1 Louisiana State University School of Medicine; and 2 Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112
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ABSTRACT |
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This study tested the hypothesis
that P2X receptor activation increases intracellular Ca2+
concentration ([Ca2+]i) in preglomerular
microvascular smooth muscle cells (MVSMC) by evoking voltage-dependent
calcium influx. MVSMC were obtained and loaded with the
calcium-sensitive dye fura 2 and studied by using single-cell
fluorescence microscopy. The effect of P2X receptor activation on
[Ca2+]i was assessed by using the P2X
receptor-selective agonist ,
-methylene-ATP and was compared with
responses elicited by the endogenous P2 receptor agonist ATP.
,
-Methylene-ATP increased [Ca2+]i dose
dependently. Peak increases in [Ca2+]i
averaged 37 ± 11, 73 ± 15, and 103 ± 21 nM at agonist
concentrations of 0.1, 1, and 10 µM, respectively. The average peak
response elicited by 10 µM
,
-methylene-ATP was ~34% of the
response obtained with 10 µM ATP.
,
-Methylene-ATP induced a
transient increase in [Ca2+]i before
[Ca2+]i returned to baseline, whereas ATP
induced a biphasic response including a peak response followed by a
sustained plateau. In Ca2+-free medium, ATP induced a sharp
transient increase in [Ca2+]i, whereas the
response to
,
-methylene-ATP was abolished. Ca2+
channel blockade with 10 µM diltiazem or nifedipine attenuated the
response to
,
-methylene-ATP, whereas nonspecific blockade of
Ca2+ influx pathways with 5 mM Ni2+ abolished
the response. Blockade of P2X receptors with the novel P2X
receptor antagonist NF-279 completely but reversibly
abolished the response to
,
-methylene-ATP. These results
indicate that P2X receptor activation by
,
-methylene-ATP
increases [Ca2+]i in preglomerular MVSMC, in
part, by stimulating voltage-dependent Ca2+ influx through
L-type Ca2+ channels.
microvascular smooth muscle cells; adeonsine 5'-triphosphate; afferent arteriole; renal microvasculature; 8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-napthalenetrisulfonic
acid hexasodium salt; ,
-methylene-adeonsine 5'-triphosphate; nifedipine; diltiazem; nickel; calcium channels; P2X receptors
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INTRODUCTION |
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EXTRACELLULAR ATP HAS BEEN shown to be an important paracrine regulator of renal epithelial and preglomerular microvascular function (21, 24, 26, 30-33). ATP induces vasoconstriction by activating P2 receptors on preglomerular microvascular smooth muscle cells (MVSMC) (14, 20, 26). This family of P2 receptors is divided into two major groups, classified as P2X and P2Y receptor subtypes (1, 16, 35). Previous studies from our laboratories have shown that inactivation of P2 receptors on preglomerular microvessels inhibits autoregulatory behavior (21, 30, 32). Activation of P2X and P2Y receptors on MVSMC stimulates an increase in intracellular calcium concentration ([Ca2+])i by distinct calcium signaling pathways (22, 27). P2X receptors function as ligand-gated, transmembrane cation channels that allow influx of extracellular cations, including calcium (1, 12, 13, 15, 16, 35). In contrast, P2Y receptors are coupled to G proteins and increase [Ca2+]i, in part, by stimulating mobilization of calcium from intracellular stores (1, 12, 13, 16, 35).
Although the capacity of the kidney to autoregulate renal blood flow has been recognized for many years, the mechanisms by which renal autoregulation occurs remain unclear. Certainly, autoregulatory responses are accomplished through myogenic and tubuloglomerular feedback (TGF)-mediated adjustments in preglomerular resistance (3). TGF is believed to be a major regulatory system coupling changes in distal tubular flow with preglomerular resistance through the actions of the macula densa. We have proposed that ATP, released from the macula densa, serves as the chemical messenger linking the macula densa with regulation of afferent arteriolar resistance through ATP-dependent activation of P2X receptors that are heavily expressed along the preglomerular but not the postglomerular microvasculature (7, 21, 32, 33). This hypothesis is derived from the striking similarities between ATP-mediated afferent arteriolar vasoconstriction and pressure-mediated autoregulatory adjustments in afferent arteriolar diameter. Both stimuli alter afferent arteriolar diameter with similar temporal profiles (21). Both stimuli rely on calcium influx through voltage-gated calcium channels (22, 25, 33). Finally, inactivation of P2 receptors inhibits autoregulatory adjustments in afferent arteriolar diameter in response to increasing renal perfusion pressure (21, 30) or increasing distal tubular perfusion (32).
The purpose of this study was to evaluate the calcium signaling
pathways involved in the preglomerular smooth muscle response to P2X
receptor activation. Freshly isolated MVSMC were exposed to the
selective P2X agonist ,
-methylene-ATP. Studies were designed to
establish the calcium signaling pathways used by P2X receptors by
evaluating the role of extracellular calcium and L-type calcium channels in the response to P2X receptor activation. Additional studies
were performed to determine the effect of P2X receptor blockade on the
response to
,
-methylene-ATP. Responses elicited by the P2X
receptor agonist were compared with responses evoked by the endogenous
ligand ATP, which activates both P2X and P2Y receptors.
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METHODS |
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Tissue Preparation and Renal MVSMC Isolation
All studies were performed in compliance with the guidelines and practices dictated by the Tulane University Advisory Committee for Animal Resources. Suspensions of MVSMC were prepared as previously described (22). For each suspension of MVSMC (n = 33), one male Sprague-Dawley CD-VAF rat (250 to 375 g; Charles River Laboratories; Wilmington, MA) was anesthetized with pentobarbital sodium (40 mg/kg ip), and the abdominal aorta was cannulated via the superior mesenteric artery. Ligatures were placed around the abdominal aorta at sites proximal and distal to the renal arteries. The kidneys were cleared of blood by perfusion with ice-cold low-calcium physiological salt solution (PSS; pH 7.35) of the following composition (in mM): 125 NaCl, 5 KCl, 1 MgCl2, 10 glucose, 20 HEPES, and 0.1 CaCl2 as well as 6% BSA (22, 23) followed by an identical solution containing 1% Evans blue.The kidneys were removed and decapsulated, and the renal medullary tissue was removed. The cortical tissue was sieved (180-µm mesh), and the retentate was washed with ice-cold low-calcium PSS. The vascular tissue remaining on the sieve was transferred to an enzyme solution containing 0.075% collagenase (Boehringer Mannheim, Indianapolis, IN), 0.02% dithiothreitol (Sigma, St. Louis, MO), 0.2% soybean trypsin inhibitor (type 1-S, Sigma), and 0.1% BSA dissolved in low-calcium PSS and incubated for 30 min at 37°C. The vascular tissue was transferred to a nylon mesh (70-µm mesh) and washed with ice-cold low-calcium PSS. The retained vascular tissue was transferred to a petri dish containing ice-cold low-calcium PSS for collection of interlobular arteries with attached afferent arterioles. The vascular segments were placed in a solution containing 0.075% papain (Sigma) and 0.02% dithiothreitol in low-calcium PSS. The tissue was incubated at 37°C for 15 min and centrifuged (2,000 g for 50 s), and the tissue pellet was transferred to a solution containing 0.3% collagenase and 0.2% soybean trypsin inhibitor in low-calcium PSS at 37°C. After 15 min, the mixture was triturated and centrifuged (500 g for 5 min). The cell pellet was resuspended in 1 ml Dulbecco's minimum essential medium (Sigma) supplemented with 20% fetal calf serum (Whittaker Bioproducts, Walkerville, MD), 100 U penicillin, and 200 µg streptomycin (Sigma). Cell suspensions were stored on ice until used.
Fluorescence Measurements in Single MVSMC
Experiments were performed by using standard microscope-based fluorescence spectrophotometry techniques (Photon Technology, Lawrenceville, NJ) as previously described (22, 23). The excitation wavelengths were set at 340 and 380 nm, and the emitted light was collected at 510 ± 20 nm. Fluorescence intensity was collected (5 data points/s) and analyzed with the aid of Photon Technology software. Calibration of the fluorescence data was accomplished in vitro according to the method used by Grynkiewicz et al. (19).Measurement of [Ca2+]i in single MVSMC was performed as described previously (22, 23, 27). Suspensions of MVSMC were loaded with fura 2-acetoxymethyl ester (fura 2-AM; 10 µM; Molecular Probes, Eugene, OR), and an aliquot of cell suspension was transferred to the perfusion chamber (Warner Instrument, Hamden, CT), and the chamber was mounted to the stage of a Nikon Diaphot inverted microscope. The cells were superfused at 35°C with a control PSS solution of the following composition (mM): 125 NaCl, 5 KCl, 1 MgCl2, 10 glucose, 20 HEPES, 1.8 CaCl2, and 0.1 g/l BSA. For each experiment, fluorescence data were collected from a single cell after background subtraction. A new coverslip was used for each experiment.
Experimental Approach
Series 1.
MVSMC were exposed to ,
-methylene-ATP to determine the effect of
P2X receptor activation on [Ca2+]i. At the
concentrations used here,
,
-methylene-ATP is selective for the
P2X1 and P2X3 purinoceptor subtypes
(35). Concentration-response data were obtained by
exposing MVSMC to PSS solutions containing
,
-methylene-ATP
concentrations of 0.1, 1, and 10 µM. Fura 2 fluorescence was
monitored in these cells under control conditions (0-100 s) during
exposure to
,
-methylene-ATP (100-300 s) and during the
recovery period, during which
,
-methylene-ATP was removed from
the bathing solution (300-600 s). Agonist-mediated responses were
evaluated by determining the magnitude of the peak and late-phase
[Ca2+]i achieved. Peak responses were defined
as the maximum [Ca2+]i attained in the first
150 s of agonist exposure. Sustained responses were calculated by
averaging [Ca2+]i over the final 50 s of
agonist exposure. Similar experiments were performed with 10 µM ATP
to obtain control data for the endogenous ligand.
Series 2.
Studies were performed to determine the role of extracellular calcium
on the increase in [Ca2+]i induced by
,
-methylene-ATP. The contribution of calcium influx to the
response was determined by exposing single cells to 10 µM
,
-methylene-ATP while they were being bathed in nominally calcium-free PSS (22, 23). Previous studies have shown
that [Ca2+]i remains unchanged when MVSMC are
subjected to strong depolarizing conditions while being bathed in
calcium-free PSS (23). Fura 2 fluorescence was monitored
in these cells under control conditions (0-100 s), during exposure
to calcium-free PSS (100-150 s), and during subsequent exposure to
,
-methylene-ATP (150- 350 s). These responses were compared
with responses obtained from similar cells challenged in normal-calcium
PSS. Additional control cells were studied under identical conditions,
except that these cells were challenged with 10 µM ATP, and the
responses were compared with those obtained with
,
-methylene-ATP.
Series 3.
The contribution of calcium influx to the MVSMC response to
,
-methylene-ATP was further evaluated under conditions in which the extracellular calcium concentration remained within the
physiological range. For these experiments, cells were challenged with
,
-methylene-ATP while being bathed in a PSS solution containing 5 mM Ni2+. Ni2+ was used as a nonselective
calcium channel antagonist (2). Fura 2 fluorescence was
monitored in these cells under control conditions (0-100 s),
during exposure to 5 mM Ni2+ in the presence of 1.8 mM
Ca2+ (100-150 s) and during subsequent exposure to
,
-methylene-ATP in combination with Ni2+ and normal
Ca2+(150-350 s). These responses were compared with
responses obtained from similar cells challenged in normal-calcium PSS
without added Ni2+.
Series 4.
Additional experiments were performed to assess the role of L-type
calcium channels in the MVSMC response to ,
-methylene-ATP. For
these experiments, cells were challenged with
,
-methylene-ATP while being bathed in a PSS solution containing the L-type calcium channel antagonists diltiazem or nifedipine. Previous studies have
established that diltiazem is an effective inhibitor of ATP- and
KCl-mediated increases in [Ca2+]i in these
cells (22, 23). Control studies were performed to verify
the ability of 10 µM nifedipine to block the increase in
[Ca2+]i induced by KCl. Exposure of cells to
90 mM KCl resulted in a peak change in calcium of 31 nM
(n = 3 cells) in 1 µM nifedipine whereas the response
was further suppressed to 20 nM (n = 4 cells) in 10 µM nifedipine. Fura 2 fluorescence was monitored in these cells under
control conditions (0-100 s), during exposure to 10 µM diltiazem
in the presence of 1.8 mM Ca2+ (100-150 s), and during
subsequent exposure to
,
-methylene-ATP in combination with
diltiazem and normal Ca2+(150-350 s). Identical
studies were performed by using 10 µM nifedipine instead of diltiazem
to control for nonspecific interactions between the calcium channel
antagonists and P2X receptors. These responses were compared with
responses obtained from similar cells challenged in normal-calcium PSS
without calcium channel blockade.
Series 5.
Previous studies suggest that preglomerular vasoconstrictor responses
to ,
-methylene-ATP may be mediated through activation of P2X
receptors (10, 14, 18, 32). Therefore, we employed a
novel, selective P2X receptor antagonist
{8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-napthalenetrisulfonic acid hexasodium salt; NF-279; Tocris Cookson} to assess the
involvement of P2X receptors in the MVSMC response to
,
-methylene-ATP (11, 29). For these experiments
(n = 23), fura 2 fluorescence was monitored under
control conditions (0-100 s), during exposure to 20 µM NF-279
(100-300 s), and during subsequent exposure to
,
-methylene-ATP in combination with NF-279 (300-400 s). Five cells were subjected to a washout period of 300 s to remove NF-279 and
,
-methylene-ATP from the bath. After the washout period, cells were exposed to 10 µM
,
-methylene-ATP a second time.
Statistical Analysis
Data are presented as means ± SE. Within-group comparisons of peak [Ca2+]i with baseline [Ca2+]i were analyzed using ANOVA for repeated measures. Differences in baseline [Ca2+]i and steady-state [Ca2+]i between treatment groups were analyzed by ANOVA. Post hoc tests were performed using Tukey's test. Statistical probabilities of <0.05 (P < 0.05) are considered significantly different. ![]() |
RESULTS |
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Experiments were performed to establish the concentration-response
profile for ,
-methylene-ATP. Figure
1 presents representative traces
depicting the changes in [Ca2+]i elicited by
increasing concentrations (0.1, 1, and 10 µM) of
,
-methylene-ATP. Exposure of MVSMC to
,
-methylene-ATP
evoked a concentration-dependent increase in
[Ca2+]i that typically included a rapid peak
response followed by a gradual return to steady-state levels similar to
baseline. Figure 2 presents the average
responses in series 1 experiments. Baseline [Ca2+]i was similar across all three
treatment groups. The peak [Ca2+]i elicited
by each concentration of
,
-methylene-ATP was significantly different from baseline and averaged 37 ± 11, 73 ± 15, and
103 ± 21 nM, respectively. In contrast, the steady-state
[Ca2+]i was not significantly different from
the respective baseline [Ca2+]i at each
,
-methylene-ATP concentration tested.
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Figure 3 shows typical traces for cells
treated with 10 µM ,
-methylene-ATP (A) and 10 µM
ATP (B). Resting [Ca2+]i averaged
99 ± 6 nM for cells treated with
,
-methylene-ATP (n = 49 cells) and 77 ± 4 nM for the ATP-treated
cells (n = 31 cells). The peak
[Ca2+]i achieved by cells treated with
,
-methylene-ATP averaged 180 ± 17 nM, which was
significantly lower than the peak [Ca2+]i
attained in cells treated with ATP (315 ± 39 nM). The temporal pattern of the response to
,
-methylene-ATP is different from responses elicited by ATP. The average response elicited by
,
-methylene-ATP includes a rapid rise in
[Ca2+]i, but the peak response is followed by
a more rapid decline in [Ca2+]i. The typical
ATP response also includes a rapid increase in [Ca2+]i to a peak value, followed by a
sustained plateau phase sometimes exhibiting periods of
[Ca2+]i oscillation (Fig. 3B).
[Ca2+]i returns to baseline after ATP is
removed from the bathing medium. The magnitude of the steady-state
[Ca2+]i averaged 10 ± 2 nM above the
baseline (P < 0.05) for cells treated with
,
-methylene-ATP and 32 ± 6 nM for cells treated with ATP.
The magnitude of the steady-state [Ca2+]i in
,
-methylene-ATP-treated cells is significantly smaller (P < 0.05) than for cells treated with ATP.
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Previous studies have shown that ATP increases
[Ca2+]i in MVSMC by stimulating
Ca2+ influx from the extracellular medium and by
mobilization of Ca2+ from intracellular stores (22,
27). Experiments were performed to compare the contribution of
extracellular Ca2+ to the increase in
[Ca2+]i stimulated by ,
-methylene-ATP
and ATP. Typical responses to
,
-methylene-ATP and ATP are
presented in Fig. 4. Figure 4A shows the response of a single cell to 10 µM
,
-methylene-ATP while the cell was being bathed in Ca2+-free medium.
Removal of Ca2+ from the extracellular medium abolished the
response to
,
-methylene-ATP. [Ca2+]i
averaged 109 ± 15 nM in normal-calcium buffer and 108 ± 15 nM during exposure to calcium-free conditions (n = 12 cells). During exposure to 10 µM
,
-methylene-ATP, peak and
steady-state [Ca2+]i averaged 124 ± 15 and 91 ± 11 nM, respectively. These
[Ca2+]i values are not significantly
different from those for control or calcium-free
[Ca2+]i. Control cells (n = 5) challenged with 10 µM
,
-methylene-ATP in normal-calcium
conditions exhibited typical increases in
[Ca2+]i from a baseline of 101 ± 12 to
peak and steady-state [Ca2+]i of 183 ± 40 and 97 ± 10 nM, respectively. Figure 4B shows the response of a single cell to 10 µM ATP while the cell was being bathed in calcium-free medium. In contrast to the effect of calcium removal on the response elicited by
,
-methylene-ATP, cells
treated with ATP exhibited significant increases in
[Ca2+]i. For the cells in this treatment
group (n = 13), the baseline [Ca2+]i averaged 90 ± 5 nM under
control conditions and 91 ± 5 nM when cells were exposed to
calcium-free medium. Subsequent exposure to 10 µM ATP induced a sharp
increase in [Ca2+]i to a peak value of
348 ± 47 nM before a rapid decline to 89 ± 5 nM. In
contrast, paired control cells (n = 13) exposed to 10 µM ATP in normal-calcium medium exhibited an increase in
[Ca2+]i from a baseline of 76 ± 5 nM to
a peak of 455 ± 95 nM, before the level stabilized to a
concentration of 88 ± 5 nM.
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P2X receptor activation involves opening a ligand-gated cation channel
that directly increases [Ca2+]i and causes
membrane depolarization (15, 35). Therefore, studies were
performed to assess the Ca2+ influx pathways involved in
the MVSMC response to ,
-methylene-ATP. Cells were exposed to
,
-methylene-ATP while being bathed in control buffer containing
1.8 mM Ca2+ plus either Ni2+, diltiazem, or
nifedipine. As shown in the representative traces presented in Fig.
5, 5 mM Ni2+ (A)
and 10 µM diltiazem (B) blocked or attenuated the response of these cells to 10 µM
,
-methylene-ATP, respectively. In
Ni2+-treated cells (n = 11),
[Ca2+]i averaged 119 ± 5 and 117 ± 5 nM during exposure to control and Ni2+-containing
solutions, respectively, and remained unchanged when challenged with 10 µM
,
-methylene-ATP. Similarly, in diltiazem-treated cells
(n = 16), [Ca2+]i averaged
122 ± 9 and 117 ± 11 nM during exposure to control and
diltiazem-containing solutions, respectively. Subsequent exposure to 10 µM
,
-methylene-ATP increased [Ca2+]i
by only 27 ± 9 nM. Similarly, in nifedipine-treated cells
(n = 15), exposure to
,
-methylene-ATP increased
[Ca2+]i by only 41 ± 7 nM. The peak
responses observed in the presence of diltiazem and nifedipine are
significantly smaller than control responses (P < 0.05).
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Presently, there are approximately seven P2X receptor subtypes that
have been cloned and expressed (15, 16, 35). Evidence has
shown that the P2X1 receptor subtype is heavily expressed along the preglomerular microvasculature (7), and
,
-methylene-ATP is known to be a potent agonist of
P2X1 receptors (15, 16, 35). Therefore, we
tested the hypothesis that
,
-methylene-ATP elevates
[Ca2+]i in MVSMC by activation of P2X
receptors. In these experiments, cells were pretreated with the novel
P2X receptor antagonist NF-279 before and during exposure to
,
-methylene-ATP. In five experiments, cells were challenged with
,
-methylene-ATP in the presence of NF-279; the bath solution was
then switched to the control buffer, and the cells were washed for
300 s. The washed cells were exposed a second time to
,
-methylene-ATP but this time without the P2X receptor blocker. A
typical example of one of these experiments is shown in Fig.
6. Preincubation with NF-279 abolished
the increase in [Ca2+]i normally observed in
response to
,
-methylene-ATP. In contrast, removal of NF-279 from
the bathing medium resulted in an increase in
[Ca2+]i when the cell was challenged a second
time with
,
-methylene-ATP. This observation verifies the
responsiveness of the cells and confirms the effectiveness of the P2X
receptor blockade with NF-279. Analysis of data collected from 23 cells
demonstrates that [Ca2+]i averaged 65 ± 3 and 66 ± 3 nM during the control and NF-279 period,
respectively. Consistent with the example shown in Fig. 6, the
[Ca2+]i averaged 74 ± 3 and 68 ± 3 nM during the peak and steady-state periods of exposure to
,
-methylene-ATP. These [Ca2+]i values
are not significantly different from those for the control and NF-279
periods.
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DISCUSSION |
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Renal hemodynamic control is accomplished by local adjustments in intrarenal vascular resistance (3). The majority of these resistance adjustments are preglomerular and occur at the level of the afferent arterioles (3). Numerous neural, humoral, and paracrine agents have been shown to exert some influence on renal vascular resistance (3, 33). Recently, interest has turned to the potential involvement of extracellular nucleotides as physiological regulators of renal vascular resistance (8, 24, 30, 33, 38). We and others have shown that exposure of the renal vasculature to extracellular nucleotides results in rapid and reversible alterations in renal vascular resistance, renal microvascular diameter, and renal perfusion (9, 14, 24-26, 30, 38, 39). Other studies have begun to investigate the intracellular signaling pathways involved in the preglomerular smooth muscle response to P2 receptor stimulation (22, 25, 27). The present studies were performed to take a more focused look at the signaling events initiated after activation of P2X receptors known to be expressed by MVSMC (7).
P2 receptors were first defined by Burnstock (5) in 1978. Since then, P2 receptors have grown into a large family of receptors divided into two basic categories (1, 6, 16, 17, 35). P2X
receptors are described as ligand-gated channels whereas P2Y receptors
are G protein-regulated receptors (1, 35). Previous studies have shown that renal microvascular and MVSMC responses to P2
receptor stimulation with ATP involve the activation of both P2X and
P2Y receptor subtypes (20, 22). In addition, evidence
suggests that each receptor type activates different calcium signaling
pathways (22). The present report focuses on the calcium
signaling pathways involved in the MVSMC response to P2X receptor
activation with the P2 agonist ,
-methylene-ATP. This stable ATP
analog is reported to be selective for P2X1 and P2X3 receptors at the agonist concentrations used here
(1, 12, 13, 35).
,
-Methylene-ATP is described as
weak or inactive at the P2X2, P2X4,
P2X5, and P2X6 receptors (15, 35)
and is either inactive or requires concentrations in excess of 100 µM to activate different splice variants of the P2X2 receptor
(4, 37).
,
-Methylene-ATP is also a very poor agonist
of P2Y receptors (35). Immunohistochemical studies have
shown the P2X1 receptor to be highly expressed along the
preglomerular microvasculature (7) but not the
postglomerular microvasculature. Interestingly, the microvascular
segments that stain positively for P2X1 receptors also
vasoconstrict when exposed to ATP or
,
-methylene-ATP
(26).
Stimulation of P2X receptors activates an inwardly directed
nonselective cation current, which can contribute to the elevation of
[Ca2+]i (1, 12, 13, 15, 34, 35).
The present studies were performed to test the hypothesis that exposure
of MVSMC to ,
-methylene-ATP would result in an elevation of
[Ca2+]i through activation of calcium influx
pathways. The data demonstrate that P2X receptor activation with
,
-methylene-ATP results in a concentration-dependent elevation of
[Ca2+]i. Furthermore, the magnitude and time
course of the response to
,
-methylene-ATP are markedly different
from those evoked by an equimolar concentration of ATP.
,
-Methylene-ATP increased [Ca2+]i by
~84% whereas an identical concentration of ATP increased [Ca2+]i by 309%. The response to
,
-methylene-ATP was transient whereas the response to ATP
exhibited a sustained elevation of [Ca2+]i.
These data demonstrate that both ATP and
,
-methylene-ATP increase
[Ca2+]i but suggest that the responses occur
by activation of different calcium signaling mechanisms and/or
different receptor subtypes.
Reliance on calcium influx for the increase in
[Ca2+]i is confirmed by exposing cells to
,
-methylene-ATP while they are being bathed in nominally
calcium-free medium or by blocking endogenous calcium influx pathways.
The data in Fig. 4 clearly demonstrate that removal of calcium from the
extracellular medium completely abolishes the calcium response evoked
by
,
-methylene-ATP. Similarly, nonspecific blockade of calcium
influx pathways by the addition of Ni2+ to the
extracellular medium while a physiological concentration of
extracellular calcium is maintained completely eliminated the increase
in [Ca2+]i in response to
,
-methylene-ATP exposure. Therefore, the increase in
[Ca2+]i found to occur under control
conditions involves activation of a nickel-sensitive calcium influx
pathway rather than the release of calcium from intracellular stores.
These data are consistent with previous observations that the afferent
arteriolar vasoconstrictor response elicited by ,
-methylene-ATP could be totally blocked when calcium was removed from the
extracellular medium (25). In those studies, EGTA was
added to the bathing medium and the blood perfusate to reduce the
concentration of free calcium in the extracellular environment.
Exposure of afferent arterioles to 1 µM
,
-methylene-ATP during
low-calcium conditions eliminated the vasoconstrictor response normally
observed. Returning the extracellular calcium concentration to
physiological levels, by the addition of excess calcium, restored the
afferent arteriolar vasoconstrictor response on subsequent exposure of
these arterioles to
,
-methylene-ATP. These data support the
argument that renal microvascular responses to
,
-methylene-ATP
require the influx of calcium from the extracellular environment.
Although the studies described above establish the requisite role of
calcium influx in the calcium signaling response to
,
-methylene-ATP, they do not address the specific nature of the
influx pathway responsible for the response. Previous studies have
shown that the sustained phase of the afferent arteriolar
vasoconstriction elicited by
,
-methylene-ATP can be blocked with
the L-type calcium channel antagonists diltiazem or felodipine, whereas
the initial vasoconstriction was significantly attenuated
(25). Furthermore, ATP-mediated elevation of
[Ca2+]i is markedly attenuated by the calcium
channel blocker diltiazem (22, 27). Therefore, we
investigated the possibility that L-type calcium channels might be
involved in the calcium response to
,
-methylene-ATP-mediated P2X
receptor activation. Calcium channel blockade with nifedipine or
diltiazem attenuated the increase in [Ca2+]i
induced by
,
-methylene-ATP. Despite the presence of calcium channel blockers,
,
-methylene-ATP still induced a small transient calcium response that was not observed under calcium-free conditions or
in the presence of extracellular Ni2+. Therefore, the
calcium channel blocker data suggest that P2X receptor activation by
,
-methylene-ATP stimulates a nickel-sensitive calcium influx
pathway that is partially dependent on the activation of L-type calcium
channels to effect the elevation of [Ca2+]i.
These data are consistent with the hypothesis that
,
-methylene-ATP is activating the ligand-gated, nonselective
cation channel that is structurally integrated into the P2X receptor
(15, 35). Activation of this cation channel could lead to
membrane depolarization and activate voltage-operated calcium channels.
It is interesting to note that the residual increase in
[Ca2+]i observed during calcium channel
blockade corresponds with the residual transient vasoconstriction that
is observed when afferent arterioles are challenged with
,
-methylene-ATP during calcium channel blockade but is absent
when calcium is removed from the bathing medium.
An alternative explanation could be that Ni2+, diltiazem,
and nifedipine all interfere with the binding of ,
-methylene-ATP to the receptor and thus impair the response to agonist stimulation. This possibility seems unlikely given the broad disparity in the structures of the agents concerned and the net impact each had on
,
-methylene-ATP-mediated responses. Ni2+ has been
used to examine membrane currents evoked by many different agonists in
many different cell types. There have not been any implications from
those studies that Ni2+ directly interferes with agonist
binding. The calcium channel blockers used in the present study are
well-established agents, whose selectivity for L-type calcium channels
has been well characterized. In the present report, small residual
responses were observed in response to
,
-methylene-ATP exposure.
The magnitude of the response was qualitatively larger in the
nifedipine-treated cells compared with the diltiazem-treated cells, but
this difference was not statistically significant. The overriding
observation in these studies is that removal of calcium from the
extracellular medium or general blockade of calcium influx pathways
with a high concentration of Ni2+ resulted in complete
blockade of the
,
-methylene-ATP-mediated increase in calcium.
Selective blockade of L-type calcium channels with two structurally
dissimilar calcium channel blockers resulted in partial inhibition of
the calcium response. These observations indicate that stimulation of
P2X receptors with
,
-methylene-ATP activates a calcium influx
pathway that relies, in part, on the opening of L-type calcium channels
as well as one or more additional influx pathways.
Interestingly, ATP, which activates both P2X and P2Y receptors, increases [Ca2+]i by stimulating both calcium influx through voltage-gated L-type calcium channels and calcium mobilization (22, 27). These findings are confirmed in the present study by the demonstration that [Ca2+]i increases transiently in cells treated with ATP while they are being bathed in calcium-free medium. Thus experimental evidence supports the expression of multiple P2 receptor subtypes and isoforms by preglomerular MVSMC.
Immunohistochemical evidence has demonstrated that the preglomerular
microvasculature of the rat stained heavily for expression of
P2X1 receptors whereas no evidence of staining was observed on the postglomerular efferent arteriole (7).
Autoradiographic data also support the existence of binding sites for
[3H]-labeled ,
,-methylene-ATP along the
interlobular arteries and afferent arterioles but not postglomerular
efferent arterioles (7). Interestingly, this
immunohistochemical distribution and autoradiographic profile mirrors
the functional assessment of the preglomerular and postglomerular
responsiveness to P2 receptor stimulation (26). In those
studies, only the preglomerular microvascular segments (arcuate and
interlobular arteries and afferent arterioles) responded to ATP with
rapid, biphasic vasoconstrictor responses (26). The
postglomerular efferent arteriole was unaffected by ATP treatment
(26). In addition, pharmacological assessment of afferent
arteriolar responsiveness to a variety of P2 agonists revealed that the
P2X1 agonist
,
-methylene-ATP was the most potent
agonist tested (20). These observations support the
involvement of P2X1 receptors in the renal microvascular
response to extracelluar ATP.
In the present report, we evaluated a newly developed receptor
antagonist, NF-279, which is purported to be a highly potent antagonist
at human P2X1 receptors (11, 28, 29, 36) and may also be effective against P2X7 receptors
(28). In addition, recent data generated in Xenopus
laevis oocytes expressing rat P2X receptors suggest that
higher concentrations may have some inhibitory properties at
P2X2, P2X3, and P2X4 receptors
(36). However, electrophysiological studies indicate that
P2X2 and P2X4 receptors are unresponsive to 10 µM ,
-methylene-ATP (11, 29). Exposure of freshly
isolated MVSMC to NF-279 had no effect on baseline
[Ca2+]i but completely eliminated the
increase in [Ca2+]i associated with exposure
to
,
-methylene-ATP. In addition, when NF-279 was removed from the
bathing medium and
,
-methylene-ATP was reapplied, cells that were
previously unresponsive to
,
-methylene-ATP now responded with an
increase in [Ca2+]i, although the response
was noticeably broader. We cannot be certain as to the reason for the
broader response, but several possibilities can be considered. The
simplest explanation would be that the NF-279 was not completely washed
from the bathing solution or dissociated from the receptors during the
washout period. Alternatively, the reversibility of NF-279 blockade of P2X receptors on preglomerular smooth muscle may be incomplete, resulting in a partial retention of P2X receptor blockade under the
conditions used here. Finally, the cellular P2 receptors could have
undergone partial desensitization during the first exposure to
,
-methylene-ATP. This would lead to a blunted, and perhaps slower, response during the subsequent exposure. Nevertheless, restoration of responsiveness to
,
-methylene-ATP after washout of
the NF-279 confirms that these cells are responsive to
,
-methylene-ATP but that blockade of P2X receptors with NF-279
prevented
,
-methylene-ATP from stimulating a response. This
observation strongly supports the contention that increases in
[Ca2+]i induced by
,
-methylene-ATP
occur through the selective activation of P2X receptors.
In summary, the data presented here provide in vitro evidence that
exposure of freshly isolated preglomerular MVSMC to
,
-methylene-ATP results in a prompt, concentration-dependent
elevation of intracellular calcium concentration.
,
-Methylene-ATP
elevates calcium by stimulating the influx of extracellular calcium
through a nickel-sensitive, voltage-dependent pathway involving L-type
calcium channels. The response elicited by
,
-methylene-ATP is
markedly different from the responses elicited by ATP alone and is
completely and reversibly blocked using a selective P2X receptor
antagonist. The results of these studies are in agreement with the
hypothesis that P2X receptor activation vasoconstricts preglomerular
microvessels by stimulating L-type calcium channel-dependent elevation
in [Ca2+]i.
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ACKNOWLEDGEMENTS |
---|
The authors thank Elizabeth LeBlanc for excellent technical assistance in these studies.
![]() |
FOOTNOTES |
---|
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 and DK-38226). E. W. Inscho is an Established Investigator of the American Heart Association.
Address for reprint requests and other correspondence: E. W. Inscho, Dept. of Physiology CL#3140, Medical College of Georgia, 1120 15th St., Augusta, GA 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.
Received 20 June 2000; accepted in final form 19 February 2001.
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