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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

P2X Receptor-Stimulated Calcium Responses in Preglomerular Vascular Smooth Muscle Cells Involves 20-Hydroxyeicosatetraenoic Acid

Vascular Biology Center (X.Z., J.D.I.) and Department of Physiology, Medical College of Georgia, Augusta, Georgia (E.W.I., J.D.I.); and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F., V.R.G.)

Received for publication April 29, 2004
Accepted August 16, 2004.

	Abstract

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The current study tested the hypothesis that endogenous 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the increase in intracellular calcium ([Ca²⁺]_i) elicited by P2X receptor activation in renal microvascular smooth muscle cells. Vascular smooth muscle cells obtained from rats were loaded with fura-2 and studied using standard single cell fluorescence microscopy. Basal renal myocyte [Ca²⁺]_i averaged 96 ± 5 nM. ATP (10 and 100 µM) increased vascular smooth muscle cell [Ca²⁺]_i by 340 ± 88 and 555 ± 80 nM, respectively. The cytochrome P450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), significantly attenuated the peak myocyte [Ca²⁺]_i responses to 10 and 100 µM ATP. ATP (100 µM) increased vascular smooth muscle cell [Ca²⁺]_i by 372 ± 93 and 163 ± 55 nM in the presence of DDMS or 20-HEDE, respectively. The P2X receptor agonist, ,-methylene-ATP (10 µM), increased myocyte [Ca²⁺]_i by 78 ± 12 nM, and this response was significantly attenuated by DDMS (40 ± 15 nM). In contrast, the vascular smooth muscle cell [Ca²⁺]_i evoked by the P2Y agonist, UTP (100 µM), was not altered by DDMS or 20-HEDE. The effect of 20-HETE on [Ca²⁺]_i was also assessed, and the peak increases in [Ca²⁺]_i averaged 62 ± 12 and 146 ± 70 nM at 20-HETE concentrations of 1 and 10 µM, respectively. These results demonstrate that 20-HETE plays a significant role in the renal microvascular smooth muscle cell [Ca²⁺]_i response to P2X receptor activation.

20-Hydroxyeicosatetraenoic acid (20-HETE), a metabolite of the arachidonic acid cytochrome P450 (P450) pathway, plays an important role in the regulation of renal vascular and tubular function (Ma et al., 1993; Harder et al., 1994; Imig et al., 1996; Hercule and Oyekan, 2000). 20-HETE inhibits vascular smooth muscle potassium channels, resulting in membrane depolarization and subsequent activation of L-type Ca²⁺ channels, leading to vasoconstriction of the afferent arteriole (Lange et al., 1997; Gebremedhin et al., 1998; Imig et al., 1999, 2000). Interestingly, P2X receptor inactivation or P450 hydroxylase inhibition significantly attenuates pressure-mediated afferent arteriolar vasoconstrictor responses (Imig et al., 1999; Inscho et al., 2003). Our previous studies showed that either P450 inhibition or 20-HETE antagonism attenuated the initial vasoconstriction and abolished the sustained vasoconstriction evoked by the P2 receptor agonist ATP (Zhao et al., 2001). In addition, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) also attenuated the initial vasoconstrictor response and abolished the sustained vasoconstrictor response to the P2X receptor agonist, ,-methylene-ATP (Zhao et al., 2001). These data suggest that the P450 metabolite 20-HETE participates in the afferent arteriolar response to activation of P2X receptors. Based on these studies, we hypothesized that endogenous 20-HETE contributes to the increase in [Ca²⁺]_i elicited by P2X receptor activation in renal microvascular smooth muscle cells.

	Materials and Methods

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Tissue Preparation and Renal Microvascular Smooth Muscle Cell Isolation
All studies were performed in compliance with the guidelines and practices dictated by the Medical College of Georgia Advisory Committee for Animal Resources. Suspensions of preglomerular microvascular smooth muscle cells were prepared with a modification of the method previously described (White et al., 2001). Male Sprague-Dawley CD-VAF rats (250–375 g; Charles River Laboratories Inc., Wilmington, MA) were anesthetized with pentobarbital sodium (40 mg/kg i.p.), and the abdominal cavity was exposed to permit cannulation of the abdominal aorta via the superior mesenteric artery. Ligatures were placed around the abdominal aorta at sites proximal and distal to the left and right renal arteries, respectively. The kidneys were cleared of blood by perfusion of the isolated aortic segment with an ice-cold low-calcium physiological salt solution (PSS; pH 7.35) of the following composition: 125 mM NaCl, 5.0 mM KCl, 1.0 mM MgCl₂, 10.0 mM glucose, 20.0 mM HEPES, 0.1 mM CaCl₂, and 6% bovine serum albumin. After the kidneys were rinsed of blood, the perfusate was changed to a similar solution containing 1% Evans Blue in low-calcium PSS.

The kidneys were resected from the animal and decapsulated, and the renal medullary tissue was removed. The cortical tissue was pressed through a sieve (180 µm), and the sieve retentate was washed several times with ice-cold low-calcium PSS. The vascular tissue remaining on the sieve was transferred to an enzyme solution containing 0.075% collagenase (Roche Diagnostics, Mannheim, Germany), 0.02% dithiothreitol (Sigma-Aldrich, St. Louis, MO), and 0.1% bovine serum albumin dissolved in low-calcium PSS, and this mixture was incubated at 37°C for 30 min. The vascular tissue was removed from the enzyme solution and transferred to a nylon mesh (70 µm), where it was vigorously rinsed with ice-cold low-calcium PSS. The mesh containing the retained vascular tissue was transferred to a Petri dish containing ice-cold low-calcium PSS. Segments of interlobular artery with attached afferent arterioles were collected by microdissection using a stereoscope and transferred to a 10-ml dissociation flask. The rinse solution was decanted from the selected vascular segments and replaced with an enzyme solution containing 0.075% papain (Sigma-Aldrich) and 0.02% dithiothreitol (Sigma-Aldrich) in low-calcium PSS. The tissue was incubated at 37°C for 15 min before being collected by centrifugation (2000g for 50 s). The tissue pellet was transferred to an enzyme solution containing 0.3% collagenase (Roche Diagnostics) and 0.2% soybean trypsin inhibitor (type 1-S; Sigma-Aldrich) in low-calcium PSS at 37°C. After a 15-min incubation period, the mixture was gently triturated and quickly centrifuged (500g for 5 min) to collect the dispersed cells. The supernatant was discarded, and the cells were gently resuspended in 1.0 ml Dulbecco's minimum essential medium (Sigma-Aldrich), supplemented with 20% fetal calf serum (Whittaker Bioproducts, Walkersville, MD), and 100 U/ml penicillin and 200 µg/ml streptomycin (Sigma-Aldrich). Cell suspensions were stored on ice until use.

Fluorescence Measurements in Single Microvascular Smooth Muscle Cells
Experiments were performed using a standard microscope-based fluorescence spectrophotometry system (Photon Technology International, Monmouth Junction, NJ) as previously described (White et al., 2001). The excitation wavelengths were set at 340 and 380 nm, and the emitted light was collected at 510 ± 20 nm (Photon Technology International). Measurements of fluorescence intensity were collected at 5 data points per second and analyzed with the aid of the Photon Technology International software. Calibration of the fluorescence data were accomplished as previously described (White et al., 2001).

Measurement of [Ca²⁺]_i in single microvascular smooth muscle cells was performed as described previously (White et al., 2001). Suspensions of freshly isolated renal microvascular cells were loaded with the calcium-sensitive fluorescent probe, fura-2/acetoxymethyl ester (4.0 µM; Molecular Probes, Eugene, OR). An aliquot of cell suspension was transferred to the perfusion chamber (Warner Instruments, Hamden, CT) and mounted to the stage of a Nikon Diaphot inverted microscope. The cells were continuously superfused (1.3 ml/min) with a 1.8 mM calcium PSS solution of the following composition: 125 mM NaCl, 5.0 mM KCl, 1.0 mM MgCl₂, 10.0 mM glucose, 20.0 mM HEPES, 1.8 mM CaCl₂, and 0.111 g/l bovine serum albumin. For each experiment, a single microvascular cell was isolated in the optical field by positioning the adjustable sampling window directly over the cell of interest. Neighboring cells and debris are thus excluded from the sampling field, allowing fluorescence emission to be measured only from the background subtracted area, and a new coverslip of cells was used for each experiment.

Experimental Approach
Series 1. Experiments were performed to assess the role of 20-HETE in the microvascular smooth muscle cell response to ATP. For these experiments, cells were challenged with ATP while being bathed in a PSS solution containing the P450 hydroxylase inhibitor DDMS (Wang et al., 1998; Imig et al., 1999), or the 20-HETE antagonist 20-HEDE (Alonso-Galicia et al., 1999; Zhao et al., 2001). Fura-2 fluorescence was monitored in these cells under control conditions (0–100 s), during exposure to 25 µM DDMS in the presence of 1.8 mM Ca²⁺ (100–150 s), during subsequent exposure to ATP (10 or 100 µM) in combination with DDMS and 1.8 mM Ca²⁺ (150–350 s), and during the recovery period, during which ATP and DDMS were removed from the bathing solution (350–600 s). Identical studies were performed using 3 µM 20-HEDE instead of DDMS. These responses were compared with responses obtained from similar cells challenged in normal calcium PSS without P450 hydroxylase inhibition or 20-HETE antagonism.

Series 2. Studies were performed to further determine the effect of P450 hydroxylase inhibition and 20-HETE antagonism on the increase in [Ca²⁺]_i induced by the P2X receptor agonist, ,-methylene-ATP. Fura-2 fluorescence was monitored in these cells under control conditions (0–100 s), during exposure to DDMS or 20-HEDE in the presence of 1.8 mM Ca²⁺ (100–150 s), during subsequent exposure to ,-methylene-ATP (10 µM) in combination with DDMS or 20-HEDE and 1.8 mM Ca²⁺ (150–350 s), and during the recovery period (350–600 s). These responses were compared with responses obtained from similar cells challenged with ,-methylene-ATP alone.

Series 3. Experiments were performed to assess the role of 20-HETE in the renal myocyte response to the P2Y receptor agonist UTP. Fura-2 fluorescence was monitored in these cells under control conditions (0–100 s), during exposure to DDMS or 20-HEDE in the presence of 1.8 mM Ca²⁺ (100–150 s), during subsequent exposure to UTP (100 µM) in combination with DDMS or 20-HEDE and 1.8 mM Ca²⁺ (150–350 s), and during the recovery period (350–600 s). These responses were compared with responses obtained from similar cells challenged with UTP alone.

Series 4. Additional experiments were performed to assess the direct effect of 20-HETE on [Ca²⁺]_i in renal microvascular smooth muscle cells. Fura-2 fluorescence was monitored in these cells under control conditions (0–150 s), during exposure to 20-HETE (1, 10 µM) in 1.8 mM Ca²⁺ (150–350 s), and during the recovery period (350–600 s).

Statistical Analysis
Data are presented as means ± S.E. Within-group comparisons of peak [Ca²⁺]_i with baseline [Ca²⁺]_i were analyzed using ANOVA for repeated measures. Differences between treated and untreated groups of cell [Ca²⁺]_i values were analyzed by ANOVA followed by Newman-Keuls multiple range test. Statistical probabilities of <0.05 (p < 0.05) are considered significantly different.

	Results

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P450 Hydroxylase Inhibition and 20-HETE Antagonism on Renal Microvascular Smooth Muscle Cell [Ca²⁺]_i Response to ATP. To test the hypothesis that 20-HETE may be involved in the microvascular smooth muscle cell response to ATP, we investigated the effect of DDMS and 20-HEDE on the [Ca²⁺]_i response evoked by ATP. Figures 1 and 2 present representative traces depicting the changes in [Ca²⁺]_i elicited by ATP (10 and 100 µM) in the presence or absence of DDMS or 20-HEDE. Consistent with previous data, exposure to ATP evoked a rapid peak response followed by a steady-state plateau. DDMS (25 µM) or 20-HEDE (3 µM) significantly attenuated the peak renal myocyte [Ca²⁺]_i responses to 10 and 100 µM ATP. Figure 3 presents the average responses in the series 1 experiment. Baseline was similar among all these groups and basal microvascular smooth muscle cell [Ca²⁺]_i averaged 96 ± 5 nM (n = 70). ATP (10 and 100 µM) increased renal myocyte [Ca²⁺]_i by 340 ± 88 and 555 ± 80 nM, respectively. The administration of 25 µM DDMS or 3 µM 20-HEDE had no effect on baseline [Ca²⁺]_i but significantly attenuated the peak renal myocyte [Ca²⁺]_i responses to 10 and 100 µM ATP. In the presence of DDMS, the peak renal myocyte [Ca²⁺]_i responses to 10 and 100 µM ATP were 172 ± 77 and 372 ± 93 nM. In addition, 20-HEDE decreased the peak renal myocyte [Ca²⁺]_i responses to 10 and 100 µM ATP to 131 ± 44 and 163 ± 55 nM. These data demonstrate that P450 hydroxylase inhibition and 20-HETE antagonism attenuated the initial [Ca²⁺]_i response to P2 receptor activation.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Response of intracellular Ca2+ to 10 µM ATP during P450 hydroxylase inhibition with DDMS or 20-HETE antagonism with 20-HEDE. A, typical response of a renal microvascular smooth muscle cell to 10 µM ATP in the presence and absence of DDMS (25 µM). B, typical response of cell to 10 µM ATP in the presence and absence of 20-HEDE (3 µM). Solid horizontal bar represents the period of exposure to ATP.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Response of intracellular Ca2+ to 100 µM ATP in the presence of DDMS or 20-HEDE. A, typical response of a microvascular smooth muscle cell to 100 µM ATP in the presence and absence of DDMS (25 µM). B, typical response of a cell to 100 µM ATP in the presence and absence of 20-HEDE (3 µM). Solid horizontal bar represents the period of exposure to ATP.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Effect of DDMS and 20-HEDE on the intracellular Ca2+ concentration response to 10 and 100 µM ATP. ATP was administered [10 µM (A) or 100 µM (B)] in the absence or presence of 25 µM DDMS or 3 µM 20-HEDE. Peak changes in [Ca2+]i were determined in the first 150 s of agonist exposure. Data present average changes in [Ca2+]i obtained from 15 to 21 cells from three to nine tissue dissociations for each group. *, significant decrease in [Ca2+]i compared with respective ATP alone.

P450 Hydroxylase Inhibition and 20-HETE Antagonism on Renal Microvascular Smooth Muscle Cell [Ca²⁺]_i Response to P2X Receptor Activation. The second series of experiments were performed using the P2X receptor-selective ATP analog ,-methylene-ATP to further examine the contribution of 20-HETE to P2 receptor-mediated calcium signaling, and the results of those studies were presented in Figs. 4 and 5. Figure 4 shows typical traces depicting the changes in [Ca²⁺]_i evoked by ,-methylene-ATP in the presence or absence of DDMS or 20-HEDE. Exposure of microvascular smooth muscle cells to 10 µM ,-methylene-ATP evoked an increase in [Ca²⁺]_i that typically included a rapid peak response followed by a gradual return to steady-state level similar to baseline. DDMS significantly attenuated the renal myocyte [Ca²⁺]_i response to ,-methylene-ATP. Figure 5 presents the average responses in these experiments. ,-Methylene-ATP (10 µM) increased vascular smooth muscle cell [Ca²⁺]_i by 80 ± 12 nM (n = 18 cells). DDMS significantly reduced this response to 40 ± 15 nM (n = 19 cells, p < 0.05); however, 20-HEDE decreased this response by only 13%.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Response of intracellular Ca2+ to ,-methylene-ATP during P450 hydroxylase inhibition with DDMS (A) or 20-HETE antagonism with 20-HEDE (B). The typical responses of microvascular smooth muscle cells to 10 µM ,-methylene-ATP in the presence and absence of 25 µM DDMS or 3 µM 20-HEDE is shown. Solid horizontal bars represent the period of exposure to 10 µM ,-methylene-ATP.

View larger version (12K): [in this window] [in a new window]   Fig. 5. Effect of DDMS and 20-HEDE on the intracellular Ca2+ concentration response to ,-methylene-ATP. ,-Methylene-ATP (10 µM) was administered in the presence or absence of 25 µM DDMS. Peak changes in [Ca2+]i were determined in the first 150 s of agonist exposure. Data present average changes in [Ca2+]i obtained from a minimum of 19 cells from five tissue dissociations for each group. Values are mean ± S.E. *, significant difference from control ,-methylene-ATP response.

P450 Hydroxylase Inhibition and 20-HETE Antagonism on Renal Microvascular Smooth Muscle Cell [Ca²⁺]_i Response to P2Y Receptor Activation. The P2Y receptor agonist UTP was used to assess the role of 20-HETE in the renal myocyte [Ca²⁺]_i response to P2Y receptor activation. The results of series 3 experiments are presented in Figs. 6 and 7. UTP (100 µM) caused a rapid increase in [Ca²⁺]_i that reached a peak (822 ± 170 nM, n = 18 cells), followed by a gradual recovery to a steady-state [Ca²⁺]_i (65 ± 19 nM, n = 18 cells) that is significantly greater than baseline (p < 0.05). Microvascular smooth muscle cell [Ca²⁺]_i responses evoked by UTP were not altered by DDMS or 20-HEDE. The peak increases in [Ca²⁺]_i elicited by UTP were 691 ± 172 (n = 15 cells) and 964 ± 156 nM (n = 16 cells) in the presence of DDMS or 20-HEDE (Fig. 7), respectively. These data suggest that 20-HETE is not involved in the P2Y receptor-mediated renal microvascular smooth muscle cell calcium signaling.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Response of intracellular Ca2+ to UTP during P450 hydroxylase inhibition with DDMS or 20-HETE antagonism with 20-HEDE. The typical responses of microvascular smooth muscle cells to 100 µM UTP in the presence and absence of 25 µM DDMS (A) or 3 µM 20-HEDE (B). Solid horizontal bars represent the period of exposure to 100 µM UTP.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Effect of DDMS and 20-HEDE on the intracellular Ca2+ concentration response to UTP. UTP (100 µM) was administered in the presence of 25 µM DDMS or 3 µM 20-HEDE. Peak changes in [Ca2+]i were determined in the first 150 s of agonist exposure. Data present average changes in [Ca2+]i obtained from a minimum of 19 cells from five tissue dissociations for each group. Values are mean ± S.E.

Renal Microvascular Smooth Muscle Cell [Ca²⁺]_i Response to 20-HETE. Figure 8 presents a representative trace depicting the change in [Ca²⁺]_i elicited by 10 µM 20-HETE. Exposure of renal myocytes to 20-HETE evoked an increase in [Ca²⁺]_i. The peak [Ca²⁺]_i elicited by 20-HETE (1 and 10 µM) averaged 62 ± 12 and 146 ± 70 nM, respectively and was significantly different from their respective baseline calcium concentrations.

View larger version (15K): [in this window] [in a new window]   Fig. 8. Effect of 20-HETE on the intracellular calcium concentration in renal microvascular smooth muscle cells. Characteristic response to 10 µM 20-HETE is shown.

	Discussion

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The current studies were performed to determine whether P450 hydroxylase metabolite, 20-HETE, is involved in the [Ca²⁺]_i response in the renal microvascular smooth muscle cells to purinoceptor activation. Consistent with our previous studies (Inscho et al., 1999a; White et al., 2001), ATP and UTP elicited a biphasic response including a peak response followed by a steady-state plateau, whereas ,-methylene-ATP evoked a transient increase in [Ca²⁺]_i that quickly returned to baseline. The P450 hydroxylase inhibitor DDMS and the 20-HETE antagonist 20-HEDE had no effect on baseline renal microvascular smooth muscle cell [Ca²⁺]_i; however, the addition of DDMS or 20-HEDE significantly attenuated the initial [Ca²⁺]_i responses evoked by 10 and 100 µM ATP. In addition, DDMS also markedly reduced the [Ca²⁺]_i response induced by the P2X receptor agonist ,-methylene-ATP. P450 hydroxylase inhibition and 20-HETE antagonism did not alter the renal myocyte [Ca²⁺]_i responses to the P2Y receptor agonist UTP. These data demonstrate that 20-HETE plays a role in the [Ca²⁺]_i response evoked by P2X receptor activation.

Renal hemodynamic control is accomplished by local adjustments in intrarenal vascular resistance. The majority of these resistance adjustments is preglomerular and occurs at the level of the afferent arterioles (Arendshorst and Navar, 1993). Myogenic and tubuloglomerular feedback-mediated adjustments in preglomerular resistance are the major contributors to renal blood flow autoregulation. Tubuloglomerular feedback is believed to be a major regulatory system, coupling changes in distal tubular flow with preglomerular resistance through the actions of the macula densa. ATP, released from the macula densa, serves as the chemical messenger linking the macula densa with regulation of afferent arteriolar resistance. Regulation of afferent arteriolar resistance involves ATP-dependent activation of P2X receptors that are heavily expressed along the preglomerular but not the postglomerular microvasculature (Mitchell and Navar, 1993; Inscho et al., 1996; Navar et al., 1996; Chan et al., 1998; Inscho, 2001; Bell et al., 2003). Inactivation of P2 receptors on preglomerular microvessels inhibits autoregulatory behavior (Inscho et al., 1996; Majid et al., 1999). More recent data suggest a specific role for P2X₁ receptors in the afferent arteriolar autoregulatory response. Pressure-dependent afferent arteriolar diameter responses were abolished by pharmacological blockade of P2X receptors in rats and deletion of P2X₁ receptors in mice (Inscho et al., 2003). Taken together, these studies strongly support the postulate that P2X₁ receptor activation plays a critical role in mediating afferent arteriolar autoregulatory adjustments.

The cellular signaling mechanisms responsible for P2 receptor vasoconstriction are not well defined. ATP-mediated afferent arteriolar vasoconstriction is largely dependent on the influx of extracellular Ca²⁺ and the sustained vasoconstriction is maintained by Ca²⁺ influx through voltage-dependent L-type Ca²⁺ channels (Inscho et al., 1995, 1999a; Navar et al., 1996; Inscho and Cook, 2002). P2 receptor activation results in the release of arachidonic acid from membrane phospholipids in glomerular mesangial cells and rat astrocytes (Pfeilschifter, 1990; Schulze-Lohoff et al.,1992; Bolego et al., 1997). Our previous studies showed that P450 inhibition or 20-HETE antagonism significantly attenuated both the initial and sustained afferent arteriolar constrictor responsiveness to ATP (Zhao et al., 2001). These data demonstrate that the P450 metabolite 20-HETE participates in the afferent arteriolar response to P2 receptor activation. To determine whether 20-HETE contributes to the afferent arteriolar response to P2 receptor activation by influencing the microvascular smooth muscle cell calcium signaling, we further investigated the effect of DDMS or 20-HEDE on the renal myocyte [Ca²⁺]_i response to ATP. Our studies showed that both DDMS and 20-HEDE significantly attenuated the initial [Ca²⁺]_i responses to ATP. These data suggest that 20-HETE is involved in the renal microvascular smooth muscle cell [Ca²⁺]_i response to P2 receptor activation.

P2 receptors were first defined by Burnstock in 1978 (Burnstock, 1978). Since then, P2 receptors have grown into a large family of receptors divided into two major categories classified as P2X and P2Y (Abbracchio and Burnstock, 1994; Fredholm et al., 1994, 1997; Ralevic and Burnstock, 1998). P2X receptors are described as having two membrane-spanning domains and function as ligand-gated channels (Abbracchio and Burnstock, 1994; Ralevic and Burnstock, 1998). P2Y receptors have seven membrane-spanning domains and function as G protein-coupled receptors (Abbracchio and Burnstock, 1994; Ralevic and Burnstock, 1998). Previous studies have shown that renal microvascular smooth muscle cell [Ca²⁺]_i responses to P2 receptor stimulation with ATP involve the activation of both P2X and P2Y receptor subtypes (Inscho et al., 1999a; White et al., 2001). Each receptor type activates different calcium signaling pathways. Exposure of renal myocytes to the P2X-selective agonist ,-methylene-ATP results in an elevation of [Ca²⁺]_i through activation of calcium influx pathways (White et al., 2001). The magnitude and time course of the response to ,-methylene-ATP are markedly different from those evoked by an equimolar concentration of ATP (White et al., 2001). The average peak response elicited by 10 µM ,-methylene-ATP was 34% of the response obtained with 10 µM ATP. The response to ,-methylene-ATP was transient whereas the response to ATP exhibited a sustained elevation of [Ca²⁺]_i. In the current study, DDMS markedly reduced the [Ca²⁺]_i response to the P2X receptor agonist ,-methylene-ATP. The inability of 20-HEDE to significantly decrease the renal microvascular smooth muscle cell Ca²⁺ response to ,-methylene-ATP is perplexing. These data are not consistent with the afferent arteriolar diameter responses to ATP and ,-methylene-ATP or the renal myocyte Ca²⁺ response to ATP in the presence of 20-HEDE. Even though there was a slight trend for a decreased ,-methylene-ATP response in cells treated with 20-HEDE, we have no real compelling explanation for the lack of a 20-HEDE effect in this experimental setting. Overall, the majority of afferent arteriolar and renal myocyte experiments would suggest that endogenous 20-HETE may contribute to the P2X receptor-mediated afferent arteriolar vasoconstriction by influencing calcium influx in preglomerular microvascular smooth muscle cells.

P2Y receptor activation stimulates vascular smooth muscle cell [Ca²⁺]_i in a strikingly different way. The P2Y receptor agonist UTP is purported to interact primarily with G protein-regulated P2Y receptors and is reported to activate phospholipase C (Dubyak and el-Moatassim, 1993; Abbracchio and Burnstock, 1994; Conigrave and Jiang, 1995). Previous studies showed that the mechanisms by which UTP and ATP elevate [Ca²⁺]_i seem to be substantially different although they stimulate similar increases in [Ca²⁺]_i overall (Inscho et al., 1999a; Inscho and Cook, 2002). ATP used both calcium influx and calcium mobilization, whereas the response to UTP seems to arise almost exclusively from the release of calcium from intracellular stores. Removal of calcium from the extracellular medium or blockade of calcium influx through L-type calcium channels had no perceptible effect on the magnitude or time course of UTP-mediated increases in [Ca²⁺]_i. This suggests that binding of UTP to its receptor stimulates a signal transduction cascade designed to access stored calcium. On the basis of findings generated by other investigators, UTP-mediated activation of the phospholipase C/inositol trisphosphate/diacylglycerol pathway represents the most likely signal transduction mechanism. In our current study, stimulation of the cells with UTP induced a rapid increase in intracellular Ca²⁺ levels, followed by a slow decrease to basal levels or cytoplasmic Ca²⁺ oscillation. P450 hydroxylase inhibition and 20-HETE antagonism did not alter the initial microvascular smooth muscle cell [Ca²⁺]_i response to UTP. Whether P450 hydroxylase inhibition or 20-HETE antagonism affects the oscillatory response is still not clear. Taken together, these data suggest that 20-HETE is not involved in the P2Y receptor-activated calcium signaling pathway, which is consistent with our previous finding that 20-HETE is not involved in the P2Y receptor-mediated preglomerular vasoconstriction.

Previous studies have implicated the arachidonic acid metabolite 20-HETE as one paracrine factor mediating tubuloglomerular feedback and autoregulatory related signals to the afferent arterioles. 20-HETE is produced by renal vascular smooth muscle cells and is a potent constrictor that depolarizes vascular smooth muscle cells by blocking calcium-activated potassium channels (Imig et al., 1996; Maier and Roman, 2001). Inhibition of 20-HETE formation blocks the myogenic response of isolated renal arterioles in vitro (Maier and Roman, 2001). In our experiments, we observed the influence of DDMS and 20-HEDE on the renal microvascular smooth muscle cell Ca²⁺ response to ATP and found that DDMS or 20-HEDE significantly decreased the peak [Ca²⁺]_i response. Interestingly, P2 receptor activation liberates arachidonic acid from membrane phospholipids (Pfeilschifter, 1990), which would provide the substrate for P450 hydroxylase enzymes and 20-HETE production. Additional experiments demonstrated that renal microvascular smooth muscle cell Ca²⁺ levels increased in response to 20-HETE. The Ca²⁺ response to 20-HETE was rapid in onset and had a time course and magnitude similar to that of P2X receptor activation. 20-HETE has been previously shown to increase intracellular Ca²⁺ levels in smooth muscle cells from canine renal arteries (Ma et al., 1993). Our laboratory has also demonstrated that the afferent arteriolar constrictor response to 20-HETE involves inactivation of potassium channels that results in smooth muscle cell depolarization and Ca²⁺ channel activation (Imig et al., 1996). Taken together, these results suggest that P450 hydroxylase metabolite 20-HETE is produced by renal microvascular smooth muscle cells and contributes to the renal preglomerular microvascular response to ATP.

In summary, both DDMS and 20-HEDE attenuated the initial [Ca²⁺]_i response evoked by the P2 receptor agonist ATP. In addition, DDMS also attenuated the renal myocyte [Ca²⁺]_i response to the P2X receptor agonist, ,-methylene-ATP. In contrast, DDMS and 20-HEDE had no effect on [Ca²⁺]_i to the P2Y receptor agonist UTP. These data demonstrate that the P450 metabolite 20-HETE is involved in the renal microvascular smooth muscle cell calcium response to P2X receptor activation.

	Acknowledgements

 
We thank Benjamin C. Hauschild and Thuy T. Kim for excellent technical assistance with these experiments.

	Footnotes

 
This work was supported by National Institutes of Health Grants (HL-59699, DK-38226, and DK-44628), Robert A. Welch Foundation, and American Heart Association. John D. Imig is an Established Investigator of the American Heart Association. Xueying Zhao is the recipient of an American Heart Association Southeast Affiliate Postdoctoral Fellowship.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.070797.

ABBREVIATIONS: [Ca²⁺]_i, intracellular calcium; P450, cytochrome P450; HETE, hydroxyeicosatetraenoic acid; DDMS, N-methylsulfonyl-12,12-dibromododec-11-enamide; HEDE, hydroxyeicosa-6(Z),15(Z)-dienoic acid; PSS, physiological salt solution; ANOVA, analysis of variance.

Address correspondence to: Dr. John D. Imig, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2500. E-mail: jdimig{at}mail.mcg.edu

	References

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Abbracchio MP and Burnstock G (1994) Purinoceptors: are there families of P_2X and P_2Y purinoceptors? Pharmacol Ther 64: 445-475.[CrossRef][Medline]
Alonso-Galicia M, Falck JR, Reddy KM, and Roman RJ (1999) 20-HETE agonists and antagonists in the renal circulation. Am J Physiol 277: F790-F796.
Arendshorst WJ and Navar LG (1993) Renal circulation and glomerular hemodynamics, in Diseases of the Kidney (Schrier RW and Gottschalk C eds) pp 65-117, Little, Brown and Company, Boston, MA.
Bell PD, Lapointe JY, Sabirov R, Hayashi S, Peti-Peterdi J, Manabe K, Kovacs G, and Okada Y (2003) Macula densa cell signaling involves ATP release through a maxi anion channel. Proc Natl Acad Sci USA 100: 4322-4327.[Abstract/Free Full Text]
Bolego C, Ceruti S, Brambilla R, Puglisi L, Cattabeni F, Burnstock G, and Abbracchio MP (1997) Characterization of the signalling pathways involved in ATP and basic fibroblast growth factor-induced astrogliosis. Br J Pharmacol 121: 1692-1699.[CrossRef][Medline]
Burnstock G (1978) A basis for distinguishing two types of purinergic receptor, in Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach (Bolis L and Straub RCO eds) pp 107-118, Raven Press Ltd., New York.
Chan CM, Unwin RJ, Bardini M, Oglesby IB, Ford AP, Townsend-Nicholson A, and Burnstock G (1998) Localization of P2X₁ purinoceptors by autoradiography and immunohistochemistry in rat kidneys. Am J Physiol 274: F799-F804.
Conigrave AD and Jiang L (1995) Review: Ca²⁺-mobilizing receptors for ATP and UTP. Cell Calcium 17: 111-119.[CrossRef][Medline]
Dubyak GR and el-Moatassim C (1993) Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol Cell Physiol 265: C577-C606.[Abstract/Free Full Text]
el-Moatassim C, Dornand J, and Mani JC (1992) Extracellular ATP and cell signaling. Biochim Biophys Acta 1134: 31-45.[Medline]
Eltze M and Ullrich B (1996) Characterization of vascular P2 purinoceptors in the rat isolated perfused kidney. Eur J Pharmacol 306: 139-152.[CrossRef][Medline]
Evans RJ, Surprenant A, and North RA (1998) P2X receptors: Cloned and expressed, in The P2 Nucleotide Receptors (Turner JT, Weisman GA, and Fedan JS eds) pp 43-61, Humana Press, Inc., Totowa, NJ.
Fredholm BB, Abbracchio MP, Burnstock G, Daly JW, Harden TK, Jacobson KA, Leff P, and Williams M (1994) Nomenclature and classification of purinoceptors. Pharmacol Rev 46: 143-156.[Medline]
Fredholm BB, Abbracchio MP, Burnstock G, Dubyak GR, Harden TK, Jacobson KA, Schwabe U, and Williams M (1997) Towards a revised nomenclature for P1 and P2 receptors. Trends Pharmacol Sci 18: 79-82.[Medline]
Gebremedhin D, Lange AR, Narayanan J, Aebly MR, Jacobs ER, and Harder DR (1998) Cat cerebral arterial smooth muscle cells express cytochrome P450 4A2 enzyme and produce the vasoconstrictor 20-HETE which enhances L-type Ca²⁺ current. J Physiol (Lond) 507: 771-781.[Abstract/Free Full Text]
Harder DR, Gebremedhin D, Narayanan J, Jefcoat C, Falck JR, Campbell WB, and Roman RJ (1994) Formation and action of a P-450 4A metabolite of arachidonic acid in cat cerebral microvessels. Am J Physiol Heart Circ Physiol 266: H2098-H2107.[Abstract/Free Full Text]
Hercule HC and Oyekan AO (2000) Cytochrome P450 /-1 hydroxylase-derived eicosanoids contribute to endothelin (A) and endothelin (B) receptor-mediated vasoconstriction to endothelin-1 in the rat preglomerular arteriole. J Pharmacol Exp Ther 292: 1153-1160.[Abstract/Free Full Text]
Imig JD, Falck JR, and Inscho EW (1999) Contribution of cytochrome P450 epoxygenase and hydroxylase pathways to afferent arteriolar autoregulatory responsiveness. Br J Pharmacol 127: 1399-1405.[CrossRef][Medline]
Imig JD, Pham BT, LeBlanc EA, Reddy KM, Falck JR, and Inscho EW (2000) Cytochrome P450 and cyclooxygenase metabolites contribute to the endothelin-1 afferent arteriolar vasoconstrictor and calcium responses. Hypertension 35: 307-312.[Abstract/Free Full Text]
Imig JD, Zou AP, Stec DE, Harder DR, Falck JR, and Roman RJ (1996) Formation and actions of 20-hydroxyeicosatertraenoic acid in rat renal arterioles. Am J Physiol Regul Integr Comp 270: R217-R227.
Inscho EW (2001) P2 receptors in regulation of renal microvascular function. Am J Physiol Renal Physiol 280: F927-F944.[Abstract/Free Full Text]
Inscho EW and Cook AK (2002) P2 receptor-mediated afferent arteriolar vasoconstriction during calcium blockade. Am J Physiol Renal Physiol 282: F245-F255.[Abstract/Free Full Text]
Inscho EW, Cook AK, Imig JD, Vial C, and Evans RJ (2003) Physiological role for P2X1 receptors in renal microvascular autoregulatory behavior. J Clin Investig 112: 1895-1905.[CrossRef][Medline]
Inscho EW, Cook AK, Mui V, and Miller J (1998) Direct assessment of renal microvascular responses to P2-purinoceptor agonists. Am J Physiol Renal Physiol 274: F718-F727.[Abstract/Free Full Text]
Inscho EW, Cook AK, and Navar LG (1996) Pressure-mediated vasoconstriction of juxtamedullary afferent arterioles involves P2-purinoceptor activation. Am J Physiol Renal Fluid Electrolyte Physiol 271: F1077-F1085.[Abstract/Free Full Text]
Inscho EW, LeBlanc EA, Pham BT, White SM, and Imig JD (1999a) Purinoceptor-mediated calcium signaling in preglomerular smooth muscle cells. Hypertension 33: 195-200.[Abstract/Free Full Text]
Inscho EW, Ohishi K, Cook AK, Belott TP, and Navar LG (1995) Calcium activation mechanisms in the renal microvascular response to extracellular ATP. Am J Physiol Renal Fluid Electrolyte Physiol 268: F876-F884.[Abstract/Free Full Text]
Inscho EW, Schroeder AC, Deichmann PC, and Imig JD (1999b) ATP-mediated Ca²⁺ signaling in preglomerular smooth muscle cells. Am J Physiol Renal Physiol 276: F450-F456.[Abstract/Free Full Text]
Lange A, Gebremedhin D, Narayanan J, and Harder D (1997) 20-hydroxyeicosatetraenoic acid-induced vasoconstriction and inhibition of potassium current in cerebral vascular smooth muscle is dependent on activation of protein kinase C. J Biol Chem 272: 27345-27352.[Abstract/Free Full Text]
Ma YH, Gebremedhin D, Schwartzman ML, Falck JR, Clark JE, Masters BS, Harder DR, and Roman RJ (1993) 20-Hydroxyeicosatetraenoic acid is an endogenous vasoconstrictor of canine renal arcuate arteries. Circ Res 72: 126-136.[Abstract/Free Full Text]
Maier KG and Roman RJ (2001) Cytochrome P450 metabolites of arachidonic acid in the control of renal function. Curr Opin Nephrol Hypertens 10: 81-87.[Medline]
Majid DS, Inscho EW, and Navar LG (1999) P2 purinoceptor saturation by adenosine triphosphate impairs renal autoregulation in dogs. J Am Soc Nephrol 10: 492-498.[Abstract/Free Full Text]
Mitchell KD and Navar LG (1993) Modulation of tubuloglomerular feedback responsiveness by extracellular ATP. Am J Physiol Renal Fluid Electrolyte Physiol 264: F458-F466.[Abstract/Free Full Text]
Navar LG, Inscho EW, Majid SA, Imig JD, Harrison-Bernard LM, and Mitchell KD (1996) Paracrine regulation of the renal microcirculation. Physiol Rev 76: 425-536.[Abstract/Free Full Text]
Pfeilschifter J (1990) Extracellular ATP stimulates polyphosphoinositide hydrolysis and prostaglandin synthesis in rat renal mesangial cells. Involvement of a pertussis toxin-sensitive guanine nucleotide binding protein and feedback inhibition by protein kinase C. Cell Signal 2: 129-138.[CrossRef][Medline]
Ralevic V and Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50: 413-492.[Abstract/Free Full Text]
Schulze-Lohoff E, Zanner S, Ogilvie A, and Sterzel RB (1992) Extracellular ATP stimulates proliferation of cultured mesangial cells via P2-purinergic receptors. Am J Physiol Renal Fluid Electrolyte Physiol 263: F374-F383.[Abstract/Free Full Text]
Wang MH, Brand-Schieber E, Zand BA, Nguyen X, Falck JR, Balu N, and Schwartzman ML (1998) Cytochrome P450-derived arachidonic acid metabolism in the rat kidney: characterization of selective inhibitors. J Pharmacol Exp Ther 284: 966-973.[Abstract/Free Full Text]
White SM, Imig JD, Kim TT, Hauschild BC, and Inscho EW (2001) Calcium signaling pathways utilized by P2X receptors in freshly isolated preglomerular MVSMC. Am J Physiol Renal Physiol 280: F1054-F1061.[Abstract/Free Full Text]
Zhao X, Inscho EW, Bondlela M, Falck JR, and Imig JD (2001) The CYP450 hydroxylase pathway contributes to P2X receptor-mediated afferent arteriolar vasoconstriction. Am J Physiol Heart Circ Physiol 281: H2089-H2096.[Abstract/Free Full Text]

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