Hilgers, R. H., Todd, J. Jr & Webb, R. C. Increased PDZ-RhoGEF/RhoA/Rho kinase signaling in small mesenteric arteries of angiotensin II-induced hypertensive rats. J. Hypertens. 25, 1687-1697

Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912-3000, USA.
Journal of Hypertension (Impact Factor: 4.72). 09/2007; 25(8):1687-97. DOI: 10.1097/HJH.0b013e32816f778d
Source: PubMed


The phosphorylation of myosin light chain (MLC) maintains the contracted state of vascular smooth muscle. Dephosphorylation results in relaxation and is determined by the activity of myosin light chain phosphatase (MLCP), which is negatively regulated by Rho kinase.
We tested whether an increased Rho kinase activity, and hence a decreased contribution of MLCP, results in an increased contractility of small fourth-order mesenteric arteries (MA) during the early onset of angiotensin II (Ang II)-induced hypertension (Ang II-14d).
Calcium sensitivity was similar, but contractile tension in response to [Ca]ex (5 mmol/l) in endothelium-denuded and depolarized MA was greater, in Ang II-14d rats compared to sham-operated normotensive (SHAM) and Ang II-1d. The Rho kinase inhibitor Y-27,632 caused a significantly greater inhibition of the contractile response to various agents (phenylephrine, norepinephrine, U46,619 and K) in MA of Ang II-14d compared to SHAM. Protein expression levels of the GDP/GTP exchange factor PDZ-RhoGEF, which co-immunoprecipitated with RhoA, were increased in MA of Ang II-14d compared to SHAM. RhoA translocation was greater in U46,619 (1 micromol/l)-stimulated MA of Ang II-14d compared to SHAM. Expression levels of Rho kinase beta were higher in MA of Ang II-14d. The MLCP inhibitor calyculin A (100 nmol/l) caused a greater contraction in MA of SHAM compared to Ang II-14d. Phosphorylation of the target subunit of MLCP (MYPT1) was enhanced in U46,619-stimulated MA of Ang II-14d compared to SHAM.
This is the first study demonstrating enhanced PDZ-RhoGEF/RhoA/Rho kinase signaling during hypertension at the level of resistance-sized arteries. This enhanced signaling leads to increased MLCP phosphorylation, resulting in vascular hyper-reactivity.

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    • " Ca 2þ sensitization . To clarify the interaction between the retina derived relaxation and these cellular targets , the response to RRF on the retinal artery was evaluated in the presence of an inhibitor of MLCK , namely , ML - 7 ( 10 À5 M , 30 min ) ( Mandela and Ordway , 2006 ) or an inhibitor of MLCP , namely Calyculin A ( 10 À7 M , 30 min ) ( Hilgers et al . , 2007 ) . Furtherly , to verify the impact of RRF on Ca 2þ sensi - tization , the retina derived response obtained in the arteries pre - treated with MLCP inhibitor , Calyculin A , was compared with that of Rho kinase inhibitor ,"
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    ABSTRACT: Retinal relaxing factor (RRF) has recently been identified as a novel paracrine regulator of retinal circulation acting differently from well known mediators of the endothelium and the retina. Herein, we aimed to characterize the relaxing mechanism of the retina, i.e. RRF, by evaluating the role of Ca(+2)-dependent and -independent signaling mechanisms as well as inward rectifier K(+) (Kir) channels. Retinal relaxation was determined by placing a piece of retinal tissue just on top of the precontracted bovine retinal arteries mounted in a wire myograph. The retina produced a complete relaxation response, which display a biphasic character, in depolarized arteries contracted by L-type Ca(2+) channel agonist, Bay k 8644. Blockade of L-type Ca(2+) channel by nifedipine, inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid or removal of extracellular Ca(2+) did not influence the prominent relaxation to the retina. Originally, retinal relaxation was found to be unaffected from the inhibition of myosin light chain kinase by ML7, whereas, completely abolished in the presence of myosin light chain phosphatase (MLCP) inhibitor, Calyculin A. Moreover, the inhibition of Rho kinase by its putative inhibitor, Y-27632 displayed comparable relaxant effects to RRF in retinal arteries precontracted either by prostaglandin F2α or K(+), and augmented the moderate response to the retina in K(+) precontracted arteries. In addition, retinal relaxation was significantly inhibited and lost its biphasic character in the presence of Kir channel blocker, Ba(2+). Our results suggested that inhibition of Ca(2+) sensitization through the activation of MLCP, possibly via interfering with Rho kinase, and the opening of Kir channels are likely to be involved in the inhibitory influence of RRF on the retinal arteries. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Feb 2015 · Experimental Eye Research
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    • "The mediated regulation of vascular tone through MLCP and upstream molecules (monomeric G protein RhoA/Rho kinase) is known as calcium (Ca2+) sensitization.3–5 As well as acting directly on vascular smooth muscle as a potent vasoconstrictor in a Ca2+-dependent manner, Ang II also activates the RhoA/Rho kinase signaling transduction pathway and increases Ca2+ sensitization of VSMCs through Rho guanine nucleotide exchange factors (RhoGEFs).1,6–8 The RhoGEFs are a large family of structurally related proteins comprising ~70 members in the human genome.9 "
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    ABSTRACT: Studies to explore angiotensin II (Ang II) and its downstream signaling pathways via Rho guanine nucleotide exchange factors (RhoGEFs) and RhoA signaling are crucial to understanding the mechanisms of smooth muscle contraction leading to hypertension. This study aimed to investigate the Ang II-induced expression of RhoGEFs in vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs) and to identify the possible regulator associated with hypertension. Cultured VSMCs of the aorta from SHRs and Wistar-Kyoto (WKY) rats were treated with or without Ang II or Ang II plus Ang II type 2 receptor antagonists. The expression levels of RhoGEF messenger RNA (mRNA) and protein were determined. To evaluate the changes of aortic ring contractile force in response to Ang II, a nonviral carrier system was adopted to deliver the leukemia-associated RhoGEF (LARG) small interfering RNA via nanoparticles into aortic rings. The baseline mRNA levels of three RhoGEFs in cultured VSMCs of WKY rats did not increase with age, but they were significantly higher in 12-week-old SHRs than in 5-week-old SHRs. Expression levels of LARG mRNA were higher in SHRs than in age-matched WKY rats. The baseline LAGR protein of 12-week-old SHRs was about four times higher than that of WKY rats of the same age. After Ang II-stimulation, LAGR protein expression was significantly increased in 12-week-old WKY rats but remained unchanged in 12-week-old SHRs. LARG small interfering RNA was successfully delivered into aortic rings using nanoparticles. LARG knockdown resulted in 12-week-old SHRs showing the greatest reduction in aortic ring contraction. There were differences in age-related RhoGEF expression at baseline and in response to Ang II-stimulation between SHRs and WKY rats in this study. Nanotechnology can assist in studying the silencing of LARG in tissue culture. The findings of this study indicate that LARG gene expression may be associated with the genesis of hypertension in SHRs.
    Full-text · Article · Dec 2012 · International Journal of Nanomedicine
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    • ", leukocyte homeostasis (Girkontaite et al., 2001; Francis et al., 2006); regulation of apoptosis in thymocytes (Harenberg et al., 2005) Rho LARG Signal transduction downstream of thrombin receptors (Wang et al., 2004); growth cone collapse (Swiercz et al., 2002; Hata et al., 2009); induction of salt-induced hypertension in vascular smooth muscle (Ying et al., 2006; Wirth et al., 2008) Rho PDZ-RhoGEF Signal transduction downstream of LPA receptors (Wang et al., 2004); Ca 2ϩ sensitization in smooth muscle (Derewenda et al., 2004); neutrophil polarization (Wong et al., 2007); neurite retraction (Togashi et al., 2000); angiotensin II induced contraction in vascular smooth muscle cells (Hilgers et al., 2007; Ying et al., 2009) Rho Lbc Promotion of cardiac hypertrophy (Appert-Collin et al., 2007; Carnegie et al., 2008) Rho G␣ q/11 p63RhoGEF G␣ q/11 -dependent activation of RhoA in smooth muscle cells (S. Lutz, personal communication) Rho TrioC In C. elegans, the C-terminal DH/PH domain of UNC-73 promotes acetylcholine vesicle release at neuromuscular junctions (Williams et al., 2007) and regulation of pharynx pumping, speed of locomotion, and egg-laying (Steven et al., 2005) Rho KalirinC Neurite extension and neuronal morphology (Penzes et al., 2001) Rho G␤␥ P-Rex1 ROS generation and rate of chemotaxis in neutrophils (Dong et al., 2005; Welch et al., 2005); Purkinje cell morphology and cerebellar function (Donald et al., 2008) Rac P-Rex2 Purkinje cell morphology and cerebellar function (Donald et al., 2008) "
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    ABSTRACT: Activation of certain classes of G protein-coupled receptors (GPCRs) can lead to alterations in the actin cytoskeleton, gene transcription, cell transformation, and other processes that are known to be regulated by Rho family small-molecular-weight GTPases. Although these responses can occur indirectly via cross-talk from canonical heterotrimeric G protein cascades, it has recently been demonstrated that Dbl family Rho guanine nucleotide exchange factors (RhoGEFs) can serve as the direct downstream effectors of heterotrimeric G proteins. Heterotrimeric Galpha(12/13), Galpha(q), and Gbetagamma subunits are each now known to directly bind and regulate RhoGEFs. Atomic structures have recently been determined for several of these RhoGEFs and their G protein complexes, providing fresh insight into the molecular mechanisms of signal transduction between GPCRs and small molecular weight G proteins. This review covers what is currently known about the structure, function, and regulation of these recently recognized effectors of heterotrimeric G proteins.
    Full-text · Article · Oct 2009 · Molecular pharmacology
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