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
ABSTRACT 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.
- SourceAvailable from: Selçuk Takir
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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 ,"
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.Experimental Eye Research 02/2015; 132. DOI:10.1016/j.exer.2015.02.003 · 3.02 Impact Factor
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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) "
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.Molecular pharmacology 10/2009; 77(2):111-25. DOI:10.1124/mol.109.061234 · 4.12 Impact Factor
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- ") . Thus , MYPT1 Thr696 is unlikely to be a physiological target of the RhoA / ROCK signalling pathway , although it may be involved in pathological conditions ( Seko et al . 2003 ; Guilluy et al . 2005 ; Hilgers et al . 2007 ) . Wooldridge et al . ( 2004 ) showed in permeabilized ileum smooth muscle that PKG could directly phosphorylate MYPT1 at Ser695 and this phosphorylation blocked the adjacent Thr696 phosphorylation , suggesting a cGMP - induced prevention of phosphorylated Thr696 - induced MLCP inhibition and Ca 2+ sensitization . Recently , Nakamura e"
ABSTRACT: Nitric oxide (NO) from endothelium is a major mediator of vasodilatation through cGMP/PKG signals that lead to a decrease in Ca(2+) concentration. In addition, NO-mediated signals trigger an increase in myosin light chain phosphatase (MLCP) activity. To evaluate the mechanism of NO-induced relaxation through MLCP deinhibition, we compared time-dependent changes in Ca(2+), myosin light chain (MLC) phosphorylation and contraction to changes in phosphorylation levels of CPI-17 at Thr38, RhoA at Ser188, and MYPT1 at Ser695, Thr696 and Thr853 in response to sodium nitroprusside (SNP)-induced relaxation in denuded rabbit femoral artery. During phenylephrine (PE)-induced contraction, SNP reduced CPI-17 phosphorylation to a minimal value within 15 s, in parallel with decreases in Ca(2+) and MLC phosphorylation, followed by a reduction of contractile force having a latency period of about 15 s. MYPT1 phosphorylation at Ser695, the PKG-target site, increased concurrently with relaxation. Phosphorylation of RhoA, MYPT1 Thr696 and Thr853 differed significantly at 5 min but not within 1 min of SNP exposure. Inhibition of Ca(2+) release delayed SNP-induced relaxation while inhibition of Ca(2+) channel, BK(Ca) channel or phosphodiesterase-5 did not. Pretreatment of resting artery with SNP suppressed an increase in Ca(2+), contractile force and phosphorylation of MLC, CPI-17, MYPT1 Thr696 and Thr853 at 10 s after PE stimulation, but had no effect on phorbol ester-induced CPI-17 phosphorylation. Together, these results suggest that NO production suppresses Ca(2+) release, which causes an inactivation of PKC and rapid CPI-17 dephosphorylation as well as MLCK inactivation, resulting in rapid MLC dephosphorylation and relaxation.The Journal of Physiology 06/2009; 587(Pt 14):3587-603. DOI:10.1113/jphysiol.2009.172189 · 4.54 Impact Factor