Rho-kinase inhibition reduces pressure-mediated autoregulatory adjustments in afferent arteriolar diameter

Abstract

Preglomerular resistance is regulated by calcium influx- and mobilization-dependent mechanisms; however, the role of Rho-kinase in calcium sensitization in the intact kidney has not been carefully examined. Experiments were performed to test the hypothesis that Rho-kinase inhibition blunts pressure-mediated afferent arteriolar autoregulatory behavior and vasoconstrictor responses evoked by angiotensin II and P2X₁ receptor activation. Rat kidneys were studied in vitro using the blood-perfused juxtamedullary nephron technique. Autoregulatory behavior was assessed before and during Rho-kinase inhibition with Y-27632 (1.0 μM; n = 5). Control diameter averaged 14.3 ± 0.8 μm and increased to 18.1 ± 0.9 μm (P < 0.05) during Y-27632 treatment. In the continued presence of Y-27632, reducing perfusion pressure to 65 mmHg slightly increased diameter to 18.7 ± 1.0 μm. Subsequent pressure increases to 130 and 160 mmHg yielded afferent arteriolar diameters of 17.5 ± 0.8 and 16.6 ± 0.6 μm (P < 0.05). This 11% decline in diameter is significantly smaller than the 40% decrease obtained in untreated kidneys. The inhibitory effects of Y-27632 on autoregulatory behavior were concentration dependent. Angiotensin II responses were blunted by Y-27632. Angiotensin II (1.0 nM) reduced afferent diameter by 17 ± 1% in untreated arterioles and by 6 ± 2% during exposure to Y-27632. The P2X₁ receptor agonist, α, β-methylene ATP, reduced afferent arteriolar diameter by 8 ± 1% but this response was eliminated during exposure to Y-27632. Western blot analysis confirms expression of the Rho-kinase signaling pathway. Thus, Rho-kinase may be important in pressure-mediated autoregulatory adjustments in preglomerular resistance and responsiveness to angiotensin II and autoregulatory P2X₁ receptor agonists.

renal perfusion is controlled by agonist-induced and pressure-mediated adjustments in preglomerular resistance (51). Accurate regulation of preglomerular resistance is central to the maintenance of glomerular capillary pressure. Afferent arterioles are the main vascular elements responsible for pressure-mediated autoregulatory adjustments in preglomerular resistance for the regulation of renal blood flow and glomerular filtration rate (9, 51, 53). Autoregulatory changes in renal vascular resistance are calcium dependent and involve the combined influences of the myogenic and tubuloglomerular feedback mechanisms (4, 12, 21, 28, 49, 52, 53, 58, 61). Influx of calcium from the extracellular medium is an essential signaling pathway leading to autoregulatory vasoconstriction (12, 52, 53, 58). Blockade of L-type calcium channels, or removal of calcium from the extracellular medium, inhibits autoregulatory vasoconstriction (10, 21, 23, 49, 58). These data demonstrate the essential role elevation of intracellular calcium concentration plays in autoregulatory vasoconstriction (10, 21, 23, 49, 58). Interestingly, the role of changes in calcium sensitivity to the renal autoregulatory response, or to the afferent arteriolar response to vasoconstrictor agents, has not been extensively investigated (50).

Evidence indicates that renal microvessels express P2X₁ and P2Y₂ receptors and that P2X₁ receptors are important for affecting pressure-mediated autoregulatory vasoconstriction of afferent arterioles (26, 27, 30). P2X₁ receptors stimulate afferent arteriolar vasoconstriction by stimulating calcium influx largely through voltage-dependent L-type calcium channels (25, 32–34, 67). In contrast, P2Y₂ receptors stimulate vasoconstriction mainly by mobilizing calcium from intracellular stores (25, 32). Inactivation of P2X₁ receptors blocks pressure-mediated autoregulatory vasoconstriction leading to the postulate that increases in transmural pressure lead to ATP-mediated activation of P2X₁ receptors to produce the autoregulatory vasoconstriction response (26, 30).

Elevation of intracellular calcium concentration is an important component of afferent arteriolar autoregulatory responses and in the response of these arterioles to vasoconstrictor agonists. Considerable evidence supports an essential role of intracellular calcium in the afferent arteriolar response to pressure, angiotensin II, norepinephrine, endothelin, and to P2 receptor agonists (8, 19, 20, 28, 31, 40–43, 50, 64). While increasing the intracellular calcium concentration facilitates contraction of vascular smooth muscle, studies indicate that enhancing the sensitivity of the contractile apparatus to calcium is also important (6, 38, 39, 48, 50, 60, 66). Many investigators examined the calcium signaling pathways involved in the renal vascular response to vasoconstrictor stimuli but little has been done to determine the contribution of altered calcium sensitivity in these responses (48).

The mechanisms underlying modulation of calcium sensitivity are poorly understood. Previous work focused on protein kinase C, but recently attention has shifted to the possible contribution of Rho and the Rho-associated family of proteins as a way to modulate calcium sensitivity (3, 6, 16, 35, 62, 65). Activation of a small GTP-binding protein, RhoA, stimulates Rho-kinase activation, which inhibits myosin light chain phosphatase, thus enhancing the interaction between actin and the myosin light chains (2, 45). Cavarape and colleagues (14) reported that Rho-kinase inhibition with Y-27632, or HA-1077, blunted renal microvascular vasoconstriction to the ET_B receptor agonist, IRL-1620, to the A₁ agonist, cyclopentyladenosine, or to guanylyl cyclase inhibition. Myogenic responses are blunted by Rho-kinase inhibition in hydronephrotic kidneys and in nonrenal blood vessels (50, 62). The effects on autoregulation in intact kidneys or the P2 receptor events postulated to participate in autoregulatory responses have not been investigated. Therefore, the current study was undertaken to assess the impact of Rho-kinase inhibition on pressure-mediated autoregulatory behavior and on the vasoconstriction induced by P2 receptor agonists.

METHODS

Studies were approved by the Committee on Animal Use for Research and Education at the Medical College of Georgia.

Juxtamedullary nephron preparation.

Experiments were performed in vitro using kidneys prepared for the blood-perfused juxtamedullary nephron technique, as previously described (25, 29). Ninety-eight male Sprague-Dawley rats (350–400 g) were used to complete these studies. For each experiment, two animals were anesthetized with pentobarbital sodium (40 mg/kg ip) and prepared for videomicroscopy experiments. Perfusate blood was collected and prepared, as previously described (28, 29). Briefly, blood was collected from the nephrectomized blood donor rat into a heparinized syringe (500 U). The plasma and washed erythrocyte fractions were combined to yield a reconstituted blood perfusate with a hematocrit of ∼33% as previously described (25, 29).

When the dissection was completed, the Tyrode's buffer perfusate was replaced with the reconstituted blood. The blood perfusate was stirred continuously in a closed reservoir while being oxygenated with a 95% O₂-5% CO₂ gas mixture. Perfusion pressure was continuously monitored using a pressure cannula positioned in the tip of a double-barreled perfusion cannula and connected to a pressure transducer (model TRN005, Kent Scientific) linked to a Grass Polygraph (model 7D, Grass Instrument, Quincy, MA). Perfusion pressure was fixed at 110 mmHg. The inner cortical surface of the kidney was superfused with Tyrode's buffer (37°C) containing 1.0% bovine serum albumin and the kidney was allowed to equilibrate for at least 15 min.

The perfusion chamber, containing the prepared kidney, was attached to the stage of a Nikon Optiphot-2UD microscope (Nikon, Tokyo, Japan) equipped with a Zeiss water immersion objective (×40). The tissue was transilluminated and the focused image, obtained with a Newvicon camera (NC-70, Dage-MTI, Michigan City, IN), was passed through an image processor (MFJ-1425, MFJ Enterprises, Starkville, MS) and displayed on a video monitor while being simultaneously recorded on DVD for later analysis. Vascular inside diameters were measured at a single site using an image shearing monitor (model 908, Vista Electronics, Ramona, CA). The image shearing monitor was calibrated using a stage micrometer.

Experimental protocols.

Afferent arteriolar responses were determined to changes in renal perfusion pressure, or administration of vasoactive agonists and antagonists. Autoregulatory behavior was assessed by measuring changes in afferent arteriolar diameter in response to acute elevations in renal perfusion pressure. Measurements of afferent arteriolar diameter were made continuously at 12-s intervals. Sustained afferent arteriolar diameter was calculated from the average of all measurements made during the final 2 min of each treatment period. Each protocol began with a 5-min control period to ensure a stable vessel diameter and was followed by either agonist stimulation or an increase in renal perfusion pressure to establish the control response.

Effect of Y-27632 on afferent arteriolar responses to angiotensin II and to acute increases in renal perfusion pressure.

The effect of angiotensin II, and of increasing renal perfusion pressure, on afferent arteriolar diameter was determined with and without treatment with the Rho-kinase inhibitor, Y-27632 (3, 13, 14, 65). Experiments examined afferent arteriolar responses to 1.0 nM angiotensin II followed by introduction of Y-27632 at concentrations of 0.1, 1.0, and 10 μM for a period of 20 min. In the presence of Y-27632, afferent arteriolar autoregulatory responses were measured at perfusion pressures of 100, 65, 130, and 160 mmHg and again at 100 mmHg in successive 5-min periods, followed by reassessment of the response to angiotensin II. Separate control animals were prepared to establish afferent arteriolar autoregulatory responses and responses to angiotensin II in naive controls. Separate groups of rats were used for each concentration of Y-27632. Y-27632 and angiotensin II were applied in the superfusion solution.

Effect of Y-27632 on the afferent arteriolar response to P2 receptor agonists, ATP, α, β-methylene ATP, and UTP.

P2 receptors are important for afferent arteriolar autoregulatory responses. Thus, experiments were performed to determine the effect of Rho-kinase inhibition with Y-27632 on the vasoconstriction induced by P2 receptor activation. Renal microvessels express P2X₁ and P2Y₂ receptors, which are activated by ATP. P2X₁ receptors have been implicated in mediating afferent arteriolar autoregulatory responses. Accordingly, we examined the impact of Rho-kinase inhibition on the vasoconstrictor actions of the P2X₁ agonist, α, β-methylene ATP, the P2Y₂ agonist, UTP, and the endogenous ligand for P2 receptors, ATP. Afferent arteriolar responses were determined before and during Y-27632 (1.0 μM) treatment.

Western blot analysis of renal cortex, renal medulla, and the preglomerular microvasculature.

Renal cortex, medulla, and freshly isolated preglomerular microvessels were homogenized in an ice-cold homogenization buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% deoxycholic acid, 1.0 mM EDTA, and supplemented with 1 mM phenylmethylsulfornyl fluoride, 1.0 mM Na₃VO₄, and 1× protease inhibitor cocktail. After the tissue lysates were centrifuged (10,000 g; 30 min), the supernatants were collected and protein concentrations were determined by a bicinchoninic acid kit. Lanes were loaded with equal amounts of total protein and the individual proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membrane. Membranes were blocked for 1 h with 5% milk in Tris-buffered saline containing 0.5% Tween-20 (TBST) and then incubated with monoclonal antibody, Rho-kinase α (1:1,000), or Rho-kinase β (1:1,000) overnight at 4°C. After being washed with TBST, the membranes were incubated with a horseradish peroxidase-linked secondary antibody and visualized using an enhanced chemiluminescence kit.

Statistical analysis.

Data were evaluated using a one-way ANOVA for repeated measures. Differences between group means, within each series, were determined using Newman-Kuels multiple range test. P values <0.05 were considered to indicate statistically significant differences. All values are reported as means ± SE.

Materials.

Male Sprague-Dawley rats were obtained from Charles River Laboratories (Raleigh, NC). Bovine serum albumin and Y-27632 were obtained from Calbiochem (La Jolla, CA). Bicinchoninic acid protein assay kit was purchased from Pierce (Rockford, IL). Chemiluminescence kit was purchased from Amersham (Piscataway, NJ). Protease inhibitor cocktail was purchased from Roche Applied Science (Indianapolis, IN). Rho-kinase α and Rho-kinase β antibodies were purchased from BD Biosciences (San Jose, CA). All other reagents were purchased from Sigma (St. Louis, MO).

RESULTS

Initial experiments determined the effect of Y-27632 on pressure-mediated autoregulatory responses and on the afferent arteriolar response to angiotensin II. Under control conditions, at 100 mmHg, afferent arteriolar diameter averaged 15.9 ± 0.7 μm (n = 15) and was similar across the three Y-27632 treatment groups. In the absence of Y-27632, exposure to 1.0 nM angiotensin II reduced afferent arteriolar diameter similarly by 14 ± 4, 18 ± 2, and 16 ± 2% (n = 5, P < 0.05 vs. control in each Y-27632 group), respectively (Fig. 1, left). In the presence of Y-27632, baseline vessel diameter increased in a concentration-dependent manner. Afferent arteriolar diameter increased by 10 ± 4, 22 ± 3, and 43 ± 7% in response to 100 nM, 1.0 μM, and 10 μM Y-27632 concentrations, respectively. Y-27632 treatment inhibited angiotensin II-mediated vasoconstriction at concentrations above 100 nM (Fig. 1, right). Exposure to 1.0 nM angiotensin II reduced afferent arteriolar diameter by 19 ± 4 and 6 ± 2% at Y-27632 concentrations of 100 nM and 1.0 μM and eliminated the response at the 10 μM Y-27632 concentration.

Fig. 1.

Increasing concentrations of Y-27632 inhibit the afferent arteriole response to angiotensin II (ANG II). Responses before (left) and during (right) treatment with Y-27632 are depicted. Y-27632 was administered at concentrations of 100 nM (bottom), 1 μM (middle), and 10 μM (top). Responses of individual arterioles are depicted by the plots (gray circle symbols) and mean responses are depicted by the black squares. *P < 0.05 vs. the diameter before ANG II treatment. #P < 0.05 vs. the response ANG II before Y-27632 treatment. The P values presented in each figure represent comparison of the response before and during Y-27632 treatment; n = 5 in each Y-27632 treatment group. Con, control.

Compared with control arterioles (Fig. 2, black circles), autoregulatory responsiveness was attenuated at each Y-27632 concentration examined (Fig. 2, square symbols). Significant pressure-mediated autoregulatory responses were observed in arterioles treated with 0.1 μM Y-27632 (white squares) but higher concentrations of Y-27632 virtually eliminated the autoregulatory response resulting in either a flat pressure diameter relationship (1.0 μM, gray squares) or a pressure-dependent increase in diameter consistent with a passive pressure diameter relationship (10 μM, black squares). These data suggest that Rho-kinase plays an important role in pressure-mediated autoregulatory vasoconstriction.

Fig. 2.

Increasing concentrations of Y-27632 inhibit the pressure-mediated afferent arteriolar autoregulatory response. Data are plotted as a percent of the control diameter. Control arterioles are depicted by black circles. Data from arterioles treated with Y-27632 are depicted by square symbols (0.1 μM Y-27632, white squares; 1 μM Y-27632, gray squares; and 10 μM Y-27632, black squares). The sample size for each group is indicated by numbers in parentheses. *P < 0.05 vs. diameter at 100 mmHg. #P < 0.05 vs. control diameter before Y-27632 treatment.

In separate experiments, we determined the effect of Y-27632 on the afferent arteriolar response to P2 receptor activation. Control diameters were similar across treatment groups and averaged 17.7 ± 1.1, 17.7 ± 0.6, and 16.5 ± 0.6 μm for the 1.0, 10, and 100 μM ATP groups, respectively. As shown in Fig. 3, left, ATP produced a significant and reversible afferent arteriolar vasoconstriction. Afferent diameter decreased by 13, 25, and 23% in response to 1, 10, and 100 μM ATP, respectively. Y-27632 blunted the afferent arteriolar response to ATP (Fig. 3, right). In the presence of Y-27632, afferent diameter decreased by 5, 15, and 7% in response to 1, 10, and 100 μM ATP, respectively. While 10 μM ATP still reduced diameter significantly, the magnitude of the vasoconstriction evoked by each ATP concentration was significantly reduced compared with the control responses in the absence of Y-27632.

Fig. 3.

Y-27632 inhibits afferent arteriolar responses to increasing concentrations of ATP. The afferent arteriolar response to ATP (1.0 μM, bottom; 10 μM, middle; and 100 μM, top) was determined before (left) and during (right) treatment with 1 μM Y-27632. Responses of individual arterioles are depicted by the gray symbols and the mean responses are presented by the black squares. *P < 0.05 vs. control diameter before ATP exposure. #P < 0.05 vs. magnitude of the control response before Y-27632 treatment.

Renal microvessels express multiple P2 receptor subtypes including P2X₁ and P2Y₂ (25, 29, 32, 67, 69). P2X₁ receptors, but not P2Y₂ receptors, have been implicated in mediating pressure-dependent autoregulatory responses of afferent arterioles (27, 30). Given that Y-27632 significantly inhibited autoregulatory responses, we examined the effect of Y-27632 of afferent arteriolar response to P2X₁ and P2Y₂ receptor activation. P2X₁ receptors were stimulated using the selective agonist, α, β-methylene ATP (1.0 μM), whereas UTP (10 μM) was used to selectively stimulate P2Y₂ receptors (56). These concentrations were selected based on previous studies showing that they yield vasoconstrictor responses similar in magnitude to those obtained with ATP (29). As shown in Fig. 4, left, α, β-methylene ATP (bottom) and UTP (top) significantly and reversibly reduced afferent arteriolar diameter by 8 ± 1 and 31 ± 5%, respectively. In the presence of Y-27632, the sustained response to α, β-methylene ATP was completely eliminated while the response to UTP was not significantly affected. These data suggest that Rho-kinase may exert differential influences on vasoconstrictor signals evoked by P2X receptors compared with those elicited by P2Y receptor activation.

Fig. 4.

Y-27632 inhibits the afferent arteriolar response to P2X₁ receptor stimulation, but not P2Y₂ receptor activation. P2X₁ receptors were stimulated with α, β-methylene ATP (1.0 μM, bottom) and P2Y₂ receptors were stimulated with UTP (10 μM, top). Control responses are shown on the left and responses during Y-27632 treatment are shown on the right. Responses of individual arterioles are depicted by the gray symbols and the mean responses are presented by the black squares. *P < 0.05 vs. control diameter before agonist exposure. #P < 0.05 vs. magnitude of the control response before Y-27632 treatment.

The data plotted in Fig. 5 provide a temporal profile of the vasoconstriction evoked by α, β-methylene ATP (1.0 μM; bottom), ATP (10 μM; middle), and UTP (10 μM; top) before and during treatment with Y-27632. Consistent with previous reports (29, 30), afferent arteriolar vasoconstrictor responses to ATP and α, β-methylene ATP are biphasic with a rapid initial phase that partially recovers to a stable diameter smaller than the control diameter (Fig. 5, bottom and middle). Responses to UTP and other P2Y₂ agonists are monophasic in that diameter decreases rapidly on exposure to agonists but vessel diameter quickly stabilizes at or near its nadir (Fig. 5). Baseline diameter increased significantly on introduction of Y-27632 to the bathing solution. Y-27632 had no detectable effect on the rapid initial response evoked by α, β-methylene ATP but the sustained vasoconstriction was completely eliminated (Fig. 5, bottom). Similarly, the initial phase of the response to ATP was also unaffected by Y-27632 but the magnitude of the sustained vasoconstriction was significantly blunted compared with the control condition. Interestingly, the time course and magnitude of the response evoked by UTP during Y-27632 treatment were similar to the control conditions. These data support the contention that different signaling pathways are involved in P2X vs. P2Y receptor activation and that Rho-kinase plays unique roles in those pathways.

Fig. 5.

Temporal response afferent arterioles to P2 receptor agonists before and during Y-27632 treatment. Control responses are depicted by the black symbols and responses obtained from the same arterioles during Y-27632 treatment are depicted by the white symbols. Responses obtained with UTP, ATP, and α, β-methylene ATP are presented in the top, middle, and bottom, respectively. Each symbol represents measurements taken at 12-s intervals. The response during Y-27632 administration is overlaid on the control response for comparison. *P < 0.05 vs. control diameter at 100 mmHg. #P < 0.05 vs. the diameter in the absence of Y-27632.

Figure 6 presents representative Western blots demonstrating expression of Rho-kinase isoforms in homogenates of preglomerular microvessels, renal cortex, and renal medulla. β-Actin staining is provided as a loading control. Clearly, Rho-kinase α (180 kDa) and Rho-kinase β (160 kDa) were also detected in the preglomerular microvasculature, renal cortex, and renal medulla, thus supporting the functional data provided in Figs. 1–5, which implicate Rho-kinase in renal vascular function.

Fig. 6.

Expression of Rho-kinase isoforms in rat preglomerular microvessels (MV), renal cortex (C), and renal medulla (M). Prominent bands are detected for Rho-kinase α at ∼180 kDa and Rho-kinase β at 160 kDa.

DISCUSSION

The current report establishes a central role for Rho-kinase in regulating afferent arteriolar function and extends previous work to demonstrate Rho-kinase involvement in autoregulatory responses and in responses to autoregulatory mediators (50). Western blot analysis demonstrated preglomerular expression of Rho-kinase α and Rho-kinase β as well as expression in renal cortical and medullary homogenates. The Rho-kinase inhibitor blunted or abolished afferent arteriolar responses to angiotensin II and selected P2 receptor agonists while having little effect on other P2 receptor activators. Importantly, the Rho-kinase inhibitor significantly attenuated pressure-mediated autoregulatory behavior suggesting that Rho-kinase is an important part of the renal microvascular myogenic signaling cascade.

Consistent with previous reports, Y-27632 significantly blunted afferent arteriolar vasoconstriction mediated by angiotensin II (13, 14). The magnitude of the inhibition increased as Y-27632 concentration increased from 100 nM to 10 μM. It is known that angiotensin II induces vasoconstriction of afferent arterioles by stimulating calcium influx through L-type calcium channels and by stimulating calcium release from intracellular stores (11, 18, 20, 24, 36, 46, 63, 64, 68, 71). Based on our findings, and those of others, it is now clear that a portion of the vasoconstriction induced by angiotensin II involves activation of Rho-kinase and presumably enhanced calcium sensitivity (13, 14).

Autoregulation is a critical function of afferent arterioles (37, 44, 51, 53, 61). Setting preglomerular resistance appropriately is essential for maintenance of a stable renal blood flow and glomerular filtration rate. Autoregulatory adjustments in resistance are calcium dependent and, like the angiotensin II responses, they involve both voltage-dependent calcium influx and calcium mobilization from intracellular stores (4, 12, 22, 28, 49, 52, 58). In the current study, the Rho-kinase inhibitor Y-27632 evoked a concentration-dependent increase of baseline diameter and a concentration-dependent inhibition of pressure-mediated vasoconstriction of juxtamedullary afferent arterioles. At the highest concentration tested, increasing perfusion pressure from 65 to 160 mmHg resulted in a pressure-dependent increase in diameter consistent with a passive pressure/diameter relationship. This blunting of autoregulatory behavior was apparent at each pressure step tested and is consistent with the findings of Nakamura et al. (50) who also reported blunted pressure-dependent responses in the hydronephrotic kidney. Therefore, Rho-kinase activity is an important part of the tension-generating machinery responsible for autoregulatory behavior.

In the kidney, complete autoregulatory responses reflect the combined influences of tubuloglomerular feedback and the myogenic mechanism (51, 53). The inhibition observed in this report likely represents inhibition of myogenic responses as the diameter measurements were made in the first half of afferent arteriolar length (47–52% of the arterioles length), well removed from the tubuloglomerular feedback-sensitive region. This conclusion is also supported by similar observations made in the hydronephrotic kidney, which is devoid of tubuloglomerular feedback influences (50). In addition, inhibition of myogenic behavior has also been reported for other vascular smooth muscle tissues, thus indicating that Rho-kinase involvement in myogenic behavior is not restricted to the renal microcirculation and may represent a common feature of myogenic control systems (17, 57, 62).

Autoregulatory resistance adjustments are induced by activation of ATP-sensitive P2 receptors (26, 30, 47). This is a large receptor class made up of two distinct receptor families, P2X and P2Y (56). This classification is based on important structural differences and mechanistic differences in the intracellular signal transduction pathways utilized. The ionotropic P2X receptor family includes seven unique receptor subtypes labeled P2X₁ through P2X₇ (55). P2X receptors are membrane-bound ligand-gated ion channels composed of two transmembrane domains coupled to a large extracellular loop and short intracellular tails (55). Receptor activation opens the ligand-gated channel permitting influx of a nonselective cation current producing a rapid and reversible vasoconstriction (5, 25, 55, 67).

P2Y receptors are metabotropic, membrane-bound receptors with seven membrane-spanning domains and currently include approximately eight subtypes (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄) (1, 7, 56). P2Y receptor activation can produce either vasoconstriction or vasodilation. P2Y receptors expressed by vascular smooth muscle stimulate vasoconstriction by increasing intracellular calcium concentration and/or by activating the Rho-kinase pathway (15, 25, 32, 54, 59). Based on a collection of evidence, it appears that afferent arterioles express P2X₁ and P2Y₂ receptors at a minimum, and we provided considerable evidence that P2X₁ receptors are essential for transducing increases in perfusion pressure into autoregulatory afferent arteriolar vasoconstrictor responses (26, 29, 32, 67, 69).

Because Rho-kinase inhibition blunts autoregulatory behavior, we examined the impact of Y-27632 on the afferent arteriolar response to ATP and to selective activation of P2X₁ receptors (α, β-methylene ATP) and P2Y₂ receptors (UTP). As noted in the current study, Y-27632 completely blocked the sustained vasoconstriction induced by P2X₁ receptor activation and by 1.0 μM ATP. In contrast, Y-27632 inhibition had little impact on the sustained vasoconstriction induced by P2Y₂ receptor activation with UTP. Previous studies showed that afferent arteriolar responses to lower concentrations of ATP (≤1.0 μM) were calcium influx dependent and could be completely blocked by L-type calcium channel blockade (25, 32). Virtually identical results were obtained during P2X₁ receptor activation suggesting that lower ATP concentrations induce their effects primarily through P2X₁ receptor activation. Higher concentrations of ATP induce vasoconstriction through mechanisms driven more by calcium mobilization, and these responses are mimicked by the P2Y₂ agonist UTP. These observations suggest that responses to higher ATP concentrations involve both P2X₁ and P2Y₂ receptor activation.

In the current report, Y-27632 treatment significantly attenuated ATP-mediated vasoconstriction at all ATP concentrations tested. Given that ATP is capable of activating both P2X and P2Y receptors and that these receptors evoke vasoconstriction through disparate calcium signaling mechanisms, it is possible that retention of some vasoconstrictor activity could reflect this dual receptor activation. The exact explanation for this observation remains unclear. Nevertheless, the results reflecting markedly different outcomes using the P2X₁ and P2Y₂ agonists argue that Rho-kinase exerts complex regulatory influences in the renal microcirculation. Thus, Rho-kinase activation is more than just a final common pathway linking vasoconstrictor stimuli to vasoconstrictor events. Indeed, afferent arterioles employ Rho-kinase activation as an essential component of the signaling pathway for some but not all vasoconstrictor agonists. These data demonstrate that Rho-kinase activation is an important part of the intracellular signaling mechanisms that connect P2X₁ receptor activation with vasoconstriction. In addition, these data demonstrate Rho-kinase-dependent contractile events link P2X₁ receptor activation with autoregulatory adjustments in renal vascular resistance.

Typical ATP-mediated vasoconstrictions are biphasic and manifest as a rapid initial vasoconstriction followed by a partial recovery until a sustained constriction diameter is reached. It is important to note that Y-27632 had little effect on the rapid initial vasoconstriction while having more pronounced effects on the sustained vasoconstriction. The explanation for these distinct temporal effects is unclear. Perhaps Rho-kinase is essential for the sustained response to receptor activation rather than participating in the initiating event arising from receptor activation. The refractory nature of this rapid initial vasoconstriction has been observed previously (33, 70). Calcium channel blockade, or inhibition of 20-HETE, did not affect this initial contractile event substantially, while completely blocking the sustained vasoconstriction. Interestingly, reducing the extracellular calcium concentration (<200 nM) eliminated both the initial and the sustained vasoconstriction induced by P2X₁ receptor activation (33). These data indicate that calcium influx is important for both aspects on the biphasic vasoconstriction by P2X₁ receptors. Thus, the initial vasoconstriction may largely reflect the inwardly directed, nonselective cation current that begins immediately on agonist activation of the ligand-gated P2X₁ receptor protein.

The inhibitor data provide functional evidence that Rho-kinase participates in the generation and maintenance of active tension in afferent arteriolar smooth muscle. We used Western blot analysis to demonstrate the presence of Rho-kinase isoforms in freshly isolated preglomerular microvessels, to demonstrate that the enzyme target was present, and to support the concept that Rho-kinase activation exerts important functional effects. Preglomerular microvessels as well as the renal cortex and renal medulla clearly express Rho-kinase α and β. Together, these proteins make up important elements of the Rho-signaling pathway and support the argument that inhibition of autoregulatory behavior and P2X₁ receptor-mediated vasoconstriction by Y-27632 occurs through inhibition of the Rho-kinase pathway. Abundant expression is also noted in the renal cortex and the renal medulla suggesting important roles for the Rho-kinase system in tubular function or in other vascular tissues in these kidney regions.

In summary, we confirmed previous reports from hydronephrotic kidney preparations that Rho-kinase contributes to angiotensin II- and pressure-mediated vasoconstriction of afferent arterioles (14, 50). The results of the current report extend the previous findings in several ways. The impact of Rho-kinase inhibition on autoregulatory behavior has been demonstrated in a renal preparation possessing normal vascular circuitry and an intact tubular system. In addition, we showed that the autoregulatory impairment coincides with similar impairment of autoregulatory P2X₁ receptor signaling, which is distinct from P2Y receptor-mediated effects.

GRANTS

This work was supported by grants from the American Heart Association (AHA; 95001370) and the National Institutes of Health (DK-44628, DK-38226). E. W. Inscho was an Established Investigator of the AHA during portions of this study.

Present address of Dr. L.-M. Jin: Dept. of Internal Medicine, Div. of Endocrinology, Clinical Nutrition and Vascular Medicine, Univ. of California, Davis, CA 95616.