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Am J Physiol Renal Physiol 274: F718-F727, 1998;
0363-6127/98 $5.00
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Vol. 274, Issue 4, F718-F727, April 1998

Direct assessment of renal microvascular responses to P2-purinoceptor agonists

Edward W. Inscho, Anthony K. Cook, Vy Mui, and Jason Miller

Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Studies were performed to determine the responsiveness of rat juxtamedullary afferent arterioles to receptor-selective P2-purinoceptor agonists. Experiments were performed in vitro using the blood perfused juxtamedullary nephron technique, combined with videomicroscopy. Renal perfusion pressure was set at 110 mmHg and held constant. Basal afferent arteriolar diameter averaged 22.0 ± 0.6 µm (n = 69). Stimulation with 0.1, 1.0, 10, and 100 µM ATP (n = 10) elicited a concentration-dependent vasoconstriction averaging 8 ± 2, 17 ± 2, 21 ± 4, and 23 ± 5%, respectively. A nearly identical afferent arteriolar vasoconstriction was observed in response to the P2X-selective agonist beta ,gamma -methylene ATP (n = 10); however, another P2X agonist, alpha ,beta -methylene ATP, evoked marked receptor desensitization (n = 10). Vessel diameter decreased by ~7 ± 2, 16 ± 2, 23 ± 3, and 22 ± 3%, respectively, over the same concentration range. The P2Y-selective agonist, 2-methylthio-ATP, evoked only a modest vasoconstriction, whereas UTP and adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) reduced afferent diameter markedly at concentrations >1.0 µM. Afferent arteriolar diameter decreased by 5 ± 4, 31 ± 8, and 72 ± 8% during UTP administration (n = 7) at concentrations of 1.0, 10, and 100 µM, respectively. Similarly, ATPgamma S (n = 6) decreased afferent diameter by 16 ± 2, 58 ± 8, and 98 ± 3%, respectively, over the same concentration range. Nitric oxide synthesis inhibition with Nomega -nitro-L-arginine did not significantly alter the afferent arteriolar response to ATP but did potentiate ATP-mediated arcuate artery vasoconstriction. The following data suggest the presence of multiple P2 receptors on juxtamedullary afferent arterioles and are consistent with classification of those receptors as members of the P2X- and P2Y2 (P2U)-receptor subtypes.

adenosine 5'-trisphosphate; uridine 5'-trisphosphate; alpha ,beta -methylene adenosine triphosphate; adenosine 5'-O-(3-thiotriphosphate); afferent arterioles; renal microcirculation

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

IDENTIFICATION OF SELECTIVE pharmacological agents for investigating the physiological role of P2 receptors has contributed to the increasing interest in P2 receptor physiology over the last two decades. The availability of selective agonists and antagonists has resulted in identification of numerous P2-receptor subtypes, based on functional responses. As a result, the family of P2 receptors has been subdivided into two major groups, classified as P2X and P2Y receptors (1, 3, 8, 10, 14, 15, 26). Subsequent advances have resulted in the successful cloning and expression of certain P2X- and P2Y-receptor subtypes (1, 3, 8, 10, 14-16, 26, 31, 39). Investigation into the signal transduction mechanisms activated by cloned receptors yield results that are consistent with functional responses and signal transduction mechanisms experimentally determined for these receptors (1, 3, 10).

Numerous studies have shown that the renal microvasculature is highly responsive to P2 receptor stimulation. Some studies indicate that ATP induces renal vasodilation (7, 12, 18, 27, 37), whereas others report renal vasoconstriction (7, 9, 12, 20, 21, 24, 25, 27, 28, 30, 35, 36, 40). Direct determination of renal microvascular responsiveness to ATP or other P2 agonists has revealed that P2 receptor activation results in rapid and sustained decreases in microvascular diameter (20, 21, 24, 25, 27). Other studies have shown that P2 receptor-mediated responses are uniquely distributed along preglomerular segments of the renal microvasculature (23, 25, 29). This arrangement suggests that P2 receptors play an important role in the regulation of preglomerular resistance and therefore participate in regulating renal blood flow and glomerular filtration rate. In fact, recent studies have shown that P2 receptor desensitization or pharmacological blockade significantly impairs renal vascular autoregulatory behavior and thus support the postulate that P2 receptors participate in the autoregulation of renal blood flow and glomerular filtration rate (21).

Although much has been learned regarding renal vascular responses to P2 receptor stimulation, the receptor subtypes involved remain to be identified. The purpose of the current study was to directly determine the afferent arteriolar response to a variety of selective P2-receptor agonists and to assess the relative contribution of P2X and P2Y receptors to those responses. Additional studies were performed to determine the contribution of endothelium-derived nitric oxide in modulating the renal microvascular response to ATP. Agonist response profiles were constructed to compare against similar profiles established for P2-receptor subtypes in other systems. Analysis of these data suggest that at least two P2-receptor subtypes are functionally expressed on afferent arterioles. Furthermore, nitric oxide does not significantly influence the afferent arteriolar response to ATP but does attenuate ATP-mediated arcuate artery vasoconstrictor responses.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Studies were approved by the Tulane University Advisory Committee for Animal Resources. Experiments were conducted in vitro using the blood perfused juxtamedullary nephron technique, as previously described (4, 5, 21, 25). For each experiment, two male Sprague-Dawley rats (350-400 g) were anesthetized with pentobarbital sodium (40 mg/kg ip) and pretreated for 30 min with the converting-enzyme inhibitor, enalaprilat (2 mg iv). Angiotensin converting-enzyme inhibition was imposed to reduce the influence of endogenously formed angiotensin II on vascular responses. Perfusate blood was collected and prepared as previously described (20, 21, 24, 25). Briefly, blood was collected from the nephrectomized blood donor rat into a heparin-containing syringe (500 units). The plasma and erythrocyte fractions were separated, and the leukocyte fraction was discarded. The plasma was filtered (0.2 µm) and combined with the recovered erythrocytes to yield a hematocrit of ~33%. The reconstituted blood was filtered through a 5-µm nylon mesh.

The right renal artery of the kidney donor was cannulated and perfused with a Tyrode buffer solution containing 52 g/l bovine serum albumin (Sigma Chemical, St. Louis, MO) and a complement of L-amino acids (20, 24, 25). The right renal vein was ligated and vented near the inferior vena cava to facilitate renal perfusion and to prevent mixing of the perfusate with circulating blood. The rat was exsanguinated into a heparinized syringe (500 units) via a carotid artery cannula and processed with blood collected from the blood donor rat. The perfused kidney was removed and sectioned along the longitudinal axis, leaving the intact papilla on the dorsal two-thirds portion of the kidney (5). The papilla was reflected back, and the pelvic mucosa was removed to expose the main arterial branches, renal tubules, glomeruli, and related microvasculature of juxtamedullary nephrons. Ligation of the terminal ends of the large arteries restored perfusion pressure to the perfused cortical and papillary tissue.

After completion of the microdissection procedures, the cell-free 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 by means of a pressure cannula positioned in the tip of a double-barrel perfusion cannula and connected to a Statham P23Db pressure transducer linked to a polygraph recorder (Grass Instruments, Quincy, MA). Perfusion pressure was fixed at 110 mmHg. The inner cortical surface of the kidney was continuously superfused with warmed (37°C) Tyrode buffer containing 10 g/l bovine serum albumin, and the kidney was allowed to equilibrate for at least 15 min.

The perfusion chamber containing the prepared kidney was affixed 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 was obtained with a high-resolution Newvicon camera (NC-70; Dage-MTI, Michigan City, IN). The video image was enhanced, using an image processor (MFJ-1425; MFJ Enterprises, Starkville, MS) and displayed on a video monitor while being simultaneously recorded on videotape for later analysis. Vascular inside diameters were measured at a single site, using an image shearing monitor (model 901; Instrumentation for Physiology and Medicine, San Diego, CA) calibrated with a stage micrometer. Microvessels were selected for study based on the clarity of the vascular walls and the adequacy of blood flow through the vessel lumen.

Experimental Protocols

After a 15-min equilibration period, an experimental protocol was initiated, consisting of consecutive 5-min treatment periods. Treatments were administered by bathing the tissue with a superfusate solution containing the agent to be tested. After an initial control period, the tissue was exposed to increasing concentrations of ATP or ATP analogs at concentrations of 0.1, 1.0, 10, and 100 µM. Each protocol concluded with a recovery period, during which the superfusion solution was returned to the control buffer. Vessel caliber was monitored continuously throughout the entire protocol while measurements of vascular inside diameter were obtained at 12-s intervals. Peak afferent arteriolar responses were determined for each agonist concentration by averaging the smallest luminal diameter obtained in each period in response to agonist administration. Steady-state diameter determinations were calculated from the average of all diameter measurements obtained during the final 2 min of each 5-min treatment period.

Series 1. Afferent arteriolar responses were determined for ATP, AMP, and a series of P2 receptor-selective agonists. P2X receptor-mediated responses were assessed using the P2X-selective agonists alpha ,beta -methylene ATP and beta ,gamma -methylene ATP. P2Y receptor-mediated effects were assessed using 2-methylthio-ATP, UTP, UDP, and adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S). All protocols using these agonists were conducted according to the protocol described above for ATP.

Series 2. Rat juxtamedullary afferent arterioles and interlobular arteries respond to 100 µM ATP with a large, transient vasoconstriction before partially waning to a stable diameter significantly smaller than control (25). Rat juxtamedullary arcuate arteries exhibit a large transient vasoconstriction, which gradually recovers to the control diameter (25). ATP or ATP analogs have been reported to produce an endothelium-dependent renal vasodilation in both the dog (27) and rat kidney (7, 12). Therefore, studies were performed to determine whether the partial recovery of the maximal ATP-induced renal microvascular vasoconstriction involves ATP-induced release of endothelium-derived nitric oxide.

Studies were performed in vitro, using the blood perfused juxtamedullary nephron technique, as described above. Preglomerular responses to 100 µM ATP were compared between control kidneys and kidneys pretreated with the nitric oxide synthase inhibitor, Nomega -nitro-L-arginine (L-NNA). Nitric oxide synthase inhibition was accomplished by bathing the kidney surface for a minimum of 20 min with a warmed Tyrode buffer containing 1% bovine serum albumin and 100 µM L-NNA. Previous studies from our laboratory have shown that 20 min of pretreatment with 100 µM L-NNA effectively blocked ACh-induced vasodilation of juxtamedullary afferent arterioles (32). After a stabilization period of 20 min, a 5-min control period was begun. After the control period, the bathing solution was changed to one containing 100 µM L-NNA and 100 µM ATP. After another 5-min period, during which ATP was administered continuously, the bathing solution was returned to the control solution, and the vessel was allowed to recover. The effect of L-NNA administration was assessed for arcuate and interlobular arteries and afferent arterioles. Arcuate arteries were defined as large arteries that ranged from 50 to 100 µm in diameter (25). These vessels generally branch to interlobular arteries but frequently give rise directly to afferent arterioles. Interlobular arteries are intermediate-size vessels with diameters of 30-50 µm and generally branch to form afferent arterioles. A separate set of animals was prepared without L-NNA to establish the segmental response to ATP alone.

Drugs

Enalaprilat was a gift from Merck Sharp & Dohme (Rahway, NJ). ATP, alpha ,beta -methylene ATP, beta ,gamma -methylene ATP, UTP, AMP, and adenosine were purchased from Sigma Chemical. 2-Methylthio-ATP and ATPgamma S were purchased from Research Biochemicals (Natick, MA). L-NNA was purchased from Aldrich Chemical (Milwaukee, WI).

Statistical Analysis

Within group comparisons for each experimental series were evaluated, using an analysis of variance for repeated measures. Differences between group means within each series were determined using Newman-Keuls multiple range test. Differences between group means from different experimental series were determined using t-tests for unpaired data or a one-way analysis of variance. P < 0.05 was considered statistically significant. All values are reported as means ± SE.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Sixty-nine afferent arterioles, averaging 438 ± 21 µm in length and 22.0 ± 0.6 µm in diameter, were examined in the course of this study. Diameter measurements were made an average of 214 ± 11 µm from the glomerulus, representing the midpoint (49 ± 1%) of the arteriolar length.

Afferent Arteriolar Response to ATP and AMP

The response of juxtamedullary afferent arterioles to ATP stimulation is illustrated in Fig. 1. Afferent arterioles exhibited a concentration-dependent reduction in vessel inside diameter in response to increasing concentrations of ATP. Afferent arteriolar diameter averaged 23.5 ± 1.2 µm under control conditions (n = 10 arterioles) and decreased biphasically in response to ATP administration. ATP stimulated a large but transient peak vasoconstriction of 16.6 ± 5, 39.4 ± 6.1, 49.5 ± 5.2, and 61.1 ± 7% for ATP concentrations of 0.1, 1.0, 10, and 100 µM, respectively, beginning within the first seconds of agonist exposure. This initial response reached a concentration-dependent maximum before partially waning to a stable vasoconstriction, where afferent arteriolar diameter was significantly smaller than control. Steady-state afferent arteriolar diameter declined significantly by 8 ± 2, 17 ± 2, 21 ± 4, and 23 ± 5% from control in response to ATP concentrations of 0.1, 1.0, 10, and 100 µM, respectively (P < 0.05 vs. control). Removal of ATP from the superfusate solution resulted in a rapid and complete recovery to 105 ± 1% of the control diameter.


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  		Fig. 1.   Time course (in min) of afferent arteriolar response to increasing concentrations of ATP. Data are expressed as the average vessel diameter measured at 12-s intervals. Each point represents the mean vessel diameter (in µm) of 10 arterioles. * P < 0.05 vs. control (CON); REC, recovery.

The ATP metabolites, ADP and adenosine, have been shown to produce modest vasoconstriction of juxtamedullary afferent arterioles (20, 25); however, the effects of AMP have not been directly evaluated. Therefore, studies were performed to determine the effect of AMP on afferent arteriolar diameter, and the results of those studies are shown in Fig. 2. Under control conditions, afferent diameter averaged 17.8 ± 0.8 µm. Superfusion of AMP containing solutions had little effect on afferent diameter, except at the 1.0 µM concentration, where there was small but significant vasoconstriction. In contrast to the rapid biphasic response elicited by ATP, AMP stimulated a gradual decrease in vessel diameter resembling adenosine-mediated responses (20).


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  		Fig. 2.   Time course (in min) of afferent arteriolar response to increasing concentrations of AMP. Data are expressed as the average vessel diameter measured at 12-s intervals. Each point represents the mean vessel diameter (in µm) of 10 arterioles. * P < 0.05 vs. control.

Afferent Arteriolar Response to P2X Agonists

Studies were performed using selective ATP analogs to further examine the mechanisms responsible for ATP-mediated vasoconstriction of afferent arterioles. The contribution of P2X receptors to the response was assessed using the P2X-selective agonists beta ,gamma -methylene ATP and alpha ,beta -methylene ATP. Figure 3 illustrates the afferent arteriolar response to increasing concentrations of beta ,gamma -methylene ATP. Under control conditions, afferent arteriolar diameter averaged 24.7 ± 1.6 µm (n = 10 arterioles). beta ,gamma -Methylene ATP evoked biphasic, concentration-dependent, vasoconstrictor responses at concentrations from 1.0 to 100 µM. These responses were typified by peak initial vasoconstrictions of 27.1 ± 4.0, 69.4 ± 6.8, and 66.1 ± 6.3%, which were significant for agonist concentrations of 1.0, 10, and 100 µM, respectively, followed by smaller but sustained vasoconstrictions that were completely reversible. Steady-state diameter declined significantly by 16 ± 2, 23 ± 3, and 22 ± 3% in response to the 1.0, 10, and 100 µM beta ,gamma -methylene ATP concentrations, respectively.


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  		Fig. 3.   Time course (in min) of afferent arteriolar response to increasing concentrations of beta ,gamma -methylene ATP. Data are expressed as the average vessel diameter measured at 12-s intervals. Each point represents the mean vessel diameter (in µm) of 10 arterioles. * P < 0.05 vs. control.

P2X receptors are subject to desensitization in response to repeated exposure to the P2X agonist, alpha ,beta -methylene ATP (1, 10, 13-15, 21, 33, 36). Therefore, we assessed the afferent arteriolar response to increasing concentrations of alpha ,beta -methylene ATP, and the results of those studies are presented in Fig. 4. Basal afferent arteriolar diameter averaged 21.1 ± 1.4 µm. Exposure to alpha ,beta -methylene ATP (n = 9 arterioles) elicited transient peak decreases in afferent diameter of 21.9 ± 5.2, 54.0 ± 6.8, and 32.8 ± 4.3%, which were significant for agonist concentrations of 0.1, 1.0, and 10 µM, respectively (Fig. 3). This initial vasoconstriction quickly waned, and afferent caliber returned to a diameter similar to control. Subsequent administration of higher concentrations resulted in progressively smaller initial responses and a sustained vasoconstriction only at the 10 µM concentration. The progressive disappearance of the afferent arteriolar response to alpha ,beta -methylene ATP is consistent with P2X receptor desensitization.


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  		Fig. 4.   Time course (in min) of afferent arteriolar response to increasing concentrations of alpha ,beta -methylene ATP. Data are expressed as the average vessel diameter measured at 12-s intervals. Each point represents the vessel diameter (in µm) of 10 arterioles. * P < 0.05 vs. control.

Afferent Arteriolar Response to P2Y Agonists

The role of P2Y receptors in the afferent arteriolar response to ATP was assessed using the P2Y receptor-selective ATP agonist, 2-methylthio-ATP (Fig. 5). Steady-state afferent arteriolar diameter averaged 20.5 ± 0.8 µm (n = 8 arterioles) under control conditions. 2-Methylthio-ATP elicited significant peak declines in afferent arteriolar diameter of 29.1 ± 6.5, 50.3 ± 4.0, and 35.4 ± 10.9% at 1.0, 10, and 100 µM agonist concentrations, respectively; however, these peak responses were not sustained. In the continued presence of 2-methylthio-ATP, significant sustained vasoconstrictor responses of 7 ± 3 and 9 ± 2% were observed at agonist concentrations of 0.1 and 1.0 µM, but afferent diameter returned to values similar to the control diameter at agonist concentrations of 10 and 100 µM.


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  		Fig. 5.   Time course (in min) of afferent arteriolar response to increasing concentrations of 2-methylthio-ATP. Data are expressed as the average vessel diameter measured at 12-s intervals. Each point represents the mean vessel diameter (in µm) of 8 arterioles. * P < 0.05 vs. control.

The role of P2Y2 (P2U) receptors in the afferent arteriolar response to ATP was assessed using UTP and ATPgamma S. Control afferent arteriolar diameter averaged 25.9 ± 2.2 µm (n = 7 arterioles) and increased slightly but not significantly to a diameter of 27.5 ± 2.2 µm in response to 0.1 µM UTP (Fig. 6). On increasing the superfusate UTP concentration to 1.0 µM, afferent diameter declined toward the control diameter (24.3 ± 1.7 µm). Subsequent increases in UTP concentration to 10 and 100 µM evoked a progressively greater vasoconstriction until afferent caliber reached a minimum diameter of 6.5 ± 1.2 µm and blood flow through the arterioles ceased. Removal of UTP from the superfusate resulted in a prompt vasorelaxation to 31.0 ± 3.8 µm, coincident with restoration of afferent arteriolar blood flow. The temporal pattern of UTP-mediated vasoconstrictor responses differed markedly from the pattern observed with ATP or any of the other ATP analogs described above. Instead of the biphasic change in diameter, 10 and 100 µM UTP, respectively, decreased afferent diameter to minimum values, where it quickly stabilized.


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  		Fig. 6.   Time course (in min) of afferent arteriolar response to increasing concentrations of UTP. Data are expressed as the average vessel diameter measured at 12-s intervals. Each point represents the mean vessel diameter (in µm) of 7 arterioles. * P < 0.05 vs. control.

The concentration response profile obtained with ATPgamma S closely resembled the response obtained with UTP, except that afferent diameter did not exhibit a vasodilatory tendency at the lowest concentration (Fig. 7). Afferent diameter averaged 20.9 ± 1.3 µm (n = 6 arterioles) during superfusion with control buffer. ATPgamma S concentrations of 1.0, 10, and 100 µM evoked significant, concentration-dependent vasoconstriction of 16 ± 3, 52 ± 7, and 97 ± 3%, respectively, and 100 µM ATPgamma S caused afferent arteriolar blood flow to cease. The temporal pattern of the vasoconstriction closely resembled the response elicited by UTP in that afferent diameter decreased rapidly with little or no waning.


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  		Fig. 7.   Time course (in min) of afferent arteriolar response to increasing concentrations of adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S). Data are expressed as the average vessel diameter measured at 12-s intervals. Each point represents the mean vessel diameter (in µm) of 6 arterioles. * P < 0.05 vs. control.

Certain P2Y-receptor subtypes exhibit marked sensitivity to UDP over UTP, whereas others are essentially insensitive to UDP (1). Interestingly, the afferent arteriolar response to UDP was strikingly different from the response to UTP or ATPgamma S. Figure 8 demonstrates that afferent arteriolar diameter remained essentially unchanged in the presence of UDP concentrations from 0.1 to 10 µM. Afferent diameter averaged 23.0 ± 1.2 µm (n = 8 arterioles) under control conditions and 23.0 ± 1.2, 22.1 ± 1.1, 20.6 ± 0.9, and 19.4 ± 1.1 µm at each UDP concentration tested. Only the diameter during 100 µM UDP was significantly smaller (14 ± 5%) than control.


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  		Fig. 8.   Time course (in min) of afferent arteriolar response to increasing concentrations of UDP. Data are expressed as the average vessel diameter measured at 12-s intervals. Each point represents the mean vessel diameter (in µm) of 8 arterioles. * P < 0.05 vs. control.

Dose-Response Profile of Afferent Arterioles to P2 Agonists

Agonist response profiles are frequently used to identify the receptor subtypes involved in the observed response. With this approach, two different patterns emerge in evaluating the peak changes in afferent arteriolar diameter elicited by P2 receptor-selective agonists. For example, the magnitude of the peak vasoconstriction elicited by 1.0 µM alpha ,beta -methylene ATP was significantly greater than the peak response elicited by any other P2 agonist tested at that concentration (Fig. 9). However, as the agonist concentration increased, the agonist responsible for the peak vasoconstrictor response shifted from the P2X-selective agonists to the P2Y-selective agonists. As can be seen at the 100 µM concentration, ATPgamma S induced a significantly greater peak response than any other agonist tested, and the response to UTP was significantly greater than the response from all but ATPgamma S and beta ,gamma -methylene ATP.


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  		Fig. 9.   Peak changes in microvascular diameter in response to P2 agonist administration. Data are expressed as %change in diameter compared with the control diameter. Each point represents the mean vessel diameter measured at 12-s intervals and averaged over the last 2 min of each treatment period. A: comparison of peak afferent arteriolar responses to ATP and P2X agonists. B: comparison of peak afferent arteriolar responses to ATP and P2Y agonists. alpha -beta -ATP, alpha ,beta -methylene ATP; beta -gamma -ATP, beta ,gamma -methylene ATP.

Steady-state changes in afferent diameter evoked by P2X agonists are compared in Fig. 10A. ATP and the P2X agonist, beta ,gamma -methylene ATP, evoked nearly identical sustained vasoconstriction of afferent arterioles. alpha ,beta -Methylene ATP elicited a smaller sustained vasoconstriction. This apparent decline in efficacy is probably due to P2X receptor desensitization, as previously demonstrated for rat juxtamedullary afferent arterioles (21).


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  		Fig. 10.   Steady-state changes in microvascular diameter in response to P2 agonist administration. Data are expressed as %change in diameter compared with the control diameter. Each point represents the mean vessel diameter measured at 12-s intervals and averaged over the last 2 min of each treatment period. A: comparison of steady-state afferent arteriolar responses to ATP and P2X agonists. B: comparison of steady-state afferent arteriolar responses to ATP and P2Y agonists.

A comparison of the steady-state changes in afferent diameter evoked by P2Y agonists is presented in Fig. 10B. ATPgamma S exhibited significantly greater sustained vasoconstrictions at concentrations >1.0 µM than any of the other agonists tested. UDP and 2-methylthio-ATP evoked significantly weaker vasoconstrictions at concentrations >1.0 µM, compared with ATP.

Contribution of Nitric Oxide to the Afferent Arteriolar Response to ATP

Typical preglomerular responses to 100 µM ATP administration involve a rapid initial vasoconstriction followed by either a partial recovery to a new steady-state diameter, as seen with afferent arterioles and interlobular arteries, or a complete recovery, as seen with arcuate arteries (25). Therefore, studies were performed to test the hypothesis that ATP-mediated nitric oxide production was responsible for the vasodilation observed during the recovery phase of this response. Nitric oxide synthesis inhibition with L-NNA did not significantly alter the response of afferent arterioles or interlobular arteries to ATP. ATP administration reduced afferent arteriolar diameter by 24 ± 6% in untreated control kidneys (n = 9 arterioles) and by 11 ± 3% in kidneys treated with L-NNA (n = 7 arterioles). Similarly, ATP decreased interlobular artery diameter by 16 ± 5% in untreated control kidneys (n = 7 arteries) and by 9 ± 4% in L-NNA-treated kidneys (n = 7 arteries). Only arcuate arteries exhibited an altered response to ATP as a consequence of L-NNA treatment. Consistent with previous studies (25), arcuate arteries of untreated kidneys responded to 100 µM ATP with a rapid, transient vasoconstriction of 21.0 ± 5.8% from a control diameter of 100.5 ± 11.1 µm before stabilizing at a diameter of 99.1 ± 11.9 µm. This constitutes a steady-state diameter change of 2 ± 4% from control, which is not statistically significant. In L-NNA-treated kidneys, the ATP-mediated vasoconstriction was potentiated such that the steady-state arcuate artery diameter declined significantly by 12 ± 3% from 105.4 ± 8.9 µm to 93.2 ± 9.3 µm. This ATP-mediated vasoconstriction was completely reversible on removal of ATP from the superfusate.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Previous studies have shown that isolated rabbit afferent arterioles (40) and rat juxtamedullary afferent arterioles (20, 21, 23-25) respond to ATP and other P2 agonists with a rapid and sustained vasoconstriction. This vasoconstriction is augmented rather than inhibited by the nonselective adenosine receptor antagonist, 1,3-dipropyl-8-p-sulfophenylxanthine (25). Furthermore, a 10-fold greater adenosine concentration is required to reduce afferent arteriolar diameter to the same extent as ATP (25). Neither ADP (20) nor AMP (Fig. 2) stimulate vasoconstrictor responses comparable in magnitude or time course to those evoked by ATP. In fact, the similarity between the afferent arteriolar response to AMP (Fig. 2) and the response to adenosine (25) suggests that these responses are mediated through activation of a P1 purinoceptor-dependent mechanism. Therefore, neither the hydrolysis of ATP to adenosine nor the direct activation of P1 purinoceptors by ATP can explain the afferent arteriolar vasoconstrictor responses elicited by ATP. Accordingly, the current evidence supports the conclusion that ATP vasoconstricts afferent arterioles by direct interaction with P2 purinoceptors rather than through activation of adenosine-sensitive P1 receptors. Furthermore, the current studies demonstrate that agonists selective for certain P2-purinoceptor subtypes elicit markedly different renal microvascular responses. These data suggest that juxtamedullary afferent arterioles express multiple P2-purinoceptor subtypes and are consistent with classification of those receptors as members of the P2X and P2Y receptor families.

Renal microvascular responses to ATP are characterized by a rapid initial vasoconstriction, which gradually stabilizes into a sustained vasoconstriction (Fig. 1; Refs. 20, 21, 23-25). This pattern of vasoconstriction closely parallels the temporal pressure profile observed in isolated kidneys perfused with solutions containing ATP or ATP analogs (28, 34) and also parallels the ATP-mediated increase in cytosolic calcium concentration elicited in renal (19, 22) and nonrenal (2, 29, 38) vascular smooth muscle cells. We have previously demonstrated that of the four major segments of the intrarenal microvasculature, the afferent arteriole is most responsive to ATP (25). In those studies, arcuate and interlobular arteries exhibited rapid but transient vasoconstrictions, such that arterial caliber returned to a diameter similar to or slightly smaller than control (25). In contrast, ATP evoked a sustained reduction in afferent arteriolar diameter (25, 40), whereas the diameter of efferent arterioles remained unchanged (25). These observations implicate increases in afferent arteriolar resistance as being primarily responsible for the increase in perfusion pressure noted in studies where ATP or ATP analogs have been infused intrarenally in vivo (27, 28, 35) or in vitro (7, 12); however, they do not reveal the P2-purinoceptor subtype responsible for the hemodynamic changes.

In recent years, the classification of purinoceptor subtypes has been revised, leading to some confusion in the literature concerning receptor nomenclature (1). Abbracchio and Burnstock (1) have proposed a new classification scheme, which has been endorsed by the International Union of Pharmacology (IUPHAR) Committee on Receptor Nomenclature and Drug Classification (14). At present, the IUPHAR Committee has supported reorganization of P2 purinoceptor nomenclature into two receptor families classified as P2X and P2Y. Within each family, there are several relatively distinct receptor subtypes, based on second messenger systems, receptor cloning and sequencing, agonist selectivity, and sensitivity to receptor antagonists (1, 3). For the context of this discussion, we will utilize this most recent purinoceptor classification while also referring to the older nomenclature for comparison, when appropriate.

Receptors of the P2X family have two membrane-spanning domains and appear to function as ligand-gated ion channels on activation by ligand binding (1). Investigators have described approximately seven P2X-receptor subtypes, using expression cloning approaches, second messenger systems, and rank-order agonist potency profiles (1, 3, 10). P2X receptors typically exhibit the greatest sensitivity to the P2X receptor-selective ATP analogs, alpha ,beta -methylene ATP and beta ,gamma -methylene ATP, and are prone to desensitization by alpha ,beta -methylene ATP. P2X receptor-mediated responses initiate rapidly through activation of an inwardly directed, nonselective cation current that can lead to membrane depolarization and activation of voltage-dependent calcium channels. We have previously demonstrated that vasoconstriction of juxtamedullary afferent arterioles by alpha ,beta -methylene ATP is dependent on the influx of extracellular calcium (24). Furthermore, the sustained phase of the vasoconstriction could be completely abolished by blockade of voltage-dependent calcium channels, using L-type calcium channel antagonists (24). In the current studies, afferent arteriolar responses to ATP exhibited the typical biphasic vasoconstrictor profile. Similar vasoconstrictor profiles were obtained with the P2X agonists alpha ,beta -methylene ATP and beta ,gamma -methylene ATP. The magnitude of the peak response to 0.1 µM alpha ,beta -methylene ATP was greater than the response to UTP, UDP, ATPgamma S, and beta ,gamma -methylene ATP, whereas the peak response to 1.0 µM alpha ,beta -methylene ATP was greater than any of the agonists tested. Furthermore, the responsiveness of afferent arterioles to alpha ,beta -methylene ATP declined on repeated agonist exposure, suggesting desensitization of P2X receptors.

The P2X-selective agonist beta ,gamma -methylene ATP closely mimicked the response to ATP by eliciting maximum and steady-state afferent arteriolar vasoconstrictions of similar magnitude. In addition, the temporal pattern of responses evoked by ATP and beta ,gamma -methylene ATP were nearly identical. Interestingly, the responses elicited by the P2Y agonists UTP, ATPgamma S, and 2-methylthio-ATP were typically smaller than the responses to low concentrations of P2X agonists. These observations are consistent with the activation of P2X receptors in mediating part of the afferent arteriolar response to ATP.

P2X receptors have frequently been shown to contract vascular and nonvascular smooth muscle (17, 26, 33). The contention that the rat renal microvasculature is invested with P2X receptors is supported by the work of several investigators. For example, infusion of P2X agonists into the rat kidney has produced an increase in renal perfusion pressure consistent with an intrarenal vasoconstriction (7, 9, 12, 28, 34-36). More recently, Eltze and Ullrich (12) have also reported pronounced renal vasoconstriction following intrarenal infusion of the P2X agonists alpha ,beta -methylene ATP and beta ,gamma -methylene ATP into the isolated perfused rat kidney. Infusion of ATP or beta ,gamma -methylene ATP into peritubular capillaries of the rat kidney elicited a sharp reduction in proximal tubular stop-flow pressure, reflecting a rapid increase in preglomerular resistance (28). Direct assessment of renal microvascular responses to P2X agonists revealed rapid preglomerular vasoconstrictor responses (20, 21, 24, 25), which could be desensitized with alpha ,beta -methylene ATP (15, 21). Finally, autoradiographic studies performed by Bo and Burnstock (see Ref. 29 for details) using alpha ,beta -[3H]methylene ATP have provided evidence of significant localization of alpha ,beta -[3H]methylene ATP binding along afferent arterioles in cortical sections of rat kidney. More recently, the presence and distribution of P2X1 receptors along the rat renal microvasculature have been performed, using receptor-specific antibodies directed at the COOH terminus of the P2X1 receptor (6). Those studies clearly demonstrate the expression of P2X1 receptors along the upstream intrarenal arteries, as well as afferent arterioles, but not along efferent arterioles (6). These data strongly support the functional evidence that ATP influences only preglomerular microvascular segments (25). Taken together, the data support the existence of functional P2X receptors on the rat renal microvasculature and suggest that these receptors play an important role in altering renal vascular resistance.

P2Y receptors are characterized as receptor proteins possessing seven membrane-spanning domains (1, 14). Stimulation of these receptors involves activation of regulatory G proteins, followed by activation of signal transduction pathways. According to the subclassification proposed by Abbracchio and Burnstock (1) and the functional evidence for P2 agonists in this report, renal microvascular responses to P2Y agonists can best be attributed to the P2Y2-receptor subtype, which was formerly identified as the P2U receptor (1). Based on the patterns and magnitudes of the responses elicited by the P2 agonists surveyed, five of the seven defined P2Y-receptor subtypes can be considered unlikely candidates for mediating renal microvascular responses to ATP. P2Y1, P2Y4, P2Y5, and P2Y6 receptors exhibit responsiveness to 2-methylthio-ATP, which is much greater than (P2Y4), greater than (P2Y6), and greater than or equal to ATP (P2Y1 and P2Y5) and are generally unresponsive to alpha ,beta -methylene ATP and beta ,gamma -methylene ATP (1). From the evidence presented in the current studies, 2-methylthio-ATP was clearly one of the weaker P2 agonists evaluated and frequently elicited responses that were significantly smaller than the responses to ATP. Furthermore, both alpha ,beta -methylene ATP and beta ,gamma -methylene ATP often evoked greater afferent arteriolar vasoconstrictions than 2-methylthio-ATP (Figs. 3-5). P2Y6 receptors are highly sensitive to UDP and insensitive to ATP (31). In this study, juxtamedullary afferent arterioles were almost completely unresponsive to UDP, while exhibiting marked sensitivity to UTP. In contrast, P2Y4 receptors are more sensitive to UTP than ATP, and UDP is inactive (31); however, these receptors are also highly sensitive to 2-methylthio-ATP and poorly responsive to beta ,gamma -methylene ATP (1). In the current report, the magnitudes of the afferent arteriolar responses to ATP and beta ,gamma -methylene ATP were similar and more effective than 2-methylthio-ATP in reducing afferent arteriolar diameter. Nevertheless, UTP was a more efficacious vasoconstrictor than ATP or beta ,gamma -methylene ATP at higher concentrations.

ADP and UDP are more potent agonists for P2Y3 receptors than are UTP or ATP (1, 3, 39). This rank-order potency profile is markedly different than the pattern observed for juxtamedullary afferent arterioles. The results presented here demonstrate that afferent arteriolar diameter declined rapidly in response to ATP but responded only weakly to UDP (Fig. 7) and ADP (20). Thus it is unlikely that vasoconstriction of juxtamedullary afferent arterioles involves activation of P2Y3 receptors.

P2Y7 receptors are primarily activated by diadenosine polyphosphates (1) and exhibit similar responses to ADP and UTP. In the current and previous studies (20), ADP only slightly alters afferent diameter, whereas UTP elicits pronounced monophasic responses. Thus the reported pattern of responsiveness for P2Y7 receptors differs markedly from that observed from afferent arterioles. Taken together, afferent arteriolar responses to P2Y agonists are not consistent with the involvement of P2Y1, P2Y3, P2Y4, P2Y5, and P2Y6 receptors.

Exclusion of the six receptor subtypes discussed above leaves P2Y2 receptors as the most likely P2Y-receptor subtype to be involved in the response. P2Y2 receptors were formerly classified as P2U receptors based on their significant sensitivity for UTP and the P2U-receptor agonist, ATPgamma S (11, 14). According to the current classification, P2Y2 receptors exhibit responsiveness to UTP, which is greater than or equal to ATP and ATPgamma S (1, 14). This characteristic is consistent with the steady-state responses observed in the current studies, except for the highest concentration where UTP was significantly more efficacious than ATP. Similarly, juxtamedullary afferent arterioles are extremely responsive to the stable P2U agonist, ATPgamma S, and the temporal pattern of the responses elicited by ATPgamma S and UTP were indistinguishable. P2Y2 receptors are also unresponsive to ADP or UDP (31), which is consistent with the UDP data presented here (Fig. 7) and from previously published data for ADP, using the same preparation (20). Therefore, the results presented here support the hypothesis that rat juxtamedullary afferent arterioles express a purinoceptor that closely resembles the P2Y2 (P2U)-receptor subtype.

The contention that juxtamedullary afferent arterioles express P2Y receptors is supported by the work of several investigators. Churchill and Ellis (7) reported that infusion of the P2Y agonist, 2-methylthio-ATP, vasodilated the rat isolated perfused kidney. Similarly, Eltze and Ullrich (12) reported that infusion of ATP, 2-methylthio-ATP, UTP, and ATPgamma S vasodilated the in vitro isolated perfused rat kidney. The current report differs from those cited above in that no significant afferent arteriolar vasodilation was observed with any of the agonists tested. The explanation for this observation is not readily apparent but may reflect significant differences in experimental conditions. In this study, the kidneys were perfused with blood, and the P2 agonists were delivered to the renal microvasculature through the superfusate, thus contacting afferent arteriolar smooth muscle from the adventitial side. This route of P2 agonist administration more closely mimics the physiological exposure sequence of the renal microvasculature to endogenous purine nucleotides released from sympathetic nerve varicosities or nucleotides released in a paracrine fashion from neighboring cells. Nucleotides released from these sources will be delivered to the interstitial fluid where they will interact with extracellular P2 receptors on microvascular smooth muscle, mesangial cells, or tubular and glomerular epithelium. In contrast, isolated perfused kidney experiments are performed using cell-free physiological salt solutions, and the nucleotides are administered into the perfusate, thus allowing them to gain access to the endothelium prior to encountering renal microvascular smooth muscle cells. Previous studies have shown that intrarenal administration of ATP and other ATP analogs evoke endothelium-dependent vasodilatory responses (7, 12, 27), which are converted to vasoconstrictor responses when the endothelium is denuded (12) or when the synthesis of nitric oxide is inhibited with L-NNA (7, 27) or L-NAME (12). Therefore, published evidence suggests that intravascular administration of P2 agonists leads to the generation of an endothelium-dependent vasodilator, probably nitric oxide, to evoke renal vasodilation, whereas adventitial administration or intravascular administration during endothelial nitric oxide synthesis inhibition leads to renal vasoconstriction.

The mechanism responsible for the partial recovery evident in the biphasic vasoconstrictor response produced by ATP remains to be explained. As mentioned above, activation of P2Y receptors in some vascular beds results in an endothelium-dependent vasodilation mediated by endothelium-derived nitric oxide. Several studies have shown that inhibition of nitric oxide synthase to block nitric oxide generation significantly alters renal hemodynamic responses to ATP (7, 12, 27). Inhibition of nitric oxide synthase in the rat (7, 12) or dog (27) kidney converts purinoceptor-mediated vasodilation to vasoconstriction. Similarly, Eltze and Ullrich (12) have reported that chemical removal of the intrarenal endothelium by perfusion of the kidney with detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)] converts renal vasodilatory responses to ATP and 2-methylthio-ATP and UTP to vasoconstrictor responses. Interestingly, the vasoconstrictor response to alpha ,beta -methylene ATP was unaffected by CHAPS treatment (12), whereas Churchill and Ellis (7) noted that the increase in renal vascular resistance evoked by alpha ,beta -methylene ATP was potentiated during nitric oxide synthesis inhibition. In this study, nitric oxide synthesis inhibition did not markedly alter ATP-mediated vasoconstriction of afferent arterioles or interlobular arteries but did significantly enhance the response of arcuate arteries. These results would suggest that endogenous nitric oxide production is not the mechanism responsible for the partial recovery of microvascular diameter seen following the initial vasoconstrictor response to ATP but that it may play an important role in endothelial P2 receptor responses to ATP.

In summary, we have shown that extracellular ATP evokes a biphasic vasoconstriction of the preglomerular vasculature, which is not markedly influenced by ATP-mediated generation of nitric oxide. More importantly, the results of these studies demonstrate that rat juxtamedullary afferent arterioles exhibit differential responsiveness to P2 agonists. The data are consistent with the hypothesis that this differential responsiveness involves activation of multiple P2-receptor subtypes. Based on these data, we conclude that rat juxtamedullary afferent arterioles express both P2X1 and P2Y2 receptors.

    ACKNOWLEDGEMENTS

We thank Drs. John D. Imig, Phillip J. Kadowitz, and Jeffrey S. Fedan for their helpful comments and suggestions provided during the preparation of this manuscript.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-44628 and by National Heart, Lung, and Blood Institute Grant HL-18426, the National Kidney Foundation, American Heart Association, and the Louisiana affiliate of the American Heart Association. E. W. Inscho was the recipient of the Amgen Young Investigator Award from the National Kidney Foundation and is an Established Investigator of the American Heart Association.

Address for reprint requests: E. W. Inscho, Dept. of Physiology, Tulane Univ. School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112.

Received 10 July 1997; accepted in final form 5 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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