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Am J Physiol Heart Circ Physiol 293: H541-H547, 2007. First published March 23, 2007; doi:10.1152/ajpheart.00770.2006
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Rho kinase contributes to basal vascular tone in humans: role of endothelium-derived nitric oxide

E. Büssemaker,1 Frank Pistrosch,1 Sarah Förster,1 Kay Herbrig,1 Peter Gross,1 Jens Passauer,1 and Ralf P. Brandes2

1Medizinische Klinik III, Nephrologie, Universitätsklinikum Dresden, Dresden, Germany; and 2Institut für Kardiovaskuläre Physiologie, J. W. Goethe Universität, Frankfurt, Germany

Submitted 18 July 2006 ; accepted in final form 21 March 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Our objective was to determine the role of the Rho-associated kinase (ROK) for the regulation of FBF (FBF) and to unmask a potential role of ROK for the regulation of endothelium-derived nitric oxide (NO). Moreover, the effect of fasudil on the constrictor response to endothelin-1 was recorded. Regarding background, phosphorylation of the myosin light chain (MLC) determines the calcium sensitivity of the contractile apparatus. MLC phosphorylation depends on the activity of the MLC kinase and the MLC phosphatase. The latter enzyme is inhibited through phosphorylation by ROK. ROK has been suggested to inhibit NO generation, possibly via the inhibition of the Akt pathway. In this study, the effect of intra-arterial infusion of the ROK inhibitor fasudil on FBF in 12 healthy volunteers was examined by venous occlusion plethysmography. To unmask the role of NO, fasudil was infused during NO clamp. As a result, fasudil markedly increased FBF in a dose-dependent manner from 2.34 ± 0.21 to 6.96 ± 0.93 ml/100 ml forearm volume at 80 µg/min (P < 0.001). At 1,600 µg/min, fasudil reduced systolic, diastolic, and mean arterial pressure while increasing heart rate. Fasudil abolished the vasoconstrictor effect of endothelin-1. The vascular response to fasudil (80 µmol/min) was blunted during NO clamp (104 ± 18% vs. 244 ± 48% for NO clamp + fasudil vs. fasudil alone; data as ratio between infused and noninfused arm with baseline = 0%, P < 0.05). In conclusion, 1) basal peripheral and systemic vascular tone depends on ROK; 2) a significant portion of fasudil-induced vasodilation is mediated by NO, suggesting that vascular bioavailable NO is negatively regulated by ROK; and 3) the constrictor response to endothelin involves the activation of ROK.

forearm blood flow

REGULATION OF BASAL VASCULAR tone is dependent on the balance of vasoconstricting and vasodilating principles. Vasoconstriction is mediated by at least four different mechanisms that tightly interact: the sympathetic nervous system, the renin-angiotensin-aldosterone system, endothelin-1 (ET), and the myogenic response (4, 6, 11, 13, 15, 18, 32, 34).

Smooth muscle cell contraction is initiated by the phosphorylation of the myosin light chain (MLC), which then allows the interaction of myosin and smooth muscle actin. The degree of MLC phosphorylation is dependent on the activities of the MLC kinase and the MLC phosphatase. The MLC kinase is a calcium/calmodulin-activated enzyme. The MLC phosphatase, which, in contrast, attenuates constriction, is calcium independent but inhibited by phosphorylation through the RhoA-dependent kinase (ROK) (30). Activation of ROK, therefore, potentiates the constrictor response at a given intracellular calcium concentration, a process termed "calcium sensitization." Cell culture experiments and studies using isolated vessel preparations demonstrated that the constrictor effects of endothelin, angiotensin II, and the generation of myogenic tone are mainly mediated by ROK activation (7, 8, 26, 29). Hence, three of the above-mentioned major vasoconstrictor mechanisms involve calcium sensitization and ROK activation.

In several animal models, activation of ROK has been suggested to be involved in the development of hypertension since inhibition of this enzyme by the selective ROK inhibitor Y-27632 elicited a pronounced antihypertensive effect (33). The central role of ROK in the control of smooth muscle constrictor tone, however, may suggest that this enzyme also has a major function in the control of vascular tone under physiological conditions.

Besides its profound effects on vascular smooth muscle cells, ROK is also involved in the regulation of endothelial nitric oxide (NO) synthase (NOS). In human endothelial cells, ROK negatively regulates phosphorylation of endothelial NOS through inhibition of protein kinase B/Akt (21). Moreover, inhibition of ROK leads to a rapid phosphorylation and an activation of Akt via the phosphatidylinositol 3-kinase, finally resulting in an increased NO production (35). These data suggest an important role of ROK in the regulation of endothelial NOS (eNOS) in the peripheral circulation of healthy subjects. The study presented here aimed to test whether ROK regulates basal vascular tone in healthy, normotensive male subjects. For this purpose, the local effect of the selective ROK inhibitor fasudil on forearm blood flow (FBF) was studied by venous occlusion plethysmography (10). Data obtained by this method largely reflect skeletal muscle blood flow, which is governed by myogenic tone and autoregulation. Moreover, this vascular region is highly responsive to angiotensin II and ET (5, 28). In all but one protocol where we intended to clarify whether increasing the dose of fasudil elicited systemic effects, we chose locally active doses to exclude counterregulating mechanisms such as the activation of the sympathetic nervous system. To examine the role of ROK for the regulation of NO, we tested the effect of fasudil after blockade of NO by the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). Moreover, the role of ROK for the ET-mediated forearm constriction was tested.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Subjects. Twelve normotensive, healthy, nonsmoking male subjects with an empty medical history were enrolled in the study. Not all subjects participated in each protocol of the study. Written informed consent was given by all of the subjects. The protocol was approved by the local ethics committee of the Universität Carl Gustav Carus, Germany. The investigation conforms with the principles outlined in the Declaration of Helsinki. All experiments were conducted in Dresden, Germany.

General procedures. The study was performed with the subjects in the supine position. They were asked to abstain from eating and drinking for 4 h before participation in any of the study protocols. Blood pressure and heart rate were measured throughout each protocol at 10-min intervals at the calf (using an automated device; Dinamap, Critikon). In the first protocol, blood pressure was measured every 5 min.

Measurement of FBF. FBF was measured simultaneously in both arms by venous occlusion plethysmography in control subjects as described previously (24). The pressure of the congesting cuffs of both upper arms was set at 40 mmHg. Mercury-in-Silastic strain gauges were wrapped around the widest parts of the forearms and connected to a calibrated venous occlusion plethysmograph (Gutmann Medizinelektronik, Eurasburg, Germany). The brachial artery of the nondominant arm was cannulated for drug infusion with a 27-gauge needle (Cooper's Needle Works). Data are given as the ratio between infused and noninfused arms with the baseline set at 0%.

After cannulation of the brachial artery saline was infused for 20 min to establish baseline conditions. Individual measurements of FBF lasting 10 s were made every 15 s for 2.5 min during each dose of agent administered. The blood flow of the hands was excluded by a wrist cuff inflated to a suprasystolic pressure (220 mmHg) during each measurement period. Blood pressure was measured at baseline and at the end of each infusion period. During each protocol, the infusion rate was kept constant at 1 ml/min. At the end of each FBF measurement, blood pressure was determined at the calf as described in General procedures.

Protocols. The first protocol aimed to test the time-dependent effect of the specific ROK inhibitor fasudil on FBF (10). Therefore, fasudil was infused at a dose of 80 µg/min over 60 min.

In a second protocol, graded doses of fasudil (10, 20, 40, and 80 µg/min) were infused over 30 min each. After each period of infusion, FBF and mean arterial pressure were measured as described in General procedures.

To clarify whether fasudil could also exert systemic hemodynamic effects, we infused a 20-times higher dose (1,600 µg/min) of fasudil over a period of 30 min into the brachial artery. Heart rate and blood pressure were measured at 10-min intervals throughout the infusion period.

In the third protocol, we tested the effect of fasudil on the ET-mediated decrease of FBF. For this purpose, ET was infused at a dose of 5 pmol/min for 60 min. FBF and blood pressure were measured every 10 min throughout the period of infusion. At a different occasion at least 5 days apart, ET (5 pmol/min) was coinfused with fasudil (80 µg/min, started 30 min before coinfusion with ET), and FBF was studied every 5 min for 60 min. In a control experiment that was performed in the same subjects, we used norepinephrine (NE) as a constrictor. First, NE was infused in incremental doses (60, 120, and 240 nmol/min). At a different occasion at least 5 days apart, we coinfused NE (60, 120, and 240 nmol/min) with fasudil (80 µg/min, started 30 min before coinfusion with NE) in the same subjects.

The fourth protocol was designed to unmask the effect of ROK on NO in the forearm circulation. In this protocol we looked at the effects of fasudil under "NO clamp" conditions. The NO clamp is established by blockade of endogenously generated NO using the inhibitor L-NMMA [16 µmol/min, a dose that has been previously shown to fully block the endothelial NO release in the forearm circulation (12)] given into the brachial artery and concomitant infusion of graded doses of the NO donor sodium nitroprusside to restore baseline flow. In this setting L-NMMA infusion was started 5 min before the application of sodium nitroprusside and maintained throughout the protocol. After restoration of baseline blood flow, fasudil was infused at a dose of 80 µg/min for 30 min. The data obtained under NO-clamp conditions were compared with the effect of fasudil (80 µg/min over 30 min) obtained on a different occasion in the same subjects. These experiments were performed at least 5 days apart from each other.

Drugs. L-NMMA and endothelin were from Clinalfa (Läufelfingen, Switzerland); fasudil (Eril Injection S) was from Asahi Kasei (Osaka, Japan). The agents were dissolved in a physiological saline solution.

Statistics. Each FBF determination consisted of 10 single FBF measurements. The final five blood flow recordings for each infusion step were used to calculate mean FBF. As indicated in Measurement of FBF, results are presented as the change in the ratio of the infused to the noninfused control arm with the baseline set at 0%. The results showing the effect of increasing doses of fasudil (10–80 µg/min) on FBF as described in the second protocol are presented as absolute flow data of the infused arm. Results are presented as means ± SE. Comparison of group characteristics was performed using Student's t-test. Dose-response curves were analyzed by two-way analysis of variance (ANOVA) for repeated measurements. For the NO-clamp experiments, a post hoc analysis (Bonferroni) was performed to evaluate the contribution of NO to the fasudil-induced vasodilatation of the human forearm at each of the time periods evaluated. Values of P < 0.05 were considered statistically significant.


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Baseline characteristics. All healthy subjects studied were normotensive as defined by the guidelines of the European Society of Cardiology/European Society of Hypertension (9). Baseline characteristics of the study population are given in Table 1. Basal FBF did not differ in any of the protocols. No systemic effects of drug infusion were observed as demonstrated by constant blood pressure and stable FBF in the contralateral, noninfused arm when fasudil was infused at infusion rates up to 80 µg/min.


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  		Table 1. Subject characteristics

 
Fasudil increases the blood flow in the forearm of healthy subjects. Fasudil increased FBF in a dose- and time-dependent manner. As shown in Fig. 1A, fasudil at a dose of 80 µg/min induced a sustained increase in FBF that reached its maximum within 25–30 min. At this dose of fasudil, we did not detect any systemic effect as indicated by a constant flow in the contralateral, noninfused arm and by unaffected arterial blood pressure and heart rate (Table 2). Fasudil infused at increasing doses elicited a dose-dependent increase in FBF (Fig. 1B). Increasing the infusion rate of fasudil to 1,600 µg/min further increased FBF in the infused arm (from 2.68 ± 0.60 at baseline to 15.00 ± 2.05 ml/min x 100 ml forearm volume, P = 0.002). Interestingly, at this dose, the FBF of the contralateral arm increased significantly (from 2.88 ± 0.52 at baseline to 4.1 ± 0.75 ml/min x 100 ml forearm volume, P = 0.001), suggesting a systemic hemodynamic effect. A concomitant decrease in systolic, diastolic, and mean arterial pressure was observed, and this effect was accompanied by an increase in heart rate (Fig. 2).


Figure 1
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  		Fig. 1. Changes in forearm blood flow (FBF) in response to intra-arterial infusion of fasudil. A: fasudil was infused over a period of 60 min at a constant rate of 80 µg/min with measurements of FBF performed every 5 min. Data are presented as the ratio between infused and noninfused arm [I-to-C ratio (I/C ratio)] with baseline = 0%. Values are given as means ± SE; n = 7 subjects. P < 0.001 for testing by ANOVA for repeated measurements. B: increasing doses of fasudil were infused into the forearm in 12 normotensive, healthy subjects. Each dose was infused over 30 min. Values are given as means ± SE of the absolute FBF of the infused arm; n = 12 subjects. The flow in the contralateral noninfused arm that served as a control did not change throughout the protocol. P < 0.001 for testing by ANOVA for repeated measurements.

 

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  		Table 2. Responses of FBF in all protocols

 

Figure 2
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  		Fig. 2. Effect of a high dose of fasudil on systemic hemodynamics. Increasing the infusion rate of fasudil to 1,600 µg/min decreased systolic (SBP) and diastolic (DBP) blood pressure and mean arterial pressure (MAP). This was accompanied by an increase in heart rate (HR). Fasudil was infused over 30 min. Data are given as the change from baseline (in mmHg for SBP, DBP, and MAP and in beats/min for HR) at 30 min. Values are means ± SE; n = 9 subjects. *P < 0.05; **P < 0.01. Comparison was by ANOVA for repeated measurements.

 
The ET-mediated reduction in FBF is sensitive to fasudil. Infusion of ET caused a dose-dependent, sustained decrease in FBF (Fig. 3A). In the presence of fasudil (80 µg/min; the ET-mediated reduction in FBF was abolished; Fig. 3B). However, when compared with ET, the vasoconstrictive effect of NE (Fig. 3C) was slightly attenuated but maintained in the presence of fasudil (Fig. 3D). The effects in these experiments were confined to the infused forearm since the flow in the contralateral arm, as well as blood pressure and heart rate, remained unchanged.


Figure 3
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  		Fig. 3. Effect of fasudil on endothelin-1 (ET) and norepinephrine (NE)-induced vasoconstriction in normotensive, healthy subjects. Effect of fasudil (80 µg/min; B and D) on the vasoconstrictive effect of ET (5 pmol/min; A) and NE (60, 120, and 240 nmol/min; C) in the forearm circulation of 6 normotensive, healthy subjects is shown. ET was infused at a dose of 5 pmol/min for 60 min. On a different occasion, ET (5 pmol/min) was coinfused with fasudil (80 µg/min) and FBF was recorded. In a control experiment that was performed in the same subjects, we used NE as a constrictor. NE was infused in incremental doses (60, 120, and 240 nmol/min). On a different occasion, we coinfused NE (60, 120, and 240 nmol/min) with fasudil (80 µg/min) in the same subjects. Data are given as I/C-ratio with baseline = 0%. Values are given as means ± SE; n = 6 subjects. **P < 0.01 and *P < 0.05 for two-way ANOVA for repeated measurements.

 
The fasudil-induced increase in FBF is partially mediated by the activation of NO. Under complete blockade of the eNOS with L-NMMA, we detected a significant attenuation of the fasudil-induced increase in FBF compared with infusion of fasudil without preceding NOS inhibition (Fig. 4). Post hoc analysis showed that NO contributed equally to the fasudil-induced dilation at each of the time periods evaluated (54.5 ± 12.8% at 10 min, 59.4 ± 15.58% at 20 min, and 48.7 ± 15.6% at 30 min for the ratio between the NO-independent and the overall fasudil-induced increase in FBF; P = not significant). No systemic effects of the infused drugs were detected during this protocol.


Figure 4
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  		Fig. 4. Role of nitric oxide (NO) for the fasudil-induced increase in FBF in normotensive, healthy subjects. Effect of fasudil on FBF under blockade of the endothelial NO synthase and restoration of basal FBF by infusion of the NO donor sodium nitroprusside (NO clamp) is shown. In this experimental setting, the endothelial NO synthase was clamped by NG-monomethyl-L-arginine (16 µmol/min) and restoration of basal FBF was achieved by infusing graded doses of the NO donor sodium nitroprusside. This procedure allows the testing of the effect of fasudil (80 µg/min for 30 min) under a constant NO level equal to that produced by the vascular endothelium under resting conditions and thus at physiological constrictor tone. Data obtained under these conditions (NO clamp) are compared with infusion of fasudil alone (control). Data are given as the I/C-ratio with baseline = 0%. Values are given as means ± SE; n = 7 subjects. *P < 0.05 for comparison between the two groups by two-way ANOVA for repeated measurements. In the post hoc analysis, we did not detect any differences between the NO-independent (NO clamp) and the overall (control) fasudil-induced increase in FBF at each period of infusion (P = not significant).

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
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In the present study, the Rho-kinase inhibitor fasudil increased FBF in healthy normotensive male subjects in a dose-dependent fashion. Strikingly, this effect appeared to be mediated by not only a direct reduction in smooth muscle constrictor tone but also an increase in endothelial NO production, since the fasudil-induced increase in FBF was markedly attenuated by the coinfusion of the NOS inhibitor L-NMMA. Moreover, we demonstrated that the vasoconstrictor effect of endothelin is exclusively mediated via ROK, since fasudil completely prevented the vasoconstrictive effects of endothelin. To underline the importance of ROK for the regulation of vascular tone under physiological conditions, we further increased the dose of fasudil infused. Indeed, we could demonstrate a systemic hemodynamic effect indicated by a decrease in systemic blood pressure and an increase in heart rate.

The current literature comprises mostly studies that were designed to look at the effect of Rho kinase inhibition in pathological situations such as arterial hypertension, coronary artery disease, or cardiac failure (17, 19, 22). The role of ROK inhibition for this vascular bed tested remains to be clarified in healthy control subjects since results published are conflicting: Masumoto and coworkers (19) showed in a study designed to test the role of Rho kinase in patients with arterial hypertension that fasudil did not increase FBF in the doses used but slightly decreased forearm vascular resistance. In another study designed to test the effect of fasudil in cardiac failure, the same authors (17) did not show any effect of fasudil on FBF in healthy control subjects. Hence, the contribution of ROK to basal vascular tone in the forearm circulation has not been adequately examined under physiological conditions. Besides, the studies mentioned were performed in a Japanese population. Ethnic differences have already been shown for studies with endothelin antagonists, and endothelin is an important stimulator of ROK.

Physiological relevance of Rho kinase for the basal arterial tone of the human forearm circulation. Our study was designed to unravel the role of ROK for the regulation of vascular tone in a caucasian, healthy, male cohort. Regarding the role of ROK for the regulation of FBF, our data are compelling and show a major contribution of ROK to basal flow in this particular vascular bed. When compared with the above-mentioned studies that mainly addressed the role of ROK under pathological conditions (fasudil up to 25.6 µg/min), we infused a considerably higher dose of fasudil (up to 80 µg/min). Fasudil infused at 80 µg/min results in a local forearm concentration well above the EC50 as given for cell culture systems, indicating that the dose we used should have been adequate to selectively inhibit ROK in the forearm circulation (10).

Regulation of total peripheral resistance and systemic blood pressure is primarily mediated by resistance-sized arteries that can be specifically examined by venous occlusion plethysmography in the human forearm. To extend our understanding of the role of ROK to the systemic hemodynamics, we further increased the doses of fasudil used. Indeed, fasudil at a high dose (1,600 µg/min) decreased diastolic, systolic, and mean arterial pressure, and this was accompanied by an increase in heart rate. This is the first compelling evidence that ROK not only controls local but also systemic hemodynamics in normotensive subjects.

Given that vascular resistance under resting conditions in the forearm is mainly determined by the tone of the arterioles in the skeletal muscles, we conclude that ROK largely contributes to the development of spontaneous tone in this vascular bed.

Fasudil-induced dilation of the human forearm: a role for endothelium-derived NO. We show that the fasudil-induced increase in FBF is partially dependent on the activation of endothelium-derived NO. To our knowledge these are the first data providing evidence for a role of ROK in the regulation of the eNOS activity in human subjects.

Our results are in line with previous observations made in cultured endothelial cells. In those experiments, protein kinase B/Akt was negatively regulated by ROK. Increasing ROK activity downregulated protein kinase B/Akt and thereby inhibited eNOS phosphorylation at serine-1177 (21). The observation that inhibition of ROK leads to a rapid activation of the phosphatidylinositol 3-kinase/Akt pathway further substantiates these data (35). In contrast, a recent study performed in healthy humans and in smokers failed to show an effect of the NOS inhibitor L-NMMA on the fasudil-induced increase of FBF in both groups (23). However, in this study the authors infused fasudil for only 5 min before the determination of FBF. Since our data clearly show that the vasodilating effect of fasudil was maximal not before 25–30 min of infusion, this could explain the discrepancy between our study and that of Noma and coworkers (23). Moreover, in cell culture experiments, thrombin-induced, ROK-mediated dephosphorylation of eNOS occurs within 15 min (21). It can therefore be assumed that 30 min but not 5 min of fasudil infusion in vivo would unmask the putative NOS-inhibiting effect of ROK. Interestingly, in our experimental setting, we could show that NO contributed equally to the fasudil-induced dilation at each time point during the infusion period as indicated by the post hoc analysis.

To assess the effect of endothelium-derived NO, we used the NO-clamp technique, which is designed to exclude changes in endogenous NO generation (25, 31). The great advantage of this method is that basal flow and constrictor tone are maintained at the physiological level in the forearm by infusion of sodium nitroprusside. Therefore, vasoconstriction-induced alterations in responsiveness to vasodilators are excluded. However, the sodium nitroprusside-corrected blood flow under NO-clamp conditions was slightly higher than the basal FBF in the control experiments. Although this difference did not reach statistical significance, it could have confounded the results. We looked at this possibility by correlating the sodium nitroprusside-corrected FBF in the group that received L-NMMA to the maximal response to fasudil. We did not find a significant correlation (R = 0.091; P = 0.72), indicating that the modest increase in basal FBF did not result in reduced sensitivity to fasudil.

Noma and coworkers (23) did not restore basal flow conditions before infusing fasudil, a fact that might contribute to the controversial results shown by their study and ours. It is known from bioassay experiments that endothelium-derived NO antagonizes the vasoconstrictor effect of ROK by activation of myosin phosphatase (3). If the basal NO-mediated tone is not restored, one would underestimate the contribution of eNOS to ROK-mediated effects because the absent inhibitory effect of (basal) NO on smooth muscle ROK activity masks the actual differences. Furthermore, in their study, they used L-NMMA at a dose of 8 µmol/min, whereas a recent study showed that L-NMMA should be administered at a dose of 16 µmol/min to completely block the endothelial NO production (12). This seems to be of special importance when vasodilators such as fasudil are tested, given the diluting effect that occurs during the increase of FBF. Whether ethnic differences also contribute to the discrepancies between the two in vivo studies remains unclear.

Fasudil and ET-mediated constriction in the human forearm. We and others have previously shown that ET contributes to basal vascular tone in the forearm (5, 15). ET is known to be an activator of ROK (1, 2, 8, 15, 20). Indeed, in the present study, we demonstrate that fasudil completely prevented the ET-induced decrease in FBF. However, in the presence of fasudil, NE still exerted a yet attenuated vasoconstrictive effect. Initially, it has been proposed that NE mainly exerts vasoconstriction via depolarization of vascular smooth muscle cells and a consecutive increase of intracellular calcium, whereas ET predominantly acts via activation of ROK (16, 20). However, recent reports imply that this dichotomic view can no longer be supported (27). The reason for the different sensitivity of NE- and ET-induced constrictions to fasudil cannot be explained with the data presented here. It is likely that the different cellular compartmentalization and connectivity between membrane receptors and signal transduction mechanisms in vascular smooth muscle cells in various organs contribute to the diversity of vascular responses to both agonists and blocking agents in different segments of the circulation. Taken together, an exquisite sensitivity of ET-induced constriction to ROK inhibition in the forearm vascular bed can be demonstrated in this setting.

Limitations. Human in vivo studies have major limitations since the experimental setting often does not allow direct insights into the mechanisms involved. Here we show clearly that infusion of the Rho-kinase inhibitor fasudil dose-dependently affected local peripheral and also systemic hemodynamics. In this in vivo study we cannot give direct evidence that fasudil as shown in numerous cell culture and animal studies actually decreased ROK activity. However, a recent in vivo study looking at the chronic effect of ROK inhibition by oral administration of fasudil in patients with coronary artery disease showed that the decrease in ROK activity correlated to the increase in flow-mediated dilation. Since flow-mediated dilation depends on eNOS activity, their results strongly suggest that the degree of ROK inhibition induced by fasudil correlates to the restoration of NO bioavailability (22).

Perspectives. Our data show that the activation of ROK represents an important mechanism to maintain peripheral and probably systemic vascular resistance in healthy men. We also demonstrate a contribution of endogenous NO to the vasodilator effects of fasudil. This implies that ROK physiologically inhibits NO, a mechanism shown here in human subjects for the first time. Pathological situations with an increased ROK activity such as arterial hypertension should benefit from ROK inhibition (19). Future studies should not only focus on the acute effects of ROK inhibition but also investigate the potentially positive effects of chronic inhibition of ROK, especially regarding the upregulation of NOS, which has already been shown on the cellular level. In this view, the improvement of flow-induced dilation after chronic ROK inhibition with fasudil in patients with coronary artery disease is promising (22). Since the ROK inhibitor fasudil inhibits ET-mediated constriction, patients with increased ET activity should profit particularly from fasudil treatment. In this context, evaluating the role of ROK for renal hemodynamics in kidney disease appears to be attractive (14). To test this hypothesis, further studies are needed. The present study was focussed on acute effects of ROK inhibition. Possibly chronic inhibition of ROK could provide additional beneficial effects through an altered vascular gene expression that will be independent of the direct effects on vascular resistance.


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E. Büssemaker was supported by an Else Kröner-Fresenius-Stiftung (Bad Homburg, Germany) Grant P15/2004F.


   ACKNOWLEDGMENTS
 
We are indebted to Jürgen Örtel for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Büssemaker, Univ. of Dresden, Medizinische Klinik III, Nephrologie, Fetscherstrasse 74, 01307 Dresden, Germany (e-mail: eckhart.buessemaker{at}ukmuenster.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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