Author Affiliations: Department of Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China (Chen W, Liu DJ, Qin J, Huo YM, Xu J and Wu ZY); Department of Surgery, Xinjiang Kashi District Second People’s Hospital, Xinjiang 844000, China (Sang JY)

Corresponding Author: Zhi-Yong Wu, MD, PhD, Department of Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China (Tel: 86-21-68383752; Email: zhiyongwu@gmail.com)

 

© 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(13)60047-8

 

Acknowledgement: We thank Associate Professor Dr. Dong Sun (Department of Physiology, New York Medical College, Valhalla, New York, USA) and Professor Chang-Dong Yan (Department of Physiology, Xuzhou Medical College, Xuzhou, China) for technical support and providing us with the vessel perfusion system.

Contributors: XJ and WZY proposed the study. CW, SJY, LDJ, QJ and HYM performed the research. CW wrote the first draft. All authors contributed to the design and interpretation of the study and to further drafts. WZY is the guarantor.

Funding: This study was supported by a grant from the National Natural Science Foundation of China (30972920).

Ethical approval: This study was approved by the Ethic Committee of the Renji Hospital, School of Medicine, Shanghai Jiaotong University.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: The increased β-arrestin-2 and its combina-tion with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models.

 

METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl₄ were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the α₁ adrenergic receptor, G proteins, β-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of α₁ adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation.

 

RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The dose-response curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC₅₀ was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the α₁ adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and β-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the α₁ adrenergic receptor and β-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used.

 

CONCLUSIONS: β-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.

 

(Hepatobiliary Pancreat Dis Int 2013;12:295-304)

 

KEY WORDS: portal hypertension; desensitization; G-protein-coupled receptors; β-arrestin-2; Rho associated coiled-coil forming protein kinase

Liver cirrhosis is associated with the development of hyperdynamic circulation and portal hyper-tension. The increase in portal pressure is triggered by persistent splanchnic vasodilation and increased intrahepatic vascular resistance. Although the levels of vasoconstrictors such as norepinephrine and angiotensin-II (AT-II) increase in the blood circulation and are accompanied by enhanced sympathetic excita-bility in portal hypertension, the splanchnic artery remains dilated. Hyporeactivity of this artery in response to vasoconstrictors plays a key role in blood vessel dilation and hyperdynamic circulation.^{[1, 2]}

 

Contraction of arterial smooth muscle cells is mainly mediated by the phosphorylation of myosin light chains (MLC), which is subject to the dual regulation of MLC kinase (MLCK) and MLC phosphatase (MLCP), and the contractile force increases with increasing phosphorylation of MLC (P-MLC) content.^[3] Contractile pathways in smooth muscle cells are activated after stimulation of G-protein-coupled receptors (GPCRs) by vasoconstrictors. Two of the best characterized and most ubiquitous vasopressor receptors are the α₁ adrenoceptor and the AT-II type 1 receptor. Both are coupled to heterotrimeric G proteins containing Gαq/11, Gα12 and Gα13 subunits. After receptor activation by catecholamines or AT-II, these Gα proteins dissociate from their β/γ-subunits and the receptor, and subsequently regulate MLCK and MLCP through calcium-dependent and calcium sensitization pathways. The latter is also known as the RhoA/Rho associated coiled-coil forming protein kinase (ROCK) pathway.^{[4, 5]}

 

Under normal circumstances, GPCRs are activated by vasoconstrictors. The activated GPCRs recruit more β-arrestin-2 protein under the influence of G protein-coupled receptor kinase 2 (GRK-2), thereby hindering the transmission of contractile signals and bringing about desensitization.^[6-8] β-arrestin-2-mediated receptor desensitization is due to high-intensity and prolonged stimulation in the circulation. Significantly high levels of endogenous vasoconstrictor such as catecholamine and increased affinity between GPCRs and β-arrestin-2, which enhances receptor desensitization and decreases receptor resensitization, may partially result in arterial hyporesponsiveness.^{[4, 9]}

 

In the present study, patients with cirrhosis due to hepatitis B and cirrhotic rat models induced by CCl₄ were used to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models.

 

 

Healthy male Sprague-Dawley rats weighing 120 to 130 g were provided by the Experimental Animal Center of the School of Medicine of Shanghai Jiaotong University. The following materials were used in the study: GRK-2 inhibitor methyl ([5-nitro-2-furyl]vinyl)-2-furoate (Cal-biochem, USA), rabbit anti-ROCK-1 antibody (CST, USA), normal rabbit IgG (sc-2027), rabbit anti-moesin antibody (sc-6410), rabbit anti-phospho-moesin (Thr 558) antibody (sc-12895), mouse anti-β-arrestin-2 antibody (sc-6387), mouse anti-GRK2 antibody (sc-166284), rabbit anti-α₁ adrenergic receptor antibody (sc-31358), rabbit anti-Gαq/11 antibody (sc-392), rabbit anti-Gα12 antibody (sc-409), and rabbit anti-Gα13 antibody (sc-410). The antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

 

The patients with cirrhosis due to hepatitis B were three males and two females, with an age range of 33-60 years. All these patients were confirmed with histories of hematemesis and melena but without complications in the cardiovascular system, lung and brain. Surgical treatment was suggested by gastroscope examination and spiral computed tomography angiography which confirmed the cirrhosis and esophagus fundus ventriculi varicosity. Some of these patients only received anti-hepatitis B virus treatment. The control group included three males and two females, with an age range of 30-58 years. Two patients were diagnosed with small calculus in the common bile duct, and three patients with a benign choledochal cyst. These patients did not take any drugs one month before operation and were confirmed without abnormalities of the liver and other organs in terms of function and morphology through computed tomography or magnetic resonance imaging. The fibrosis or cirrhosis was eliminated by liver biopsy. The present study was approved by the local ethics committee.

 

A 60% CCl₄ oil solution was injected intramuscularly at a dose of 0.4 mL per 100 g body weight, twice a week, combined with 5% ethanol as drinking water. Within 14 to 16 weeks, the rats developed cirrhotic nodules along with portal hypertension. When the rats presented with ascites, exposure to CCl₄ and alcohol was stopped for 7 days. Age-matched, same volume of oil injected rats served as controls.

 

The groups of cirrhotic and control rats were randomized into the control and treatment sub-groups: CS group: control rats treated with saline; CG group: control rats treated with GRK-2 inhibitor; PS group: cirrhotic rats treated with saline; and PG group: cirrhotic rats treated with GRK-2 inhibitor. Animals in the treatment groups received a single injection of GRK-2 inhibitor methyl([5-nitro-2-furyl]vinyl)-2-furoate (200 µg/ kg, i.v.), while their controls received an equal volume of saline. It was proved by previous studies that this dosage was effective in inhibiting the activity of GRK-2.^{[10, 11]} Then, at 60 minutes after the treatment with GRK-2 inhibitor or saline injection, the animals were anesthetized and the experiments were performed subsequently.

 

Before operation, all of the patients received inhalation anesthesia and equilibrium liquid for rehydration. All these patients did not take any kind of vasoactive agents before the collection of specimens. During the operation, heart rate was controlled between 60-100/min, mean arterial pressure was kept between 80-100 mmHg and oxygen saturation was above 98%. Moreover, artery blood gas was monitored regularly in order to make sure all the indices were in normal range during the operation. Portal vein pressure was measured as described in our previous study.^[12]

 

The rats were anesthetized with ketamine through intramuscular injection (250 mg/kg) and then fastened. An incision was made at the midline of the abdomen. After exposure of the portal vein, a 22G catheter filled with heparin saline was inserted directly into the portal vein and the pressure was measured.

 

Following the measurement of the portal vein pressure, we removed the mesenteric arteries as well as the mesentery. All of the specimens from patients were separated from above the first-order mesenteric arteries and the mesentery beside the jejunum one meter away from the suspensory ligament of the duodenum, which would be sure not to impact local blood supply.

 

All of the specimens were divided into two groups. For the first group, we preserved them in 3-(N-morpholino) propanesulfonic acid (MOPS)-buffered physiological salt solution (MOPS-PSS) (0-4 ^{mmol/L), dextrose (5 mmol/L), pyruvate (2mmol/L), EDTA (0.02 mmol/L), and MOPS (3mmol/L).[13] These specimens were used to dissect the third-order arterioles. For the second group, all of the mesenteric arteries were drawn out and frozen in liquid nitrogen immediately or transferred to MOPS-PSS and incubated at 37 �� for 30 minutes. Norepinephrine was then added to the MOPS-PSS at a final concentration of 10 µmol/L. The samples were incubated for another 20 minutes and then stored in liquid nitrogen for later use. Alternatively, the specimens were incubated in MOPS-PSS for 20 minutes at 37 �� without stimulation by norepinephrine and then frozen in liquid nitrogen.} ��; pH 7.4) contained NaCl (145 mmol/L), KCl (5 mmol/L), CaCl₂ (2 mmol/L), MgSO₄ (1 mmol/L), NaH₂PO₄ (1

 

The third-order arterioles in the mesentery were sharply dissected with a dissecting microscope and transferred to a vascular perfusion system containing MOPS-PSS (37 ��; pH 7.4). A glass micropipette contain-ing MOPS-PSS (top diameter, 50 µm) was inserted into one end of an arteriole and fixed with 11-0 single-strands. Blood was flushed out at a perfusion pressure of 8 mmHg and the other end of the blood vessel was kept open. We then inserted another glass micropipette into the other end of the lumen and tied it with 11-0 suture.

 

The vascular diameter was measured with a microscope camera system and displayed on a computer screen. The diameter change was determined by arteriole imaging and the data were recorded in a computer. The diameter of each blood vessel under a pressure of 80 mmHg for 30 minutes was recorded as the baseline. According to the cumulative concentration method, norepinephrine was added to the vascular tank to a concentration ranging from 10^-7 to 10^-5 mol/L, and the contraction rate of the blood vessel was recorded under different concentrations. Cumulative dose-response curves for norepinephrine were drawn on the basis of the contraction rate, i.e., the ratio of intravascular diameter change to maximum diameter. The vasoconstriction rate and the logarithm of the norepinephrine concentration were used as the vertical axis and the abscissa, respectively. We compared the differences in the curves among the groups and recorded the concentration of norepinephrine when the contraction rate was 50% (i.e., EC₅₀).

 

The expression of the following proteins in the mesenteric artery was detected by Western blot: α₁ adrenergic receptor, Gαq/11, Gα12, Gα13, β-arrestin-2, ROCK-1, moesin and p-moesin. To determine the change in ROCK-1 activity, phosphorylation of the ROCK substrate moesin was detected by Western blot. Since ROCK is capable of phosphorylating threonine at position 558 in moesin, its activity change can be examined with a site-specific antibody via Western blot^[9,14,15] as described in our previous studies.^{[16, 17]} In brief, frozen mesenteric arteries were homogenized in 1 mL of tissue lysis buffer. The homogenate was moved to Eppendorf tubes and centrifuged at 4 ��, 10 000 g for 15 minutes. The protein concentration was determined by using a BCA Protein Assay Kit (Pierce, USA). Forty micrograms of total protein was loaded into each well of a 10% SDS-polyacrylamide gel after being denatured at 100 �� for 5 minutes. After electrophoresis, the proteins were transferred to a nitrocellulose membrane at 250 mA for 30 minutes. The membrane was blocked in 5% milk in Tris-buffered saline (TBS) for 1 hour at room temperature. The primary antibody was added and incubated with the membrane over night at room temperature, and then the membrane was washed 3 times with TBS/Tween 20. A secondary antibody was then added to the membrane and it was incubated at room temperature with gentle agitation. Two hours later, the membrane was washed three times with TBS/Tween 20 for 10 minutes per wash. The bands were visualized using an enhanced chemiluminescence kit after being exposed on X-ray film. β-actin was used as an internal reference, then protein quantitative analysis was performed by employing the digital imaging software (Kodak, USA).

 

Protein extracts were incubated with normal rabbit IgG (50 µL) in an ice bath for 1 hour, pre-cleared by incubation with protein A-agarose beads (Bio-Rad, USA) at 4 �� for 30 minutes, and centrifuged at 12 000 ^{β-arrestin-2 .} g at 4 �� for 10 minutes. Then, 80 µL of the supernatant (about 50 µg protein) was incubated with the adrenergic receptor antibody at 4 �� for 2 to 4 hours, and then washed in lysis buffer following centrifugation at 1000 g for 5 minutes. The precipitates were then stored at -80�� or used for Western blot to detect

 

Data were presented as mean±SEM and analyzed using SPSS 16.0. The variables were compared by one-way ANOVA or Student's t test. A P value of <0.05 was considered to be statistically significant. The change in the reactivity of the mesenteric arteriole in response to norepinephrine was presented as a dose-response curve, which was fitted by nonlinear regression analysis (Graph Pad Software Inc., San Diego, CA., USA), and EC₅₀ values were calculated from the fitted curve.

 

 

The portal vein pressure increased significantly in the cirrhotic rats compared to that in the controls (15.1±1.6 vs 5.9±0.9 mmHg, n=7-8, P<0.01), and there was no significant difference between GRK-2 inhibitor-treated and untreated rats group (cirrhosis or control).

 

The portal vein pressure was significantly increased in the cirrhotic patients compared with the controls (25.6±1.8 vs 13.7±2.0 mmHg, n=5, P<0.01).

 

To confirm the arteriole hypocontractility to norepinephrine of cirrhotic rats and the effect of GRK-2 inhibitor, the contractility of isolated arteriole to norepinephrine was assessed in the control and cirrhotic rats with or without treated with GRK-2 inhibitor. As shown in Fig. 1, compared with the CS and CG groups, the dose-response curve of the mesenteric arteriole in response to norepinephrine shifted to the right and the EC₅₀ increased in the cirrhotic rats. However, in mesenteric arterioles from the cirrhotic rats treated with GRK-2 inhibitor, the dose-response curve shifted toward the control group and EC₅₀ was decreased (P<0.01).

 

Compared with the control group, the dose-response curve of the mesenteric arteriole in response to norepinephrine shifted to the right and the EC₅₀ was increased in the patients with cirrhosis (P<0.01; Fig. 2).

 

The expressions of α₁ adrenergic receptor, G proteins, GRK-2, and β-arrestin-2 in rat mesenteric arteries were compared among the four groups by Western blot. There were no significant differences in protein expressions of the α₁ adrenergic receptor, Gαq/11, Gα12, and Gα13 (P>0.05; Fig. 3). However, GRK-2 and β-arrestin-2 were significantly increased in the cirrhotic group compared with those in the control group with or without treatment by GRK-2 inhibitor. GRK-2 inhibitor did not change the expression of GRK-2 in the mesenteric artery; GRK-2 inhibitor significantly decreased the expression of β-arrestin-2 (P<0.01; Fig. 3).

 

There were no significant differences in expressions of the α₁ adrenergic receptor, Gαq/11, Gα12, and Gα13 (P>0.05; Fig. 4). Protein GRK-2 and β-arrestin-2 were significantly increased in the cirrhotic group compared with the control group (P<0.01; Fig. 4).

 

The interaction between the α₁ adrenergic receptor and β-arrestin-2 in rat and human mesenteric arteries was investigated by co-immunoprecipitation, and the results showed that the adrenergic receptor co-immunoprecipitated with β-arrestin-2. Moreover, the interaction of the α₁ adrenergic receptor and β-arrestin-2 was much stronger in the cirrhotic rats than in the control group. However, in the cirrhotic rats, GRK-2 inhibitor interrupted the interaction between the α₁ adrenergic receptor and β-arrestin-2 (P<0.01; Fig. 5). Very similar findings were obtained in human mesenteric arteries. The binding of β-arrestin-2 to the α₁ adrenergic receptor was higher in mesenteric arteries from patients with cirrhosis than that in mesenteric arteries of the control group (P<0.01; Fig. 5).

 

The protein expressions of ROCK-1, moesin, and p-moesin were compared among the four groups in rats. Moesin is a substrate of ROCK and its phosphorylation was analyzed in order to detect ROCK activity. Western blot showed that the expression of ROCK-1, moesin, and p-moesin were not significantly different in the four groups (P>0.05; Fig. 6).

 

We also detected the expressions of ROCK-1, moesin and p-moesin in human mesenteric arteries with Western blot. Interestingly, the expression levels of ROCK-1 and p-moesin were significantly decreased in mesenteric arteries in patients with cirrhosis compared to those in the control group (P<0.01), while the expression level of mosein was not significantly changed (P>0.05; Fig. 7).

 

We compared the protein expression and activity of ROCK-1 among the four groups with or without stimulation by norepinephrine. The results suggested that the expression of ROCK-1 was not significantly changed after stimulation with norepinephrine (P>0.05; Fig. 8). The p-moesin but not moesin increased significantly after norepinephrine stimulation in CS and CG rats (P<0.01; Fig. 9). The cirrhotic rats showed no changes in the expression of moesin and p-moesin after stimulation with norepinephrine (P>0.05). However, GRK-2 inhibitor sensitized the response of p-moesin to norepinephrine (P<0.01; Fig. 9).

 

 

The complexity of the body's environment including neural, humoral, and mechanical factors is related to vascular reactivity. Therefore, studies on vascular reactivity should be carried out by eliminating interfering factors as much as possible. In the present study, mesenteric arterioles were isolated and fixed in a perfusion tank that was filled with MOPS-PSS to provide a stable environment for vascular metabolism. This also helped to maintain the intravascular pressure and thereby eliminate the influence of fluid flow on the vascular response. The intravascular pressure remained at 80 mmHg for two reasons. First, the vascular diameter increases with the elevation of pressure but remains unchanged when the pressure is kept at 80 mmHg.^[18] Second, 80 mmHg is close to the mean arterial blood pressure of humans and rats.

 

In previous studies on the mechanism that is responsible for vascular reactivity, aortic rings hypo-contractility was investigated both in bile duct ligation (BDL) or CCl₄ induced cirrhotic models.^{[9, 19, 20]} Ferlitsch et al^[21] reported that the responses of forearm arteries to norepinephrine and AT-II were decreased in patients with cirrhosis. The mean portal vein pressure of our cirrhotic rats was around 15 mmHg, which was in line with the diagnostic standard of portal hypertension in rats. Our data suggested that mesenteric arteriole sensitivity and contractility in response to norepinephrine were decreased in both cirrhotic patients and rats, indicating the hyporesponsiveness of splanchnic and peripheral vessels to vasoconstrictors. Although the hyporeactivity of aortic rings to vaso-constrictor has been will documented,^{[9, 19, 20, 22, 23]} the splanchnic vasculature exhibits more hyporeactivity to vasoconstrictor. It is a microvessel with a diameter of 10 to 150 µm that is essential in regulating vascular resistance, not the conduit vessel like aorta. In our study, the third-order mesenteric artery was used to study vascular reactivity, the diameter of which was just inside this range. In addition, a microscopic amplification system was applied in order to precisely observe the small changes in the splanchnic capillary bed that occurs under the effect of a vasoconstrictor.

 

GPCRs comprise a large protein family and more than 1000 members have been discovered in recent years, including the adrenergic receptor, the AT-II receptor and the M-type acetylcholine receptor. These proteins connect to their corresponding ligands and couple with G proteins to transmit extracellular signals into cells and produce specific effects. The persistent existence of an agonist will result in receptor desensitization, which is mainly mediated by two major proteins: GRK-2 and β-arrestins. GRK-2, which is actually a group of protein kinases that specifically recognize and phosphorylate agonist-activated GPCRs, has attracted the interest of researchers. As a ubiquitous GRK family member, GRK-2 appears to play a central, integrative role in signal transduction pathways known to modulate intracellular effectors involved in the function of blood vessels. The phosphorylation of GPCR mediated by GRK-2 promotes the binding of β-arrestin-2 to GPCR. β-arrestin-2 is ubiquitously expressed and uncouples the receptor from G proteins, leading to interruption of various signal transduction pathways such as RhoA/ROCK.

 

In the current study, the protein levels of the α₁ adrenergic receptor and related G-protein subunits showed no significant differences in the cirrhotic patients and rats compared with the controls. However, the expression of β-arrestin-2, which is closely related to α₁ adrenergic receptor desensitization,^{[8, 24]} increased significantly, whereas the corresponding G proteins exhibited no change. This suggests that the decreased reactivity of the mesenteric artery to contractors was the desensitization and not the quantity change of receptor and G proteins. In addition, the interaction between the α₁ adrenergic receptor and β-arrestin-2 has been reported in a previous study by co-immunoprecipitation.^[25] We found that in the mesenteric arteries in rats with cirrhosis, the binding capacity increased, which indicates a stronger interaction between the two proteins. This phenomenon was also consistent with the finding of the studies focusing on human mesenteric arteries. The increased binding of β-arrestin-2 to GPCRs such as the α₁ adrenergic receptor should inhibit the dissociation of Gα, Gβ, and Gγ, thereby leading to the uncoupling of receptors from G proteins and resulting in desensitization. GRK-2 causes phosphorylation and activation in GPCRs, β-arrestin-2 bound with GPCRs neutralizes GPCRs over activation due to GRK-2 stimulation.^[6] Such phenomenon was observed not only in isolated arteries but also in cytological studies. However, the exact mechanism of this phenomenon remains to be elucidated.^{[26, 27]}

 

Studies^{[28, 29]} have shown that ROCK is a critical mediator of vasocontraction. Our study did not show significant difference in both ROCK protein expression and reactivity among the four groups of rats. To further investigate the effects of enhanced desensitization on the calcium sensitization pathway, the changes in ROCK protein expression and its activity in rats were detected upon stimulation with norepinephrine (10 µmol/L).

 

In our study, norepinephrine stimulation did not change the protein expression of ROCK and moesin but the stimulation increased p-moesin significantly. This finding suggests that it was the activity, not the expression of ROCK, that was affected by norepinephrine. As mentioned above, ROCK activity can be determined by the level of threonine phosphorylation at position 558. In this study, p-moesin expression was significantly increased in the control group after norepinephrine stimulation but not in the cirrhotic group. Therefore reduced smooth muscle contractility may be a result of the increased β-arrestin-2 expression and its stronger interaction with the α₁ adrenergic receptor, which hindered G-protein-dependent signaling and prevented the vasoconstriction due to the activation of ROCK. In this study, a highly selective GRK-2 inhibitor was applied to inhibit GRK-2 activity but not the expression level.^[11] When the activity of GRK-2 was inhibited, the expression of β-arrestin-2 protein was significantly decreased in rat mesenteric arteries and therefore, its interaction with the α₁ adrenergic receptor was decreased, which released the inhibition of smooth muscle contractility, and recovered the sensitivity of smooth muscle to norepinephrine. This further confirmed the role of receptor desensitization by β-arrestin-2 in inhibiting the transmission of contracting signals.

 

In the present study, increased expression of β-arrestin-2 and decreased activity of ROCK protein were all observed in the cirrhotic patients and rat models induced by CCl₄, but the exact mechanisms of vascular hypocontractility were not exactly the same. The expression and activity of ROCK in mesenteric arteries were the same in rats, whereas ROCK expression and activity were significantly decreased in the patients with cirrhosis compared to those in the controls. A down-regulation of ROCK also contributes to vascular hypocontractility in the cirrhotic rats induced by BDL.^[19] Overexpression of β-arrestin-2 might impair contractile signaling in vascular smooth muscle via different mechanisms (Fig. 10). Firstly, binding of β-arrestin-2 to GPCRs causes desensitisation of the G-protein-dependent signaling pathway, including the calcium sensitization pathway. Secondly, enhanced interaction between GPCRs and β-arrestin-2 might result in overactivation of the β-arrestin-2-dependent signaling pathway, and subsequently exaggerated β-arrestin-2-mediated extracellular signal-regulated kinase (ERK) activation may be responsible for post-transcriptional downregulation of ROCK expression. Indeed, pharmacological ERK inhibition such as sorafenib in BDL rats results in up-regulation of ROCK expression and contractility.^[30] The lack in CCl₄ rats of ROCK expression down-regulation found in BDL rats and patients with cirrhosis may demonstrate differences in the β-arrestin-2/ROCK regulation in arteries. Although the existence of apparently very similar features of β-arrestin-2 expression increasing in cirrhosis, the β-arrestin-2 mediated ERK pathway was not over-stimulated in CCl₄ rats, whereas the over-activation of ERK pathway significantly inhibited the expression of ROCK in BDL rats and patients with cirrhosis. This finding remains to be further investigated.

 

In conclusion, we found that β-arrestin-2 expression and its interaction with the α₁ adrenergic receptor are upregulated in the mesenteric arteries in cirrhotic patients and rats. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These results may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.

 

 

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