Published online before print December 20, 2004, doi: 10.1161/01.HYP.0000152701.97426.5f
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(Hypertension. 2005;45:233.)
© 2005 American Heart Association, Inc.
Scientific Contributions |
Thiazide-Like Diuretics Attenuate Agonist-Induced Vasoconstriction by Calcium Desensitization Linked to Rho Kinase
From the Department of Hypertension and Endocrinology (Z.Z., S.Z., T.C., D.L., L.W.), Daping Hospital, Center for Hypertension and Metabolic Disease, Third Military Medical University, Chongqing Hypertension Institute, Chongqing, China; and Charite Campus Benjamin Franklin (M.T.), Berlin, Germany.
Correspondence to Dr Zhiming Zhu, Department of Hypertension and Endocrinology, Daping Hospital, Third Military Medical University, Chongqing 400042, PR China. E-mail zhuzm{at}yahoo.com
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Abstract |
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Lowering blood pressure using thiazide-like diuretics, including chlorthalidone and hydrochlorothiazide, has been proven to be effective in clinical studies. However, the mechanisms by which thiazide-like diuretics lower blood pressure are still poorly understood. To evaluate whether thiazide-like diuretics cause calcium desensitization in smooth muscle cells, we measured their effects on agonist-induced increase of blood pressure in Wistar rats in vivo and on agonist-induced vasoconstriction of aortic rings, DNA synthesis, and protein synthesis, RhoA, Rho kinase, and intracellular calcium in vascular smooth muscle cells in vitro. Thiazide-like diuretics significantly attenuated angiotensin II–induced or norepinephrine-induced increase of systolic blood pressure in rats. Thiazide-like diuretics inhibited agonist-induced vasoconstriction of aortic rings in a concentration-dependent manner in the presence and absence of endothelium. The inhibitory effects of thiazide-like diuretics were similar to that of the specific Rho kinase inhibitor Y27632. RT-PCR and immunoblotting showed that RhoA and Rho kinase were significantly reduced in vascular smooth muscle cells after administration of thiazide-like diuretics. In contrast, thiazide-like diuretics did not affect protein tyrosine phosphatase-2 (SHP-2) expression. Agonist-induced changes of intracellular calcium were not affected by thiazide-like diuretics. The study indicates that thiazide-like diuretics inhibit agonist-induced vasoconstriction by calcium desensitization in smooth muscle cells linked to the Rho–Rho kinase pathway.
Key Words: diuretics • kinase • vasoconstriction
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Introduction |
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Thiazide-like diuretics have been the cornerstone in hypertension management for several years. Recent data from the Antihypertensive and Lipid-Lowering to prevent Heart Attack Trial (ALLHAT) showed that blood pressure lowering using thiazide-like diuretics had beneficial effects, including fewer events for all cardiovascular diseases, stroke, and heart failure compared with an angiotensin-converting enzyme inhibitor.1 Thiazide-like diuretics, including chlorthalidone and hydrochlorothiazide, lower blood pressure by decreasing peripheral resistance rather than by their diuretic effect.2 Although it has been suggested that thiazide-induced vasodilation is mediated by opening of calcium-activated potassium channels or by inhibition of carbonic anhydrase,3–5 the precise mechanisms by which thiazide-like diuretics inhibit vasoconstriction and vascular growth are still unclear.
Rho is a member of the Ras family of small GTP-binding proteins and cycles between a GDP-bound inactive state and a GTP-bound active state. Rho is involved in regulation of actin/myosin-dependent contractility in smooth muscle cells.6 Smooth muscle myosin ATPases are activated after phosphorylation of regulatory myosin light chains by a calcium–calmodulin-dependent myosin light chain kinase, and they are inactivated after dephosphorylation by the calcium-dependent myosin light chain phosphatase. Activation of Rho and its effector Rho kinase inhibits the myosin light chain phosphatase by phosphorylation of the myosin phosphatase–targeting subunit, thereby decreasing activity of myosin phosphatase. Inhibition of the myosin light chain phosphatase causes calcium sensitization (ie, increasing the contraction of vascular smooth muscle cells in the absence of an increase of intracellular calcium concentration).7 Hence, long-term inhibition of the Rho–Rho kinase pathway may cause vasodilation and reduce blood pressure because it can be observed for thiazide-like diuretics.
The aim of the present study was to investigate whether thiazide-like diuretics cause calcium desensitization in smooth muscle cells. The study shows that thiazide-like diuretics inhibit agonist-induced vasoconstriction by calcium desensitization after affecting the Rho–Rho kinase pathway.
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Methods |
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Hemodynamic Measurements
All experiments using male Wistar rats were performed as approved by the animal care and use committee. Rats were randomly administered chlorthalidone hydrochlorothiazide, Rho kinase inhibitor Y27632,8 or placebo for control through gavage for 1 week. Hemodynamic measurements were done according to Symons et al9 after intravenous infusion of angiotensin II or norepinephrine.10 Vasoconstriction of aortic rings was measured as described previously.11,12 Vascular smooth muscle cells were obtained from thoracic aortas and cultured as described.13,14 DNA synthesis and protein synthesis were measured according to Touyz et al.15 Cytosolic calcium concentrations were measured using the fluorescent dye technique.16
RT-PCR and Immunoblotting of RhoA and Rho Kinase
Expression of RhoA and Rho kinase mRNA was assessed by RT-PCR according to Hyvelin et al.17 Immunoblotting was performed using specific antibodies by standard procedures.16 Immunoblots of protein tyrosine phosphatase SHP-2 were performed as described by Wakino et al.18
Statistics
All values reported are mean±SD. Comparisons between groups were analyzed using 1-way ANOVA with Bonferroni post hoc test. Two-sided P values <0.05 were considered to indicate statistical significance.
An expanded Materials and Methods section is available online at http://www.hypertensionaha.org.
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Results |
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Thiazide-Like Diuretics Attenuate Agonist-Induced Blood Pressure Increase in Rats
Effects of thiazide-like diuretics on agonist-induced blood pressure increase were measured in Wistar rats that had been given placebo for control, chlorthalidone, or hydrochlorothiazide for 1 week. Under control conditions, angiotensin II administration significantly increased systolic blood pressure from 111±10 mm Hg to 137±20 mm Hg, whereas norepinephrine administration significantly increased systolic blood pressure from 111±10 mm Hg to 134±12 mm Hg (each n=5; P<0.05 by ANOVA). As shown in Figure 1, the angiotensin II–induced increase of systolic blood pressure was significantly attenuated from 26±17 mm Hg in the control group to 6±7 mm Hg in the chlorthalidone group or to 2±1 mm Hg in the hydrochlorothiazide group (each n=5; P<0.05 by ANOVA). The norepinephrine-induced increase of systolic blood pressure was also significantly attenuated from 24±8 mm Hg in the control group to 2±13 mm Hg in the chlorthalidone group or to 3±10 mm Hg in the hydrochlorothiazide group (each n=5; P<0.05 by ANOVA). In rats given the specific Rho kinase inhibitor Y27632 for 1 week, the angiotensin II- or norepinephrine-induced increase of systolic blood pressure was significantly attenuated to 6±7 mm Hg or 1±9 mm Hg, respectively (each n=5; P<0.05 by ANOVA). These data indicate that thiazide-like diuretics attenuate agonist-induced increase of blood pressure in vivo. The inhibitory effects of thiazide-like diuretics were similar to those observed after inhibition of the Rho–Rho kinase pathway.
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Figure 2 shows the concentration-dependent reduction of angiotensin II–induced vasoconstriction by thiazide-like diuretics. In the presence of endothelium, incubation of segments from rat aorta with 1 µmol/L chlorthalidone or 1 µmol/L hydrochlorothiazide significantly reduced the angiotensin II–induced vasoconstriction to 22±11% or to 22±18%, respectively (each n=6; P<0.01 by ANOVA). Chlorthalidone or hydrochlorothiazide also significantly reduced the angiotensin II–induced vasoconstriction in the presence of the NO synthase inhibitor NG-nitro-L-arginine-methylester to 45±9% or to 41±8%, respectively (each n=4; P<0.05 by ANOVA). Further, the inhibitory effect of thiazide-like diuretics could also observed in the absence of endothelium, indicating that thiazide-like diuretics directly affect vascular smooth muscle cells.
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) of endothelium. Data are mean±SD; each n=3.The inhibitory effects of thiazide-like diuretics could also be observed when the vasoconstriction was induced by norepinephrine. In the presence of endothelium, incubation of segments from rat aorta with 1 µmol/L chlorthalidone or 1 µmol/L hydrochlorothiazide significantly reduced the norepinephrine-induced vasoconstriction to 67±4% or to 53±4%, respectively (each n=6; P<0.01 by ANOVA). In contrast, the initial vasoconstriction induced by potassium chloride was not significantly affected by chlorthalidone or hydrochlorothiazide (82±14% or 83±28%, respectively; each n=6; P=NS). Further, the barium-induced vasoconstriction was not significantly affected by chlorthalidone or hydrochlorothiazide (97±18% or 90±35%; each n=3; P=NS), probably because barium directly causes depolarization and hence vasoconstriction.19
Next, the effects of thiazide-like diuretics were compared with those of the specific Rho kinase inhibitor Y27632. In the presence or absence of endothelium, incubation of segments from rat aorta with 1 µmol/L Y27632 significantly reduced the angiotensin II–induced vasoconstriction to 19±5% or 18±6% (each n=5; P<0.01 by ANOVA). In the presence or absence of endothelium, incubation of segments from rat aorta with 1 µmol/L Y27632 significantly reduced the norepinephrine-induced vasoconstriction to 36±6% or 46±8% (each n=5; P<0.01 by ANOVA). The dose-dependent relaxing effect of Y27632 on segments from aorta contracted with norepinephrine is shown in Figure 2E, indicating that the Rho–Rho kinase system is activated after norepinephrine administration. In the presence or absence of endothelium, incubation of segments from rat aorta with 1 µmol/L Y27632 did not significantly change the barium-induced vasoconstriction (98±12% or 80±25%; each n=3; P=NS).
These data indicate that the angiotensin II- and norepinephrine-induced vasoconstriction in vitro is inhibited by thiazide-like diuretics similar to Rho kinase inhibition.
Effects of Thiazide-Like Diuretics on Angiotensin II–Induced DNA Synthesis, Protein Synthesis, and Intracellular Calcium in Vascular Smooth Muscle Cells
The effects of thiazide-like diuretics on angiotensin II–induced stimulation of DNA synthesis, measured by [3H] thymidine incorporation, and protein synthesis, measured by [3H] leucine incorporation, were investigated (Figure 3). Chlorthalidone and hydrochlorothiazide dose-dependently reduced angiotensin II–induced DNA synthesis and protein synthesis. In the presence of 100 µmol/L chlorthalidone or 100 µmol/L hydrochlorothiazide, the angiotensin II–induced DNA synthesis was reduced to 54±6% or to 47±5% (each n=6; P<0.01 by ANOVA), respectively. In the presence of 100 µmol/L chlorthalidone or 100 µmol/L hydrochlorothiazide, the angiotensin II–induced protein synthesis was reduced to 81±7% (n=6) or to 73±3% (each n=6; P<0.01 by ANOVA), respectively.
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Next, agonist-induced changes of cytosolic calcium were measured in vascular smooth muscle cells using the fluorescent dye technique. Chlorthalidone or hydrochlorothiazide did not significantly change the angiotensin II–induced calcium increase in vascular smooth muscle cells (chlorthalidone 89±11% of control; hydrochlorothiazide 89±35% of control; each n=6; P=NS). Further, chlorthalidone or hydrochlorothiazide did not significantly change the norepinephrine-induced calcium increase in vascular smooth muscle cells (chlorthalidone 81±16% of control; hydrochlorothiazide 85±13% of control; each n=6; P=NS).
Thiazide-Like Diuretics Affect the Rho–Rho Kinase Pathway
Because the agonist-induced vasoconstriction was significantly inhibited by thiazide-like diuretics, whereas calcium increase was not significantly changed, we hypothesized that calcium desensitization in smooth muscle cells might occur after administration of thiazide-like diuretics. Because a reduction of RhoA and Rho kinase is associated with calcium desensitization, we investigated the effects of thiazide-like diuretics on RhoA and Rho kinase using RT-PCR and immunoblotting. Compared with control conditions, RhoA mRNA and Rho kinase mRNA were significantly reduced after administration of thiazide-like diuretics. Compared with control conditions, RhoA mRNA was significantly reduced in cultured vascular smooth muscle cells after incubation with chlorthalidone or hydrochlorothiazide to 20±12% or to 41±12%, respectively (each n=5; P<0.05 by ANOVA). The Rho kinase mRNA was also significantly reduced after incubation with chlorthalidone or hydrochlorothiazide to 63±18% or to 66±25%, respectively (each n=5; P<0.05 by ANOVA; Figure 4). Compared with control conditions, RhoA expression was significantly reduced in cultured vascular smooth muscle cells after incubation with chlorthalidone or hydrochlorothiazide to 41±15% or to 38±17%, respectively (each n=5; P<0.05 by ANOVA). The expression of Rho kinase was also significantly reduced after incubation with chlorthalidone or hydrochlorothiazide to 30±3% or to 36±3%, respectively (each n=5; P<0.05 by ANOVA; Figure 5). Next, measurements of membrane-associated and cytosolic fractions of RhoA in the absence and presence of thiazide-like diuretics were performed. In the presence of chlorthalidone or hydrochlorothiazide, the membrane-associated RhoA was slightly reduced to 78±9% or to 91±62%, respectively (each n=5; P=NS). On the other hand, in the presence of chlorthalidone or hydrochlorothiazide, the cytosolic RhoA was reduced to 29±4% or to 34±4% of control, respectively (each n=3; P<0.05 by ANOVA). Administration of hydrochlorothiazide did not significantly affect protein tyrosine phosphatase SHP-2 expression in vascular smooth muscle cells (78±89% or 112±116%; each n=5; P=NS; Figure 5), respectively. These data indicate that the effects of thiazide-like diuretics on the Rho–Rho kinase pathway are not related to protein tyrosine phosphatase SHP-2.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study indicates that thiazide-like diuretics, including chlorthalidone and hydrochlorothiazide, attenuate agonist-induced vasoconstriction by calcium desensitization after affecting the Rho–Rho kinase pathway. The agonist-induced calcium increase in vascular smooth muscle cells was similar in the presence or absence of thiazide-like diuretics, whereas the agonist-induced increase of systolic blood pressure and vasoconstriction was significantly reduced. Calcium desensitization was first recognized in smooth muscle cells in experiments showing a decline in force during maintained depolarization, whereas the calcium increase was unchanged.20 One important mechanism leading to calcium desensitization is the inhibition of the Rho–Rho kinase pathway. In the present study, the reduced agonist-induced vasoconstriction in the presence of chlorthalidone or hydrochlorothiazide was similar to that observed in the presence of the specific Rho kinase inhibitor Y27632. In addition, preincubation with thiazide-like diuretics significantly reduced RhoA and Rho kinase in vascular smooth muscle cells. Reduced Rho kinase causes reduced phosphorylation of myosin phosphatase–targeting subunit at its inhibitory sites, thereby increasing myosin phosphatase activity. The increased myosin phosphatase activity while myosin light chain kinase activity remains unchanged leads to decreased phosphorylation of myosin regulatory light chain, causing calcium desensitization of the contractile apparatus and finally reduced contraction of smooth muscle cells.6,7
Changes of Rho kinase–mediated regulation of vascular tone appears to be a likely cause of increased vascular resistance in hypertension. Vascular Rho kinase mRNA levels are increased in spontaneously hypertensive rats, stroke-prone spontaneously hypertensive rats, and angiotensin II–induced hypertensive rats.21–23 Moreover, inhibition of the Rho–Rho kinase pathway reduces blood pressure in several hypertensive animal models.24 Inhibition of the Rho–Rho kinase pathway also reduces vasoconstriction in human left internal mammary artery.12 Therefore. it can be assumed that the inhibitory effect of thiazide-like diuretics on the Rho–Rho kinase pathway observed in the present study mediates their well-known antihypertensive effects in humans. When activated, RhoA moves to the membrane fraction from the cytosolic fraction. We showed that membrane-associated fractions of RhoA were slightly reduced, whereas cytosolic fractions of RhoA were significantly reduced in the presence of thiazide-like diuretics. These findings may indicate that thiazide-like diuretics predominantly affect RhoA expression rather than translocation of RhoA to the membrane. It has been reported recently that peroxisome proliferator-activated receptor 
ligands inhibit the Rho–Rho kinase pathway by inducing protein tyrosine phosphatase SHP-2.18 In the present study, we showed that thiazide-like diuretics did not affect protein tyrosine phosphatase SHP-2 expression. The effects of thiazide-like diuretics and peroxisome proliferator-activated receptor ligands on the Rho–Rho kinase pathway are apparently mediated by different mechanisms.
Chlorthalidone showed a dose-dependent inhibition of angiotensin II–induced vasoconstriction of aortic rings. A chlorthalidone concentration of 1 µmol/L significantly reduced angiotensin II–induced vasoconstriction. In addition, a chlorthalidone concentration of 100 µmol/L significantly reduced DNA synthesis, protein synthesis, and Rho kinase expression. The concentrations used in the present study can be achieved clinically in patients. Mean serum concentrations of chlorthalidone in patients were 
40 µmol/L, as reported in the literature.25 These findings indicate that the inhibitory effects of thiazide-like diuretics on vasoconstriction attributable to calcium desensitization linked to the Rho–Rho kinase pathway may also be an important mechanism in hypertensive patients. In support of that assumption, Pickkers et al showed that hydrochlorothiazide increases human forearm blood flow.4 Furthermore, the Rho kinase inhibitor fasudil increased forearm blood flow and decreased forearm vascular resistance in hypertensive patients stronger than in normotensive control subjects, probably because of increased Rho kinase expression in hypertensive patients.26 These findings are supported by the fact that the antihypertensive action of diuretics is related to initial blood pressure.27
In the kidney, chlorthalidone and hydrochlorothiazide exert their diuretic action by binding to the sodium–chloride cotransporter (NCC) in the distal convoluted tubule.28 NCC is selectively expressed in the kidney and apparently does not play a significant role for vessel wall function.29,30 Furthermore, it is unlikely that the effects of thiazide-like diuretics on vasoconstriction are also mediated by NCC because NCC knockout mice show normal blood pressure.31 In addition, the hydrochlorothiazide-induced vasodilation in humans could also be observed in patients with Gitelman syndrome, indicating that the absence of NCC does not alter the vasodilatory effect of hydrochlorothiazide.4
Activation of RhoA and Rho kinase has been shown to play a role in angiotensin II–induced hypertrophic changes in vascular smooth muscle cells.32 Moreover, oral treatment with the Rho kinase inhibitor fasudil blocked the angiotensin–induced hypertrophic changes of vascular smooth muscle cells.33 Taking these results into consideration, the findings of the present study that chlorthalidone and hydrochlorothiazide reduce expression of RhoA and Rho kinase indicate that thiazide-like diuretics reduce vasoconstriction and vascular hypertrophy by affecting the Rho–Rho kinase pathway.
Perspectives
The present study showed a novel mechanism by which thiazide-like diuretics inhibit vasoconstriction and vascular growth. Thiazide-like diuretics attenuated agonist-induced increase of blood pressure and vasoconstriction by calcium desensitization after affecting the Rho–Rho kinase pathway. Further research based on thiazide-like diuretics or structurally related substances may help identify more specific inhibitors of the Rho–Rho kinase system to establish novel and clinically relevant antihypertensive drugs.
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Acknowledgments |
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This study was supported by grants for Natural Science Foundation of China (30400179) and Major State Research Development Program of China (2000056901).
Received November 10, 2004; first decision November 17, 2004; accepted November 29, 2004.
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