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Basic Science Aspects

THE ROLE OF CALCIUM DESENSITIZATION IN VASCULAR HYPOREACTIVITY AND ITS REGULATION AFTER HEMORRHAGIC SHOCK IN THE RAT

Xu, Jing; Liu, Liangming

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doi: 10.1097/01.shk.0000161387.23817.36
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Abstract

INTRODUCTION

After severe trauma or shock, including hemorrhagic, endotoxic, and septic shock, vascular reactivity to vasoconstrictors and vasodilators is greatly reduced. 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 membranes (1, 2). However, recovering the function of the K+ and Ca2+ channels and polarization state of the cell membrane cannot restore the vascular reactivity completely (1, 2). Our previous study showed that calcium overload and calcium desensitization (the decrease of force/Ca2+ ratio) coexisted in the myocardium cell after severe trauma or shock, which resulted in less reactivity of the myocardium cell to the traditional cardiotonics, which increase the intracellular calcium concentration ([Ca2+]i) (3, 4). In addition, calcium overload was also found in VSMC after shock, but whether calcium desensitization exists in the vascular smooth muscle cell after hemorrhagic shock, and what roles does calcium desensitization play in vascular hyporeactivity are still unknown. To elucidate this problem and get more understanding about the mechanisms of the occurrence of vascular hyporeactivity and the regulation of calcium sensitivity, the superior mesenteric artery (SMA) from hemorrhagic shock rats was used in the present study to investigate possible calcium desensitization of vascular smooth muscle after shock, the role of calcium desensitization in vascular hyporeactivity, and the regulatory effects of Rho-kinase, protein kinase C (PKC), and protein kinase G (PKG) on calcium sensitivity of vascular smooth muscle after hemorrhagic shock.

MATERIALS AND METHODS

Instrumentation

Seventy-two Wistar rats of both sexes, weighing 226 ± 25.3 g, were fasted for 12 h but allowed water 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 (30 mg/kg) and the right femoral arteries were catheterized with PE tubing (outer diameter of 0.965 mm, inner diameter of 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 heparin, and the rats were injected with heparin (500 U/kg) through the tubing after catheterization. After completion of the surgical procedure, the rats were allowed to stabilize for 10 min, and were then hemorrhaged rapidly (within 10 min) from the right femoral arterial catheter until the MAP dropped to 30 mmHg. They were maintained at this level for 2 h by additional blood removal as necessary. Rats in the sham-operated group received the identical operations as the shock group, but without hemorrhage. Finally, a laparotomy was performed, and the SMA was obtained from the sham or shocked rats. After cleaning off the adhering tissues carefully, each SMA was cut into two rings of 2 to 3 mm in length for the experiments. One ring was used for the measurement of vascular reactivity and the other was used for the measurement of calcium sensitivity.

This study was approved by the Research Council and Animal Care and Use Committee of Research Institute of Surgery, Daping Hospital, the Third Military Medical University. All experiments 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.

Experimental protocol

The experiment was conducted in three parts. In the first part, we observed whether calcium desensitization existed in the hyporesponsive blood vessels. SMA rings from sham-operated rats (n = 8) and shock rats (n = 8) were used to observe the vascular reactivity. The other 16 SMA rings also from the sham-operated and shock rats were used to determine the calcium sensitivity.

In the second part, using the experimental setup of part 1, we tested whether Angiotensin II (Ang-II), insulin, the calcium sensitivity-regulating agents, and a relatively selective inhibitor of Rho-kinase, Fasudil, affected vascular reactivity through regulation of calcium sensitivity. Two sets of SMA rings from shock rats were divided into three groups (n = 8/group/set): Ang-II group, insulin group, and Fasudil + Ang-II group. The first set was used to measure the vascular reactivity, and the second set was used to measure calcium sensitivity.

The third part determined the regulatory effects of Rho-kinase, PKC, and PKG on the calcium sensitivity of SMA after hemorrhagic shock and their relationship to myosin light chain phosphatase (MLCP). The Rho-kinase agonist, Ang-II, and inhibitor, Fasudil, the PKC agonist, phorbol 12-myristate 13-acetate (PMA) and inhibitor, staurosporine, the PKG agonist, 8Br-cGMP and inhibitor, KT-5823, and MLCP inhibitor, Calyculin A, were used as tool agents. SMA rings from the shock group were randomized into nine groups of eight rings per group: Ang-II group, Fasudil group, Calyculin A + Ang-II group, PMA group, staurosporine group, Calyculin A + PMA group, 8Br-cGMP group, KT-5823 group, and Calyculin A + 8Br-cGMP group.

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, and 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 ring was determined by a Power Laboratory System via a force transducer (AD Instruments, Castle Hill, Australia). After 2 h of equilibration, artery rings in the insulin group and the Ang-II group were incubated with insulin (100 nmol/L) or Ang-II (1 nmol/L) for 10 min, and artery rings in the Fasudil + Ang-II group were incubated with Fasudil (1 μmol/L) for 10 min, followed by Ang-II (1 nmol/L) for 10 min, and then the vascular reactivity of SMA to NE (10−9, 10−8, 10−7, 10−6, and 10−5 mol/L) was determined.

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, and 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, artery rings in the insulin, Ang-II, Fasudil, PMA, staurosporine, 8Br-cGMP, and KT-5823 groups were incubated with insulin (100 nmol/L), Ang-II (1 nmol/L), Fasudil (1 μmol/L), PMA (0.1 μmol/L), staurosporine (100 nmol/L), 8Br-cGMP (100 μmol/L), and KT-5823 (1 μmol/L), respectively, for 10 min. Artery rings in the Fasudil + Ang-II group were incubated with Fasudil (1 μmol/L) for 10 min, followed by Ang-II (1 nmol/L) for 10 min, and artery rings in the Calyculin A + Ang-II group, the Calyculin A + PMA group, and the Calyculin A + 8Br-cGMP group were incubated with Calyculin A (10−7 mol/L) for 10 min, followed by Ang-II (1 nmol/L), PMA (0.1 μmol/L), and 8Br-cGMP (100 μmol/L) for 10 min, then reactivity to Ca2+ (3 × 10−5, 10−4, 3 × 10−4, 10−3, 2 × 10−3, 6 × 10−3, 10−2, and 3 × 10−2 mol/L) was determined.

Statistical analysis

The cumulative dose-response curve of SMA to NE and Ca2+ and 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.

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

RESULTS

The changes of vascular reactivity and calcium sensitivity of SMA after hemorrhage shock

The cumulative dose-response curve of SMA to NE in shock group was significantly shifted to the right as compared with the sham-operated group (Fig. 1). Its Emax was significantly decreased (0.898 ± 0.012 g/mg) compared with the control group (1.358 ± 0.007 g/mg; P < 0.01), and its pD2 was decreased to 7.077 ± 0.015 from 7.187 ± 0.044 of the control group (P < 0.05; Figs. 1 and 2A). In addition, the cumulative dose-response curve of SMA to Ca2+ in the shock group was also shifted to the right, and the Emax and pD2 of Ca2+ were significantly reduced (P < 0.05, P < 0.01; Fig. 3). The Emax was reduced to 0.377 ± 0.014 g/mg from 1.004 ± 0.031 g/mg of the control group (P < 0.01), and the pD2 was reduced to 2.557 ± 0.065 from 2.814 ± 0.027 of the control group (P < 0.05; Figs. 2C and 3). There were no differences between male and female rats in vascular reactivity and calcium sensitivity after hemorrhagic shock.

Fig. 1
Fig. 1:
The contractile response of SMA to NE after hemorrhage shock, and the effect of Ang-II, insulin, and Ang-II plus Fasudil (*P< 0.05 and **P< 0.01 compared with sham-operated group, ^P < 0.05 and ^^P < 0.01 compared with shock group, and##P < 0.01 compared with Ang-II group).
Fig. 2
Fig. 2:
The representative curve of raw data. (A) The contractile response of SMA to NE in sham-operated and shock groups. (B) Effect of Ang-II, insulin, and Ang-II plus Fasudil on vascular reactivity of SMA to NE after hemorrhagic shock. (C) The change of calcium sensitivity of SMA after hemorrhagic shock. (D) Effect of Ang-II, insulin, and Ang-II plus Fasudil on the calcium sensitivity of SMA from hemorrhagic shock rat.
Fig. 3
Fig. 3:
The change of calcium sensitivity of SMA after hemorrhage shock, and the effect of Ang-II, insulin, and Ang-II plus Fasudil (*P < 0.05 and **P < 0.01 compared with sham-operated group, ^P < 0.05 and ^^P < 0.01 compared with shock group, and#P < 0.05 compared with Ang-II group).

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

Compared with the shock group, Ang-II (1 nmol/L) enhanced the contractile response of SMA to NE (at 10−7, 10−6, and 10−5 mol/L; P < 0.01) and made the cumulative dose-response curve of NE shift to the left. The Emax of NE was significantly increased from 0.898 ± 0.012 g/mg of the shock group to 1.224 ± 0.046 g/mg (P < 0.01). In contrast, insulin (100 nmol/L) shifted the cumulative dose-response curve of NE to the right and weakened the contractile response of SMA to NE. Pretreatment with Fasudil (1 μmol/L) nearly abolished the vascular responsiveness to NE (Figs. 2B and 3).

Ang-II (1 nmol/L) treatment also shifted the cumulative dose-response curve of Ca2+ to the left and increased the contractile response of Ca2+ (at 6 × 10−3, 10−2, 3 × 10−2 mol/L; P < 0.05, P < 0.01). The Emax was increased from 0.377 ± 0.014 g/mg of the shock group to 0.630 ± 0.025 g/mg (P < 0.01). Insulin (100 nmol/L) shifted the cumulative dose-response curve of Ca2+ to the right and decreased the contractile response of Ca2+ (at 10−3, 2 × 10−3, 6 × 10−3, 10−2, and 3 × 10−2 mol/L; P < 0.05). The Emax of Ca2+ was decreased from 0.377 ± 0.014 g/mg of the shock group to 0.205 ± 0.014 g/mg (P < 0.01). Fasudil pretreatment (1 μmol/L) abolished the Ang-II (1 nmol/L) induced increase of calcium sensitivity (at 10−3, 2 × 10−3, 6 × 10−3, 10−2, and 3 × 10−2 mol/L; P < 0.05) and made the cumulative dose-response curve of Ca2+ shift to the right. Its Emax was decreased from 0.630 ± 0.025 g/mg of the Ang-II group to 0.256 ± 0.007 g/mg (P < 0.05; Figs. 2D and 3), whereas pD2 did not change significantly.

Effects of Rho-kinase, PKC, and PKG on the calcium sensitivity of SMA after hemorrhagic shock and their relationship to MLCP

Fasudil (1 μmol/L), the relatively selective antagonist of Rho-kinase, further decreased shock-induced decrease of calcium sensitivity of SMA. Its Emax was decreased to 0.242 ± 0.009 g/mg from 0.377 ± 0.014 g/mg of shock group (P < 0.01), Calyculin A (10−7 mol/L) pretreatment promoted Ang-II (1 nmol/L) induced the increase of calcium sensitivity (at 10−3, 2 × 10−3, 6 × 10−3, 10−2, and 3 × 10−2 mol/L; P < 0.05), and made the cumulative dose-response curve of Ca2+ shift to the left. Its Emax was increased from 0.630 ± 0.025 g/mg of the Ang-II group to 0.902 ± 0.052 g/mg (P < 0.01; Figs. 4 and 5A).

Fig. 4
Fig. 4:
Effect of Ang-II, Fasudil, and Ang II plus Calyculin A on the calcium sensitivity of SMA from hemorrhagic shock rat (*P< 0.05 and **P< 0.01 compared with shock group, ^P < 0.05 and ^^P < 0.01 compared with Ang-II group).
Fig. 5
Fig. 5:
The representative curve of raw data. (A) Effect of Ang-II, Fasudil, and Ang-II plus Calyculin A on the calcium sensitivity of SMA from hemorrhagic shock rat. (B) Effect of PMA, staurosporine, and PMA plus Calyculin A on the calcium sensitivity of SMA from hemorrhagic shock rat. (C) Effect of 8Br-cGMP, KT-5823, and 8Br-cGMP plus Calyculin A on the calcium sensitivity of SMA from hemorrhagic shock rat.

PMA (0.1 μmol/L), the PKC agonist, significantly increased the calcium sensitivity of SMA compared with shock rats, and shifted the cumulative dose-response curve of SMA to Ca2+ to the left. The Emax was increased from 0.377 ± 0.014 g/mg of the shock group to 0.595 ± 0.037 g/mg (P < 0.05). Staurosporine (100 nmol/L), the PKC antagonist, shifted the cumulative dose-response curve of Ca2+ to the right and reduced the calcium sensitivity of SMA from shock rats significantly. The Emax was reduced from 0.377 ± 0.014 g/mg of the shock group to 0.230 ± 0.012 g/mg (P < 0.01). Calyculin A (10−7 mol/L) pretreatment promoted the PMA- (0.1 μmol/L) induced increase of calcium sensitivity (at 10−3, 2 × 10−3, 6 × 10−3, 10−2, and 3 × 10−2 mol/L; P < 0.05) and made the cumulative dose-response curve of Ca2+ shift to the left. Its Emax was increased from 0.595 ± 0.037 g/mg PMA group to 0.998 ± 0.075 g/mg (P < 0.05; Figs. 5B and 6).

Fig. 6
Fig. 6:
Effect of PMA, staurosporine, and PMA plus Calyculin A on the calcium sensitivity of SMA from hemorrhagic shock rat (*P < 0.05 and **P < 0.01 compared with shock group, ^P < 0.05 and ^^P< 0.01 compared with PMA group).

8Br-cGMP (100 μmol/L), the PKG agonist, made the cumulative dose-response curve of Ca2+ shift to the right and decreased the calcium sensitivity of SMA from shock rats significantly. The Emax was decreased to 0.256 ± 0.012 g/mg from 0.377 ± 0.014 g/mg of the shock group (P < 0.05). In contrast, KT-5823 (1 μmol/L), the PKG antagonist, shifted the cumulative dose-response curve of Ca2+ to the left and enhanced the calcium sensitivity of SMA significantly compared with shock rats. The Emax was enhanced from 0.377 ± 0.014 g/mg of the shock group to 0.624 ± 0.051 g/mg (P < 0.05). Calyculin A (10−7 mol/L) pretreatment weakened the 8Br-cGMP (100 μmol/L) induced decrease of calcium sensitivity (at 10−2 and 3 × 10−2 mol/L; P < 0.05) and made the cumulative dose-response curve of Ca2+ shift to the left. Its Emax was increased from 0.256 ± 0.012 g/mg 8Br-cGMP group to 0.377 ± 0.014 g/mg(P < 0.05; Figs. 5C and 7), whereas pD2 did not change significantly.

Fig. 7
Fig. 7:
Effect of 8Br-cGMP, KT-5823, and 8Br-cGMP plus Calyculin A on the calcium sensitivity of SMA from hemorrhagic shock rat (*P< 0.05 compared with shock group, ^P< 0.05 and ^^P< 0.01 compared with 8Br-cGMP group).

DISCUSSION

Previous studies have demonstrated that cardiac dysfunction is closely related to calcium overload and calcium desensitization of myocardial cells after severe trauma or shock. Calcium overload was also found in VSMC after shock, but whether calcium desensitization exists in the hyporesponsive blood vessels and contributes to the vascular hyporeactivity is unknown. Our study showed that vascular reactivity and calcium sensitivity were decreased significantly in SMA after hemorrhagic shock, and the calcium sensitivity regulating agents Ang-II (1 nmol/L) and insulin (100 nmol/L) affected vascular reactivity through improving or weakening calcium sensitivity. It was suggested that the vasculature after shock was desensitized to calcium, which played an important role in the onset of vascular hyporeactivity after shock.

Many molecules such as Rho-kinase, PKC, and PKG participate in the regulation of vascular calcium sensitivity under normal conditions (5), but whether these molecules are also involved in the regulation of calcium sensitivity of VSMC after hemorrhagic shock is still unknown. Our experiments determined the effect of the agonist and antagonist of Rho-kinase, PKC, and PKG on the calcium sensitivity of SMA after hemorrhagic shock. The results showed that the agonists of Rho-kinase and PKC, Ang II and PMA, and the inhibitor of PKG, KT-5823, increased the calcium sensitivity of SMA. On the other hand, the inhibitors of Rho-kinase and PKC, Fasudil and staurosporine, and the agonist of PKG, 8Br-cGMP, decreased the calcium sensitivity of SMA, which indicated that Rho-kinase and PKC may upregulate the calcium sensitivity of vascular smooth muscle, and PKG may down-regulate it after hemorrhagic shock.

Ang-II is a multifunctional hormone that regulates the cardiovascular functions through many intracellular signaling events by acting at AT1 and AT2 receptors (6). It can regulate calcium sensitivity through activating Rho-kinase (7, 8), yet it is not a specific Rho-kinase agonist because other factors may contribute to this process such as calcium influx and mobilization, and so on. To exclude this possibility, we adopted Ang-II at a concentration of 1 nmol/L in the present study. At this concentration, the increase of [Ca2+]i is only 100 to 200 nmol/L, which is about the level of resting status and far less than that stimulated by K+ (120 mmol/L) (9). Therefore, improvement of vascular reactivity of SMA by Ang-II can be mainly considered induced by its calcium sensitization. In addition, we included a Fasudil + Ang-II group to observe if a Rho-kinase inhibitor, Fasudil, can antagonize Ang-II-induced increase of vascular reactivity and calcium sensitivity of SMA. The results showed that preincubation with Fasudil (1 μmol/L) almost completely abolished the effect of Ang-II on vascular reactivity and calcium sensitivity. These results suggested that Ang-II (1 nmol/L) can increase calcium sensitivity and vascular reactivity through Rho-kinase.

Rho-kinase, PKC, and PKG play important roles in the regulation of calcium sensitivity of vascular smooth muscle in hemorrhagic shock, yet their precise mechanisms are not clear. Basic research in normal VSMC showed that Rho-kinase may phosphorylate MBS, the 110/130-kDa myosin-bound regulatory subunit of MLCP to inhibit the activity of MLCP and reduce the dephosphorylation of phosphorylated 20-kDa myosin light chain (MLC20) (10, 11). PKC may also inhibit MLCP through phosphorylating PKC-dependent phosphatase inhibitor of 17kDa (CPI-17), a smooth muscle-specific protein inhibitor of MLCP (12-14). PKG may phosphorylate Telokin (kinase-related protein of 17kDa, KRP), which has a similar amino acid sequence as myosin light chain kinase (MLCK) at COOH terminal with 156 amino acids, and promotes the dephosphorylation of MLC20 through an indistinct pathway (15, 16). The increase of phosphorylated MLC20 is closely associated with the calcium sensitization. Therefore, we propose that Rho-kinase, PKC, and PKG regulate the calcium sensitivity possibly through regulating MLCP and influencing the phosphorylation balance of MLC20 in VSMC after hemorrhagic shock. Our study indicated that Calyculin A, the MLCP inhibitor, promoted Ang-II and PMA induced the increase of calcium sensitivity and weakened the 8Br-cGMP induced the decrease of calcium sensitivity, suggesting that Rho-kinase and PKC up-regulating the calcium sensitivity of vasculature after hemorrhagic shock were possibly by inhibiting MLCP and PKG down-regulating the calcium sensitivity of vasculature was possibly through activating MLCP.

The sensitivity of SMA to Ca2+ was determined under depolarizing conditions (120 mM K+) of the cell membrane in the present study. Under this condition, the voltage-operated calcium channels are open, therefore increases in calcium concentration in the bath can lead to a simultaneous increase of [Ca2+]i in the VSMC (17). This approach is not only convenient, but also practicable. Furthermore, it would not cause any loss of intracellular molecules because the integrity of the cellular membrane is maintained. An alternate method is available to assay calcium sensitivity, in which arterial rings become permeable after treatment with β-escin (or α-toxin, Triton X-100, etc.) and internal Ca2+ stores are depleted irreversibly with A-23187. After such treatment, Ca2+ can cross the cell membrane freely (18), but this method can cause the leakage of intracellular small molecules and even some protein such as CaM (19) and CPI-17 (20). Therefore, the method for calcium sensitivity determination under depolarizing condition was chosen in the present study and achieved satisfactory results.

Many studies showed that there existed gender differences in the response to shock or other stresses. Losonczy et al. (21) reported that male rats were more sensitized to LPS-induced shock. It appeared the blood pressure of males was reduced more than females. Rivier (22) used two stresses, mild electrofoot shock (a neurogenic stress) and acute alcohol injection (a systemic stress) to investigate the influence of gender and circulating sex steroids on ACTH and corticosterone released by adult rats. They found that both types of stresses significantly increased plasma levels of ACTH and corticosterone. After exposure to electroshock, intact females secreted more ACTH than intact males, and the difference was abolished by ovariectomy. Gender differences in corticosterone response were sometimes, but not always, present. In contrast, males released more ACTH than females when acutely injected with alcohol, while there was no obvious effect of sex on corticosterone secretion. Our results did not show the gender difference in vascular reactivity and calcium sensitivity after hemorrhagic shock. More studies need to confirm these results because this work was only a preliminary study.

In conclusion, this study demonstrated that after hemorrhagic shock, the SMA was desensitized to calcium, which contributed to the development of vascular hyporeactivity. Rho-kinase, PKC, and PKG appear to be involved in the regulation of calcium sensitivity of vascular smooth muscle after hemorrhagic shock and the mechanism of their effects may be related to MLCP.

ACKNOWLEGMENT

The authors thank Dr. Michael A. Dubick of the U.S. Army Institute of Surgical Research for his editorial assistance in the preparation of this manuscript.

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Keywords:

Hemorrhagic shock; vascular hyporeactivity; calcium sensitivity; calcium desensitization; Rho-kinase; PKC; PKG; MLCP; rats

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