1. Volume 130, Issue 3, Pages 838-854 (March 2006)
Defective RhoA/Rho-Kinase Signaling Contributes to Vascular Hypocontractility and Vasodilation in Cirrhotic Rats 
Martin Hennenberg, Erwin Biecker, Jonel Trebicka, Kerstin Jochem, Qi Zhou, Martina Schmidt, Karl H. Jakobs, Tilman Sauerbruch, Jörg Heller 
Gastroenterology 
Volume 130, Issue 3, Pages (March 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Terms and Conditions

2. Figure 1
Contractile pathways in vascular smooth muscle. Agonist activation of vasopressor receptors coupled to Gαq/11 and Gα12/13-types of G-proteins stimulates the PLC/IP3 and the RhoA/Rho-kinase pathways, both resulting in increased MLC phosphorylation and thus contraction of the vascular smooth muscle cell. Contraction induced by high molar KCl occurs exclusively via activation of Ca2+/calmodulin-dependent MLC kinase after depolarization of the vascular smooth muscle cell.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

3. Figure 2
Contraction of isolated aortic rings from sham-operated and BDL rats in response to 10 μmol/L methoxamine (A) and 80 mmol/L KCl (B) and effect of inhibition of endogenous NO production by 200 μmol/L L-NAME on contraction. (A) †P < .05 vs sham without L-NAME and BDL with L-NAME, n = 10–11 for sham-operated, and n = 11 for BDL rats. (B) Differences between groups are not significant, n = 9–10 for sham-operated and n = 10 for BDL rats.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

4. Figure 3
mRNA expression of RhoA (A) and Rho-kinase (B) in aortas from sham-operated (n = 7) and BDL rats (n = 7), as determined by quantitative RT-PCR. Lower ΔCT values denote higher mRNA levels.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

5. Figure 4
Protein expression of RhoA (A) and Rho-kinase (B) in aortas from sham-operated (n = 12) and BDL rats (n = 12), as determined by immunoblots with specific antibodies. Shown are representative Western blots (left panels) and densitometric quantification of all experiments (right panels), with the densitometric units (d.u.) found in the sham set to 100%.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

6. Figure 5
Activation of RhoA in aortas from sham-operated (n = 14) and BDL rats (n = 8) without (−) and with (+) stimulation with 100 μmol/L methoxamine, determined by pull down of GTP-RhoA with GST-Rhotekin coupled to glutathione Sepharose beads. The upper panels show representative Western blots of the levels of total RhoA and GTP-RhoA, whereas, in the lower panels, densitometric quantification of GTP-RhoA from all experiments is presented, with the level of GTP-RhoA found in unstimulated samples set to 100%.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

7. Figure 6
Membrane and cytosolic content of RhoA in aortas from sham-operated (n = 8) and BDL rats (n = 4) without (−) and with (+) stimulation with 100 μmol/L methoxamine, determined by ultracentrifugation of aortic homogenates and subsequent Western blots. Note that samples for sham-operated and BDL rats were run in separate gels, and, therefore, different exposure times were used for the development of the Western blots in these experiments. The upper panel shows representative Western blots of RhoA in the membrane and cytosolic fractions, whereas, in the lower panels, densitometric quantification of membrane-associated RhoA from all experiments is presented, with the level of membrane-associated RhoA found in unstimulated samples set to 100%.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

8. Figure 7
Membrane and cytosolic content of RhoA in aortas from sham-operated (n = 4) and BDL rats (n = 4) without (−) and with (+) stimulation with 80 mmol/L KCl. The procedure was the same as that for Figure 6. Shown are representative Western blots (upper panels) and densitometric quantification of membrane-associated RhoA from all experiments (lower panels), with the level of membrane-associated RhoA found in unstimulated samples set to 100%.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

9. Figure 8
Basal, constitutive phosphorylation state of moesin in aortas from sham-operated (n = 8) and BDL rats (n = 8). Shown is a representative experiment (upper panels) and densitometric quantification of P-moesin from all experiments (lower panels), with the level of P-moesin found in aortas from sham-operated rats set to 100%.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

10. Figure 9
Phosphorylation of moesin in aortas from sham-operated (n = 4) and BDL rats (n = 4) without (−) and with (+) stimulation with 100 μmol/L methoxamine. P-moesin and total moesin levels were detected by Western blot analysis using a site- and phospho-specific anti-moesin and a nonphospho-specific anti-moesin antibody, respectively. The upper panels show representative Western blots of total and P-moesin, whereas, in the lower panels, densitometric quantification of P-moesin from all experiments is presented, with the level of P-moesin found in unstimulated samples set to 100%.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

11. Figure 10
Methoxamine stimulates moesin phosphorylation in intact rat aortic smooth muscle, which is mediated by Rho-kinase. Two of 3 samples of intact rat aorta (from sham-operated animals, n = 6) were stimulated for 10 minutes with 100 μmol/L of the α1-adrenoceptor agonist methoxamine. Prior to stimulation with methoxamine, 1 sample was preincubated with 1 μmol/L of the Rho-kinase inhibitor Y (20 minutes). Methoxamine alone induced a significant increase in aortic moesin phosphorylation, which was completely blocked by Y Thus, in the intact rat aorta, the phosphorylation state of moesin reflects the activity of Rho-kinase. Shown are representative Western blots (upper panels) and densitometric quantification of P-moesin from all experiments (lower panels), with the level of P-moesin found in unstimulated samples set to 100%.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

12. Figure 11
Phosphorylation of moesin in aortas from sham-operated (n = 4) and BDL rats (n = 4) without (−) and with (+) stimulation with 80 mmol/L KCl. The procedure was the same as that given for Figure 9. Shown are representative Western blots (upper panels) and densitometric quantification of P-moesin from all experiments (lower panels), with the level of P-moesin in unstimulated samples set to 100%.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

13. Figure 12
Relaxation of isolated aortic rings by Y-27632, concentration-response curves, and effect of inhibition of endogenous NO production. Aortic rings from sham-operated and BDL rats were precontracted with 10 μmol/L methoxamine (A–D) or 80 mmol/L KCl (E and F). Thereafter, cumulative concentration-response curves for relaxation by Y were constructed. (A) Intact aortic rings (without L-NAME, with intact endothelium), n = 9 for sham-operated, n = 10 for BDL rats. (B) In vitro preincubation with 200 μmol/L L-NAME, n = 9 for sham-operated, n = 11 for BDL rats. (C) Endothelium-denuded aortic rings, n = 7 for sham-operated, n = 5 in BDL rats. (D) Aortic rings from chronic L-NAME-treated rats, n = 6 for sham-operated, n = 5 for BDL rats. (E) Intact aortic rings (without L-NAME, with intact endothelium), precontracted with 80 mmol/L KCl, n = 7 for sham-operated, n = 8 for BDL rats. (F) Aortic rings precontracted with 80 mmol/L KCl, after in vitro preincubation with 200 μmol/L L-NAME, n = 5 for sham-operated, n = 9 for BDL rats.
Gastroenterology  , DOI: ( /j.gastro )
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14. Figure 13
Effects of a bolus injection of Y (1 mg/kg) on MAP in anesthetized sham-operated (n = 13) and BDL rats (n = 12). P < .05 for sham vs BDL, 2-way ANOVA, †P < .05 vs BDL.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions