Extracellular ATP Stimulates NO Production in Rat Thick
Ascending Limb
Guillermo Silva, William H. Beierwaltes, Jeffrey L. Garvin
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Abstract—NO produced by NO synthase (NOS) 3 acts as an autacoid to regulate NaCl absorption in the thick ascending
limb. ATP induces NO production by NOS 3 in endothelial cells. We hypothesized that extracellular ATP activates NOS
in thick ascending limbs through P2 receptors. To test this, we measured intracellular NO production using the
NO-selective fluorescent dye DAF-2 in suspensions of rat medullary thick ascending limbs. We found that ATP
increased DAF-2 fluorescence in a concentration-dependent manner, reaching saturation at ⬇200 ␮mol/L with an EC50
of 37 ␮mol/L. The increase was blunted by 74% by the nonselective NOS inhibitor L-␻-nitro-arginine-methyl-ester
(2 mmol/L; 60⫾7 versus 16⫾6 arbitrary fluorescence units; P⬍0.02; n⫽5). In the presence of the P2 receptor antagonist
suramin (300 ␮mol/L), ATP-induced NO production was reduced by 64% (101⫾11 versus 37⫾5 arbitrary fluorescence
units; P⬍0.002; n⫽5). Blocking ATP hydrolysis with a 5⬘-ectonucleotidase inhibitor, ARL67156 (30 ␮mol/L) enhanced
the response to ATP and shifted the EC50 to 0.8 ␮mol/L. In the presence of ARL67156, the EC50 of the P2X-selective
agonist ␤,␥-methylene-adenosine 5⬘-triphosphate was 4.8 ␮mol/L and the EC50 for the P2Y–selective agonist UTP was
40.4 ␮mol/L. The maximal responses for both agonists were similar. Taken together, these data indicate that ATP
stimulates NO production in the thick ascending limb primarily through P2X receptor activation and that ATP
hydrolysis may regulate NO production. (Hypertension. 2006;47[part 2]:563-567.)
Key Words: nitric oxide 䡲 kidney 䡲 receptors, purinergic 䡲 signal transduction
T
he thick ascending limb of Henle’s loop is a waterimpermeable segment of the nephron. It generates the
corticomedullary osmotic gradient necessary for urine concentration and absorbs 30% of the filtered NaCl load. NaCl
absorption by the thick ascending limb is regulated by several
factors,1 including NO.2 NO decreases NaCl absorption by
inhibiting the Na⫹/K⫹/2Cl⫺ cotransporter.3 NO synthase
(NOS) 3 is the primary source of NO in the thick ascending
limb.4 Factors known to enhance NOS 3 activity in the thick
ascending limb also stimulate NOS 3 activity in endothelial
cells. These include endothelin 1,5 ␣2-adrenergic receptor
agonists,6 and luminal flow.7
ATP is a paracrine/autocrine factor released by several
different cells in response to many stimuli.8 –10 The effects of
extracellular ATP are mediated by a family of transmembrane
purinergic (P2) receptors including P2X and P2Y receptors.11
P2 receptors are expressed in several types of cells in
different structures in the kidney, including epithelial cells
along the nephron12 and in the microvasculature.13 They are
involved in basic functions such as ion transport14,15 and
regulation of afferent arteriole diameter.16,17 Stimulation of
both P2X18 and P2Y19 receptors has been shown to induce
NO production. The thick ascending limb expresses P2
receptors,12,20 but their role in NO production is unknown.
The effects of extracellular ATP are limited via hydrolysis
by 5⬘-ectonucleotidases. These enzymes are expressed in the
plasma membrane of several nephron segments.21 This family
of enzymes has a high affinity for ATP and other nucleotides.
Thus, the expression and activity of 5⬘-ectonucleotidases in
the thick ascending limb could modify the response to ATP.
We hypothesized that extracellular ATP stimulation in the
thick ascending limb stimulates NO production by activation
of P2 receptors, and this response is modified by the 5⬘ectonucleotidases expressed by this nephron segment.
Methods
Medullary Thick Ascending Limb
Tubule Suspensions
This study was approved by the Henry Ford Hospital review
committee. All studies were conducted in accordance with the
National Institutes of Health Guide for the Care and Use of
Laboratory Animals. Male Sprague-Dawley rats weighing 200 to
250 g (Charles River Breeding Laboratories, Kalamazoo, MI) were
fed a diet containing 0.22% sodium and 1.1% potassium (Purina) for
ⱖ7 days and then anesthetized with ketamine (100 mg/kg body
weight IP) and xylazine (20 mg/kg body weight IP). Medullary thick
ascending limb suspensions were prepared according to Herrera and
Garvin.22 In brief, kidneys were perfused via retrograde perfusion of
the aorta with 40 mL of HEPES-buffered physiological saline
Received September 29, 2005; first decision October 23, 2005; revision accepted November 17, 2005.
From the Division of Hypertension and Vascular Research (G.S., W.H.B., J.L.G.), Henry Ford Hospital and Department of Physiology (W.H.B.,
J.L.G.), School of Medicine, Wayne State University, Detroit, Mich.
Correspondence to Jeffrey L. Garvin, Division of Hypertension and Vascular Research, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, MI 48202.
E-mail [email protected]
© 2006 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000197954.93874.ef
563
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March 2006 Part II
containing (in mmol/L): 130 NaCl, 4 KCl, 2.5 NaH2PO4, 1.2 MgSO4,
2 calcium dilactate, 5.5 glucose, 6 D-alanine, 1 trisodium citrate, and
10 HEPES plus 0.1% collagenase A (Sigma-Aldrich, St. Louis, MO)
and 100 U of heparin. The inner stripe of the outer medulla was cut
from coronal slices of the kidney, minced, and incubated at 37°C for
30 minutes in 0.1% collagenase A and was agitated and gassed with
100% O2 every 5 minutes. Tissue was pelleted by centrifugation at
100g for 2 minutes, resuspended in cold HEPES-buffered physiological saline, and stirred on ice for 30 minutes to release the tubules.
The suspension was filtered through a 250-␮m nylon mesh and
centrifuged at 100g for 2 minutes. The pellet was washed, centrifuged again, and resuspended in 1 mL of cold HEPES-buffered
physiological saline.
Measurement of Intracellular NO by DAF-2 DA
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Intracellular NO production by thick ascending limbs was measured
using the NO-selective fluorescent dye DAF-2 DA (Calbiochem).
Suspensions of medullary thick ascending limbs were loaded by
adding 10 ␮mol/L DAF-2 DA to the suspension and incubating them
at 37°C for 20 minutes. Tubules were spun at 100g for 2 minutes; the
pellet was washed 3 times and resuspended in 1 mL of HEPESbuffered physiological saline gassed with 100% oxygen at 37°C. All
experiments were performed in the presence of L-arginine
(100 ␮mol/L), the substrate for NO synthase. Once loaded, the
tubules were placed in the chamber of a fluorometer at 37°C. After
20 minutes of equilibration, measurements were taken for 10 s once
every minute for a 5-minute baseline. Then, ATP or another agonist
was added and fluorescence measured for 20 minutes, taking the last
5 minutes as the experimental period. The dye was excited with a
low-pressure mercury arc lamp using a 488-nm filter. Emitted
fluorescence was filtered using a 510-nm high-pass filter and
measured with a photomultiplier tube. Data were collected using
DATAquisition acquisition software v1.46 (DATAQ) and an analogue chart recorder.
Figure 2. Effect of ATP on DAF-2 fluorescence in the presence
of the NOS inhibitor L-NAME (n⫽5).
To investigate whether ATP stimulates NO production in the
thick ascending limb, we first examined concentration dependence. Treatment of medullary thick ascending limb suspensions with ATP (Sigma-Aldrich) at concentrations of 0.5, 20,
50, 100, 250, and 500 ␮mol/L increased DAF-2 fluorescence
in a concentration-dependent manner (Figure 1). The response showed saturation at ⬇200 ␮mol/L and an EC50 of
37 ␮mol/L. The vehicle effect was subtracted from the effect
of each concentration.
To show that the effect on DAF-2 fluorescence was caused
by an increased NO production, we tested the ability of the
NO synthase inhibitor, L-␻-nitro-arginine-methyl-ester (LNAME; 2 mmol/L; Sigma-Aldrich) to block the increase
caused by ATP. Medullary thick ascending limb suspensions
were pretreated with L-NAME for 20 minutes. In the absence
of L-NAME, ATP (100 ␮mol/L) increased DAF-2 fluorescence by 60⫾7 arbitrary fluorescence units (AFUs), corrected
for the vehicle effect. In the presence of L-NAME, ATP
increased DAF-2 fluorescence by only 16⫾6 AFU (P⬍0.02
versus control; n⫽5), an inhibition of 74% (Figure 2). Taken
together, these data indicate that ATP induces NO production
in the thick ascending limb by activating NOS.
Because ATP is a selective ligand of P2 receptors, we
investigated the possible role of P2 receptor activation in
ATP-induced NO production. For this, we tested the ability of
ATP to stimulate NO production in the presence of a generic
P2 receptor antagonist, suramin (Sigma-Aldrich). In the
Figure 1. Concentration dependence of ATP-induced DAF-2
fluorescence. EC50⫽37 ␮mol/L.
Figure 3. Effect of ATP on NO production in the presence of
suramin, a P2 receptor antagonist (n⫽5).
Statistics
Data are reported as mean⫾SEM. Differences in means were
analyzed using an unpaired t test. 95% CIs were calculated for EC50
and maximum responses of the concentration-response curves for
UTP and ␤,␥-methylene-adenosine 5⬘-triphosphate (␤,␥-Me-ATP).
Statistical analysis was performed by the Department of Biostatistics
and Epidemiology of Henry Ford Hospital.
Results
Silva et al
ATP Stimulates NO Production in THAL
565
(UTP; Sigma-Aldrich) by generating concentration–response
curves for both agonists and calculating the EC50s and
maximum responses. We found that the EC50 for ␤,␥-Me ATP
was 4.8 ␮mol/L, with a 95% CI of 2.8 to 6.8 ␮mol/L. The
maximum response was 79 AFU with a 95% CI of 74 to 84
AFU. In contrast, the EC50 for UTP was 40.4 ␮mol/L with a
95% CI of 26 to 54 ␮mol/L. The maximum response was 78
AFU with a 95% CI of 70 to 86 AFU (Figure 5). These data
indicate that ATP stimulates NO production primarily by
activating the P2X receptors in the thick ascending limb.
Discussion
Figure 4. Concentration dependence of ATP-induced NO production in the presence of the 5⬘-ectonucleotidase inhibitor
ARL67156. EC50⫽0.8 ␮mol/L.
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absence of suramin, ATP (100 ␮mol/L) increased NO production by 101⫾12 AFU. After treating medullary thick
ascending limb suspensions with suramin (300 ␮mol/L) for
20 minutes, we found that ATP only increased NO production
by 37⫾5 AFU (P⬍0.002 versus control; n⫽5), a decrease of
65% (Figure 3). These data indicate that ATP-induced NOS
activation is primarily mediated by P2 receptors.
To investigate which P2 receptor mediates ATP-stimulated
NO production, we first tested whether ATP was undergoing
hydrolysis, because the P2Y–selective agonist UTP could also
be hydrolyzed. We measured NO production induced by ATP
concentrations of 0, 0.5, 1, 10, and 50 ␮mol/L in the presence of
a selective 5⬘-ectonucleotidase inhibitor, 6-N,N-diethyl-␤-␥dibromomethylene- D -adenosine-5-triphosphate (ARL67156; Sigma-Aldrich; 30 ␮mol/L). When
hydrolysis was blocked, saturation was reached at ⬇5 ␮mol/L
with an EC50 of 0.8 ␮mol/L (Figure 4). The EC50 value obtained
was ⬎40 times more sensitive than the EC50 value observed
during hydrolysis (Figure 1), indicating that ATP is hydrolyzed
by 5⬘-ectonucleotidases in the extracellular space of the thick
ascending limb. The maximal responses obtained between both
concentration–response curves were not statistically different.
Because of this endogenous hydrolysis, we ran the remaining
experiments in the presence of the inhibitor ARL67156.
The kidney expresses both P2X and P2Y receptors.12,14,17,23
To determine which receptor mediates ATP-induced NO
production, in the presence of ARL67156 (30 ␮mol/L) we
examined the relative efficacy of a P2X-selective agonist
␤,␥-Me-ATP (Sigma-Aldrich) and a P2Y-selective agonist
ATP has been shown to stimulate NO production in several
cell types.18,24 We have reported previously that several
factors that stimulate NO production in endothelial cells also
enhance NO generation in thick ascending limbs.5–7 Consequently, we investigated whether ATP stimulates NO production by thick ascending limbs and which receptors are
involved. We found that ATP enhanced thick ascending limb
NO production in a concentration-dependent manner as
measured by the NO-sensitive dye DAF-2. The EC50 was
37 ␮mol/L, and saturation occurred at ⬇200 ␮mol/L. To
show that the change in fluorescence was because of activation of NOS by ATP, we added the NO synthase inhibitor
L-NAME, which blunted the effect of ATP on NO production
by 74%. These data indicate that ATP stimulates NOS
activity in thick ascending limbs.
In the thick ascending limb, NOS is activated by endothelin
1,5 ␣2-adrenergic receptor agonists,6 and luminal flow.7 Interestingly, these same stimuli have been demonstrated to
produce similar effects in other cells, including chondrocytes25 and vascular endothelial cells.26,27 Given that ATP
stimulates NOS in neurons19 and vascular endothelium,24,28 it
is not surprising that ATP would also stimulate NOS in the
thick ascending limb.
The actions of ATP are limited via hydrolysis by 5⬘ectonucleotidases.29 To determine which P2 receptor mediates the effects of ATP in the thick ascending limb, we first
investigated whether ATP was being hydrolyzed by 5⬘ectonucleotidases, because the selective P2Y agonist (UTP)
could also be hydrolyzed by these enzymes. For this, we
generated a second concentration–response curve in the presence
of the 5⬘-ectonucleotidase inhibitor ARL67156. We found that
the ATP concentration–response curve was shifted to lower
concentrations of the agonist. When 5⬘-ectonucleotidases were
inhibited, the EC50 was 0.8 ␮mol/L, and saturation occurred
at ⬇5 ␮mol/L. These data indicate that ATP is hydrolyzed by
Figure 5. (A) Calculated EC50 for the
P2Y–selective agonist UTP and the P2Xselective agonist ␤,␥-Me-ATP on NO
production. (B) Calculated maximal
responses for UTP and ␤,␥-Me-ATP on
NO production. 95% CIs are shown.
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5⬘-ectonucleotidases in the plasma membrane of thick ascending limbs.
ATP usually acts by binding to P2 receptors. This family of
receptors is composed of both P2X and P2Y receptors.11 To
study whether P2 receptors are responsible for NOS activation in the thick ascending limb, we measured the effect of
ATP on intracellular NO production in the presence of the P2
receptor antagonist suramin and observed a 65% decrease in
NO production. These data indicate that extracellular ATP
stimulates the P2 receptors expressed by the thick ascending
limb and mediates NOS activation.
Both P2X and P2Y receptors are expressed in the thick
ascending limb.12,20 Pharmacological characterization of the P2
receptors involved in NO production is possible because there
are selective agonists for both receptors. UTP has been shown to
be a selective P2Y receptor agonist.30 On the other hand,
␤,␥-Me-ATP has been shown to be highly selective for P2X
receptors.31,32 The dissociation constant (KD) of ␤,␥-Me-ATP for
P2X receptors is ⬇10 ␮mol/L.31,33 In contrast the KD of UTP for
these receptors is ⱖ10-fold greater. However, UTP has a KD of
⬇3 ␮mol/L for P2Y receptors.34 –36 Our data show that the EC50
for ␤,␥-Me-ATP in stimulating NO production is ⬇5 ␮mol/L,
whereas that for UTP is ⬎40 ␮mol/L. The maximum response
caused by both agents was similar. These data indicate that ATP
enhances NO production primarily by activating P2X receptors.
ATP regulates epithelial transport in the lung,14 gastrointestinal tract,37 and kidney.14,15 We found that ATP acting
via the P2X receptor stimulates NO production in the thick
ascending limb. Because we reported previously that NO
inhibits NaCl absorption in this segment,3,5,38,39 ATP may
also inhibit NaCl absorption in the thick ascending limb. This
hypothesis is supported by data showing that in MDCK cells,
ATP inhibits Na⫹/K⫹/Cl⫺ cotransport.40
Although the effect of ATP on NaCl absorption has not
been investigated in the medullary thick ascending limb, it
has been studied in other nephron segments. In vivo microperfusion experiments showed that ATP inhibited bicarbonate reabsorption in the proximal tubule.15 These effects
may be mediated by NO, which likewise reduced bicarbonate
absorption in this segment.2 ATP also reduces transport in
collecting duct cells, inhibiting sodium absorption via activation of P2Y41– 43 or both P2X and P2Y receptors.44
In conclusion, we found that ATP acts as a paracrine factor
in the thick ascending limb of Henle’s loop, inducing NO
production via activation of P2X receptors. ATP is hydrolyzed in the plasma membrane by 5⬘-ectonucleotidases,
indirectly regulating the amount of NO produced in the thick
ascending limb. ATP may be an important regulator of NaCl
absorption by the thick ascending limb.
Perspectives
The effect of ATP on NO production is likely to reduce NaCl
absorption.3 The ability of ATP to stimulate NO production
may be significant when interstitial ATP concentrations are
elevated. This occurs during autoregulation of renal blood
flow45 and is at least partly attributable to release of ATP by
the macula densa during tubuloglomerular feedback.46 ATP
levels may also increase during the early stages of diabetes,
because glucose enhances ATP release by mesangial cells.47
Additionally, ATP may mediate cross-talk between the vasa
recta and thick ascending limbs, because endothelial cells
release ATP in response to many stimuli.8 –10 Finally, ATP
may be released from the renal nerves.48
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Hypertension. 2006;47:563-567; originally published online December 27, 2005;
doi: 10.1161/01.HYP.0000197954.93874.ef
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