The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca

Abstract-Rho kinases (ROCKs) are the first and the best-characterized effectors of the small G-protein RhoA. In addition to their effect on actin organization, or through this effect, ROCKs have been found to regulate a wide range of fundamental cell functions such as contraction, motility, proliferation, and apoptosis. Abnormal activation of the RhoA/ROCK pathway has been observed in major cardiovascular disorders such as atherosclerosis, restenosis, hypertension, pulmonary hypertension, and cardiac hypertrophy. This review, based on recent molecular, cellular, and animal studies, focuses on the current understanding of ROCK signaling and its roles in cardiovascular physiology and pathophysiology. (Circ Res. 2006;98:322-334.)Key Words: Rho kinase Ⅲ cardiovascular diseases Ⅲ Rho-GTP-binding proteins Ⅲ signal transduction R hoA is one of the best-known members of the Rho protein family that, in addition to its effect on actin organization or through this effect, regulate a wide range of fundamental cell functions such as contraction, motility, proliferation, and apoptosis. 1 RhoA acts as a molecular switch that cycles between an inactive GDP-bound and an active GTP-bound conformation interacting with downstream targets (effectors) to elicit cellular responses. Rho kinases (ROCKs) are the first and the bestcharacterized RhoA effectors. However, ROCKs can be considered more generally as Rho effectors because they also bind other Rho proteins such as RhoB and RhoC. 2 Since their discovery in 1996, ROCKs have been extensively studied, leading to the publication of Ͼ1300 articles, many of which focus on ROCK functions in the cardiovascular system. The interest for ROCKs in the heart and vessels has been further reinforced by the observation that the beneficial cardiovascular effects of statins result, at least in part, from the inhibition of ROCKs. 3 Indeed, by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase, statins reduce cholesterol synthesis but also prevent the formation of geranylgeranylpyrophosphate required for membrane translocation and activation of RhoA, the main upstream activator of ROCKs. In this review, we describe the current understanding of ROCK signaling and its roles in cardiovascular physiology and pathophysiology.

Hypertension is one of the most frequent pathologies in the industrialized world. Although recognized to be dependent on a combination of genetic and environmental factors, its molecular basis remains elusive. Increased activity of the monomeric G protein RhoA in arteries is a common feature of hypertension. However, how RhoA is activated and whether it has a causative role in hypertension remains unclear. Here we provide evidence that Arhgef1 is the RhoA guanine exchange factor specifically responsible for angiotensin II-induced activation of RhoA signaling in arterial smooth muscle cells. We found that angiotensin II activates Arhgef1 through a previously undescribed mechanism in which Jak2 phosphorylates Tyr738 of Arhgef1. Arhgef1 inactivation in smooth muscle induced resistance to angiotensin II-dependent hypertension in mice, but did not affect normal blood pressure regulation. Our results show that control of RhoA signaling through Arhgef1 is central to the development of angiotensin II-dependent hypertension and identify Arhgef1 as a potential target for the treatment of hypertension.

The aim of this work was to investigate the coupling of human urotensin II (hU-II) to RhoA activation and regulation of RhoA-dependent functions. The use of the Rho-kinase inhibitor Y-27632 and the development of a membrane-permeant RhoA inhibitor (TAT-C3) allowed us to demonstrate that hU-II induced arterial smooth muscle contraction, actin stress fiber formation, and proliferation through the activation of the small GTPase RhoA and its downstream effector Rho-kinase.T he human homologue of the fish dodecapeptide urotensin II (hU-II) has been recently cloned. 1 Prepro-U-II mRNA was highly expressed in spinal cord but also found in the adrenal glands, kidney, and spleen. 1,2 hU-II-like immunoreactivity was detected in the vasculature and a diffuse staining was observed in the heart. 3 hU-II induced vasoconstriction of arteries from both rat and human. [3][4][5] With a potency Ϸ6-to 28-fold greater than endothelin-1 in nonhuman primate arteries, hU-II is the most potent mammalian vasoconstrictor identified so far. 3 hU-II has been defined as the ligand for the orphan receptor GPR14, 2,3 predominantly expressed in cardiovascular tissues. 3 Recombinant GPR14 coupled to Ca 2ϩ mobilization, and hU-II has been reported to produce a phospholipase C-dependent increase in inositol phosphates. 6 However, the intracellular signaling pathways of hU-II are not fully established.The small GTPase RhoA is now recognized as a major regulator of smooth muscle (SM) contraction involved in the control of arterial tone. 7 Thus, we postulate that hU-II should activate RhoA and regulate RhoA-dependent functions in vascular smooth muscle cells (SMCs). Materials and Methods Tension MeasurementsWistar rats (Janvier, France) were stunned and then killed by cervical dislocation. Isometric tension of endothelium-denuded arterial rings of thoracic aorta from the 2-cm portion proximal to the carotid bifurcation and pulmonary artery was measured as previously described. 8 Measurement of RhoA DistributionEndothelium-denuded aortic rings were stimulated with 0.1 mol/L hU-II. When maximal tension was raised, rings were rapidly frozen in liquid nitrogen then homogenized in lysis buffer. Membrane and cytosolic fractions were prepared and analyzed by Western blot using a mouse monoclonal anti-RhoA antibody (Santa Cruz Biotechnology) as previously described. 8 All experiments were approved by the local ethics committee. Plasmid Constructions and TAT-C3 Protein PurificationcDNA encoding for Clostridium botulinum C3 exoenzyme was cloned in frame, in the C-terminal of the HIV TAT protein transduction domain (AA 47-57) in vector pTAT-HA (kindly provided by S. Dowdy, Washington University, St. Louis, Mo). 9 Recombinant TAT-C3 protein was produced in Escherichia coli and purified as previously described. 9 SMC Culture and Actin StainingRat SMCs from the proximal segment of thoracic aorta were isolated by enzymatic dissociation and cultured as previously described. 8 Polymerized (F) actin was stained with FITC-conjugated phalloidin (5 g/mL) and Texas Red-la...

SUMMARY1. The action of carbachol, which activates muscarinic receptors, was studied in single patch-clamped cells where free internal calcium concentration in the cell (Call) was estimated using the emission from the dye Indo-1. Cells were dialysed with potassium-free caesium solution to block any Ca2+-activated K+-current. 2. Carbachol applied to the cell evoked an initial peak in CaF+ followed by a smaller sustained rise (plateau) upon which several oscillations in Ca2+ were often superimposed; the changes in inward, cationic current (icarb) followed changes in Ca2+ closely. Calcium entry blocker did not affect these responses.3. The initial peak in Ca2+ produced by carbachol was due to calcium store release:it was essentially unchanged at + 50 mV, and abolished by prior application of caffeine (10 mM) to the cell or by inclusion of heparin (which blocks D-myoinositol 1,4,5-trisphosphate receptors) in the pipette. In contrast, the rise in CaF+ produced by ATP in rabbit ear artery smooth muscle cells was unaffected by caffeine or heparin as it was due to calcium entry into the cell. 4. The later sustained rise (plateau) in Ca2+ produced by carbachol was due to the entry of calcium into the cell down its electrochemical gradient as it was affected by changing the cell membrane potential or the calcium concentration of the bathing solution. As the sustained rise in CaF+ produced by caffeine had similar properties, it was suggested that depletion of calcium stores can evoke an increased calcium entry into the cell through some pathway.5. The cationic current evoked by carbachol was strongly dependent on Ca2+. It was small if any rise in Ca2+ due to calcium store release was prevented by the inclusion of heparin in the pipette solution and increased greatly if calcium entry was provoked through voltage-dependent channels by applying a depolarizing pulse or if calcium was released from stores by caffeine.6. In the longitudinal muscle of guinea-pig small intestine, activation of muscarinic receptors by carbachol results in the opening of cationic channels; the resulting depolarization increases the frequency of action potential discharge and this determines the degree of contraction. Muscarinic receptor activation opens cationic channels by two mechanisms: release of stored calcium increases Ca2+ and this strongly potentiates a primary mechanism which may involve a G-protein.MS 9086

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Arterial hypertension is a common health problem that affects 25% of the adult population in industrialized societies, and is a major risk factor for myocardial infarction and stroke. However, the pathogenesis of hypertension, as well as the basic mechanisms of blood-pressure control, are insufficiently understood. Although the development of hypertension is complex, involving many different mechanisms, including dysregulation of the autonomic nervous system, renal function, and the balance between water and electrolytes, and increased vascular tone and the resulting rise in peripheral vascular resistance are major determinants of the elevated arterial pressure in hypertension. Since the discovery of the essential role of RhoA and its downstream target, Rho kinase, in the regulation of vascular tone, as well as the antihypertensive effect of a Rho kinase inhibitor, much evidence has accumulated to implicate activation of Rho family proteins in the pathogenesis of hypertension. RhoA remains the most-analyzed member of the Rho proteins in the context of vascular physiology and hypertension, but evidence is accumulating that also points to a role of Rac1 in arterial pathophysiology. In this Review, we discuss progress in our understanding of the role of Rho proteins and their regulators in the pathogenesis of high blood pressure.

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