSO4 · 7H2O, and 11.1 Glucose at pH 7.4. After 10 to 20 min for equilibration, the contractile responses of artery rings to Ca2+ (3 × 10−5, 1 × 10−4, 3 × 10−4, 1 × 10−3, 2 × 10−3, 6 × 10−3, 1 × 10−2, and 3 × 10−2 mol/L) in sham-operated and shock groups were determined. The incubation time of artery rings with treatment agents in different groups was the same to the method described in the measurement of vascular reactivity.

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Measurement of Rho kinase activity in mesenteric artery

Immunoprecipitation of Rho kinase-

The mesenteric artery was homogenized with lysis buffer (composition: Tris-HCl, (pH = 7.4) 50 mmol/L; NaCl, 400 mmol/L; EGTA, 2 mmol/L; EDTA, 1 mmol/L; dithiothreitol, 1 mmol/L; phenylmethylsulfonyl fluoride, 10 μmol/L; leupeptin, 10 μg/mL; pepstatin, 1 μg/mL; benzamidine, 1 mmol/L), homogenate was centrifuged at 8000g for 10 min at 4°C, and the supernatant was collected.

Five hundred micrograms of lysate proteins was incubated with protein G Sepharose (15 μL) at 4°C for 30 min and centrifuged at 12000g for 5 min at 4°C to remove lysis buffer. Supernatant was incubated with anti-Rho kinase antibody (6 μg/tube) at 4°C for 2 h, then incubated with protein G Sepharose (40 μL) at 4°C for 1 h. The compound were washed 2 times by buffer A (composition: Tris-HCl (pH = 8.0), 2 mmol/L; NaCl, 0.14 mol/L; NaN3, 0.025%), and buffer B (composition: Tris-HCl (pH = 8.0), 2 mmol/L; NaCl, 0.14 mol/L; NaN3, 0.025%; Triton-X100, 0.1%), and once by buffer C (composition: Tris-HCl (pH, 6.8), 50 mmol/L) and then centrifuged at 200g for 5 min at 4°C. The protein G anti-Rho kinase antibody and antigen were collected (15).

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Rho kinase activity measurement-

Activity of Rho kinase was assayed as described previously (16, 17). Briefly, the immunoprecipitates and 15 μL of myosin (0.1 mg/mL) were added into the 25 μL of enzyme buffer (composition: Tris-HCl (pH = 7.5), 20 mmol/L; KCl, 100 mmol/L; dithiothreitol, 0.1 mmol/L; MgCl2, 5 mmol/L; EDTA, 1 mmol/L; microcysin-LR 1 μmol/L, [γ-32P]-ATP 100 nmol/L) and incubated at 30°C for 5 min. Twenty-five microliters of aliquots of the reaction mixture was spotted on phosphocellulose paper, followed by extensive washing with phosphate-buffered saline, and the incorporation of 32P was determined by liquid scintillation spectroscopy.

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Statistical analysis

The cumulative concentration-response curve of SMA to NE and Ca2+, the maximal contraction (Emax) and pD2(−log[EC50]) of NE and Ca2+ were used to evaluate the vascular reactivity and calcium sensitivity of the blood vessels observed. Rho kinase activity in sham-operated group was taken as 100%; other groups were expressed as the percentage of sham-operated group.

All data are presented as mean ± SD of n observations. The difference between experimental groups was analyzed by one-way analysis of variance, followed by posthoc Tukey test. P < 0.05 was considered significant.

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RESULTS

Changes of vascular reactivity and calcium sensitivity of SMA after hemorrhagic shock

As compared with the sham-operated group, the cumulative dose-response curves of SMA to NE at early shock (immediate and 30 min after shock) were significantly shifted to the left, their Emax were significantly increased (P < 0.05 or P < 0.01) (Table 1, Fig. 1). Whereas the cumulative dose-response curves of SMA to NE at late shock (1 h and 2 h after shock) were significantly shifted to the right, Emax were significantly decreased (P < 0.05 or P < 0.01). The pD2 of NE at immediate, 30 min, and 1 h after shock was decreased significantly compared with sham-operated group (P < 0.05).

Table 1

Table 1

Fig. 1

Fig. 1

The cumulative dose-response curves of SMA to Ca2+ at early shock (immediate and 30 min after shock) were also significantly shifted to the left; their Emax were significantly increased (P < 0.05 or P < 0.01) (Table 1, Fig. 2). Whereas the cumulative dose-response curves of SMA to Ca2+ at late shock (1 h and 2 h after shock) were significantly shifted to the right, Emax were significantly decreased (P < 0.05 or P < 0.01). The pD2 of Ca2+ at 30 min and 2 h after shock were decreased significantly (P < 0.05).

Fig. 2

Fig. 2

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Changes of Rho kinase activity after hemorrhagic shock and its correlation to vascular reactivity and calcium sensitivity

As compared with the sham-operated group, the activity of Rho kinase in mesenteric artery was significantly increased at shock immediate(P < 0.05), but it was significantly decreased at 1 and 2 h after shock (P < 0.05 or P < 0.01) (Fig. 3). It was positively correlated with the changes of vascular reactivity and calcium sensitivity, the correlation coefficient between the activity of Rho kinase and vascular reactivity was 0.9624, and it was 0.9704 between Rho kinase activity and calcium sensitivity.

Fig. 3

Fig. 3

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Effects of Ang-II, Y-27632, and insulin on vascular reactivity and calcium sensitivity of SMA after hemorrhagic shock

Insulin (100 nmol/L) significantly decreased shock-induced increase of the contractile response of SMA to NE (at 1 × 10−4 and 1 × 10−5 mol/L) and Ca2+ (at 1 × 10−3, 2 × 10−3, 6 × 10−3, 1 × 10−2, 3 × 10−2 mol/L) (P < 0.05 or P < 0.01) at early stage, and made the cumulative dose-response curves shift to the right. The Emax of NE and Ca2+ were significantly decreased (P < 0.01) (Table 2, Figs. 4, 5).

Table 2

Table 2

Fig. 4

Fig. 4

Fig. 5

Fig. 5

As compared with the 2-h shock group, Ang-II (1 nmol/L) enhanced the contractile response of SMA to NE (at 1 × 10−6, 1 × 10−5, 1 × 10−4 mol/L) and Ca2+ (at 2 × 10−3, 6 × 10−3, 1 × 10−2, 3 × 10−2 mol/L) (P < 0.05 or P < 0.01) at 2 h after shock (P < 0.05 or P < 0.01) and made the cumulative dose-response curves of NE and Ca2+ shift to the left. The Emax of NE and Ca2+ were significantly increased (P < 0.01) (Table 2, Figs. 4, 5). Y-27632 (10 μmol/L) decreased the contractile response of SMA to NE and Ca2+ at 2 h after shock and made the cumulative dose-response curve of NE and Ca2+ shift to the right. Y-27632 pretreatment (10 μmol/L) abolished Ang-II (1 nmol/L), induced the increase of calcium sensitivity (P < 0.05), and made the cumulative dose-response curve of SMA to NE and Ca2+ shift to the right. Their Emax were significantly decreased (P < 0.05) (Table 2, Figs. 4, 5).

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DISCUSSION

Many studies have demonstrated that after severe trauma or shock, or the associated systemic inflammatory response syndrome and multiple organ dysfunction syndrome, the vascular reactivity to vasoconstrictors and vasodilators is greatly reduced (1, 2). This reduced vascular reactivity plays an important role in the incidence, development, and outcome of the shock and interferes with the therapy for shock, especially interferes with the application of vasoactive agents. Our previous studies showed that calcium desensitization existed in the vascular smooth muscle cell after hemorrhagic shock, which contributed to the incidence of vascular hyporeactivity, Rho kinase had some regulatory effect on calcium sensitivity of VSMC during shock (6). But how the Rho kinase activity of VSMC changes after hemorrhagic shock and if it is involved in the biphasic change of vascular reactivity and calcium sensitivity shock are unclear.

Our present study indicated that vascular reactivity and calcium sensitivity were significantly increased at early shock (immediate and 30 min after shock), but they were significantly decreased at late shock (1 and 2 h after shock). Meanwhile, Rho kinase activity in mesenteric artery was also significantly increased at early shock, but decreased at late shock (1 and 2 h after shock). The changes of vascular reactivity and calcium sensitivity after hemorrhagic shock were positively correlated with the changes of Rho kinase activity. Angiotensin II (1 nmol/L), with the effect of Rho kinase stimulation, could increase shock-induced decrease of vascular reactivity and calcium sensitivity, and insulin, with Rho kinase inhibitory effect, could decrease early shock-induced increase of vascular reactivity and calcium sensitivity. It was suggested that the biphasic change of Rho kinase activity after hemorrhagic shock anticipated in the biphasic response of vascular reactivity and calcium sensitivity. Rho kinase played an important role in the occurrence of vascular hyporeactivity and calcium sensitivity after hemorrhagic shock. Rho kinase may regulate the vascular reactivity through calcium sensitivity regulation.

The mechanisms responsible for the changes of Rho kinase activity after shock are unknown. But from previous studies, it was speculated that the increase of Rho kinase activity at early shock may be related to the stimulation of agonists such as ET-1 and Ang-II. Basic research demonstrated that ET-1 and Ang-II were increased significantly at early shock and could lead to the activation of Rho kinase. The decreased Rho kinase activity at late shock may be related to many factors such as nitric oxide or high concentration of some agonist stimulation such as Ang-II. Nitric oxide, significantly increased at late shock, can phosphorylate RhoA via protein kinase G, and then result in the inactivation of Rho kinase. Prolonged stimulation of high concentration of agonists can decrease the activity of Rho kinase. Schmitz et al. (18) reported that Ang-II may induce the activation of phosphatidylinsitol-3-kinase and tyrosine kinase in VSMC and then activate the Rac. Activated Rac can lead to the inhibition of Rho kinase (18, 19). But the precise mechanisms for the regulation of Rho kinase activity after shock need further study.

Angiotensin II is a multifunctional hormone, it can regulate calcium sensitivity via activating Rho kinase, but Ang-II is not a specific Rho kinase agonist (20, 21). Our previous studies demonstrated that Ang-II at a concentration of 1 nmol/L regulated calcium sensitivity of VSMC mainly through activation of Rho kinase (6), so we adopted Ang-II at the concentration of 1 nmol/L in the present study to see if upregulation of Rho kinase activity can increase the decreased vascular reactivity and calcium sensitivity. The results found that Ang-II at the concentration of 1 nmol/L may increase the decreased vascular reactivity and calcium sensitivity. To further testify that Ang-II at a concentration of 1 nmol/L regulates calcium sensitivity of VSMC mainly through Rho kinase, Y-27632, a specific Rho kinase inhibitor, was used in the present study. Y-27632 inhibits Rho kinase by competing with adenosine triphosphate for binding to the catalytic site of the Rho kinase (21). It is an important pharmacological tool for elucidating the importance of Rho kinase not only in pathogenesis of systemic cardiovascular disorders but also in pulmonary hypertension and so on. Y-27632 inhibited the contraction of rat arteries by more than 80% when used at the concentration as low as 10−5 mol/L (22). So in present study, 10−5 mol/L of Y-27632 was selected. The experiment demonstrated that this dosage of Y-27632 can antagonize Ang-II-induced increase of vascular reactivity and calcium sensitivity. The results further demonstrated that Ang-II at the concentration of 1 nmol/L increased both calcium sensitivity and vascular reactivity mainly through Rho kinase.

Some studies showed that insulin could inhibit Rho kinase activity. Begum (23) reported that insulin could rapidly stimulate MLCP and simultaneously inhibit RhoA/Rho kinase in vascular smooth muscle. Insulin at the concentration of 100 nmol/L inhibits Rho kinase activity by more than 40% (13). Our previous studies demonstrated that insulin at the concentration of 100 nmol/L decreased vascular reactivity and calcium sensitivity through inhibiting Rho kinase activity, so we adopted insulin at the concentration of 10−7 mol/L to see if downregulation of Rho kinase activity can decrease the vascular reactivity and calcium sensitivity. The results showed that inhibition of Rho kinase activity by insulin can decrease the vascular reactivity and calcium sensitivity. Insulin inactivates Rho kinase mainly by blocking RhoA and its translocation to the membrane fraction via increasing cGMP/cGK-1 (mediated RhoA phosphorylation and decreased geranylgeranylation).

The mechanism that Rho kinase regulates the vascular reactivity and calcium sensitivity of VSMC after shock is not clear. Basic research demonstrated that Rho kinase regulates the cellular contraction and calcium sensitivity mainly through MLCP, protein kinase C-dependent phosphatase inhibitor of 17 kDa (CPI-17), or phosphorylating MLC20 (20-kDa myosin light chain) directly. Many studies demonstrated that Rho kinase induced calcium sensitization via inhibiting MLCP activity in VSMC after hypoxia. Wang et al. (24) found that MLCP activity in VSMC decreased at the beginning of hypoxia, but increased at later of hypoxia; Y-27632 can eliminate hypoxia-induced the change of MLCP activity. The precise mechanism of Rho kinase regulating vascular reactivity and calcium sensitivity after shock needs further study.

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CONCLUSION

Rho kinase activity was significantly increased at early shock and significantly decreased at late shock; it was closely related to the biphasic changes of vascular reactivity and calcium sensitivity after hemorrhagic shock. It played an important role in the occurrences of vascular hyporeactivity and calcium desensitization. Rho kinase-stimulating agent may have some improving effects on vascular hyporeactivity. More efforts should be made to further elucidate the precise mechanism that Rho kinase regulates the calcium sensitivity after hemorrhagic shock and probes good approaches to restore the decreased vascular reactivity induced by shock or trauma.

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Keywords:

Hemorrhagic shock; vascular reactivity; calcium sensitivity; Rho kinase activity; angiotensin II; insulin; Y-27632

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