Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112; and Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912-3000
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ABSTRACT |
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Experiments were performed to
determine the role of L-type calcium channels on the afferent
arteriolar vasoconstrictor response to ATP and UTP. With the use of the
blood-perfused juxtamedullary nephron technique, kidneys were perfused
at 110 mmHg and the responses of arterioles to ,
-methylene ATP,
ATP, and UTP were determined before and during calcium channel blockade
with diltiazem.
,
-Methylene ATP (1.0 µM) decreased arteriolar
diameter by 8 ± 1% under control conditions. This response was
abolished during calcium channel blockade. In contrast, 10 µM UTP
reduced afferent arteriolar diameter to a similar degree before
(20 ± 4%) and during (14 ± 4%) diltiazem treatment.
Additionally, diltiazem completely prevented the vasoconstriction normally observed with ATP concentrations below 10 µM and attenuated the response obtained with 10 µM ATP. These data demonstrate that L-type calcium channels play a significant role in the vasoconstrictor influences of
,
-methylene ATP and ATP but not UTP. The data also
suggest that other calcium influx pathways may participate in the
vasoconstrictor response evoked by P2 receptor activation. These
observations support previous findings that UTP-mediated elevation of
intracellular calcium concentration in preglomerular vascular smooth
muscle cells relies primarily on calcium release from intracellular
pools, whereas ATP-mediated responses involve both voltage-dependent
calcium influx, through L-type calcium channels, and the release of
calcium from intracellular stores. These results support the argument
that P2X and P2Y receptors influence the diameter of afferent
arterioles through activation of disparate signal transduction mechanisms.
afferent arterioles; calcium channels; cytosolic calcium; renal
microcirculation; P2X receptors; P2Y receptors; adenosine
5'-triphosphate; uridine 5'-triphosphate; ,
-methylene adenosine
5'-triphosphate; cadmium
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INTRODUCTION |
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PREVIOUS STUDIES SUGGEST
THAT renal microvascular smooth muscle expresses both P2X and P2Y
receptors (2, 8, 10, 13, 16, 25, 34, 39-41). This
finding is supported by more recent studies focused on determining the
calcium signaling pathways involved in P2X and P2Y receptor activation,
using freshly isolated afferent arterioles (16) and
vascular smooth muscle cells obtained from rat preglomerular vascular
segments (24, 41). Published reports using a collection of
experimental approaches have established the presence of specific P2
receptor subtypes in the renal vasculature. Chan and co-workers
(7) have shown pronounced expression of P2X1
receptors along the preglomerular microvasculature. These immmunohistochemical data are supported by functional studies demonstrating responsiveness of the renal microvasculature to ,
-methylene ATP (13, 19, 22, 24-26, 33, 37).
P2X3 receptors, which also respond to
,
-methylene ATP
(36), could also be present and could contribute to the
renal microvascular response to ATP stimulation.
P2Y2 receptors have also been strongly implicated in
influencing the diameter of rat afferent arterioles (13, 19, 22, 24). These studies showed marked renal vasoconstriction in
response to treatment with UTP or the UTP analog ATP--S but little
or no response to UDP or 2-methylthio ATP. This agonist profile is consistent with the activation of P2Y2 receptors.
Isolated microvascular smooth muscle cell experiments revealed that ATP
and selective P2X and P2Y agonists increase intracellular calcium
concentration through disparate calcium signaling mechanisms. Selective
P2Y receptor activation increases intracellular calcium concentration
primarily through mobilization of calcium from intracellular stores
(24, 27). P2X receptor activation with ,
-methylene ATP increases intracellular calcium concentration largely through activation of voltage-dependent calcium influx pathways (27, 41). Interestingly, the endogenous ligand ATP, which activates both P2X and P2Y receptors, increases intracellular calcium
concentration by stimulating voltage-dependent calcium influx and
calcium mobilization from intracellular stores (24, 27,
41).
The purpose of the present study was to extend the calcium signaling
observations made with freshly isolated preglomerular smooth muscle
cells (24, 41) to functional responses obtained in the
intact renal microvasculature from which they were derived. Experiments
were performed to determine the role of L-type calcium channels in P2X
and P2Y receptor-mediated vasoconstriction of afferent
arterioles. We determined the effect of calcium channel blockade on the
afferent arteriolar vasoconstriction induced by P2X receptor
activation with ,
-methylene ATP, by P2Y receptor activation with
UTP, and by nonselective P2 receptor activation with the endogenous
ligand ATP.
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METHODS |
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Studies were approved by the Tulane University Advisory Committee for Animal Resources and by the Committee on Animal Use for Research and Education at the Medical College of Georgia. Experiments were conducted in vitro, using the blood-perfused juxtamedullary nephron technique, as previously described (6, 21, 22). Two male Sprague-Dawley rats (350-400 g) were used for each experiment. Rats were anesthetized with pentobarbital sodium (40 mg/kg ip) and pretreated for 30 min with the converting enzyme inhibitor enalaprilat (2 mg iv) (21, 22). Perfusate blood was collected and prepared, as previously described (21, 22). Briefly, blood was collected from the nephrectomized blood donor rat into a heparinized syringe (500 U). The plasma and erythrocyte fractions were separated, and the leukocyte fraction was discarded. The erythrocytes underwent two saline washes before being combined with the filtered (0.2-µm exclusion) plasma to obtain a hematocrit of ~33%. The reconstituted blood was filtered through 5-µm nylon mesh and saved for later use.
The right renal artery of the kidney donor was cannulated and perfused with a Tyrode buffer solution containing 5.2% bovine serum albumin (Calbiochem, La Jolla, CA) and a complement of L-amino acids (Sigma, St. Louis, MO) (26). 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 with the use of a heparinized syringe (500 U) via a carotid artery cannula, and blood was processed with blood collected from the blood donor rat. The perfused kidney was removed and sectioned along the longitudinal axis, leaving the papilla intact on the dorsal two-third portion of the kidney (6). The papilla was reflected out of the visual field, and the pelvic mucosa was removed to expose the main arterial branches, renal tubules, glomeruli, and related microvasculature of the juxtamedullary nephrons. Ligation of the terminal ends of the large arteries restored intravascular 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, using a pressure cannula positioned in the tip of a double-barreled 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 1% bovine serum albumin, and the kidney was allowed to equilibrate for at least 15 min.
The perfusion chamber, containing the prepared kidney, was positioned on 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 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). The displacement of the video image on the image-shearing monitor was calibrated with a stage micrometer (smallest division = 2 µm). Microvessels were selected for study on the basis of the clarity of the vascular walls and the adequacy of blood flow through the vessel lumen.
Experimental Protocols
P2 receptor-mediated microvascular responses were determined with the use of ATP, UTP, andSeries 1: effect of repeated exposure of juxtamedullary afferent
arterioles to P2 agonists.
Prolonged exposure to extracellular ATP can result in desensitization
of P2 receptors in some tissues, and high concentrations of
,
-methylene ATP are frequently used to desensitize P2X receptors to ATP-evoked responses (1, 11). Because the experimental protocols require comparison of afferent arteriolar responses obtained
during two separate periods of
,
-methylene ATP, ATP, or UTP
treatment, desensitization of P2 receptor-mediated responses could be a
confounding variable. Therefore, time control experiments were
performed to establish the reproducibility of the afferent arteriolar
vasoconstrictor response produced by duplicate agonist applications,
spaced 10 min apart. After the initial control period, the tissue was
exposed to a solution containing 1.0 µM
,
-methylene ATP, 10 µM ATP, or 10 µM UTP, and the change in the afferent arteriolar diameter was determined. After 5 min of exposure, the agonist was
removed from the bathing medium, and the vessel was allowed to recover
in control buffer for 10 min before the same agonist solution was
reintroduced. The time course and magnitude of the afferent arteriolar
responses to the P2 agonist were compared between the first and second applications.
Series 2: effect of calcium channel blockade on the afferent
arteriolar response to P2 agonists.
Studies were performed to assess the role of L-type calcium channels in
the afferent arteriolar response to selective P2X and P2Y receptor
activation. Experiments followed the basic protocol described above and
involved a control period followed by a 5-min exposure to 1.0 µM
,
-methylene ATP, 10 µM ATP, or 10 µM UTP to assess the
control response. Subsequently, each vessel underwent a 5-min recovery
period in control buffer before being exposed to 10 µM diltiazem.
Five minutes later, the superfusate was changed to one containing 10 µM diltiazem plus 1.0 µM
,
-methylene ATP (P2X), 10 µM ATP
(P2X and P2Y), or 10 µM UTP (P2Y), and the afferent arteriolar
response to P2 receptor activation was reassessed.
Series 3: effect of Cd2+ on the
afferent arteriolar response to P2 agonists.
Studies were performed to assess the role of calcium influx in the
afferent arteriolar response to P2X and P2Y receptor activation. Experiments followed the basic protocol described above for the diltiazem series, except that 3 mM Cd2+, an inorganic
calcium channel blocker, was used to block a broader range of calcium
influx pathways. Experiments were performed, starting with a control
period, followed by a 5-min exposure to 55 mM KCl, 1.0 µM
,
-methylene ATP, 10 µM UTP, or 1 or 10 µM ATP to assess the
control response. Subsequently, each vessel underwent a 5-min recovery
period in control buffer before being exposed to a similar solution
containing 3 mM CdCl2. Five minutes later, the superfusate
was changed to one containing 3 mM Cd2+ plus 55 mM KCl, 1.0 µM
,
-methylene ATP (P2X), 10 µM UTP (P2Y), or 1 or 10 µM
ATP (P2X and P2Y), and the afferent arteriolar response to P2 receptor
activation was reassessed.
Series 4: effect of mefenamic acid on the afferent arteriolar
response to P2X receptor activation.
A final set of experiments was performed to determine the effect of the
purported nonselective cation channel blocker mefenamic acid on the
afferent arteriolar response to P2X receptor stimulation with
,
-methylene ATP. Experiments were performed as described above,
except that mefenamic acid was used in place of diltiazem. Control
experiments were performed to confirm that the concentration of
mefenamic acid used did not interfere with L-type calcium channel function.
Statistical Analysis
Data were evaluated with the use of a one-way analysis of variance for repeated measures. Differences between group means within each series were determined using a Newman-Kuels multiple-range test. P values <0.05 were considered to indicate statistically significant differences. All values are reported as means ± SE. ![]() |
RESULTS |
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The afferent arterioles used in the present study averaged 517 ± 18 µm in length (n = 85). Diameter measurements were obtained near the midpoint of the arteriolar length or ~256 ± 9 µm of the glomerulus. At this site, baseline afferent arteriolar diameter averaged 19.5 ± 0.4 µm.
The reproducibility of juxtamedullary afferent arteriolar responses to
repeat applications of ,
-methylene ATP, ATP, and UTP is
illustrated in Fig. 1. Arterioles treated
with
,
-methylene ATP averaged 17.1 ± 1.4 µm
(n = 3) during the initial control period. Application
of 1.0 µM
,
-methylene ATP evoked a rapid decline in vessel
diameter of 56 ± 11% to a minimum diameter of 7.7 ± 2.4 µm (Fig. 1A) within the first 20-30 s. This response was followed by a partial recovery to a new steady-state diameter of
14.7 ± 1.0 µm (86 ± 2% of control; P < 0.05 vs. control). Afferent caliber rapidly returned to the control
diameter of 17.1 ± 1.4 µm on removal of
,
-methylene ATP
from the bathing medium. The second application of
,
-methylene
ATP, administered 10 min after termination of the first exposure,
resulted in a virtually identical response, with afferent diameter
declining by 59 ± 11% to a minimum diameter of 7.1 ± 2.2 µm within the first 30 s and stabilizing at 14.8 ± 1.1 µm (86 ± 2% of control; P < 0.05 vs.
control). The response to the second application of
,
-methylene
ATP was not significantly different from the first.
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Similar experiments were performed using ATP or UTP. Baseline diameter
averaged 16.6 ± 0.3 µm in the ATP-treated group
(n = 3). As shown in Fig. 1B, the first and
second applications of 10 µM ATP stimulated an initial decrease in
afferent diameter of 43 ± 7 and 43 ± 7%, respectively. The
initial responses gradually waned until stable diameters 17 ± 1 and 17 ± 1% smaller than control, respectively, were reached.
Both ATP treatments induced a significant reduction in afferent
arteriolar diameter (P < 0.05 vs. control). No
significant difference was observed between the first and second responses to ATP. Similarly, the first application of 10 µM UTP (Fig.
1C; n = 6) reduced afferent arteriolar
diameter from a baseline diameter of 24.8 ± 0.9 to 14.9 ± 2.0 µm (59 ± 7% of control; P < 0.05). The
second treatment reduced afferent diameter to 58 ± 8% of the
control diameter. Consistent with previous observations (22), the afferent arteriolar response to UTP was more
monophasic than responses induced by ,
-methylene ATP or ATP, such
that the initial vasoconstriction was only slightly greater than the steady-state diameter. As with
,
-methylene ATP and ATP, both the
time course and the magnitude of the first and second responses to UTP
were nearly identical. These data demonstrate that repeat applications
of
,
-methylene ATP, ATP, and UTP cause comparable vasoconstrictor
responses with no evidence of receptor desensitization or tachyphylaxis.
Previous studies have suggested that preglomerular smooth muscle cells
express multiple P2 receptor subtypes that are coupled to different
calcium signaling pathways (22, 24). Therefore, studies
were performed to assess the effect of calcium channel blockade on the
afferent arteriolar response to selective P2X and P2Y receptor
activation. The results of experiments examining the effect of calcium
channel blockade on the afferent arteriolar response to the P2X agonist
,
-methylene ATP are presented in Fig.
2.
,
-Methylene ATP (1.0 µM)
administration induced a sharp reduction in afferent arteriolar caliber
of 72 ± 6% from a stable control diameter of 21.7 ± 1.9 µm to a minimum diameter of 6.5 ± 1.9 µm within 30 s.
Afferent caliber partially recovered to a stable diameter of 19.9 ± 1.7 µm, representing a sustained vasoconstriction of 8 ± 1%
(P < 0.05 vs. control). Removal of
,
-methylene-ATP from the superfusate resulted in complete
recovery to a diameter similar to control (21.7 ± 1.8 µm).
Exposure to 10 µM diltiazem caused 19 ± 3% vasorelaxation to a
stable diameter of 22.5 ± 1.9 µm, which is significantly
greater than the control diameter (P < 0.05).
Subsequent addition of
,
-methylene ATP to the superfusion solution, in the continued presence of 10 µM diltiazem, evoked a
rapid initial vasoconstriction of 41 ± 5%, which was
significantly blunted compared with control (P < 0.05). Furthermore, there was no evidence of a sustained
vasoconstriction in the presence of diltiazem. Afferent diameter
returned to 25.5 ± 1.9 µm, which is similar to the diameter
obtained with diltiazem alone. These data are in excellent agreement
with the results of an earlier report (25).
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Similar experiments were performed to determine the effect of calcium
channel blockade on the afferent arteriolar response to P2Y receptor
activation with 10 µM UTP. This concentration was selected on the
basis of previous work that determined 10 µM UTP to be the lowest
concentration to induce a significant reduction in afferent arteriolar
diameter (22). As shown in Fig.
3, UTP administration stimulated a
monophasic vasoconstriction of afferent arterioles that was rapidly
reversible on returning the superfusate to the control medium. UTP
reduced the diameter of afferent arterioles by 20 ± 4%, from a
control diameter of 21.4 ± 0.7 µm to a stable diameter of
17.1 ± 0.8 µm (P < 0.05 vs. control). When the
superfusate was returned to the control solution, the diameter of the
afferent arterioles returned to values similar to those of control.
Administration of 10 µM diltiazem led to a significant vasorelaxation
to a stable diameter of 25.4 ± 0.8 µm (P < 0.05 vs. control). In the continued presence of diltiazem, the
vasoconstrictor response to 10 µM UTP averaged 14 ± 4% at a
stable arteriolar diameter of 21.6 ± 0.9 µm (P < 0.05 vs. control). The magnitude of the UTP-mediated
vasoconstriction during calcium channel blockade tended to be slightly
smaller than the control response, but this difference was not
statistically significant (P > 0.05; n = 7 arterioles).
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Studies suggest that rat juxtamedullary afferent arterioles express
functional P2X and P2Y receptors and that ATP alters afferent arteriolar caliber by activating both receptor subtypes (22, 23,
25). The calcium signaling mechanisms by which ATP alters afferent arteriolar function have not been thoroughly investigated. Therefore, experiments were performed to determine the effect of
calcium channel blockade on the response of afferent arterioles to ATP.
Experiments were performed using three ATP concentrations (0.1, 1.0, and 10 µM), which have been shown to elicit minimum, intermediate,
and maximum vasoconstriction of afferent arterioles, respectively (Fig.
4) (22, 26). As shown in
Fig. 4A, 0.1 µM ATP evoked a modest reduction in afferent
arteriolar diameter from 19.7 ± 1.3 µm during the control
period, which then reached a stable diameter of 17.8 ± 1.3 µm
(P < 0.05 vs. control). Diltiazem treatment increased
vessel diameter and completely blocked the response to 0.1 µM ATP.
The diameter of afferent arterioles examined averaged 24.2 ± 1.4 and 24.2 ± 1.4 µm during the diltiazem and the diltiazem+ATP
periods, respectively.
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A similar response was observed in arterioles challenged with 1.0 µM
ATP (Fig. 4B). In this group, ATP stimulated a biphasic vasoconstrictor response similar to that observed with
,
-methylene ATP. Afferent arteriolar diameter decreased initially
by 30 ± 4%, from a control diameter of 20.2 ± 1.0 to
14.1 ± 0.7 µm (P < 0.05), before stabilizing
at a diameter of 18.1 ± 0.9 µm (P < 0.05 vs.
control). Subsequent treatment with diltiazem markedly blunted the
initial response and completely abolished the sustained response.
Afferent diameter decreased transiently from an average of 24.1 ± 1.1 µm (P < 0.05 vs. control) during diltiazem alone to a minimum diameter of 22.8 ± 0.9 µm, before reaching a
stable level of 24.1 ± 1.1 µm in response to the second
exposure to ATP. The diameter during the ATP+diltiazem period was not
significantly different from that during treatment with diltiazem
alone. Thus the magnitude of the second response to 1.0 µM ATP was
significantly smaller than the response to the first exposure.
The effect of calcium channel blockade on the afferent arteriolar response to 10 µM ATP is illustrated in Fig. 4C. Control diameter averaged 22.0 ± 1.2 µm and decreased rapidly, but transiently, to a minimum diameter of 9.9 ± 1.5 µm in response to ATP. The diameter stabilized at 14.3 ± 0.9 µm, which is significantly smaller than the control diameter (P < 0.05 vs. control). Diltiazem administration increased afferent diameter to 26.0 ± 1.5 µm (P < 0.05 vs. control), and the second ATP exposure decreased the diameter initially to 16.0 ± 1.5 µm, before a sustained diameter of 25.5 ± 0.9 µm was reached. During both treatment periods, 10 µM ATP produced a significant reduction in afferent arteriolar diameter (P < 0.05 vs. control); however, the magnitude of the first ATP-mediated response was significantly greater than the magnitude of the response obtained during calcium channel blockade (P < 0.05 vs. the second response). These data suggest that vasoconstriction induced by low concentrations of ATP are dependent on activation of L-type calcium channels, whereas higher concentrations of ATP can elicit vasoconstriction through activation of L-type calcium channel-dependent and -independent pathways.
Additional experiments were performed to assess the role of calcium
influx on the afferent arteriolar vasoconstriction induced by P2
receptor activation. Calcium influx pathways were nonselectively blocked by the addition of 3 mM cadmium chloride (Cd2+) to
the superfusate solution (Figs. 5 and
6). Under these conditions, the
K+-induced vasoconstriction of afferent arterioles was
blocked (Fig. 6, n = 3). During the control period, 55 mM KCl reduced afferent arteriolar diameter by 33 ± 2%, from
15.3 ± 0.6 to 10.3 ± 0.7 µm (P < 0.05).
Addition of 3 mM Cd2+ to the superfusate increased
arteriolar diameter by 39 ± 0.3%. Subsequent exposure to 55 mM
KCl, in the continued presence of Cd2+, did not result in a
significant vasoconstriction. Afferent diameter averaged 21.3 ± 2 with Cd2+ alone and 22.6 ± 1.4 µm in the presence
of Cd2+ and KCl. These data establish that extracellular
Cd2+ blocks depolarization-induced afferent arteriolar
vasoconstriction.
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Similar experiments were performed to determine the effect of
Cd2+ blockade of calcium influx pathways on afferent
arteriolar vasoconstrictor responses to P2 receptor activation. Figures
5A and 6 present the effect of Cd2+ on the
afferent arteriolar response to ,
-methylene ATP
(n = 5). Control administration of
,
-methylene TP
evoked a typical biphasic vasoconstrictor response that decreased
arteriolar diameter by 11 ± 1%, from 15.8 ± 0.4 to
14.0 ± 0.4 µm (P < 0.05). During superfusion
with Cd2+, the transient vasoconstriction was attenuated
and the sustained vasoconstrictor response was blocked. Afferent
diameter averaged 18.1 ± 0.6 with Cd2+ alone and
18.4 ± 0.7 µm during simultaneous exposure to Cd2+
and
,
-methylene ATP.
In contrast to the effect of Cd2+ on the response to
,
-methylene ATP, the vasoconstriction elicited by UTP
administration was retained (Figs. 5B and 6). In the absence
of Cd2+, UTP significantly reduced the diameter of afferent
arterioles by ~30 ± 5%, from 16.3 ± 0.5 to 11.4 ± 1.0 µm (P < 0.05; n = 7). In the
presence of the Cd2+, the response to UTP was attenuated
compared with the control response, but UTP still evoked a significant
reduction in afferent arteriolar diameter of 8 ± 2%, from
24.3 ± 1.5 to 22.3 ± 1.3 µm (P < 0.05).
The effect of superfusion with Cd2+ on the response to ATP administration is summarized in Fig. 6. Treatment with ATP concentrations of 1 (n = 4) or 10 µM (n = 6) reduced afferent diameter by 16 ± 3 and 18 ± 1%, respectively. Cadmium treatment increased afferent diameter by an average of 45 ± 7% and completely blocked the sustained vasoconstrictor response to both ATP concentrations. In the presence of Cd2+, afferent arteriolar diameter averaged 22.4 ± 1.8 and 22.5 ± 1.9 µm before and during treatment with 1.0 µM ATP and 23.0 ± 0.4 and 23.1 ± 0.5 µm before and during treatment with 10 µM ATP.
KCl, ,
-methylene ATP, UTP, and both concentrations of ATP
produced significant reductions in afferent arteriolar diameter during
the control response (Figs. 5 and 6). Also shown in Fig. 6 are the
average transient responses observed with each agonist (B).
All agonists examined elicited transient vasoconstrictions. These
transient responses were statistically significant during the control
period and during simultaneous exposure to Cd2+.
Nevertheless, the transient responses observed during Cd2+
treatment were substantially attenuated compared with the control responses.
P2X receptor-mediated responses involve activation of a nonselective
cation channel that allows the influx of extracellular sodium and
calcium (1, 11, 12, 36). Mefenamic acid is purported to
block nonselective cation channels (14, 15, 28). Excessively high concentrations of mefenamic acid may also block L-type
calcium channels (27). Experiments were performed to assess the effect of mefenamic acid on the afferent arteriolar response
to P2X receptor stimulation with ,
-methylene ATP. Control experiments were performed to determine a mefenamic acid concentration that would not significantly alter afferent arteriolar L-type calcium
channel function. As shown in Fig.
7A, increasing concentrations of mefenamic acid were applied to three afferent arterioles that were
preconstricted with 55 mM KCl. Administration of KCl decreased arteriolar diameter by 36 ± 4%, from 20.0 ± 1.8 to
12.0 ± 1.3 µm. Progressive addition of increasing
concentrations of mefenamic acid (1-100 µM) had little or no
effect on afferent diameter. However, exposure to 1,000 µM mefenamic
acid attenuated the magnitude of the KCl-mediated vasoconstriction of
these arterioles, suggesting the possible inhibition of L-type calcium
channel function.
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In a subsequent series of control experiments, we examined the effect of 100 µM mefenamic acid pretreatment on KCl-mediated vasoconstriction of afferent arterioles. This concentration was chosen on the basis of the previous series indicating that mefenamic acid did not attenuate an existing KCl-mediated vasoconstriction. As shown in Fig. 7B, control administration of KCl resulted in a 49 ± 3% reduction in afferent diameter from 21.1 ± 0.8 to 10.8 ± 0.7 µm. This response was completely reversed on removal of KCl from the superfusate solution. Administration of 100 µM mefenamic acid did not alter resting afferent arteriolar diameter nor did it attenuate the vasoconstrictor response elicited by KCl. These data indicate that 100 µM mefenamic acid does not impair voltage-dependent afferent arteriolar vasoconstriction.
Experiments were performed to determine the effect of mefenamic acid on
the afferent arteriolar vasoconstriction induced by 1.0 µM
,
-methylene ATP. As shown in Fig.
8, control administration of
,
-methylene ATP elicited a typical biphasic response. After a
recovery period, administration of 100 µM mefenamic acid did not
alter the initial vasoconstriction to
,
-methylene ATP but significantly attenuated the magnitude of the sustained
vasoconstriction by ~66%. Under control conditions, 1 µM
,
-methylene ATP evoked an initial and sustained reduction in
afferent diameter of 55 ± 10 and 18 ± 1%, respectively. In
the presence of mefenamic acid, the initial and sustained responses to
,
-methylene ATP averaged 57 ± 11 and 6 ± 1%,
respectively.
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DISCUSSION |
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P2 receptors are a large category of receptors that are divided
into two major families, termed P2X and P2Y (1, 3, 17, 36). Stimulation of P2X receptors induces contraction of
vascular smooth muscle by directly activating a ligand-gated,
nonselective cation channel (1, 3, 11, 12, 17, 36).
Activation of the cation current can lead to depolarization of vascular
smooth muscle and activation of voltage-dependent Ca2+
channels. In this report, blockade of L-type calcium channels attenuated the initial rapid vasoconstriction and abolished the sustained vasoconstriction induced by the P2X agonist ,
-methylene ATP. This effect of L-type calcium channel blockade is consistent with
an earlier report (25). These data also support recent observations that calcium channel blockade attenuates the increase in
intracellular calcium concentration observed in freshly isolated preglomerular vascular smooth muscle cells stimulated with
,
-methylene ATP (41). Administration of the divalent
cation Cd2+ also blocked the sustained vasoconstriction
evoked by
,
-methylene ATP and ATP but did not abolish the
vasoconstriction elicited by UTP, further supporting the suggestion
that
,
-methylene ATP, ATP, and UTP induced vasoconstriction of
juxtamedullary afferent arterioles by activating varied signal
transduction mechanisms.
P2Y receptors are G protein-regulated receptors coupled to phospholipase C in vascular smooth muscle cells and mesangial cells (1, 3, 17, 18, 35, 36, 38). Activation of P2Y receptors can lead to generation of inositol 1,4,5-trisphosphate and the release of calcium from intracellular stores (18, 35, 38). In the present report, stimulation of P2Y receptors with UTP resulted in a monophasic vasoconstriction that was essentially unaltered by calcium channel blockade. The time course and magnitude of the vasoconstrictor responses were similar before and during calcium channel blockade, and the magnitude was only attenuated in the presence of Cd2+. The finding that UTP-mediated renal microvascular vasoconstriction is retained during calcium channel blockade agrees well with previous reports that calcium channel blockade or removal of Ca2+ from the extracellular medium did not significantly alter UTP-induced increases in intracellular calcium concentration in preglomerular smooth muscle cells (24). In addition, these data lend additional support to the argument that at least two distinct P2 receptors are expressed by preglomerular microvascular smooth muscle (13, 19, 22, 24).
ATP is believed to be the endogenous ligand for P2 receptors. We have
postulated that extracellular ATP might function as the extracellular
messenger that evokes autoregulatory adjustments in afferent arteriolar
resistance (19, 23, 31, 33, 34). For this hypothesis to be
correct, ATP-mediated afferent arteriolar vasoconstrictor responses
must involve activation of L-type calcium channels (5, 25, 30,
32, 34). In the present report, we have shown that afferent
arteriolar vasoconstriction, mediated by low concentrations of ATP, are
completely abolished by calcium channel blockade. In contrast, afferent
arteriolar vasoconstrictor responses induced by high concentrations of
ATP were relatively insensitive to calcium channel blockade. This
pattern suggests that activation of P2 receptors with low
concentrations of ATP stimulates voltage-dependent calcium influx,
perhaps through activation of P2X receptors. It is interesting to note
that increases in intracellular calcium concentration, mediated by high
concentrations of ATP, are reduced by ~50% in the presence of
calcium channel blockers or during removal of extracellular calcium
(24). These observations suggest that low concentrations
of ATP bind preferentially to P2X receptors, stimulating L-type calcium
channel-dependent afferent arteriolar vasoconstriction. It is also
possible that higher concentrations of ATP activate both P2X and P2Y
receptors, leading to both voltage-dependent and voltage-independent
calcium influx as well as mobilization of calcium from intracellular
stores. This suggestion is supported by the retention of a significant vasoconstriction with 10 µM ATP despite the continued presence of
diltiazem. It is further supported by the observation that a high
concentration of ATP increases the intracellular calcium concentration
in preglomerular smooth muscle cells incubated in a nominally
calcium-free solution with a nearly identical time course and magnitude
as those observed during calcium channel blockade (24).
Stimulation of P2X receptors with ,
-methylene ATP, under similar
circumstances, essentially abolished the rise in intracellular calcium
normally observed under control conditions (41).
Despite being able to block or attenuate the sustained vasoconstriction
of afferent arterioles exposed to ,
-methylene ATP and low
concentrations of ATP, calcium channel blockade did not eliminate the
initial phase of the response. It is presumed that the rapid response
arises from stimulation of ligand-gated P2X receptors with the
subsequent activation of a nonselective cation channel. To test this
hypothesis, we examined the effect of the purported nonselective cation
channel blocker mefenamic acid (14, 28) on the response.
Pretreatment with mefenamic acid had no discernable effect on the time
course or magnitude of the initial vasoconstriction elicited by
,
-methylene ATP, but it attenuated the sustained
vasoconstriction. These data suggest that a mefenamic acid-sensitive
nonselective cation current contributes to the agonist-induced
depolarization of afferent arteriolar smooth muscle and facilitates the
opening of L-type calcium channels. However, the mechanism responsible
for the rapid initial vasoconstriction appears to completely
insensitive to mefenamic acid treatment. It is further noted that the
initial vasoconstriction induced by
,
-methylene ATP was
attenuated by about the same degree by Cd2+ treatment as it
was by diltiazem, whereas the effect on ATP and UTP was more
pronounced. For example, Cd2+ treatment reversed or
attenuated the vasoconstriction induced by
,
-methylene ATP and to
1.0 µM ATP in a pattern comparable to that observed with diltiazem.
In addition, superfusion with Cd2+ attenuated or abolished
the transient and sustained vasoconstrictor responses evoked by 10 µM
ATP, respectively, while only attenuating the response to UTP. Taken
together, these observations suggest that other signal transduction
pathways may be involved in mediating the transient and sustained
vasoconstrictor responses observed during P2 receptor activation. These
pathways could include nonselective cation channels, non-L-type
voltage-dependent calcium channels, or store-operated calcium influx
pathways. Further studies will need to be performed to better elucidate
the mechanisms of this response.
Curiously, diltiazem markedly reduced the increase in intracellular
calcium concentration observed in freshly isolated preglomerular smooth
muscle cells in response to ,
-methylene ATP (41),
whereas a transient vasoconstriction was still observed in the present studies with the use of intact arterioles. The reasons for this discrepancy are unclear but may arise, in part, from differences in the
"activation state" that exist between isolated smooth muscle cells
compared with vascular smooth muscle cells in intact, pressurized preglomerular microvessels. Vascular smooth muscle cells in intact vascular segments are stretched and depolarized compared with isolated
cells. Tension-generating signal transduction pathways are already
activated, whereas isolated cells are not exposed to a workload and
thus function at a different level of activation. This notion is
supported by the observation that calcium channel blockers induce a
prompt vasorelaxation of pressurized afferent arterioles (4, 5,
25), but they do not alter basal intracellular calcium
concentration in freshly isolated preglomerular smooth muscle cells
(2231, 2564, 3058). This difference may result in altered response
kinetics between the two experimental conditions and underscores the
importance of examining signal transduction mechanisms in both preparations.
The contribution of P2 receptors to the overall regulation of renal
hemodynamics and autoregulatory behavior is still unclear. Activation
of L-type calcium channels plays a major role in the regulation of
preglomerular resistance in response to both agonist stimulation
(4, 5, 29, 30, 34), autoregulation, and tubuloglomerular
feedback signals (6, 19, 30, 32, 34). Similarly, the
mobilization of calcium from intracellular stores also contributes
significantly to agonist-induced and pressure-mediated afferent
arteriolar vasoconstriction (9, 21, 29). In the present
report, the data establish the central role L-type calcium channels
play in mediating the afferent arteriolar response to P2X receptor
activation by either the P2X agonist ,
-methylene ATP or low
concentrations of the endogenous ligand ATP. Results of this study are
also consistent with previous observations implicating a P2Y receptor
subtype in evoking afferent arteriolar vasoconstriction through a
L-type calcium channel-independent pathway, presumably involving
IP3-dependent mobilization of calcium from intracellular stores (24, 36). However, the specific P2 receptor
subtypes involved remain to be identified. Previous work supports the
postulate that P2X1 and P2Y2 receptors are
expressed by the preglomerular vasculature (7, 19, 22,
23). Chan and co-workers (7) provided solid
immunohistochemical evidence depicting strong distribution of
P2X1 receptors along preglomerular microvascular segments
and no detectable staining on postglomerular vascular elements. This observation, coupled with the functional evidence demonstrating potent
afferent arteriolar vasoconstriction induced by
,
-methylene ATP
(19, 23, 25, 33), strongly supports the hypothesis that
P2X1 receptors are major contributors to the renal
microvascular responses to P2X receptor agonists. Interestingly,
P2X1 receptors are known to desensitize when repeatedly
challenged by an agonist (36). Deliberate attempts to
desensitize P2X1 receptors on juxtamedullary afferent
arterioles resulted in a marked blunting of the vasoconstriction induced by
,
-methylene ATP and abolition of the pressure-mediated autoregulatory response (23).
The possible involvement of P2X3 receptors is also an
intriguing possibility. Although definitive evidence of their presence on the renal microvasculature is lacking, P2X3 receptors
can be activated by ,
-methylene ATP, and they do not desensitize
as readily as P2X1 receptors (36). These
receptors might be better suited to produce the sustained
vasoconstriction induced by
,
-methylene ATP on juxtamedullary
afferent arterioles. Another possibility is that P2X1 and
P2X3 receptors could be present as a multimeric receptor,
thus conferring properties of both receptors on the overall response
(36).
Functional evidence for the presence of various P2Y receptors in the
renal microcirculation can also be found in the literature and have
been demonstrated using the juxtamedullary nephron technique (8,
13, 19, 22, 24, 31). Under the present experimental conditions,
a strong argument can be made for the involvement of P2Y2
and/or P2Y4 receptors in the afferent arteriolar response to ATP in the rat kidney (36). This conclusion is based on
the observation that UTP and ATP--S produce a strong and sustained afferent arteriolar vasoconstrictor response that cannot be mimicked by
other P2Y agonists, such as 2-methylthio ATP (22). UTP is a good agonist for P2Y2 and P2Y4 receptors and
a poor one for P2Y1 and P2Y6 receptors
(36). The fact that rat juxtamedullary afferent arterioles
are nearly unresponsive to 2-methylthio ATP, ADP, and UDP (20,
22) essentially rules out a major role for P2Y1 and
P2Y6 receptors in this vascular bed. Rank-order potency data compiled from other tissues suggest that P2Y4
receptors exhibit comparable sensitivity to ATP and ADP; however, ADP
is a very poor agonist for juxtamedullary afferent arterioles
(20). Therefore, on the basis of the information presently
available for this vascular bed, P2Y2 receptors are the
most likely P2Y receptor subtype involved in the afferent arteriolar
response to ATP. The receptor activation and signaling mechanisms known
to be utilized by these receptors are also consistent with the
signaling mechanisms known to participate in renal preglomerular
autoregulatory behavior (21, 34).
In summary, L-type calcium channels play a major role in regulating the diameter of afferent arterioles. Blockade of L-type calcium channels induces a prompt increase in arteriolar diameter and reduced responsiveness to P2 agonists. The results of the studies presented here reveal that activation of P2 receptors on afferent arterioles induces vasoconstriction through activation of distinct signal transduction pathways. Activation of P2X receptors relies largely on activation of voltage-dependent L-type calcium channels to evoke afferent arteriolar vasoconstriction. In contrast, P2Y receptor activation stimulates vasoconstriction of afferent arterioles largely through L-type calcium channel-independent mechanisms. P2 receptor activation by low concentrations of ATP induces afferent arteriolar vasoconstriction primarily through L-type calcium channel-dependent mechanisms, whereas higher concentrations of ATP invoke additional L-type calcium channel-independent vasoconstrictor mechanisms. These mechanisms are consistent with those known to participate in autoregulatory vasoconstrictor responses and are supportive of the postulate that activation of afferent arteriolar P2 receptors plays a central role in mediating autoregulatory adjustments in afferent arteriolar resistance.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. John D. Imig for a thoughtful review of the manuscript and Lisette Bourgouis for excellent secretarial assistance.
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FOOTNOTES |
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This work was supported by grants from the American Heart Association (AHA 95001370) and the National Institute of Diabetes and Digestive and Kidney Diseases (DK-44628). E. W. Inscho is an Established Investigator of the American Heart Association. These studies were performed at the Tulane University School of Medicine and at the Medical College of Georgia.
Address for reprint requests and other correspondence: E. W. Inscho, Dept. of Physiology, School of Medicine, Medical College of Georgia, 1120 15th St., Augusta, Georgia 30912-3000 (E-mail einscho{at}mail.mcg.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published August 15, 2001; 10.1152/ajprenal.00038.2001
Received 8 February 2001; accepted in final form 24 September 2001.
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