Rho-Kinase Inhibition Blunts Renal Vasoconstriction Induced by Distinct Signaling Pathways In Vivo
Alessandro Cavarape*,
Nicole Endlich,
Roberta Assaloni*,
Ettore Bartoli,
Michael Steinhausen,
Niranjan Parekh and
Karlhans Endlich
*Department of Experimental and Clinical Pathology and Medicine (DPMSC), University of Udine, Udine, Italy; Department of Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany; Department of Clinical Pathology and Medicine, University of Piemonte Orientale, Novara, Italy; and Department of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany.
Correspondence to Dr. Alessandro Cavarape, Department of Experimental and Clinical Pathology and Medicine (DPMSC), Chair of Internal Medicine, Piazza S. Maria della Misericordia, 1, 33100 Udine, Italy. Phone: 39-0432-559815; Fax: 39-0432-42097;
ABSTRACT. In addition to intracellular calcium, which activatesmyosin light chain (MLC) kinase, MLC phosphorylation and hencecontraction is importantly regulated by MLC phosphatase (MLCP).Recent evidence suggests that distinct signaling cascades ofvasoactive hormones interact with the Rho/Rho kinase (ROK) pathway,affecting the activity of MLCP. The present study measured theimpact of ROK inhibition on vascular F-actin distribution andon vasoconstriction induced by activation/inhibition of distinctsignaling pathways in vivo in the microcirculation of the splithydronephrotic rat kidney. Local application of the ROK inhibitorsY-27632 or HA-1077 induced marked dilation of pre- and postglomerularvessels. Activation of phospholipase C with the endothelin ETBagonist IRL 1620, inhibition of soluble guanylyl cyclase with1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), or inhibitionof adenylyl cyclase with the adenosine A1 agonist N6-cyclopentyladenosine(CPA) reduced glomerular blood flow (GBF) by about 50% throughvasoconstriction at different vascular levels. ROK inhibitionwith Y-27632 or HA-1077, but not protein kinase C inhibitionwith Ro 31-8220, blunted ETB-induced vasoconstriction. Furthermore,the reduction of GBF and of vascular diameters in response toODQ or CPA were abolished by pretreatment with Y-27632. ROKinhibitors prevented constriction of preglomerular vessels andof efferent arterioles with equal effectiveness. Confocal microscopydemonstrated that Y-27632 did not change F-actin content anddistribution in renal vessels. The results suggest that ROKinhibition might be considered as a potent treatment of renalvasoconstriction, because it interferes with constriction inducedby distinct signaling pathways in renal vessels without affectingF-actin structure. E-mail: alessandro.cavarape@dpmsc.uniud.it
The primary signal for vascular smooth muscle cell (VSMC) contractionis an increase of intracellular calcium (Ca2+) levels, activatingmyosin light chain (MLC) kinase, which leads to MLC phosphorylation(1). In the past few years, evidence has accumulated that enhancementof Ca2+ sensitivity of VSMC, involving inhibition of MLC phosphatase(MLCP), contributes to VSMC contraction importantly (2). Sofar, two molecular mechanisms of MLCP inhibition have been uncovered(3). First, receptor-mediated activation of the small G proteinRhoA leads to activation of Rho kinase (ROK), which inhibitsMLCP through phosphorylation of its regulatory subunit eitherdirectly or via ZIP-like kinase (4,5). Second, protein kinaseC and isoforms have been identified to phosphorylate CPI-17,which, in the phosphorylated state, inhibits MLCP specifically(6,7). A recently developed ROK inhibitor, the pyridine derivativeY-27632, has been shown to potently inhibit the contractionof preparations from experimental animals and human arterialvessels induced by different agonists in vitro and also to correctarterial pressure to normotensive levels in different experimentalmodels of hypertension in vivo (8–11). Because of theevidence that the Rho/ROK pathway mediates tonic vasoconstriction,ROK is thought to play a crucial role in human diseases, e.g.,in hypertension and myocardial ischemia (12,13). The contributionof the ROK pathway to renal vasoconstriction is practicallyunknown.
A number of investigations have elucidated the role of a widevariety of circulating and locally produced vasoactive moleculesin regulating renal hemodynamics under normal and pathologicconditions (14). Vasoconstrictors bind to specific receptorsto activate intracellular pathways triggering contraction ofVSMC. Also in the kidney, VSMC tone is regulated by a complexnetwork of constricting and dilating intracellular signalingcascades, some of which might modulate vasomotor tone in partindependent of changes in intracellular Ca2+. Thus, renal vasoconstrictorscould increase Ca2+ sensitivity through MLCP inactivation mediatedby ROK. Conversely, VSMC relaxation mediated by an increasein intracellular cAMP and cGMP is associated with a decreaseof Ca2+ sensitivity (3,15). Evidence has accumulated that cGMP-and cAMP-dependent protein kinases can directly increase theactivity of MLCP and interfere with the Rho/ROK pathway (3).However, the role of ROK in renal vasoconstriction mediatedby distinct signaling pathways has not yet been investigated.
In addition to smooth muscle contraction, the Rho/ROK pathwaystimulates stress fiber formation and actin polymerization incultured cells (16). Accordingly, ROK inhibition with Y-27632completely abolished stress fibers in cultured cells (11). Thoughactin filament disruption has been demonstrated to severelyimpair vasoconstriction in isolated mesenteric arteries (17),F-actin distribution in vessels has not yet been examined afterROK inhibition.
The aim of this study was to assess the influence of ROK onrenal vasoconstriction at the microvascular level utilizingthe in vivo model of the split hydronephrotic rat kidney. Weemployed different approaches to induce vasoconstriction throughdistinct intracellular pathways, namely phospholipase C activation,and reduction of intracellular cGMP or cAMP levels. Drug-inducedconstrictions in different renal vascular segments and reductionsin glomerular blood flow (GBF) were measured under control conditionsand after local application of the ROK inhibitors Y-27632 andHA-1077 as compared with PKC inhibition. Moreover, we also investigatedthe effect of ROK inhibition on actin filament organizationin the renal vascular wall.
Preparation of the Hydronephrotic Kidney
The study protocols were performed on 29 female Wistar rats(210 to 240 g) in accordance with national animal protectionguidelines. The animals had free access to food and water beforethe experiments. The technique and experimental procedures havebeen previously described in detail (18). Briefly, unilateralsurgical hydronephrosis was induced by permanently ligatingthe left ureter via a flank incision during pentobarbital sodiumanesthesia (Nembutal, 60 mg/Kg intraperitoneally; Ceva, BadSegeberg, Germany). The final experiments were performed underthiobutabarbital anesthesia (Inactin, 100 mg/kg intraperitoneally;Byk Gulden, Konstanz, Germany) 2 to 3 mo after induction ofhydronephrosis. Each rat was placed on a 37°C heated surgicaltable. The trachea was intubated, and the left jugular veinwas cannulated for the continuous replacement of isotonic salineat a rate of 60 µl/min and for infusion of drugs. A polyethylenecatheter was inserted into the left femoral artery for continuousmeasurements of systemic BP. The left hydronephrotic kidneywas exposed through a flank incision and split carefully alongthe great curvature using a thermal cautery. The dorsal halfof the split kidney was sutured to a semicircular-shaped wireframe. Blood supply and innervation remained intact after thispreparation. The wire frame with the fixed split kidney wasplaced at the bottom of a specially designed Plexiglas chambersuitable for intravital transillumination microscopy. The entryof the renal hilus into the chamber was sealed with silicongrease, and the chamber was filled with 50 ml of an isotonic,isocolloidal solution (Hemaccel; Behringwerke AG, Marburg, Germany)maintained at a constant temperature of 37°C with a feedback-controlledheating system.
Videomicroscopy
The table was mounted on the microscope stage. Images of renalmicrovessels obtained through a special water-immersion objective(Ultropak UO-55; Leitz, Wetzlar, Germany) were visualized witha CCD camera, displayed on a calibrated monitor by a closedcircuit TV system, and recorded on line on videotape. The luminalvessel diameters were measured directly from the monitor andwere converted to in vivo diameters (expressed in micrometers)using a conversion factor to account for the magnification ofthe vessel images.
Vessel Segments and GBF
Measurements of the following vessel segments were carried outaccording to their branching pattern from the selected glomerulus:(1) proximal interlobular artery (near the arcuate artery);(2) distal interlobular artery (near the afferent arteriole);(3) proximal afferent arteriole (near the interlobular artery);(4) distal afferent arteriole (at a site within 100 µmfrom the glomerulus); and (5) efferent arteriole (within 50µm from the glomerulus).
Velocity of red blood cells was measured in the efferent arterioleof the selected glomerulus by using a velocity-tracking correlator(Model 102B; IPM Inc., San Diego, CA). In this on-line computingsystem two photodiodes obtain photometric signals from the movingred blood cells (19). To obtain the GBF value, the measuredred cell velocity was multiplied by the luminal cross-sectionof the efferent arteriole and corrected for the Fahreus-Lindqvisteffect (20).
Experimental Protocols
After the surgical procedure, each kidney was allowed to adaptto the tissue bath conditions for at least 60 min. The stabilityof the preparation has been demonstrated for a duration of 3h (21). Each protocol included several experimental periodsat the end of which mean arterial pressure (MAP), vascular diameters,and GBF were measured. The first period served as baseline control.
Six series of experiments were performed. In the first series,dose-response experiments for Y-27632 were conducted. The otherfive experimental series started with control measurements duringwhich the ETB receptor agonist IRL 1620 (group 1, n = 6; group2, n = 5; group 3, n = 5), the guanylyl cyclase inhibitor ODQ(group 4, n = 6) or the selective A1 adenosine receptor agonistN6-cyclopenthyladenosine (CPA) (group 5, n = 7) were locallyadministered into the tissue bath to reduce GBF from a thresholdvalue to about 50% by up to three sequentially increasing dosesof vasoconstrictors. After wash out (30 min) and baseline measurementsin the five experimental series, measurements were taken afterintrarenal inhibition of ROK by local administration of theselective inhibitors Y-27632 or HA-1077 or after intrarenalinhibition of PKC by local administration of Ro 31–8220.After control measurements, during which the renovascular parameterswere evaluated in a new baseline condition at the steady state,the effects of IRL 1620, ODQ, and CPA were assessed after intrarenalinhibition of ROK or PKC, whereby the doses of IRL 1620 andCPA were augmented in groups 1, 4, and 5 to obtain a reductionin GBF comparable to that detected during the pre-inhibitionmeasurements. Measurements were taken about 10 min after applicationof IRL 1620 and CPA and about 15 min after application of ODQ,Y-27632, HA-1077, and Ro 31–8220, when a steady statehad been reached.
For stimulation of PLC, the ETB agonist IRL 1620 was chosen,because we have demonstrated earlier that IRL 1620 induces notonly preglomerular vasoconstriction but also marked efferentarteriolar constriction (22). The effects of IRL 1620 and ofA1 receptor agonists have been shown to be fully reversibleafter washout (22–24). Furthermore, vasoconstrictionsinduced by IRL 1620 and A1 receptor agonists do not desensitizein the hydronephrotic kidney, as is the case for angiotensinII and diadenosine polyphosphates (22–26). The reversibilityof the ODQ-induced vasoconstriction was confirmed by wash out.To exclude that the renal vasculature desensitized in responseto ODQ, the protocol was reversed (Y-27632 + ODQ, washout, ODQ)in half of the experiments. We obtained similar results forODQ with the normal and the reversed protocol; therefore, datawere pooled together.
Y-27632 and HA-1077 have been reported to inhibit ROK with IC50values of 8 · 10-7 M and 2 · 10-6 M, respectively(27). Concentrations of 10-5 M Y-27632 and 2 · 10-5 MHA-1077 inhibit PKC by 2% and 14%, respectively (27). The IC50value of Ro 31–8220 for PKC inhibition is 3 · 10-8M, whereas 1 µM Ro 31–8220 reduces ROK activityby only 8% (27).
It should be noted that the concentrations of vasoactive drugs,which are locally administered into the tissue bath, need tobe about tenfold higher to elicit similar effects in the hydronephrotickidney as compared with isolated or perfused preparations. Forexample, 10-9 M ET-1 and 10-8 M angiotensin II in the tissuebath constrict afferent arterioles by about 15%, whereas a similardiameter reduction is seen with 10-10 M ET-1 and <10-9 Mangiotensin II in isolated rat afferent arterioles (28) or inisolated perfused hydronephrotic kidneys (29,30). There is mostlikely a steep concentration gradient for locally applied drugsbetween the tissue bath and the vessel lumen, because the hydronephrotickidney has a considerable thickness (200 to 400 µm) andthe concentration of drugs in the blood stream is practicallyzero.
IRL 1620 (Alexis, Germany), CPA (Boehringer Mannheim, Germany),Y-27632 (kindly provided by Yoshitomi Pharmaceutical Inc.),and HA-1077 (Alexis, Germany) were dissolved in saline. ODQand Ro 31–8220 (Alexis, Germany) were dissolved in DMSO;the final DMSO concentration in the tissue bath was below 0.1%.All the vasoactive drugs used in our experiments were administeredinto the tissue bath by local application to avoid any systemiceffect on hemodynamic parameters.
Examination of F-Actin Distribution
Hydronephrotic kidneys were prepared for videomicroscopy asdescribed above. Hydronephrotic kidneys were incubated witheither vehicle (controls) or 10-4 M Y-27632 for 30 min. Hydronephrotickidneys were then excised and fixed in 2% paraformaldehyde for2.5 h. The kidney tissue was cut in stripes, dehydrated in isopentanein liquid nitrogen, and mounted on styropor snips for cryosectioning.Kidney tissue was sectioned at 15-µm thickness. Furtherprocedures were identical to those as recently described (31).Briefly, sections were permeabilized (0.3% Triton X-100), blockedfor 1 h at room temperature in blocking solution (2% fetal bovineserum, 2% bovine serum albumin, 0.2% fish gelatin in phosphatebuffered saline), and incubated with Alexa-conjugated phalloidin(Molecular Probes, Eugene, OR) to visualize F-actin. Specimenwere viewed with a confocal laser scanning microscope (TCS-SP;Leica Microsystems, Heidelberg, Germany). To assess F-actinintensity, all images were taken with identical confocal microscopesettings for laser power, photomultiplier gain, and pinholesize.
Statistical Analyses
Data are presented as mean ± SEM. Changes in vasculardiameters and GBF are expressed as percentage changes from thepreceding control values. Paired and unpaired t test, ANOVA,and Bonferroni’s method for multiple comparisons wereused as appropriate to test for statistical significance. Thedata have been analyzed both as absolute and percent values.Values of P < 0.05 were considered statistically significant.
Absolute values for measured parameters in five experimentalgroups under control conditions before and after applicationof inhibitors are listed in Table 1. MAP was not affected bylocal administration of vasoconstrictors or inhibitors in allthe experimental groups, and it remained stable throughout theexperimental protocols if not otherwise indicated.
Table 1. Absolute values for vessel diameters, glomerular blood flow (GBF), and MAP for the different experimental groups and for the pooled data of three groupsa
Effects of ROK and PKC Inhibitors on Basal Vessel Tone
In the first series of experiments, the effect of increasingconcentrations of the ROK inhibitor Y-27632 (10-7 to 10-4 M)on the spontaneous, endogenous tone of the interlobular arterywas determined (Figure 1A). In the interlobular artery, diameterdid not change at 10-7 and 10-6 M Y-27632, but it significantlyincreased at higher concentrations (10-5 and 10-4 M). The vascularpattern of dilation in response to the ROK inhibitor Y-27632(10-4 M) was obtained from the pooled data of three groups ofexperiments (Figure 1B). Dilation was observed preferentiallyin preglomerular vessels, i.e., in interlobular arteries andin proximal afferent arterioles. Efferent arterioles also dilatedsignificantly in response to the ROK inhibitor Y-27632. Y-27632almost doubled GBF.
Figure 1. Effect of the Rho kinase (ROK) inhibitor Y-27632 on renal vessels. The ROK inhibitor Y-27632 induced a dose-dependent vasodilation in renal vessels. (A) Percent changes of the diameter of the proximal interlobular artery in response to increasing concentrations of Y-27632. (B) Percent changes of GBF and of diameters for different renal vessels in response to Y-27632 (10-4 M). ilob, interlobular artery; aff, afferent arteriole; eff, efferent arteriole; p, proximal; d, distal. Data are mean ± SEM; n = 3 (panel A) and n = 19 animals (panel B); * P < 0.05.
The structurally different ROK inhibitor HA-1077 (10-4 M) reducedthe spontaneous tone in pre- and postglomerular vessels to asimilar degree as Y-27632 (Figure 2). However, dilation of thedistal interlobular artery in response to the ROK inhibitorHA-1077 was somewhat weaker as compared with Y-27632-induceddilation. Pre- and postglomerular dilation in response to thePKC inhibitor Ro 31–8220 further revealed the involvementof PKC in the spontaneous tone of renal vessels (Figure 2).Although PKC inhibition dilated efferent arterioles as effectivelyas the ROK inhibitors, dilation of the distal interlobular arteryin response to the PKC inhibitor was clearly less.
Figure 2. Effect of ROK versus PKC inhibition on renal vessels. Percent changes of the diameter of the distal interlobular artery (ilob, d) and of the efferent arteriole (eff) in response to ROK inhibition with Y-27632 (10-4 M) or HA-1077 (10-4 M) or to PKC inhibition with Ro 31–8220 (5 · 10-6 M). Data are mean ± SEM; n = 14 to 23 vessels of n = 5 to 19 animals; * P < 0.05 significant diameter change; oP < 0.05 significant difference versus Y-27632; #P < 0.05 significant difference versus Y-27632 and HA-1077.
Effects of ETB Receptor Agonist (IRL 1620), Guanylyl Cyclase Inhibitor (ODQ), and Adenosine A1 Receptor Agonist (CPA)
The effects of local application of IRL 1620, ODQ, and CPA onluminal diameters and on GBF in hydronephrotic rat kidneys aredepicted in Figure 3. The ETB receptor agonist (IRL 1620) ata local concentration of 10-8 M constricted both pre- and postglomerularvessels and decreased GBF. The most pronounced vasoconstrictionwas observed in efferent arterioles, in agreement with earlierfindings (22). At a local concentration of 3 · 10-5 Mof the guanylyl cyclase inhibitor (ODQ), a stable reductionof GBF was reached within 15 min. The reduction in GBF was dueto a rather homogenous vasoconstriction within the renal arteriolarnetwork ranging from 11% to 19%. The adenosine A1 receptor agonist(10-5 M CPA) induced constriction selectively in preglomerularvessels. In accordance with earlier findings (23,24), the strongestvasoconstriction in response to CPA was observed in the distalsegment of the afferent arteriole in close vicinity to the glomerulus.The effects of IRL 1620, ODQ, and CPA were completely reversible,with return of hemodynamic parameters to baseline values afterwash out (Table 1).
Figure 3. Effect of vasoconstrictors that activate distinct signaling pathways in renal vessels. Percent changes of glomerular blood flow (GBF) and vessel diameters in response to the ETB receptor agonist IRL 1620 (10-8 M), to the guanylyl cyclase inhibitor ODQ (3 · 10-5 M), and to the adenosine A1 receptor agonist CPA (10-5 M). Note the different vascular patterns of constriction observed in response to the three different compounds. ilob, interlobular artery; aff, afferent arteriole; eff, efferent arteriole; p, proximal; d, distal. Data are mean ± SEM; n = 6 to 7 animals; * P < 0.05.
Effects of ETB Receptor Agonist (IRL 1620) after ROK and PKC Inhibition
In the presence of the ROK inhibitor Y-27632, the ETB agonist-inducedvasoconstriction was markedly blunted. As shown in Figure 4,the same concentration (10-8 M) of the ETB agonist (IRL 1620)as applied in the beginning induced only small changes in GBFand in vessel diameters (<6%; P > 0.05) in the presenceof the ROK inhibitor Y-27632. A tenfold higher concentration(10-7 M) of the ETB agonist (IRL 1620) was required to obtaina distinct reduction in GBF, whereas vasoconstriction stillremained weak (<17%, P > 0.05). Importantly, both efferentand preglomerular vasoconstriction in response to the ETB agonist(IRL 1620) were attenuated by the ROK inhibitor Y-27632 to asimilar extent. The structurally different ROK inhibitor HA-1077(10-4 M) blunted ETB agonist-induced vasoconstriction in pre-and postglomerular vessels to a similar degree as Y-27632 (Figure 5).In contrast, the PKC inhibitor Ro 31–8220 did notsignificantly affect ETB agonist-induced vasoconstriction (Figure 5).
Figure 4. Effect of the ROK inhibitor Y-27632 on ETB agonist-induced renal vasoconstriction. Percent changes of GBF and vessel diameters in response to the ETB receptor agonist IRL 1620 (10-8 M) in the absence and presence of the ROK inhibitor Y-27632 (10-4 M). ilob, interlobular artery; aff, afferent arteriole; eff, efferent arteriole; p, proximal; d, distal. Data are mean ± SEM; n = 6 animals; * P < 0.05 significant diameter change; oP < 0.05 significant effect of Y-27632.
Figure 5. Effect of ROK versus PKC inhibition on ETB agonist-induced renal vasoconstriction. Percent changes of the diameter of the distal interlobular artery (ilob, d) and of the efferent arteriole (eff) in response to the ETB receptor agonist IRL 1620 (10-8 M) in the absence and presence of the ROK inhibitor Y-27632 (10-4 M) or HA-1077 (10-4 M) or the PKC inhibitor Ro 31–8220 (5 · 10-6 M). Data are mean ± SEM; n = 6 to 27 vessels of n = 5 to 6 animals; * P < 0.05 significant diameter change; oP < 0.05 significant effect of inhibitor.
Effects of Guanylyl Cyclase Inhibitor (ODQ) and Adenosine A1 Receptor Agonist (CPA) after ROK Inhibition
After ROK inhibition with Y-27632, vasoconstriction inducedby the guanylyl cyclase inhibitor (3 · 10-5 M ODQ) wascompletely abolished both in preglomerular vessels and in efferentarterioles (Figure 6). Likewise, the guanylyl cyclase inhibitor(ODQ) did not induce a significant reduction of GBF in the presenceof the ROK inhibitor Y-27632.
Figure 6. Effect of the ROK inhibitor Y-27632 on renal vasoconstriction induced by guanylyl cyclase inhibition. Percent changes of GBF and vessel diameters in response to the guanylyl cyclase inhibitor ODQ (3 · 10-5 M) in the absence and presence of the ROK inhibitor Y-27632 (10-4 M). ilob, interlobular artery; aff, afferent arteriole; eff, efferent arteriole; p, proximal; d, distal. Data are mean ± SEM; n = 6 animals; * P < 0.05 significant diameter change; oP < 0.05 significant effect of Y-27632.
The same concentration (10-5 M) of the adenosine A1 agonist(CPA) as applied in the beginning did not alter vessel diametersin the presence of the ROK inhibitor Y-27632 (Figure 7). Specifically,the marked vasoconstriction located in the distal afferent arteriolein response to the adenosine A1 agonist (CPA) under controlconditions was completely abolished after ROK inhibition withY-27632. However, there was a detectable decrease in GBF, indicatingpossible vasoconstriction further upstream in larger vessels,e.g., arcuate arteries. A tenfold higher concentration of theadenosine A1 agonist (10-4 M CPA) significantly constrictedonly the proximal interlobular artery in the presence of theROK inhibitor Y-27632. At that high concentration, however,the adenosine A1 agonist (CPA) evoked systemic effects, i.e.,an increase in heart rate and severe hypotension, being mainlyresponsible for the large fall of GBF.
Figure 7. Effect of the ROK inhibitor Y-27632 on A1 agonist-induced renal vasoconstriction. Percent changes of GBF and vessel diameters in response to the adenosine A1 receptor agonist CPA (10-5 M) in the absence and presence of the ROK inhibitor Y-27632 (10-4 M). ilob, interlobular artery; aff, afferent arteriole; eff, efferent arteriole; p, proximal; d, distal. Data are mean ± SEM; n = 7 animals; * P < 0.05 significant diameter change; oP < 0.05 significant effect of Y-27632.
Effects of ROK Inhibition on F-Actin in Renal Vessels
Inhibition of ROK with Y-27632 has been shown to induce disassemblyof stress fibers in cultured cells (11). To test whether Y-27632exerted its renal vasodilator effects via changes in contentor structure of filamentous actin in renal arterioles, Y-27623treated hydronephrotic kidneys were stained for F-actin andexamined by confocal microscopy. F-actin intensity and structuredid not differ between vessels of Y-27632-treated and controltissue (Figure 8). Treatment with the ROK inhibitor Y-27632affected actin filament structure in neither vascular smoothmuscle cells (arrowheads in Figure 8) nor endothelial cells(arrows in Figure 8).
Figure 8. Effect of the ROK inhibitor Y-27632 on F-actin in renal vessels. F-actin distribution in arcuate arteries (ARC), interlobular arteries (ILOB), and afferent arterioles (AFF) of the hydronephrotic rat kidney visualized by confocal microscopy. Images were taken with identical settings of confocal parameters for control and tissue treated with the ROK inhibitor Y-27632 (10-4 M). Intensity and distribution of F-actin in renal vessels were similar for control and Y-27632 treated kidneys. Actin filaments around nuclei in smooth muscle cells are marked by arrowheads; actin filaments in endothelial cells are marked by arrows. Magnifications: x130 (ARC, Control); x300 (ARC, Y-27632), and x600 (ILOB, AFF).
In the present study, we demonstrate that inhibition of ROKblunts the constriction related to distinct signaling pathwaysin preglomerular vessels as well as in efferent arterioles.The investigation of the role of ROK in vasoconstriction invivo became possible with the discovery of specific ROK inhibitors,i.e., HA-1077 (fasudil) (32) and Y-27632 (11). Y-27632 is themost selective ROK inhibitor known, and it blocks MLC kinaseonly at concentrations above 250 µM (11). Using thesetwo ROK inhibitors it has been shown that inactivation of MLCPsignificantly contributes to regulation of vasomotor tone andblood flow in several tissues and organs (2). However, the impactof ROK inhibition on the renal vasculature has been addressedin only one study so far, at a time when the molecular targetof HA1077 was unknown (32). Intravenous bolus injection of HA1077increased renal blood flow by about 20% in anesthetized dogs(32).
Endothelin exerts potent renal vasoconstrictor effects in vivothrough activation of ETA and ETB receptors (18,22). Both ETAand ETB receptors are known to couple to several types of heterotrimericG-proteins predominantly leading to PLC activation and intracellularCa2+ mobilization. ET-1 has been demonstrated to increase theamount of GTP-RhoA (33) and to induce most effective phosphorylationof MLC and contraction in bovine aortic VSMC (9). Specifically,the ETB receptor is able to couple to G12/13, which in turnactivates RhoA (34). Our results demonstrate an important roleof ROK in mediating ETB-induced renal vasoconstriction. ETBreceptor stimulation leads to marked efferent arteriolar constriction,which is blocked by Y-27632 or HA-1077; therefore, ROK-mediatedconstriction is clearly operative in efferent arterioles. Inagreement with our findings, inhibition of the Rho/ROK pathwayblunted ET-1-induced vasoconstriction in mammary, mesenteric,penile, cerebral, and coronary arteries (8,35–38). Preventingcoronary artery vasospasm in response to ET-1, ROK inhibitionprotected against myocardial ischemia in rabbits (13,39).
The PKC inhibitor Ro 31–8220 dilated pre- and postglomerularvessels, being equally effective as ROK inhibitors in the efferentarteriole but less effective in the interlobular artery (Figure 2).Thus, part of the basal tone in vessels of the hydronephrotickidney in vivo is due to activation of PKC. Despite the reductionof basal vessel tone, PKC inhibition with Ro 31–8220 failedto prevent ETB-induced vasoconstriction in our experiments.The ability of PKC to induce constriction of afferent and efferentarterioles has been demonstrated by stimulating PKC with phorbolester and a diacylglycerol analogue (40). However, selectiveinhibitors of PKC did not affect norepinephrine- and 20-HETE-inducedconstriction of isolated interlobar and interlobular arteries,respectively (41,42), suggesting limited or selective activationof PKC by vasoconstrictors. Furthermore, the inability of Ro31–8220 to inhibit ETB-induced vasoconstriction clearlydemonstrates that the effects of ROK inhibitors Y-27632 andHA-1077 occurred independently of the PKC pathway.
The adenosine A1 receptor decreases intracellular cAMP levelsthrough Gi protein coupling and has a unique function in thekidney in that it mediates the tubuloglomerular feedback (43).Consistent with this notion, the adenosine A1 receptor agonistCPA induced marked vasoconstriction in the distal segment ofthe afferent arteriole, which was sensitive to ROK inhibitionin the present study. Besides cAMP, the cyclic nucleotide cGMPimportantly regulates renal vessel tone being the second messengerof natriuretic peptides and NO (44). Inhibition of soluble guanylylcyclase by ODQ induced a strong vasoconstriction of preglomerularvessels and efferent arterioles in our present study, comparableto the effects of NO synthesis blockade by L-NAME (45). Likewise,Trottier et al. (46) reported that ODQ completely inhibits therenal vasodilation evoked by endogenous NO. Similar to the effectof ROK inhibition on vasoconstriction elicited by reduced cAMPlevels, Y-27632 blocked renal vasoconstriction when cGMP levelswere lowered by ODQ.
There are several targets in the pathways of agonist-inducedMLC phosphorylation that can be inhibited by cAMP- and cGMP-dependentkinases (2,3). Thus, changes in intracellular cAMP and cGMPlevels result in more or less pronounced changes of vasomotortone depending on the current degree of MLC phophorylation.Our observation that decreasing cAMP or cGMP levels failed toinduce vasoconstriction in the presence of the ROK inhibitorY-27632 could be interpreted in a way that both nucleotidesinterfere with the Rho/ROK pathway. Indeed it has been shownthat both cAMP- and cGMP-dependent kinases inhibit the Rho/ROKpathway via serine phosphorylation of RhoA (47–49). Onthe other hand, in our study ROK inhibition could have loweredthe level of MLC phosphorylation to such an extent that reductionof cAMP or cGMP was no longer able to induce vasoconstrictionthrough disinhibition of pathways unrelated to the Rho/ROK pathway.In any case, our results indicate that ROK inhibition impairsvasoconstriction mediated by cAMP and cGMP pathways.
Besides MLCP, ROK also inactivates LIM kinase, thereby inhibitingactin depolymerization (16). Consequently, ROK inhibition hasbeen shown to consistently cause disassembly of stress fibersand a decrease of the F- to G-actin ratio in various culturedcell types, including VSMC (11,50). Matrougui et al. (17) demonstratedthat ROK inhibition as well as actin filament disruption bycytochalasin B abolished the contractile response of isolatedmesenteric arteries to angiotensin II. For the first time, ourexperiments provide evidence that ROK inhibition results invasodilation without affecting F-actin organization in vesselsin vivo. Additional downstream effectors of RhoA (e.g., mDia)(16), which stimulate actin polymerization, might be responsiblefor the maintenance of the actin cytoskeleton in VSMC duringROK inhibition in vivo.
In their first report on Y-27632, Uehata et al. (11) have alreadydescribed that ROK inhibition induces stronger reductions inarterial BP in hypertensive as compared with normotensive rats.Recently, this finding has been extended to humans, in thatthe increase in forearm blood flow induced by the ROK inhibitorHA-1077 was about twofold higher in hypertensive patients ascompared with normotensive subjects (12). There are two findingssuggesting a potential therapeutic benefit of ROK inhibitionin the renal vasculature. First, lovastatin reduces the levelof Rho proteins in renal vessels of hypertensive rats (51).Second, enhanced activation of phospholipase D by angiotensinII in renal VSMC of hypertensive rats is sensitive to blockadeof the Rho/ROK pathway (52). The fact that ROK inhibition bluntedvasoconstriction in preglomerular vessels as well as in efferentarterioles is of therapeutic relevance and contrasts with theaction of Ca2+ antagonists, which are without effect on L-typechannel-lacking efferent arterioles (53,54).
In conclusion, we demonstrate that ROK inhibitors blunt renalvasoconstriction evoked by distinct signaling pathways withoutaffecting vascular F-actin organization in vivo.
Acknowledgments
The authors are indebted to Rudolf Dussel for technical assistance.This work was supported in part by a grant from SocietàItaliana di Nefrologia (SIN) to Alessandro Cavarape, and bya grant (VIGONI project) from the joint Committee of the Conferenzadei Rettori delle Università Italiane (CRUI) and theDeutscher Akademischer Ausstauschdienst (DAAD) to AlessandroCavarape, Ettore Bartoli, and Karlhans Endlich.
Horowitz A, Menice CB, Laporte R, Morgan KG: Mechanisms of smooth muscle contraction. Physiol Rev 76: 967–1003, 1996[Abstract/Free Full Text]
Somlyo AP, Somlyo AV: Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522: 177–185, 2000[Abstract/Free Full Text]
Pfitzer G: Regulation of myosin phosphorilation in smooth muscle. J Appl Physiol 91: 497–503, 2001[Abstract/Free Full Text]
Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, Matsuura Y, Kaibuchi K: Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem 271: 20246–20249, 1996[Abstract/Free Full Text]
MacDonald JA, Borman MA, Muranyi A, Somlyo AV, Hartshorne DJ, Haystead TA: Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc Natl Acad Sci USA 98: 2419–2424, 2001[Abstract/Free Full Text]
Eto M, Ohmori T, Suzuki M, Furuya K, Morita F: A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C. Isolation from porcine aorta media and characterization. J Biochem 118: 1104–1107, 1995[Abstract/Free Full Text]
Eto M, Kitazawa T, Yazawa M, Mukai H, Ono Y, Brautigan DL: Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C and isoforms. J Biol Chem 276: 29072–29078, 2001[Abstract/Free Full Text]
Batchelor TJ, Sabada JR, Ishola A, Pacaud P, Munsch CM, Beech D: Rho-kinase inhibitors prevent agonist-induced vasospasm in internal mammary artery. Br J Pharmacol 132: 302–308, 2001[CrossRef][Medline]
Gohla A, Schultz G, Offermanns S: Role for G12/G13 in agonist-induced vascular smooth muscle cell contraction. Circ Res 87: 221–227, 2000[Abstract/Free Full Text]
Lucius CA, Anders A, Steusloff M, Troschka F, Hofmann K, Aktories K, Pfitzer G: Clostridium difficile toxin B inhibits carbachol-induced force and myosin light chain phosphorylation in guinea-pig smooth muscle: role of Rho proteins. J Physiol 506: 83–93, 1998[Abstract/Free Full Text]
Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S: Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990–994, 1997[CrossRef][Medline]
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 38: 1307–1310, 2001[Abstract/Free Full Text]
Sato S, Ikegaki I, Asano T, Shimokawa H: Antiischemic properties of fasudil in experimental models of vasospastic angina. Jpn J Pharmacol 87: 34–40, 2001[CrossRef][Medline]
Seguchi H, Nishimura J, Toyofuku K, Kobayashi S, Kumazawa J, Kanaide H: The mechanism of relaxation induced by atrial natriuretic peptide in the porcine renal artery. Br J Pharmacol 118: 343–351, 1996[Medline]
Kaibuchi K, Kuroda S, Amano M: Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annu Rev Biochem 68: 459–486, 1999[CrossRef][Medline]
Matrougui K, Tanko LB, Loufrani L, Gorny D, Levy BI, Tedgui A, Henrion D: Involvement of Rho-kinase and the actin filament network in angiotensin II-induced contraction and extracellular signal-regulated kinase activity in intact rat mesenteric resistance arteries. Arterioscler Thromb Vasc Biol 21: 1288–1293, 2001[Abstract/Free Full Text]
Cavarape A, Endlich K, Feletto F, Parekh N, Bartoli E, Steinhausen M: Contribution of endothelin receptors in renal microvessels in acute cyclosporine-mediated vasoconstriction in rats. Kidney Int 53: 963–969, 1998[Medline]
Wayland H, Johnson P: Erythrocyte velocity measurement in microvessels by a two slit-photometric method. J Appl Physiol 22: 333–337, 1967[Free Full Text]
Gaethgens P: Flow of blood through narrow capillaries: Rheological mechanisms determining capillary hematocrit and apparent viscosity. Biorheology 17: 183–189, 1980[Medline]
Steinhausen M, Blum M, Fleming JT, Holz FG, Parekh N, Wiegman DL: Visualization of renal autoregulation in the split hydronephrotic kidney of rats. Kidney Int 35: 1151–1160, 1989[Medline]
Endlich K, Hoffend J, Steinhausen M: Localization of endothelin ETA and ETB receptor-mediated constriction in the renal microcirculation of rats. J Physiol 497: 211–218, 1996[Medline]
Dietrich MS, Endlich K, Parekh N, Steinhausen M: Interaction between adenosine and angiotensin II in renal microcirculation. Microvasc Res 41: 275–288, 1991[CrossRef][Medline]
Endlich K, Steinhausen M: Role of kinins and angiotensin II in the vasodilating action of angiotensin converting enzyme inhibition in rat renal vessels. J Hypertens 15: 633–641, 1997[CrossRef][Medline]
Gabriels G, Endlich K, Rahn KH, Schlatter E, Steinhausen M: In vivo effects of diadenosine polyphosphates on rat renal microcirculation. Kidney Int 57: 2476–2484, 2000[CrossRef][Medline]
Davies SP, Reddy H, Caivano M, Cohen P: Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105, 2000[CrossRef][Medline]
Lanese DM, Conger JD: Effects of endothelin receptor antagonist on cyclosporine-induced vasoconstriction in isolated rat renal arterioles. J Clin Invest 91: 2144–2149, 1993[Medline]
Loutzenhiser R, Epstein M, Hayashi K, Horton C: Direct visualization of effects of endothelin on the renal microvasculature. Am J Physiol 258: F61–F68, 1990[Medline]
Loutzenhiser R, Epstein M, Hayashi K, Takenaka T, Forster H: Characterization of the renal microvascular effects of angiotensin II antagonist, DuP 753: Studies in isolated perfused hydronephrotic kidneys. Am J Hypertens 4: 309S–314S, 1991[Medline]
Welsch T, Endlich N, Kriz W, Endlich K: CD2AP and p130Cas localize to different F-actin structures in podocytes. Am J Physiol Renal Physiol 281: F769–F777, 2001[Abstract/Free Full Text]
Asano T, Suzuki T, Tsuchiya M, Satoh S, Ikegaki I, Shibuya M, Suzuki Y, Hidaka H: Vasodilator actions of HA1077 in vitro and in vivo putatively mediated by the inhibition of protein kinase. Br J Pharmacol 98: 1091–1100, 1989[Medline]
Sakurada S, Okamoto H, Takuwa N, Sugimoto N, Takuwa Y: Rho activation in excitatory agonist-stimulated vascular smooth muscle. Am J Physiol Cell Physiol 281: C571–C578, 2001[Abstract/Free Full Text]
Kitamura K, Shiraishi N, Singer WD, Handlogten ME, Tomita K, Miller RT: Endothelin-B receptors activate G13. Am J Physiol 276: C930–C937, 1999[Medline]
Matsumura Y, Kita S, Okui T: Mechanisms of endothelin-1-induced potentiation of noradrenaline response in rat mesenteric artery. Clin Exp Pharmacol Physiol 28: 36999–37003, 2001
Mills TM, Chitaley K, Wingard CJ, Lewis RW, Webb RC: Effect of Rho-kinase inhibition on vasoconstriction in the penile circulation. J Appl Physiol 91: 1269–1273, 2001[Abstract/Free Full Text]
Nakamura K, Nishimura J, Hirano K, Ibayashi S, Fujishima M, Kanaide H: Hydroxyfasudil, an active metabolite of fasudil hydrochloride, relaxes the rabbit basilar artery by disinhibition of myosin light chain phosphatase. J Cereb Blood Flow Metab 21: 876–885, 2001[CrossRef][Medline]
Sato A, Hattori Y, Sasaki M, Tomita F, Kohya T, Kitabatake A, Kanno M: Agonist-dependent difference in the mechanisms involved in Ca2+ sensitization of smooth muscle of porcine coronary artery. J Cardiovasc Pharmacol 35: 814–821, 2000[CrossRef][Medline]
Yamamoto Y, Ikegaki I, Sasaki Y, Uchida T: The protein kinase inhibitor fasudil protects against ischemic myocardial injury induced by endothelin-1 in the rabbit. J Cardiovasc Pharmacol 35: 203–211, 2000[CrossRef][Medline]
Nagahama T, Hayashi K, Ozawa Y, Takenaka T, Saruta T: Role of protein kinase C in angiotensin II-induced constriction of renal microvessels. Kidney Int 57: 215–223, 2000[CrossRef][Medline]
Fetscher C, Chen H, Schafers RF, Wambach G, Heusch G, Michel MC: Modulation of noradrenaline-induced microvascular constriction by protein kinase inhibitors. Naunyn Schmiedebergs Arch Pharmacol 363: 57–65, 2001[CrossRef][Medline]
Sun CW, Falck JR, Harder DR, Roman RJ: Role of tyrosine kinase and PKC in the vasoconstrictor response to 20-HETE in renal arterioles. Hypertension 33: 414–418, 1999[Abstract/Free Full Text]
Sun D, Samuelson LC, Yang T, Huang Y, Paliege A, Saunders T, Briggs J, Schnermann J: Mediation of tubuloglomerular feedback by adenosine: Evidence from mice lacking adenosine 1 receptors. Proc Natl Acad Sci USA 98: 9983–9988, 2001[Abstract/Free Full Text]
Gabbai FB, Blantz RC: Role of nitric oxide in renal hemodynamics. Semin Nephrol 19: 242–250, 1999[Medline]
Hoffend J, Cavarape A, Endlich K, Steinhausen M: Influence of endothelium-derived relaxing factor on renal microvessels and pressure-dependent vasodilation. Am J Physiol 265: F285–F292, 1993[Medline]
Trottier G, Triggle CR, O’Neill SK, Loutzenhiser R: Cyclic GMP-dependent and cyclic GMP-independent actions of nitric oxide in the renal afferent arteriole. Br J Pharmacol 125: 563–569, 1998[CrossRef][Medline]
Lang P, Gesbert F, Delespine-Carmagnat M, Stancou R, Pouchelet M, Bertoglio J: Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes. EMBO J 15: 510–519, 1996[Medline]
Sauzeau V, Le Jeune H, Cario-Toumanianz C, Smolenski A, Lohmann SM, Bertoglio J, Chardin P, Pacaud P, Loirand G: Cyclic GMP-dependent protein kinase signalling pathway inhibits RhoA-induced Ca2+: Sensitisation of contraction in vascular smooth muscle. J Biol Chem 275: 21722–21729, 2000[Abstract/Free Full Text]
Sawada N, Itoh H, Yamashita J, Doi K, Inoue M, Masatsugu K, Fukunaga Y, Sakaguchi S, Sone M, Yamahara K, Yurugi T, Nakao K: cGMP-dependent protein kinase phosphorylates and inactivates RhoA. Biochem Biophys Res Commun 280: 798–805, 2001[CrossRef][Medline]
Sauzeau V, Le Mellionnec E, Bertoglio J, Scalbert E, Pacaud P, Loirand G: Human urotensin II-induced contraction and arterial smooth muscle cell proliferation are mediated by RhoA and Rho-kinase. Circ Res 88: 1102–1104, 2001[Abstract/Free Full Text]
Jiang J, Sun CW, Alonso-Galicia M, Roman RJ: Lovastatin reduces renal vascular reactivity in spontaneously hypertensive rats. Am J Hypertens 11: 1222–1231, 1998[CrossRef][Medline]
Andresen BT, Jackson EK, Romero GG: Angiotensin II signaling to phospholipase D in renal microvascular smooth muscle cells in SHR. Hypertension 37: 635–639, 2001[Abstract/Free Full Text]
Fleming JT, Parekh N, Steinhausen M: Calcium antagonists preferentially dilate preglomerular vessels of hydronephrotic kidney. Am J Physiol 253: F1157–F1163, 1987[Medline]
Hansen PB, Jensen BL, Andreasen D, Skott O: Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels. Circ Res 89: 630–638, 2001[Abstract/Free Full Text]
Received for publication February 25, 2002.
Accepted for publication August 19, 2002.
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Top
Abstract
Introduction
and 
isoforms have been identified to phosphorylate CPI-17, which, in the phosphorylated state, inhibits MLCP specifically (6,7). A recently developed ROK inhibitor, the pyridine derivative Y-27632, has been shown to potently inhibit the contraction of preparations from experimental animals and human arterial vessels induced by different agonists in vitro and also to correct arterial pressure to normotensive levels in different experimental models of hypertension in vivo (8–11). Because of the evidence that the Rho/ROK pathway mediates tonic vasoconstriction, ROK is thought to play a crucial role in human diseases, e.g., in hypertension and myocardial ischemia (12,13). The contribution of the ROK pathway to renal vasoconstriction is practically unknown. 
