Departments of 1Internal Medicine, 6Physiology, and 3Neurobiology and Anatomy, University of Utah Health Sciences Center, Salt Lake City 84132; 2Nephrology Research and 5Geriatric Research, Education, and Clinical Center, Veterans Affairs Salt Lake City Health Care System, Salt Lake City, Utah 84148; and 5Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
Submitted 15 March 2003 ; accepted in final form 15 May 2003
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ABSTRACT |
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cyclooxygenases; arachidonic acid; extracellular nucleotides; arginine vasopressin; purinergic receptor
Extracellular nucleotides bind to specific subtypes of P2 purinergic receptors on cell membranes and elicit a wide variety of biological responses in several tissues. P2 receptors are classified into two families: the ion-tropic P2X and the metabotropic P2Y receptors. The former are extracellular nucleotide-activated membrane channels that allow a variety of ions and/or small molecules to enter the cells (31, 32). The P2Y receptors are G protein-coupled receptors that mostly act through the phosphoinositide signaling pathway (10, 42). Recent experimental studies have unraveled the role of extracellular nucleotides and autocrine and/or paracrine purinergic signaling in the regulation of glomerular, microvascular, and epithelial functions of the kidney in health and disease (3, 19, 27, 37, 38).
Using a pharmacological approach of measuring the agonist-stimulated rise in intracellular calcium responses, Ecelbarger et al. (12) identified and characterized the presence of the P2Y2 (previously known as P2u) receptor in rat IMCD. Kishore et al. (23) showed that extracellular nucleotides (ATP/UTP) inhibit the AVP-stimulated osmotic water permeability of in vitro microperfused rat IMCD, thus establishing a physiological role for the P2Y2 receptor in IMCD. Subsequently, Kishore et al. (24) demonstrated by molecular approaches that P2Y2 receptor mRNA and protein are expressed in rat IMCD. Immunocytochemical localization studies revealed the expression of P2Y2 receptor protein on apical and basal domains of collecting duct cells, as well as on thin limbs and vascular elements (24).
A rise in intracellular calcium, such as that induced by agonist stimulation of the P2Y2 receptor in the medullary collecting duct, is known to be frequently associated with enhanced arachidonic acid metabolism. It has been demonstrated that, in several types of tissues or cells (e.g., guinea pig ileum and uterus, bovine aortic smooth muscle cells, rabbit heart and tracheal epithelial cells, isolated rat dura mater, thymic epithelial cells and astrocytes, porcine or human endothelial cells, and mouse peritoneal macrophages), agonist activation of P2Y receptors resulted in stimulation of arachidonic acid metabolism and production of prostanoids. In most of these tissues or cells, especially those of nonendothelial nature, the predominant prostanoid produced was PGE2 (1, 2, 9, 11, 14, 16, 20, 28, 33, 34, 39, 44).
Experiments conducted on Madin-Darby canine kidney-D1 cells also revealed
that agonist stimulation of the P2Y2 receptor results in activation
of cytosolic phospholipase A2 (cPLA2) and release of
arachidonic acid in a protein kinase C- and MAPK-dependent fashion
(43). However, to the best of
our knowledge, no studies are available on the synthesis and release of
prostanoids by renal medullary collecting duct cells after purinergic
stimulation. Initial experiments conducted by Ecelbarger et al.
(12) demonstrated that when
terminal IMCD segments of rat were exposed to indomethacin, a nonspecific
inhibitor of cyclooxygenases (COX), for 5 min and then challenged with ATP,
the signals displayed a reduced intracellular calcium peak. This finding
suggests that, in rat IMCD, purinergic activation stimulates arachidonic acid
metabolism and enhanced production of COX products, which apparently
potentiate the intracellular calcium response. On the basis of these
observations, we hypothesized that COX products of arachidonic acid
metabolism, such as PGE2, are formed and released as a result of
agonist activation of the P2Y2 receptor in rat IMCD. To test this
hypothesis, we conducted studies on freshly prepared rat IMCD suspensions and
examined whether the basal and adensoine
5'-O-(3-thiotriphosphate) (ATPS)-stimulated release of
PGE2 was affected by nonspecific inhibitors of COX or COX-1- or
COX-2-specific inhibitors.
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MATERIALS AND METHODS |
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The animal experiments were conducted according to the protocol approved by
the Institutional Animal Care and Use Committee of the Veterans Administration
Salt Lake City Health Care System. Specific pathogen-free male Sprague-Dawley
rats (Harlan, Indianapolis, IN) were housed two or three per cage in the
Veterinary Medical Unit of the Veterans Administration Salt Lake City Health
Care System, which is an American Association for Accreditation of Laboratory
Animal Care-accredited and US Department of Agriculture-approved animal
facility. The rats were maintained in pathogen-free state and fed ad libitum a
commercial rodent diet and had free access to drinking water. The rats were
acclimated to the housing conditions for 5 days before the experiments
were conducted. The rats weighed 220400 g (mean 292 g) at the time of
euthanasia.
Agents
ATPS (95.7% purity) and arachidonyltrifluoromethyl ketone (AACOCF3,
97% purity) were purchased from Calbiochem-Novabiochem (La Jolla, CA), UTP
(97% purity) and DMSO (99.9% purity) from Sigma Chemical (St. Louis, MO),
2-acetoxyphenylhept-2-ynyl sulfide (APHS, 98% purity),
2-fluoro-
-methyl-(1,1'-biphenyl)-4-acetic acid (flurbiprofen, 99%
purity), 2-[(1-oxopentyl)oxy]-benzoic acid (valeroyl salicylate, 99% purity),
and N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398,
99% purity) from Cayman Chemical (Ann Arbor, MI), collagenase B from Roche
Molecular Biochemicals (Indianapolis, IN), and bovine testes hyaluronidase
from Worthington Biochemical (Lakewood, NJ). All other chemicals used were of
the highest purity available.
Preparation of Fractions Enriched in IMCD
Fractions enriched in IMCD were prepared from rat kidney inner medullae
essentially as described previously
(24,
26). Briefly, rats were
euthanized by pentobarbital sodium overdose, and both kidneys were removed
rapidly. The kidneys were chilled in ice-cold phosphate-buffered saline, and
the inner medullae or papillae were dissected on ice and transferred to an
isotonic HEPES-buffered physiological solution of the following composition
(in mM): 135 NaCl, 0.5 KCl, 0.1 Na2HPO4, 0.3 sodium
acetate, 0.12 NaSO4, 2.5 CaCl2, 1.2 MgSO4, 5
HEPES, and 5.5 D-glucose (pH 7.4, 311 ± 9
mosmol/kgH2O). This solution was oxygenated by bubbling in 95%
O2-5% CO2. For each experiment, depending on body weight
of the rats, renal papillae from 610 rats were pooled to obtain a
sufficient quantity of the IMCD preparation for 36 incubations. The pooled
papillae were minced with a razor blade and digested at 37°C with
collagenase B (3 mg/ml) and hyaluronidase (600 U/ml) in the same
HEPES-buffered physiological solution for 4050 min with continuous
oxygenation. Halfway through the digestion process, DNase I (GIBCO-BRL,
Gaithersburg, MD) was added to the digestion mixture to a final concentration
of 1 U/ml to digest stray DNA released from broken cells. The digestion
mixture was intermittently aspirated into and pushed through a glass Pasteur
pipette to disperse the tubules into a uniform suspension. After digestion of
the papillary tissue to a uniform suspension, the IMCD fraction was separated
from the non-IMCD elements (thin limbs and vasculature) by sedimentation by
low-speed centrifugation and repeated washings. The final pellet was suspended
in the oxygenated HEPES-buffered physiological solution to a protein
concentration of
1 mg/ml and kept on ice for
30 min to allow for
recovery of IMCD cells from stress. This preparation consisted of mostly IMCD
segments or sheets of IMCD cells and very few other non-IMCD elements.
Representative IMCD fractions were assessed for enrichment and viability by
immunoblotting for collecting duct-specific water channel protein aquaporin-2
(AQP2) and by binding of ethidium homodimer-1 to the cellular DNA of dead
cells, respectively.
Incubation of IMCD Preparations
Fractions enriched in IMCD were incubated with or without the addition of
inhibitors of COX and/or nucleotides (ATPS or UTP), and the amount of
PGE2 released from the cells was assayed. Briefly, the IMCD
suspensions prepared and kept on ice for 30 min for recovery from stress as
described above were aliquoted into 1.5-ml plastic microtubes kept on ice.
Nucleotide stock solutions (10x final incubation concentration) were
prepared in the same oxygenated HEPES-buffered physiological solution used for
the preparation of IMCD suspensions. Stock solutions of the various inhibitors
were dissolved in DMSO for preparation at a very high concentration (several
millimolar, depending on the solubility), aliquoted, and frozen at
20°C. These stock solutions were diluted freshly before use with
the HEPES-buffered physiological solution to give 10x final incubation
concentrations. These dilutions resulted in 0.050.3% of DMSO in the
final incubations. The aliquots of IMCD suspensions were warmed to 37°C
for 5 min on heat blocks before the agents were added. When nucleotides alone
were used without any inhibitors, the incubations were started immediately
after the 5-min warm-up period and lasted for 10 min unless otherwise
specified. When the inhibitors were used, the IMCD, after the 5-min warm-up
period, were preincubated with the inhibitors for 5 or 15 min, depending on
the type of the inhibitor, and incubated for another 10 min after the addition
of nucleotides. To control incubations that did not contain any inhibitor
and/or nucleotide, equal volumes of the vehicle (incubation buffer) were
added, so that all the incubations had a final volume of 200 µl. All
incubations were carried out in triplicate. To stop the reactions, chilled
HEPES-buffered physiological solution (200 µl) was added and the tubes were
kept on ice for a few minutes. The tubes were centrifuged at 8,000 g
for 10 min in a cold room (4°C), and 350 µl of the supernatant from
each tube were transferred to a fresh tube, frozen, and stored at
80°C until assayed for the PGE2 content. The pellets
with the remaining 50 µl of the incubation buffer were frozen at
20°C for protein assay.
Assay of PGE2
PGE2 content in the supernatants from the incubations described above was determined according to the instructions of the manufacturer using the PGE2 enzyme-linked immunoassay (EIA) kit-monoclonal (catalog no. 514010, Cayman Chemical). The absorbance of the product was read spectrophotometrically at 405 nm in a microplate reader (Molecular Devices, Menlo Park, CA). According to the manufacturer, this assay system has a specificity of 100% to PGE2 and PGE2 ethanolamide and 43 and 18.7% to PGE3 and PGE1, respectively. Other prostanoids have specificities of 0.011%. For our assays, the frozen supernatants were thawed on ice and diluted with EIA buffer to yield final dilutions of 1:200 and 1:400 with respect to the original incubation. The assays were run on 50 µl of these diluted samples. The raw data from the plate reader were stored in a computer and analyzed using Soft MaxPro software (Molecular Devices). Protein pellets were thawed at room temperature, and cellular proteins were precipitated and delipidated by addition of methanol. After the separation of methanol by centrifugation and drying, the protein pellets were dissolved in 0.05 N NaOH. Aliquots of the clear solutions thus obtained were assayed for the protein content by Coomassie Plus protein assay reagent kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. The concentrations of PGE2 in the incubations were normalized with the corresponding protein contents and expressed as nanograms of PGE2 released per milligram of protein.
Immunoblotting for AQP2 Protein
Representative samples of whole inner medullae (collagenase- and hyaluronidase-digested tissue), IMCD-enriched fraction (pellet after low-speed centrifugations), and non-IMCD fractions (pooled supernatants from the low-speed centrifugations) were assessed for the AQP2 protein content by immunoblotting. Briefly, the cellular elements in these fractions were sedimented by centrifugation and washed free of soluble proteins. The final pellets were suspended in homogenization buffer containing protease inhibitors, homogenized, assayed for protein content, solubilized in Laemmli buffer, and immunoblotted for AQP2 protein according to the standard procedures established in our laboratory (13, 24).
Cell Viability Assay
The effect of various inhibitors and DMSO on the viability of IMCD was assessed using the ethidium homodimer-1 dead cell stain as outlined by the manufacturer (Molecular Probes). Only dead or dying cells with damaged (30) membranes are stained by this fluorescent DNA stain, which has been used as an indicator of viability of cultured neurons (8) with the methodology described below for IMCD suspensions. IMCD preparations suspended in the HEPES-buffered physiological solution with or without the added agents were incubated with 2 µM ethidium homodimer-1 for 60 min at room temperature. A positive control for cell staining was run in parallel using 1% Triton X-100 to permeate cells for staining. The incubated suspensions were examined under a fluorescent microscope using filters for Texas red dye (excitation at 495 nm and emission at 635 nm). The samples were coded so that the investigator performing the microscopic examination did not know the identities of the agents being examined. Images were captured with a digital imaging system using Image-Pro Plus (Media Cybernetics, Silver Spring, MD), and the images were grouped using Adobe Photoshop (Adobe Systems, San Jose, CA).
Statistical Analysis
Values are means ± SE. Unless otherwise stated, data were analyzed by analysis of variance followed by assessment of differences between the means of the groups by Tukey-Kramer's multiple comparison test or Bonferroni's multiple comparison test. P < 0.05 was considered significant.
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RESULTS |
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Efficacy of cell separation technique. Representative samples from an IMCD preparation were assessed for enrichment of collecting ducts using the collecting duct-specific marker protein AQP2 water channel. Renal medullary suspension (whole IM) was fractionated into collecting duct-enriched (IMCD) and non-collecting duct (non-IMCD) fractions. Figure 1 shows an immunoblot prepared from SDS-polyacrylamide gels loaded with an equal amount of protein from each of these fractions and probed with AQP2 antibody. The IMCD fraction was enriched severalfold in the collecting duct-specific AQP2 protein compared with the starting material (whole IM; Fig. 1, left and middle lanes). Conversely, the non-IMCD fraction showed very little contamination of collecting duct cells (Fig. 1, right lane). This demonstrates the efficacy of the cell separation technique.
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Cell viability. We assessed the viability of the cellular elements in the IMCD preparations using ethidium homodimer-1, a DNA stain that will stain only dead or dying cells (see MATERIALS AND METHODS). Representative examples from one such assay are shown in Fig. 2. Control preparations (vehicle) contained very few cells stained with ethidium homodimer-1, indicating that most of the cells in the preparation were intact and viable. When DMSO was added to a final concentration of 0.3%, the proportion of cells that were stained with ethidium homodimer-1 was comparable to that in vehicle controls. On the other hand, permeabilization of cells with 1% Triton X-100 resulted in staining of all cellular elements with ethidium homodimer. This established that the cellular elements in IMCD suspensions were viable and remained viable in the presence of DMSO, the solvent used to prepare the various inhibitors used in this study. A similar cell viability assay was performed using various inhibitors, dissolved in DMSO, at their maximum concentrations used in this study. These inhibitors also did not show any significant effect on cell viability compared with the vehicle controls (data not shown), which indicates that any of the inhibitory effects on prostanoid biosynthesis were not due to loss of cells. The dead cells in incubations with these agents, similar to the vehicle controls, were mostly single cells or clumps of a few cells, but not intact tubular segments.
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Efficiency of PGE2 Detection
PGE2 accumulation in the IMCD preparations was assessed using a commercially available EIA kit. In our hands and in our system, this assay had an intra-assay coefficient of variation of 8% as assessed by determinations on two dilutions of six control incubations of a single IMCD preparation. Thus this 8% coefficient of variation represents the variation due to incubations also, in addition to the inherent variations in the assay technique. Figure 3 shows the pooled data from the standard curves that were run on different days. The day-to-day coefficient of variation was 515% in the linear range of the standard curve (2090% binding of tracer; Fig. 3).
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Time Course of ATPS-Stimulated PGE2 Release by
IMCD
Figure 4 shows the time
course of PGE2 release by IMCD after stimulation with 100 µM
ATPS or no stimulation (vehicle controls). Even unstimulated IMCD
released moderate, but significant, amounts of PGE2 into the
medium. Significant amounts of PGE2 were released under
unstimulated conditions for up to 30 min, after which no significant increases
were observed up to 60 min. Thus there was apparently no significant increase
in the amount of PGE2 released between 30 and 60 min under
unstimulated conditions. When the IMCD were stimulated with 100 µM
ATP
S, release of PGE2 was enhanced at all time points. The
differences between the unstimulated and stimulated release became significant
as early as 10 min. Furthermore, in contrast to the unstimulated cells, the
stimulated release in Fig. 4
continued to show an increase when the cells were incubated for up to 60 min.
In experiments with longer incubations, the stimulated release of
PGE2 continued to increase approximately twofold between 60 and 120
min. Under those conditions, the unstimulated release of PGE2 also
showed a twofold increase between 60 and 120 min, without a significant
increase between 30 and 60 min (data not shown). Comparable results were
obtained when the time course experiments were conducted using a different
P2Y2 receptor agonist, UTP (100 µM), instead of ATP
S
(data not shown).
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Effect of Different Concentrations of ATPS on PGE2
Release by IMCD
The ATPS concentration-response curve for the release of PGE2 by
IMCD is shown in Fig. 5.
Because the time course response showed significant increases in the release
of PGE2 as early as 10 min after stimulation of IMCD with
ATP
S, the concentration-response curve was assessed at 10 min after
stimulation. There was a rapid and linear increase in the release of
PGE2 from IMCD after stimulation with ATP
S at concentrations
up to 25 µM. The amount of PGE2 released became significant at
25 µM, and no further significant increase in PGE2 release was
observed up to 100 µM ATP
S.
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Effect of Various COX Inhibitors on PGE2 Release by Unstimulated IMCD
Figure 6 shows the effect of various COX inhibitors on release of PGE2 by unstimulated IMCD incubated at 37°C for 5 or 15 min. The nonspecific COX inhibitors flurbiprofen and APHS caused a significant decrease in the release of PGE2 by unstimulated IMCD. The COX-2-specific inhibitor NS-398 also caused a comparable level of decrease in the release of PGE2, even at 10 µM. Increasing the concentration of NS-398 to 30 µM did not result in a further decrease in the amount of PGE2 released. On the other hand, 30 µM valeroyl salicylate, a COX-1-specific inhibitor, did not cause a significant decrease in the amount of PGE2 released. Only at the 10-fold increase in the concentration of valeroyl salicylate to 300 µM did the amount of PGE2 released significantly decrease compared with the control values. Therefore, all the COX inhibitors tested decreased the amount of unstimulated PGE2 release. The amounts of PGE2 shown in Fig. 6 that correspond to the highest concentrations of the COX inhibitors do not actually represent the amount of PGE2 release that is resistant or insensitive to COX inhibition. Rather, these bars represent the amount of PGE2 in the preparation before addition of the inhibitor. Because the procedure for preparation of IMCD suspensions is a lengthy one, cells may release PGE2 and other substances into the medium during the process. Therefore, the ability of various inhibitors to prevent the release of PGE2 after their addition to the incubation is represented as the difference between 100% and the lowest values. The minor (but not significant) differences in maximal inhibition between flurbiprofen and APHS incubations may likely represent differences in the ability of these inhibitors to reach effective concentrations in the cell and/or initial rate of COX inactivation. Figure 6 also shows the lack of effect of the DMSO solvent (0.08%) on PGE2 release by unstimulated IMCD preparations.
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Effect of Nonspecific COX Inhibition on ATPS-Stimulated
PGE2 Release by IMCD
We used 50 µM ATPS and 10 min of incubation in all the following
experiments with COX inhibitors, on the basis of the time course
(Fig. 4) and the
concentration-response curve (Fig.
5), which showed that 10 min of stimulation with 2550 µM
ATP
S caused optimal amounts of PGE2 release.
Figure 7 shows the inhibitory
effect of flurbiprofen, a nonselective competitive COX inhibitor
(5), on the ATP
S (50
µM)-stimulated release of PGE2 by IMCD. Flurbiprofen at 30 or
300 µM completely inhibited the stimulated release of PGE2 from
IMCD. We also tested the effect of APHS, a potent covalent inhibitor of COX-1
and COX-2 that is similar to aspirin
(22), on the release of
PGE2 by IMCD. As shown in Fig.
8, 10 or 30 µM APHS completely inhibited the ATP
S (50
µM)-stimulated release of PGE2 from IMCD.
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Effect of COX-1 Inhibition on ATPS-Stimulated PGE2
Release by IMCD
Figure 9 shows the effect of
valeroyl salicylate, a selective, irreversible inhibitor of COX-1
(6), on ATPS (50
µM)-stimulated PGE2 release from IMCD. Lower concentrations of
valeroyl salicylate (30 µM) produced little inhibition of the
ATP
S-stimulated PGE2 release. However, 300 µM valeroyl
salicylate completely inhibited the ATP
S-stimulated release of
PGE2 from IMCD.
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Effect of COX-2 Inhibition on ATPS-Stimulated PGE2
Release by IMCD
Figure 10 shows the effect
of NS-398, a selective competitive inhibitor of COX-2
(5), on ATPS (50
µM)-stimulated PGE2 release by IMCD. The stimulated release of
PGE2 was not inhibited by 10 and 30 µM NS-398. Although NS-398
lowered the basal release of PGE2 to
60% of the vehicle
control (Fig. 10, cf. 10 and
30 µM NS-398 with vehicle control), the relative amount of
ATP
S-stimulated PGE2 release in the presence of 10 and 30
µM NS-398 was
1.35-fold greater than the unstimulated amount
(Fig. 10, cf. 10 µM NS-398
with 10 µM NS-398 + 50 µM ATP
S and 30 µM NS-398 with 30 µM
NS-398 + 50 µM ATP
S). This relative increase in
ATP
S-stimulated PGE2 release in the presence of NS-398 was
comparable to the 1.34-fold increase observed without NS-398
(Fig. 10, cf. vehicle control
with 50 µM ATP
S).
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Comparative Stimulatory Effect of ATPS on PGE2
Release by IMCD in the Presence of COX-1 or COX-2 Inhibition
A direct comparison of the inhibition of ATPS-stimulated
PGE2 release by 300 µM valeroyl salicylate (a COX-1 inhibitor)
or 30 µM NS-398 (a COX-2 inhibitor) is presented in
Fig. 11. As shown in
Fig. 11, ATP
S could
stimulate significantly more PGE2 release in the presence of COX-2
inhibition than in the presence of COX-1 inhibition.
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Effect of Inhibition of cPLA2 on ATPS-Stimulated
PGE2 Release by IMCD
The availability of arachidonic acid is a rate-limiting factor for the
synthesis of prostanoids by COX, and cPLA2 is considered to be a
major player in the kidney for the release of arachidonic acid from membrane
phospholipids (7). Hence, we
examined the role of cPLA2 in the ATPS-stimulated
PGE2 release of PGE2 by the IMCD. Under our experimental
conditions, 30 µM AACOCF3, a cPLA2-specific inhibitor, did not
have an effect on ATP
S-stimulated PGE2 release by IMCD
preparations (data not shown).
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DISCUSSION |
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We used a model of freshly prepared IMCD fractions from collagenase- and
hyaluronidase-digested rat inner medullae. This preparation is a
well-characterized model for the study of hormonal response of IMCD. We
validated the purity and viability of the IMCD preparation by demonstrating
the enrichment of collecting duct-specific water channel protein (AQP2) and by
visually assessing the proportion of dead cells by ethidium homodimer
staining, respectively. Because earlier studies have shown that
PGE2 synthesis in IMCD is sensitive to increasing osmolality of the
medium (21), we carried out
all our experiments at 300 mosmol/kgH2O to exclude variability
due to osmolality of the medium. We also demonstrated that DMSO, the solvent
used for various inhibitors, and the inhibitors at the concentrations used in
this study do not have a significant effect on the viability of the IMCD
cells. The commercial EIA kit used for determination of PGE2 has a
high degree of reproducibility in our hands, with acceptable intra-assay and
day-to-day coefficients of variation in the linear range of standard
curve.
In our experiments, we used the specific agonists ATPS and UTP to
examine the effect of P2Y2 receptor activation in IMCD on the
release of PGE2. To the best of our knowledge, the P2Y2
receptor is the only purinergic receptor with well-characterized expression
and functional significance in rat IMCD
(12,
23,
24). Attempts to identify
other purinergic receptors by Ecelbarger et al.
(12) did not reveal an effect
of stimulation with ADP (a P2Y1 agonist), 2-methylthio-ATP (a
P2Y1 agonist), and
,
-methylene-ATP (a P2X agonist) on
the intracellular calcium rise in rat IMCD. Our in vitro microperfusion
experiments also confirmed that rat IMCD does not respond to ADP
(23). These studies apparently
rule out the possibility that P2Y1 and P2X receptors are expressed
in rat IMCD. Therefore, we focused our research efforts on understanding the
cellular and molecular mechanisms of P2Y2 receptor-mediated effects
in rat IMCD.
ATPS was the major agonist that we used to stimulate the
P2Y2 receptor because of its nonhydrolyzable nature, which prevents
the formation of adenosine, which can interact with adenosine A1
receptors in IMCD. We have also observed similar results using other
P2Y2 agonists (data not shown here). In preliminary experiments
using the same model of freshly prepared IMCD fractions where RIA was used for
the measurement of PGE2, we observed that the agonists ATP and UTP
(100 µM), but not the nonagonist ADP, stimulated comparable amounts of
PGE2 release after 10 min of incubation at 37°C. Those
preliminary studies also showed that carbachol, a muscarinic cholinergic
agonist, used as a positive control for intracellular calcium rise, had a
similar effect on the production of PGE2 by IMCD (unpublished
data).
The time course experiments clearly demonstrated that the P2Y2
agonist-stimulated release of PGE2 was significantly higher than
the unstimulated basal release from IMCD. Concentration-response experiments
showed that the PGE2 release reaches a plateau at 25 µM
ATPS, with 50% of the increase at 10 µM. This finding is consistent
with the earlier observations by Ecelbarger et al.
(12), who showed that 10 µM
ATP increased intracellular calcium by
50% of that observed at 100 µM.
In vitro microperfusion experiments conducted by us
(23) also showed that the
decrease in AVP-stimulated osmotic water permeability in the presence of 100
µM ATP is not much different from that caused by 10 µM ATP, thus
indicating that higher concentrations of agonists do not have any added
effect.
Our experiments showed that the basal or unstimulated release of PGE2 by IMCD was apparently dependent on the activity of COX-2, rather than COX-1, inasmuch as it was more sensitive to the COX-2-specific inhibition. Although there is no evidence in the literature that IMCD cells express COX-2 protein, a recent study using a combination of RT-PCR analyses of microdissected renal tubular segments and immunocytochemistry on tissue sections indicated that COX-2 mRNA, but not protein, was present in rat IMCD (41). However, these investigators could not exclude the possibility that the observed results for COX-2 mRNA were due to small amounts of residual medullary interstitial cells that remained attached to the IMCD in their preparations (41). Because medullary interstitial cells are known to express abundant amounts of COX-2 mRNA and protein (17), even a few of these cells in the IMCD preparations could result in detectable amounts of COX-2 transcripts. The same could also be said for assays for COX-2 activity with IMCD preparations, where small amounts of medullary interstitial cells could yield detectable activity. Hence, the COX-2-dependent release of PGE2 that we observed under basal conditions was possibly due to small amounts of medullary interstitial cells that remained attached tightly to the IMCD preparations. Conversely, it may be due to a yet to be identified activity of COX-2 in IMCD cells in our experimental conditions. This issue needs further investigation, and we did not address these aspects in this study.
Because the ATPS-stimulated PGE2 release by IMCD was
completely suppressed by two different types of nonspecific COX inhibitors,
namely, the competitive and covalent inhibitors flurbiprofen and APHS,
respectively, the activity of COX is required for the P2Y2
receptor-mediated release of PGE2 in IMCD. On the other hand, our
studies with differential COX inhibitors revealed that, unlike the basal or
unstimulated production, the ATP
S-stimulated PGE2 release is
dependent on COX-1, rather than COX-2, activity. These observations are
consistent with the documented expression of the P2Y2 receptor and
COX-1 in IMCD cells (24,
41). In our immunocytochemical
studies using a peptide-derived polyclonal antibody specific to the
P2Y2 receptor, we could not detect P2Y2 receptor protein
in medullary interstitial cells
(24; unpublished
observations). Hence, even if present in our IMCD preparations, the
interstitial cells may not respond to stimulation by ATP
S.
The availability of arachidonic acid is the rate-limiting step in the
synthesis of prostanoids by COX in many tissues
(18). However, our attempts to
examine the effect of inhibition of cPLA2 activity on
ATPS-stimulated PGE2 release using the specific inhibitor
AACOCF3 were not successful. This may be due to the difficulties in attaining
effective intracellular concentrations of AACOCF3 under our experimental
conditions. It is also possible that the release of arachidonic acid by IMCD
after P2Y2 receptor activation is more complex and may likely
involve phospholipases other than cPLA2. This aspect needs further
investigation; hence, it was not probed further in the present series of
experiments.
Finally, from our experiments where we could inhibit the release of PGE2 by the IMCD, it appears that the "release" is due to de novo synthesis, and not "secretion" of premade and stored PGE2. The inhibitors that we used here are known to inhibit the activities of COX and are not known to inhibit release or secretion of PGE2 from cells. Furthermore, available evidence shows that the release of PGE2 from cells, including collecting duct principal cells, is dependent on specific prostaglandin transporters (PGTs) in the cell membranes (4). Several PGTs have been cloned and characterized. These are broadly expressed in COX-positive cells and are coordinately regulated by COX. However, there is some evidence in Madin-Darby canine kidney cells that the PGTs are exocytotically inserted into the collecting duct apical membrane, where they could control the concentration of luminal prostaglandins (14). The various factors that may modulate the release of prostaglandins, such as the exocytotic insertion of PGTs into the cell membrane, are poorly understood. Further studies in this area of research will definitely shed new light in the near future on the paracrine regulation of collecting duct function.
In conclusion, our study has important physiological significance with regard to the role of purinergic regulation of medullary collecting duct function beyond the direct modulation of AVP-stimulated water permeability. Because PGE2 is known to affect the transport of water, salt, and urea in IMCD (29, 35, 36), the production and release of PGE2 after purinergic stimulation can indirectly influence handling of water, salt, and urea by the medullary collecting duct. Thus the interaction between the purinergic and prostanoid systems in IMCD, expounded here, further emphasizes the complex nature of the AVP-independent regulatory mechanisms that determine the overall function of IMCD in the renal concentration mechanism.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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This work was presented as a featured topic at the Experimental Biology 2003 Meeting, April 2003, San Diego, CA.
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FOOTNOTES |
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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.
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REFERENCES |
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