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Department of Clinical and Experimental Medicine, Clinica Medica 4, University of Padova, Italy
Paul A. Davis
Department of Internal Medicine, University of California, Davis
Michelangelo Sartori; Andrea Semplicini; Achille C. Pessina
Department of Clinical and Experimental Medicine, Clinica Medica 4, University of Padova, Italy
To the Editor:
In a recent article, Wolfrum et al1 have shown that in human cells in culture, inhibition of Rho kinase (RKO) activates Akt pathway, which they contend leads to cardiovascular protection via activation of eNOS. ROK (a downstream effector of RhoA G protein) involvement has been advanced in the pathogenesis of hypertension and atherosclerosis.2 This is based on its modulation of regulatory chain phosphorylation of myosin II which contributes to smooth muscle Ca2+ sensitization,3 increased expression of NAD(P)H oxidase,4 and induction of oxidative stress.
We would like to suggest that recent results from our ongoing studies in patients with Bartter and Gitelman syndrome (BS/GS)5 provide additional support for Wolfrum and colleagues’ conclusions as well as additional evidence for the importance of ROK in cardiovascular protection. Of direct relevance to the report of Wolfrum and coworkers1 is our recent demonstration in BS/GS patients that RhoA/Rho Kinase pathway is blunted6 and that the expression of p22phox, a subunit of the multienzymatic complex NADPH oxidase, is reduced.7 BS/GS, caused by gene defects in specific kidney transporters and ion channels, presents a puzzling clinical picture characterized by hypokalemia, sodium depletion, activation of the renin-angiotensin-aldosterone system (RAAS), with increased plasma levels of Ang II and aldosterone, yet normohypotension, reduced peripheral resistance, and hyporesponsiveness to pressor agents.8 Therefore, understanding why patients with BS/GS do not develop hypertension, in spite of high Ang II and activation of RAAS, sheds considerable light on the cellular basis of hypertension. In BS/GS specifically, the short-term Ang II signaling pathway is blunted as documented by the increased regulator of G-protein signaling-2,9 reduced Gq gene and protein expression,10,11 and reduced related downstream cellular events.9–13 The long-term signaling pathway of Ang II, which modulates the cell redox state to promote cardiovascular remodeling and atherosclerosis, is also altered in BS/GS.5–7 The reduced peripheral resistance, vascular hyporeactivity, and normohypotension typical of BS/GS patients and their collection of biochemical characteristics present a mirror image of those found in hypertension. The reduced RKO and p22phox expression noted in our recent studies6,7 occurred in the context of the increased level of the endothelial subunit of NO synthase (eNOS) mRNA14 alongside elevated urinary NO metabolites and cGMP levels.9 This, we suggest, indicates that the RKO activity of BS/GS patients is reduced with induction of Akt pathway in response to RKO downregulation.1 Our findings of reduced RKO expression in the face of increased ecNOS expression and increased NO level present in BS/GS6,9,14 exactly parallel the upregulation of NO system on ROK inhibition as shown by Wolfrum et al.1 Moreover, the induction of the Akt pathway reported by Wolfrum after ROK inhibition is also mirrored in BS/GS, as these patients have an increased expression of heme-oxygenase-1 (HO-1),7 potent antiapoptotic and antioxidant15,16 which has been shown to be under Akt control.17,18 In conclusion, our studies provide "in vivo" in a clinical condition of altered vascular tone regulation such as BS/GS5–14 confirmatory data in support of the conclusion of Wolfrum et al, derived by "in vitro" studies, that inhibition of ROK is important for cardiovascular protection.1
References
Wolfrum S, Dendorfer A, Rikitake Y, Stalker TJ, Gong Y, Scalia R, Dominiak P, Liao PK. Inhibition of Rho-kinase leads to rapid activation of phosphatidylinositol 3 kinase/protein kinase Akt and cardiovascular protection. Arterioscler Thromb Vasc Biol. 2004; 24: 1842–1847.
Masumoto A, Hirooka Y, Shimokawa H, Hironaga K, Setoguchi S, Takeshita A. Possible involvement of Rho-kinase in the pathogenesis of hypertension in humans. Hypertension. 2001; 38: 1307–1310.
Wettschureck N, Offermanns S. Rho/Rho-kinase mediated signaling in physiology and pathophysiology. J Mol Med. 2002; 80: 629–638.
Higashi M, Hiroki J, Hattori T, Mukai Y, Morikawa K, Ichiki T, Takahashi S, Takeshita A, Shimokawa H. Long-term inhibition of Rho-kinase suppresses angiotensin II–induced cardiovascular hypertrophy in rats in vivo: effect on endothelial NAD(P)H oxidase system. Circ Res. 2003; 93: 767–775.
Calo‘ L, Davis PA, Semplicini A. Bartter’s/Gitelman’s syndrome: a model for the relationships between hypertension, angiotensin II, oxidative stress and remodeling. Clin Nephrol. 2003; 59: 393–394.
Pagnin E, Davis PA, Sartori M, Semplicini A, Pessina AC, Calò LA. Rho kinase and plasminogen activator inhibitor (PAI)-1 in Bartter’s/Gitelman’s syndromes: relationship to angiotensin II signaling. J Hypertens;. 2004; 22: 1963–1969.
Calò LA, Pagnin E, Davis PA, Sartori M, Semplicini A. Oxidative stress related factors in Bartter’s and Gitelman’s syndromes: relevance for angiotensin II signalling. Nephrol Dial Transplant. 2003; 18: 1518–1525.
Calò L, Davis PA, Semplicini A. Control of vascular tone in the syndromes of Bartter and Gitelman. Crit Rev Clin Lab Sci. 2000; 37: 503–522.
Calò LA, Pagnin E, Davis PA, Sartori M, Ceolotto G, Pessina AC, Semplicini A. Increased expression of regulator of G protein signaling–2 (RGS-2) in Bartter’s/Gitelman’s syndrome: a role in the control of vascular tone and implication for hypertension. J Clin Endocrinol Metab. 2004; 89: 4153–4157.
Calò L, Ceolotto G, Milani M, Pagnin E, van den Heuvel LP, Sartori M, Sartori M, Davis PA, Costa R, Semplicini A. Abnormalities of Gq-mediated cell signaling in Bartter and Gitelman syndromes. Kidney Int. 2001; 60: 882–889.
Calo‘ L, Davis PA, Semplicini A. Reduced content of alpha subunit of Gq protein in monocytes of Bartter and Gitelman syndromes: relationship with vascular hyporeactivity. Kidney Int. 2002; 61: 353–354.
Calo L, D’Angelo A, Cantaro S, Rizzolo M, Favaro S, Antonello A, Borsatti A. Intracellular calcium signalling and vascular reactivity in Bartter’s syndrome. Nephron. 1996; 72: 570–573.
Di Virgilio F, Calo‘ L, Cantaro S, Favaro S, Piccoli A, Borsatti A. Resting and stimulated cytosolic free calcium levels in neutrophils from patients with Bartter’s syndrome. Clin Sci. 1987; 72: 483–488.
Calò L, Davis PA, Milani M, Cantaro S, Antonello A, Favaro S, D’Angelo A. Increased endothelial nitric oxide synthase mRNA level in Bartter’s and Gitelman’s syndrome. Relationship to vascular reactivity. Clin Nephrol. 1999; 51: 12–17.
Maines MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol. 1997; 37: 517–554.
Durante W. Heme oxygenase-1 in growth control and its clinical application to vascular disease. J Cell Physiol. 2003; 195: 373–382.
Lee TS, Chang CC, Zhu Y, Shyy JY. Simvastatin induces heme oxygenase-1: a novel mechanism of vessel protection. Circulation. 2004; 110: 1296–1302.
Martin D, Rojo AI, Salinas M, Diaz R, Gallardo G, Alam J, De Galarreta CM, Cuadrado A. Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol. J Biol Chem. 2004; 279: 8919–8929.
In Response:
Sebastian Wolfrum; Yoshiyuki Rikitake; James K. Liao
Vascular Medicine Research Unit, Brigham & Women’s Hospital and Harvard Medical School, Cambridge, Mass
Andreas Dendorfer; Peter Dominiak
Institute of Pharmacology and Toxicology, University of Schleswig-Holstein, Campus Lübeck, Germany
Timothy J. Stalker; Yulan Gong; Rosario Scalia
Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pa
Calò and coworkers suggest that signal pathways in patients experiencing Bartter and Gitelman syndrome may be similar to those activated by the acute inhibition of Rho-kinase as observed in our study.1 Calò and colleagues have shown that the expression of Rho-kinase mRNA is reduced in mononuclear cells of these patients. This is associated with a decreased expression of p22phox, a subunit of the NADPH oxidase, but an increased expression of eNOS and heme-oxygenase-1.2–4 Therefore, decreased activity of Rho-kinase in these patients may lead to the chronic activation of Akt and eNOS.
However, in our recent work, we investigated the short-term effects of Rho-kinase inhibition on Akt activity. The effects were observed within minutes after Rho-kinase inhibition. This time course supports a posttranslational modification, eg, phosphorylation of eNOS, by Akt, which has also been seen by Ming and coworkers.5 This, however, does not exclude chronic effects of Rho-kinase inhibition on eNOS expression. Indeed, we have previously reported that eNOS mRNA is negatively regulated by Rho GTPase and Rho-kinase. We showed that activation of Rho destabilizes eNOS mRNA stability and that inhibition of Rho-kinase by hydroxyfasudil blocks hypoxia-induced downregulation of eNOS in endothelial cells.6,7 This upregulation of eNOS expression was not mediated by activation of Akt, as demonstrated by Ming et al.5 Therefore, we believe that stabilization of eNOS mRNA by long-term Rho-kinase inhibition may explain the changes in eNOS expression observed by Calò and coworkers in patients with Bartter and Gitelman syndrome.
References
Wolfrum S, Dendorfer A, Rikitake Y, Stalker TJ, Gong Y, Scalia R, Dominiak P, Liao JK. Inhibition of Rho-kinase leads to rapid activation of phosphatidylinositol 3-kinase/protein kinase Akt and cardiovascular protection. Arterioscler Thromb Vasc Biol. 2004; 24: 1842–1847.
Pagnin E, Davis PA, Sartori M, Semplicini A, Pessina AC, Calo LA. Rho kinase and plasminogen activator inhibitor (PAI)-1 in Bartter’s/Gitelman’s syndromes: relationship to angiotensin II signaling. J Hypertens. 2004; 22: 1963–1969.
Calo LA, Pagnin E, Davis PA, Sartori M, Semplicini A. Oxidative stress-related factors in Bartter’s and Gitelman’s syndromes: relevance for angiotensin II signalling. Nephrol Dial Transplant. 2003; 18: 1518–1525.
Calo L, Davis PA, Milani M, Cantaro S, Antonello A, Favaro S, D’Angelo A. Increased endothelial nitric oxide synthase mRNA level in Bartter’s and Gitelman’s syndrome: relationship to vascular reactivity. Clin Nephrol. 1999; 51: 12–17.
Ming XF, Viswambharan H, Barandier C, Ruffieux J, Kaibuchi K, Rusconi S, Yang Z. Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells. Mol Cell Biol. 2002; 22: 8467–8477.
Takemoto M, Sun J, Hiroki J, Shimokawa H, Liao JK. Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation. 2002; 106: 57–62.
Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem. 1998; 273: 24266–24271.