P2Y2 receptor-stimulated release of prostaglandin E2 by rat inner medullary coll - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 23 下一页

返回列表

查看: 569185\|回复: 222	go P2Y2 receptor-stimulated release of prostaglandin E2 by rat inner medullary coll [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 13:45 |只看该作者 |倒序浏览 |打印

作者：Brett D. Welch, Noel G. Carlson, Huihui Shi, Leslie Myatt,  Bellamkonda K. Kishore,作者单位：Departments of Internal Medicine, Physiology, and Neurobiologyand Anatomy, University of Utah Health Sciences Center, Salt Lake City 84132; Nephrology Research and Geriatric Research, Education, and Clinical Center,Veterans Affairs Salt Lake City Health Care System, Salt Lake City, Utah84148; and De $ U/ B( N& c# n
   * e4 h3 {9 q$ Q2 G
   * ~0 ~% i; d+ K8 }2 W# t


         3 o* h3 J7 N" H! n+ U& ~9 J; F- K


         $ c) I" e# v" P+ j, A0 b2 `/ Z

      0 b7 \" _% P3 p6 ]% J4 b8 T


      【摘要】
   Extracellular nucleotides, acting through the P2Y 2 receptor andthe associated phosphoinositide-Ca 2 signaling pathway, inhibit AVP-stimulated osmotic water permeability in rat inner medullarycollecting duct (IMCD). Because a rise in intracellular Ca 2 is frequently associated with enhanced arachidonicacid metabolism, we examined the effect of activation of the P2Y 2 receptor on release of PGE 2 in freshly prepared rat IMCDsuspensions. Unstimulated IMCD released moderate, but significant, amounts ofPGE 2, which were more sensitive to cyclooxygenase (COX)-2 thanCOX-1 inhibition. Agonist activation of P2Y 2 receptor by adenosine5'- O -(3-thiotriphosphate) enhanced release of PGE 2 from IMCD in a time- and concentration-dependent fashion. Purinergic-stimulated release of PGE 2 was completely blocked bynonspecific COX inhibitors (flurbiprofen and 2-acetoxyphenylhept-2-ynyl sulfide). Differential COX inhibition studies revealed that purinergic-stimulated release of PGE 2 was more sensitive to aCOX-1-specific inhibitor (valeroyl salicylate) than a COX-2-specific inhibitor(NS-398). Thus purinergic stimulation resulted in significantly more releaseof PGE 2 in the presence of COX-2 inhibitor than COX-1 inhibitor. Ifit is assumed that increased release of PGE 2 is related to itsincreased production, our results suggest that purinergic stimulation of IMCDresults in enhanced production and release of PGE 2 in aCOX-1-dependent fashion. Because PGE 2 is known to affect transportof water, salt, and urea in IMCD, interaction of the purinergic system withthe prostanoid system in IMCD can modulate handling of water, salt, and ureaby IMCD and, thus, may constitute an AVP-independent regulatory mechanism.
      【关键词】 cyclooxygenases arachidonic acid extracellular nucleotides arginine vasopressin purinergic receptor  e) {3 Z1 W" E+ V. u
   THE MEDULLARY COLLECTING DUCT plays an important role in the regulation of body water, sodium, and acid-base balance and in the recyclingof urea. It is a complex tubular segment that responds to stimulation by avariety of hormones or autacoids or paracrine agents, such as AVP, -and -adrenergic agonists, adenosine, atrial natriuretic peptide,bradykinin, endothelin, epidermal growth factor, muscarinic cholinergicagents, and PGE 2. These agents act through a variety of signalingpathways or systems, such as adenylyl cyclase, guanylyl cyclase,phospholipases A 2 and C, and the associated protein kinases( 40 ). These diverse signalingpathways interact with each other in modulation of the overall function of theinner medullary collecting duct (IMCD). Central to this interaction is themutually inhibitory relation between the activation of adenylyl cyclase andphospholipases. Thus increased cellular cAMP content impairs activation of phospholipases A 2 and C, and, conversely, stimulation ofphospholipase C impairs AVP-stimulated adenylyl cyclase activity viaactivation of protein kinase C( 40 ).
8 R8 [! P( o+ R3 O
Extracellular nucleotides bind to specific subtypes of P2 purinergic receptors on cell membranes and elicit a wide variety of biological responsesin several tissues. P2 receptors are classified into two families: theion-tropic P2X and the metabotropic P2Y receptors. The former areextracellular nucleotide-activated membrane channels that allow a variety ofions and/or small molecules to enter the cells( 31, 32 ). The P2Y receptors are Gprotein-coupled receptors that mostly act through the phosphoinositidesignaling pathway ( 10, 42 ). Recent experimentalstudies have unraveled the role of extracellular nucleotides and autocrineand/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 ).
4 h3 _; @$ o4 l5 G1 O1 Q- Z! E
Using a pharmacological approach of measuring the agonist-stimulated risein intracellular calcium responses, Ecelbarger et al. ( 12 ) identified andcharacterized the presence of the P2Y 2 (previously known as P2u)receptor in rat IMCD. Kishore et al.( 23 ) showed that extracellularnucleotides (ATP/UTP) inhibit the AVP-stimulated osmotic water permeability ofin vitro microperfused rat IMCD, thus establishing a physiological role forthe P2Y 2 receptor in IMCD. Subsequently, Kishore et al.( 24 ) demonstrated by molecularapproaches that P2Y 2 receptor mRNA and protein are expressed in ratIMCD. Immunocytochemical localization studies revealed the expression ofP2Y 2 receptor protein on apical and basal domains of collectingduct cells, as well as on thin limbs and vascular elements( 24 ).3 D( H, g$ G) ~. K9 T( N& a% |
: z  w9 g; C! D/ T  N
A rise in intracellular calcium, such as that induced by agonist stimulation of the P2Y 2 receptor in the medullary collecting duct,is known to be frequently associated with enhanced arachidonic acidmetabolism. It has been demonstrated that, in several types of tissues orcells (e.g., guinea pig ileum and uterus, bovine aortic smooth muscle cells,rabbit heart and tracheal epithelial cells, isolated rat dura mater, thymicepithelial cells and astrocytes, porcine or human endothelial cells, and mouseperitoneal macrophages), agonist activation of P2Y receptors resulted instimulation of arachidonic acid metabolism and production of prostanoids. Inmost of these tissues or cells, especially those of nonendothelial nature, thepredominant prostanoid produced was PGE 2 ( 1, 2, 9, 11, 14, 16, 20, 28, 33, 34, 39, 44 ).

Experiments conducted on Madin-Darby canine kidney-D1 cells also revealedthat agonist stimulation of the P2Y 2 receptor results in activationof cytosolic phospholipase A 2 (cPLA 2 ) and release ofarachidonic acid in a protein kinase C- and MAPK-dependent fashion( 43 ). However, to the best ofour knowledge, no studies are available on the synthesis and release ofprostanoids by renal medullary collecting duct cells after purinergicstimulation. Initial experiments conducted by Ecelbarger et al.( 12 ) demonstrated that whenterminal IMCD segments of rat were exposed to indomethacin, a nonspecificinhibitor of cyclooxygenases (COX), for 5 min and then challenged with ATP,the signals displayed a reduced intracellular calcium peak. This findingsuggests 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 theseobservations, we hypothesized that COX products of arachidonic acidmetabolism, such as PGE 2, are formed and released as a result ofagonist activation of the P2Y 2 receptor in rat IMCD. To test thishypothesis, we conducted studies on freshly prepared rat IMCD suspensions andexamined whether the basal and adensoine5'- O -(3-thiotriphosphate) (ATP S)-stimulated release ofPGE 2 was affected by nonspecific inhibitors of COX or COX-1- orCOX-2-specific inhibitors.

MATERIALS AND METHODS! B2 O5 ?* @  m* I8 E: b6 z
0 z3 L" z! }0 T; K- [
Experimental Animals. S( s; n( s' q* I4 [
: G3 A, H6 [3 v) B9 C8 F( }. I4 k
The animal experiments were conducted according to the protocol approved bythe Institutional Animal Care and Use Committee of the Veterans AdministrationSalt Lake City Health Care System. Specific pathogen-free male Sprague-Dawleyrats (Harlan, Indianapolis, IN) were housed two or three per cage in theVeterinary Medical Unit of the Veterans Administration Salt Lake City HealthCare System, which is an American Association for Accreditation of LaboratoryAnimal Care-accredited and US Department of Agriculture-approved animalfacility. The rats were maintained in pathogen-free state and fed ad libitum acommercial rodent diet and had free access to drinking water. The rats wereacclimated to the housing conditions for 5 days before the experiments were conducted. The rats weighed 220-400 g (mean 292 g) at the time ofeuthanasia.

Agents: p( U; V6 n! v  ~
' W8 h! G, t) ~1 z/ F
ATP S (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 RocheMolecular Biochemicals (Indianapolis, IN), and bovine testes hyaluronidasefrom Worthington Biochemical (Lakewood, NJ). All other chemicals used were ofthe highest purity available.% i6 M5 ?3 L. O

Preparation of Fractions Enriched in IMCD! D  Q* o1 z, M8 ]; D. r& Q3 V
  j5 \% h0 g& ~2 y. U
Fractions enriched in IMCD were prepared from rat kidney inner medullaeessentially as described previously( 24, 26 ). Briefly, rats wereeuthanized by pentobarbital sodium overdose, and both kidneys were removedrapidly. The kidneys were chilled in ice-cold phosphate-buffered saline, andthe inner medullae or papillae were dissected on ice and transferred to anisotonic HEPES-buffered physiological solution of the following composition (in mM): 135 NaCl, 0.5 KCl, 0.1 Na 2 HPO 4, 0.3 sodiumacetate, 0.12 NaSO 4, 2.5 CaCl 2, 1.2 MgSO 4, 5HEPES, and 5.5 D -glucose (pH 7.4, 311 ± 9mosmol/kgH 2 O). This solution was oxygenated by bubbling in 95%O 2 -5% CO 2. For each experiment, depending on body weightof the rats, renal papillae from 6-10 rats were pooled to obtain asufficient quantity of the IMCD preparation for 36 incubations. The pooledpapillae were minced with a razor blade and digested at 37°C withcollagenase B (3 mg/ml) and hyaluronidase (600 U/ml) in the sameHEPES-buffered physiological solution for 40-50 min with continuous oxygenation. Halfway through the digestion process, DNase I (GIBCO-BRL,Gaithersburg, MD) was added to the digestion mixture to a final concentrationof 1 U/ml to digest stray DNA released from broken cells. The digestionmixture was intermittently aspirated into and pushed through a glass Pasteurpipette to disperse the tubules into a uniform suspension. After digestion ofthe papillary tissue to a uniform suspension, the IMCD fraction was separatedfrom the non-IMCD elements (thin limbs and vasculature) by sedimentation bylow-speed centrifugation and repeated washings. The final pellet was suspendedin the oxygenated HEPES-buffered physiological solution to a proteinconcentration of 1 mg/ml and kept on ice for 30 min to allow forrecovery of IMCD cells from stress. This preparation consisted of mostly IMCDsegments or sheets of IMCD cells and very few other non-IMCD elements. Representative IMCD fractions were assessed for enrichment and viability byimmunoblotting for collecting duct-specific water channel protein aquaporin-2(AQP2) and by binding of ethidium homodimer-1 to the cellular DNA of deadcells, respectively.

Incubation of IMCD Preparations7 Q7 k. K5 y" s6 C+ ~
) p6 Z. P/ I2 ~8 ?* O: c+ |  d2 @8 r
Fractions enriched in IMCD were incubated with or without the addition ofinhibitors of COX and/or nucleotides (ATP S or UTP), and the amount ofPGE 2 released from the cells was assayed. Briefly, the IMCDsuspensions prepared and kept on ice for 30 min for recovery from stress asdescribed above were aliquoted into 1.5-ml plastic microtubes kept on ice.Nucleotide stock solutions (10 x final incubation concentration) wereprepared in the same oxygenated HEPES-buffered physiological solution used forthe preparation of IMCD suspensions. Stock solutions of the various inhibitorswere dissolved in DMSO for preparation at a very high concentration (severalmillimolar, 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 10 x final incubationconcentrations. These dilutions resulted in 0.05-0.3% of DMSO in thefinal incubations. The aliquots of IMCD suspensions were warmed to 37°Cfor 5 min on heat blocks before the agents were added. When nucleotides alonewere used without any inhibitors, the incubations were started immediatelyafter 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-upperiod, were preincubated with the inhibitors for 5 or 15 min, depending onthe type of the inhibitor, and incubated for another 10 min after the additionof nucleotides. To control incubations that did not contain any inhibitorand/or nucleotide, equal volumes of the vehicle (incubation buffer) wereadded, so that all the incubations had a final volume of 200 µl. Allincubations were carried out in triplicate. To stop the reactions, chilledHEPES-buffered physiological solution (200 µl) was added and the tubes werekept 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 fromeach tube were transferred to a fresh tube, frozen, and stored at-80°C until assayed for the PGE 2 content. The pelletswith the remaining 50 µl of the incubation buffer were frozen at-20°C for protein assay.
& O7 S+ a- r, I& P/ l& P
Assay of PGE 2
3 r7 A& M6 c0 ]8 l
PGE 2 content in the supernatants from the incubations described above was determined according to the instructions of the manufacturer usingthe PGE 2 enzyme-linked immunoassay (EIA) kit-monoclonal (catalogno. 514010, Cayman Chemical). The absorbance of the product was readspectrophotometrically at 405 nm in a microplate reader (Molecular Devices,Menlo Park, CA). According to the manufacturer, this assay system has aspecificity of 100% to PGE 2 and PGE 2 ethanolamide and 43and 18.7% to PGE 3 and PGE 1, respectively. Otherprostanoids have specificities of 0.01-1%. For our assays, the frozensupernatants were thawed on ice and diluted with EIA buffer to yield finaldilutions of 1:200 and 1:400 with respect to the original incubation. Theassays were run on 50 µl of these diluted samples. The raw data from theplate reader were stored in a computer and analyzed using Soft MaxPro software(Molecular Devices). Protein pellets were thawed at room temperature, andcellular proteins were precipitated and delipidated by addition of methanol.After the separation of methanol by centrifugation and drying, the proteinpellets were dissolved in 0.05 N NaOH. Aliquots of the clear solutions thusobtained were assayed for the protein content by Coomassie Plus protein assayreagent kit (Pierce Biotechnology, Rockford, IL) according to themanufacturer's instructions. The concentrations of PGE 2 in theincubations were normalized with the corresponding protein contents and expressed as nanograms of PGE 2 released per milligram ofprotein.8 U1 P/ L9 c: e

Immunoblotting for AQP2 Protein

Representative samples of whole inner medullae (collagenase- andhyaluronidase-digested tissue), IMCD-enriched fraction (pellet after low-speedcentrifugations), and non-IMCD fractions (pooled supernatants from thelow-speed centrifugations) were assessed for the AQP2 protein content byimmunoblotting. Briefly, the cellular elements in these fractions weresedimented 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 Laemmlibuffer, and immunoblotted for AQP2 protein according to the standardprocedures established in our laboratory( 13, 24 ).

Cell Viability Assay

The effect of various inhibitors and DMSO on the viability of IMCD wasassessed using the ethidium homodimer-1 dead cell stain as outlined by themanufacturer (Molecular Probes). Only dead or dying cells with damaged( 30 ) membranes are stained bythis fluorescent DNA stain, which has been used as an indicator of viabilityof cultured neurons ( 8 ) withthe methodology described below for IMCD suspensions. IMCD preparationssuspended in the HEPES-buffered physiological solution with or without theadded agents were incubated with 2 µM ethidium homodimer-1 for 60 min atroom temperature. A positive control for cell staining was run in parallelusing 1% Triton X-100 to permeate cells for staining. The incubatedsuspensions were examined under a fluorescent microscope using filters forTexas red dye (excitation at 495 nm and emission at 635 nm). The samples werecoded so that the investigator performing the microscopic examination did notknow the identities of the agents being examined. Images were captured with adigital imaging system using Image-Pro Plus (Media Cybernetics, Silver Spring,MD), and the images were grouped using Adobe Photoshop (Adobe Systems, SanJose, CA).3 \* M% W7 k% m, p) c
' c$ _" q" u* s
Statistical Analysis
0 q# |4 l4 R. @5 ~! `( E6 z
Values are means ± SE. Unless otherwise stated, data were analyzedby analysis of variance followed by assessment of differences between themeans of the groups by Tukey-Kramer's multiple comparison test or Bonferroni'smultiple comparison test. P  A4 {7 J- }& I) v9 J3 [
( ~* r! o0 r- `, K4 s9 y" d7 X
RESULTS/ f* [* i$ i$ g& f. O

Characterization of IMCD Preparation. p* ]* z. X( A3 h( a4 S
! a: x7 L# b1 M
Efficacy of cell separation technique. Representative samples froman IMCD preparation were assessed for enrichment of collecting ducts using thecollecting duct-specific marker protein AQP2 water channel. Renal medullarysuspension (whole IM) was fractionated into collecting duct-enriched (IMCD)and non-collecting duct (non-IMCD) fractions. Figure 1 shows an immunoblotprepared from SDS-polyacrylamide gels loaded with an equal amount of proteinfrom each of these fractions and probed with AQP2 antibody. The IMCD fractionwas enriched severalfold in the collecting duct-specific AQP2 protein comparedwith the starting material (whole IM; Fig.1, left and middle lanes ). Conversely,the non-IMCD fraction showed very little contamination of collecting ductcells ( Fig. 1, rightlane ). This demonstrates the efficacy of the cell separationtechnique.6 g! \4 L+ S0 k$ F/ R+ g
! `  V  _+ s# Z2 I) l8 Z! u
Fig. 1. Assessment of inner medullary collecting duct (IMCD) fractions forenrichment of the collecting duct-specific water channel protein aquaporin-2(AQP2) by immunoblotting. Cellular elements in samples of whole innermedullary digest (whole IM), IMCD-enriched fractions (pellets from low-speedcentrifugations), and non-IMCD fractions (pooled supernatants from low-speedcentrifugations) were collected by centrifugation and washed free of solubleproteins. Equal amounts (5 µg) of solubilized proteins from each fractionwere immunoblotted and probed with a peptide-derived polyclonal antibody torat AQP2 protein ( 24 ).Secondary antibody was donkey anti-rabbit IgG conjugated to horseradishperoxidase. Sites of antigen-antibody interactions were visualized usingchemiluminescence reaction and captured on a light-sensitive imaging film. The29-kDa band corresponds to the nonglycosylated form; streaks that extend from35 kDa and above represent various species of glycosylated forms of AQP2protein.
8 k% ?1 a6 M$ Z( q' _" g% \" b+ ~6 c
Cell viability. We assessed the viability of the cellular elements in the IMCD preparations using ethidium homodimer-1, a DNA stain that willstain 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 cellsthat were stained with ethidium homodimer-1 was comparable to that in vehiclecontrols. On the other hand, permeabilization of cells with 1% Triton X-100resulted in staining of all cellular elements with ethidium homodimer. Thisestablished that the cellular elements in IMCD suspensions were viable andremained viable in the presence of DMSO, the solvent used to prepare thevarious inhibitors used in this study. A similar cell viability assay wasperformed using various inhibitors, dissolved in DMSO, at their maximumconcentrations used in this study. These inhibitors also did not show anysignificant effect on cell viability compared with the vehicle controls (datanot shown), which indicates that any of the inhibitory effects on prostanoid biosynthesis were not due to loss of cells. The dead cells in incubations withthese agents, similar to the vehicle controls, were mostly single cells orclumps of a few cells, but not intact tubular segments.) q* }; y5 c+ p( Z* c3 Z
- u4 f4 d: {7 E8 N  y2 l% P$ I6 |
Fig. 2. Cell viability assay using ethidium homodimer-1 (ethidium HD). IMCDsuspensions containing no added agents [vehicle control (Veh)] or 0.3% DMSO(solvent used to dissolve inhibitors) or 1% Triton X-100 (positive control forcell death) were incubated in a 24-well microplate with 2 µM ethidiumhomodimer-1, a fluorescent DNA stain, for 60 min at room temperature. At theend of the incubation period, suspensions were examined under a fluorescentmicroscope (excitation at 495 nm, emission at 635 nm), and images werecaptured digitally. Left : living and dead cellular elements in thesuspension as seen under phase contrast mode; right : fluorescence ofdead or dying cells in the corresponding fields, inasmuch as ethidiumhomodimer-1 is not permeable to intact living cells.

Efficiency of PGE 2 Detection- K, i6 G4 C! d  `1 D( l' S& V

PGE 2 accumulation in the IMCD preparations was assessed using acommercially available EIA kit. In our hands and in our system, this assay hadan intra-assay coefficient of variation of 8% as assessed by determinations ontwo dilutions of six control incubations of a single IMCD preparation. Thusthis 8% coefficient of variation represents the variation due to incubationsalso, in addition to the inherent variations in the assay technique. Figure 3 shows the pooled datafrom the standard curves that were run on different days. The day-to-daycoefficient of variation was 5-15% in the linear range of the standardcurve (20-90% binding of tracer; Fig.3 ).0 S1 K$ S4 K3 e, B9 A* `
0 X5 {) ^9 o! [/ b, P: `/ U
Fig. 3. Standard curve for determination of PGE 2 by enzyme-linkedimmunoassay (EIA). PGE 2 standards were run in each assay plate.Values obtained for percent binding were plotted against PGE 2 concentrations in standards. Values are means ± SE for 10 assays, witheach standard run in duplicate. Thus the standard curve represents day-to-dayvariation in assays that were run to generate data presented in this study. Onthe basis of the standard curve, day-to-day coefficient of variation is5-15% in the linear range of the curve (20-90% binding oftracer).# ?9 ]+ C9 z" u: |* u& Z

Time Course of ATP S-Stimulated PGE 2 Release byIMCD0 y9 _1 w3 U4 }6 L7 W
/ {9 J/ N- ~. W. R
Figure 4 shows the timecourse of PGE 2 release by IMCD after stimulation with 100 µMATP S or no stimulation (vehicle controls). Even unstimulated IMCDreleased moderate, but significant, amounts of PGE 2 into themedium. Significant amounts of PGE 2 were released underunstimulated conditions for up to 30 min, after which no significant increaseswere observed up to 60 min. Thus there was apparently no significant increasein the amount of PGE 2 released between 30 and 60 min underunstimulated conditions. When the IMCD were stimulated with 100 µM ATP S, release of PGE 2 was enhanced at all time points. The differences between the unstimulated and stimulated release became significantas early as 10 min. Furthermore, in contrast to the unstimulated cells, thestimulated 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 ofPGE 2 continued to increase approximately twofold between 60 and 120min. Under those conditions, the unstimulated release of PGE 2 alsoshowed a twofold increase between 60 and 120 min, without a significantincrease between 30 and 60 min (data not shown). Comparable results wereobtained when the time course experiments were conducted using a different P2Y 2 receptor agonist, UTP (100 µM), instead of ATP S (data not shown).( \6 p4 C  @( B) S+ u
7 L  t' a1 s2 s# M5 \/ ~/ F3 t- b
Fig. 4. Time course of release of PGE 2 by IMCD preparations underunstimulated conditions (vehicle) or after stimulation with 100 µMadenosine 5'- O -(3-thiotriphosphate) (ATP S). IMCDpreparations, suspended in oxygenated HEPES-buffered physiological solution,were warmed to 37°C, and vehicle (incubation buffer) or freshly preparedATP S solution was added to obtain a final concentration of 100 µM inthe incubation. Incubations were continued for 10-60 min. Values aremeans ± SE of triplicate incubations. *Significantly different fromcorresponding unstimulated value ( P P" |& x- @# a# q( Q! a

Effect of Different Concentrations of ATP S on PGE 2 Release by IMCD) h# Z& c/ \/ l; G4 [  n
) w) V1 m% r& P. M
The ATP S concentration-response curve for the release of PGE2 byIMCD is shown in Fig. 5.Because the time course response showed significant increases in the releaseof PGE 2 as early as 10 min after stimulation of IMCD withATP S, the concentration-response curve was assessed at 10 min afterstimulation. There was a rapid and linear increase in the release ofPGE 2 from IMCD after stimulation with ATP S at concentrationsup to 25 µM. The amount of PGE 2 released became significant at25 µM, and no further significant increase in PGE 2 release wasobserved up to 100 µM ATP S.

Fig. 5. ATP S concentration (Conc)-response curve for release ofPGE 2 by IMCD preparations. IMCD preparations, suspended inoxygenated HEPES-buffered physiological solution, were warmed to 37°C andthen challenged with 0-100 µM ATP S for 10 min at 37°C.Values are means ± SE of triplicate incubations. * P S. Tukey-Kramer's multiple comparisons test was used forall comparisons." g7 X3 i2 a0 T& L1 K5 |9 w6 l

Effect of Various COX Inhibitors on PGE 2 Release byUnstimulated IMCD

Figure 6 shows the effect ofvarious COX inhibitors on release of PGE 2 by unstimulated IMCDincubated at 37°C for 5 or 15 min. The nonspecific COX inhibitorsflurbiprofen and APHS caused a significant decrease in the release ofPGE 2 by unstimulated IMCD. The COX-2-specific inhibitor NS-398 alsocaused a comparable level of decrease in the release of PGE 2, evenat 10 µM. Increasing the concentration of NS-398 to 30 µM did not resultin a further decrease in the amount of PGE 2 released. On the otherhand, 30 µM valeroyl salicylate, a COX-1-specific inhibitor, did not causea significant decrease in the amount of PGE 2 released. Only at the10-fold increase in the concentration of valeroyl salicylate to 300 µM didthe amount of PGE 2 released significantly decrease compared withthe control values. Therefore, all the COX inhibitors tested decreased theamount of unstimulated PGE 2 release. The amounts of PGE 2 shown in Fig. 6 that correspondto the highest concentrations of the COX inhibitors do not actually representthe amount of PGE 2 release that is resistant or insensitive to COXinhibition. Rather, these bars represent the amount of PGE 2 in thepreparation before addition of the inhibitor. Because the procedure for preparation of IMCD suspensions is a lengthy one, cells may releasePGE 2 and other substances into the medium during the process.Therefore, the ability of various inhibitors to prevent the release ofPGE 2 after their addition to the incubation is represented as thedifference between 100% and the lowest values. The minor (but not significant)differences in maximal inhibition between flurbiprofen and APHS incubationsmay likely represent differences in the ability of these inhibitors to reacheffective concentrations in the cell and/or initial rate of COX inactivation. Figure 6 also shows the lack ofeffect of the DMSO solvent (0.08%) on PGE 2 release by unstimulated IMCD preparations.8 f( g8 R- i! t$ ]) l& z4 a

Fig. 6. Effect of various inhibitors of cylcooxygenases (COX) or DMSO on release ofPGE 2 by IMCD under basal conditions. IMCD preparations, suspendedin oxygenated HEPES-buffered physiological solution, were warmed to 37°Cand then challenged with nonspecific COX inhibitors [flurbiprofen or2-acetoxyphenylhept-2-ynyl sulfide (APHS)], COX-1-specific inhibitor [valeroylsalicylate (Val Sal)], COX-2-specific inhibitor (NS-398), or DMSO. Allincubations, except valeroyl salicylate, were carried out for 5 min. Valeroylsalicylate was incubated for 15 min. PGE 2 release was assayed,normalized to protein concentration, and expressed as percentage of respectivevehicle control value. Data were generated from 5 different assays, in whicheach agent was run in triplicate incubations. For valeroyl salicylate andNS-398, data from 2 different assays were pooled. Thus values are means± SE of 3 or 6 incubations. *Significantly different from correspondingcontrol value ( P) ~" P& j1 ^: C/ _/ [; K

Effect of Nonspecific COX Inhibition on ATP S-StimulatedPGE 2 Release by IMCD
2 Y( n1 C. D# d
We used 50 µM ATP S and 10 min of incubation in all the followingexperiments with COX inhibitors, on the basis of the time course( Fig. 4 ) and theconcentration-response curve ( Fig.5 ), which showed that 10 min of stimulation with 25-50 µMATP S caused optimal amounts of PGE 2 release. Figure 7 shows the inhibitoryeffect of flurbiprofen, a nonselective competitive COX inhibitor( 5 ), on the ATP S (50µM)-stimulated release of PGE 2 by IMCD. Flurbiprofen at 30 or300 µM completely inhibited the stimulated release of PGE 2 fromIMCD. We also tested the effect of APHS, a potent covalent inhibitor of COX-1and COX-2 that is similar to aspirin( 22 ), on the release ofPGE 2 by IMCD. As shown in Fig.8, 10 or 30 µM APHS completely inhibited the ATP S (50µM)-stimulated release of PGE 2 from IMCD.

Fig. 7. Effect of nonspecific COX inhibition by flurbiprofen onATP S-stimulated release of PGE 2 by IMCD. IMCD preparations,suspended in oxygenated HEPES-buffered physiological solution, were warmed to37°C and then preincubated for 5 min with or without addition offlurbiprofen to a final concentration of 30 or 300 µM. After preincubation,ATP S was added to some incubations to a final concentration of 50µM, and incubation was continued for 10 min at 37°C. PGE 2 release was determined by EIA and normalized to protein content. Values aremeans ± SE of triplicate incubations. Results are expressed aspercentage of mean values in vehicle controls (105 ng PGE 2 /mgprotein). * P P

Fig. 8. Effect of nonspecific COX inhibition by APHS on ATP S-stimulatedrelease of PGE 2 by IMCD. IMCD preparations, suspended in oxygenatedHEPES-buffered physiological solution, were warmed to 37°C and thenpreincubated with or without addition of APHS to a final concentration of 10or 30 µM. After preincubation, ATP S was added to some incubations toa final concentration of 50 µM, and incubation was continued for 10 min at37°C. PGE 2 release was determined by EIA and normalized toprotein content. Results are expressed as percentage of mean values in vehiclecontrols (185 ng PGE 2 /mg protein). Values are means ± SE oftriplicate incubations. * P3 q7 i  s" w' A' _& X

Effect of COX-1 Inhibition on ATP S-Stimulated PGE 2 Release by IMCD: Q9 r9 Z$ W" P0 |6 f

Figure 9 shows the effect ofvaleroyl salicylate, a selective, irreversible inhibitor of COX-1( 6 ), on ATP S (50µM)-stimulated PGE 2 release from IMCD. Lower concentrations ofvaleroyl salicylate (30 µM) produced little inhibition of theATP S-stimulated PGE 2 release. However, 300 µM valeroylsalicylate completely inhibited the ATP S-stimulated release ofPGE 2 from IMCD.

Fig. 9. Effect of COX-1 inhibition by valeroyl salicylate (Valeroyl Sal) onATP S-stimulated release of PGE 2 by IMCD. IMCD preparations,suspended in oxygenated HEPES-buffered physiological solution, were warmed to37°C and then preincubated for 15 min with or without addition of valeroylsalicylate to a final concentration of 30 or 300 µM. After preincubation,ATP S was added to some incubations to a final concentration of 50µM, and incubations were continued for 10 min at 37°C. PGE 2 release was determined by EIA, normalized to protein content, and expressed aspercentage of mean values obtained for vehicle controls. Values are means± SE of pooled data from 2 different experiments, where each incubationwas run in triplicate. * P P S alone or 30 µM valeroyl salicylate.*** P S. Bonferroni's multiplecomparisons test was used for all comparisons.' k. ?# N) [' d9 `1 ~

Effect of COX-2 Inhibition on ATP S-Stimulated PGE 2 Release by IMCD
! |" x" j7 m5 g7 `) v5 \" x7 D" z
Figure 10 shows the effectof NS-398, a selective competitive inhibitor of COX-2( 5 ), on ATP S (50µM)-stimulated PGE 2 release by IMCD. The stimulated release ofPGE 2 was not inhibited by 10 and 30 µM NS-398. Although NS-398lowered the basal release of PGE 2 to 60% of the vehiclecontrol ( Fig. 10, cf. 10 and30 µM NS-398 with vehicle control), the relative amount ofATP S-stimulated PGE 2 release in the presence of 10 and 30µM NS-398 was 1.35-fold greater than the unstimulated amount( Fig. 10, cf. 10 µM NS-398with 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 inATP S-stimulated PGE 2 release in the presence of NS-398 wascomparable to the 1.34-fold increase observed without NS-398( Fig. 10, cf. vehicle controlwith 50 µM ATP S).2 Q8 q: n8 ^0 p" _/ a
2 R5 l/ i( z+ W1 t9 k
Fig. 10. Effect of COX-2 inhibition by NS-398 on ATP S-stimulated release ofPGE 2 by IMCD. IMCD preparations, suspended in oxygenatedHEPES-buffered physiological solution, were warmed to 37°C and thenpreincubated for 5 min with or without addition of NS-398 to a finalconcentration of 10 or 30 µM. After preincubation, ATP S was added tosome incubations to a final concentration of 50 µM, and incubations werecontinued for 10 min at 37°C. Release of PGE 2 was determined byEIA, normalized to protein content, and expressed as percentage of mean valuesobtained for vehicle controls. Except for 30 µM NS-398 and 30 µM NS-398  50 µM ATP S, values are means ± SE of 2 experiments, whereincubations were run in triplicate. Data for 30 µM NS-398 and 30 µMNS-398 50 µM ATP S are from 1 experiment with triplicateincubations. * P P P+ l- q& P; s: z1 r& N, `. @

Comparative Stimulatory Effect of ATP S on PGE 2 Release by IMCD in the Presence of COX-1 or COX-2 Inhibition
& d) v4 C0 ]' N& x+ S. ?2 T7 {
A direct comparison of the inhibition of ATP S-stimulatedPGE 2 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 couldstimulate significantly more PGE 2 release in the presence of COX-2inhibition than in the presence of COX-1 inhibition.
( {" @8 [  ], @: m0 w
Fig. 11. Comparative stimulatory effect of ATP S on release of PGE 2 from IMCD preparations in the presence of a COX-1 inhibitor [300 µMvaleroyl salicylate (VS)] or a COX-2 inhibitor [30 µM NS-398 (NS)]. Dataare from experiments shown in Figs. 9 and 10. Ability of ATP S toovercome the effect of COX inhibitors was calculated as percent stimulation ofPGE 2 release over and above values obtained in the presence of thecorresponding inhibitor alone. Values are means ± SE of 6 (300 µMvaleroyl salicylate) and 3 (30 µM NS-398) incubations. *Median and meanvalues are significantly different from those of 300 µM valeroyl salicylateas assessed by Mann-Whitney's test and unpaired t -test with Welchcorrection, respectively.

Effect of Inhibition of cPLA 2 on ATP S-StimulatedPGE 2 Release by IMCD& w5 W7 J" {9 q2 e- @

The availability of arachidonic acid is a rate-limiting factor for thesynthesis of prostanoids by COX, and cPLA 2 is considered to be amajor player in the kidney for the release of arachidonic acid from membranephospholipids ( 7 ). Hence, weexamined the role of cPLA 2 in the ATP S-stimulatedPGE 2 release of PGE 2 by the IMCD. Under our experimentalconditions, 30 µM AACOCF3, a cPLA 2 -specific inhibitor, did nothave an effect on ATP S-stimulated PGE 2 release by IMCDpreparations (data not shown).
. B) g, e2 [& N- V2 ^, X/ k
DISCUSSION
/ d% [( O& ]) X2 D
We demonstrated that 1 ) unstimulated IMCD release moderate, butsignificant, amounts of PGE 2, which are more sensitive to COX-2than COX-1 inhibition, 2 ) activation of the P2Y 2 receptor by the agonist ATP S results in enhanced release of PGE 2 from IMCD in a time- and concentration-dependent fashion, 3 )purinergic-stimulated release of PGE 2 by IMCD is blocked bynonspecific COX inhibition, and 4 ) purinergic-stimulated release ofPGE 2 by IMCD is more sensitive to COX-1- than COX-2-specificinhibition. If it is assumed that increased release of PGE 2 by IMCDis related to its increased production, our results suggest that purinergic stimulation of IMCD causes an enhanced production and release ofPGE 2, which appear to be mediated by COX-1.9 Z: L. e  M, d- @& y+ }+ |- `7 D  R

We used a model of freshly prepared IMCD fractions from collagenase- andhyaluronidase-digested rat inner medullae. This preparation is awell-characterized model for the study of hormonal response of IMCD. Wevalidated the purity and viability of the IMCD preparation by demonstratingthe enrichment of collecting duct-specific water channel protein (AQP2) and byvisually assessing the proportion of dead cells by ethidium homodimerstaining, respectively. Because earlier studies have shown thatPGE 2 synthesis in IMCD is sensitive to increasing osmolality of themedium ( 21 ), we carried outall our experiments at 300 mosmol/kgH 2 O to exclude variabilitydue to osmolality of the medium. We also demonstrated that DMSO, the solventused for various inhibitors, and the inhibitors at the concentrations used inthis study do not have a significant effect on the viability of the IMCDcells. The commercial EIA kit used for determination of PGE 2 has a high degree of reproducibility in our hands, with acceptable intra-assay andday-to-day coefficients of variation in the linear range of standardcurve.

In our experiments, we used the specific agonists ATP S and UTP toexamine the effect of P2Y 2 receptor activation in IMCD on therelease of PGE 2. To the best of our knowledge, the P2Y 2 receptor is the only purinergic receptor with well-characterized expressionand functional significance in rat IMCD( 12, 23, 24 ). Attempts to identifyother purinergic receptors by Ecelbarger et al.( 12 ) did not reveal an effectof stimulation with ADP (a P2Y 1 agonist), 2-methylthio-ATP (aP2Y 1 agonist), and, -methylene-ATP (a P2X agonist) onthe intracellular calcium rise in rat IMCD. Our in vitro microperfusionexperiments also confirmed that rat IMCD does not respond to ADP( 23 ). These studies apparently rule out the possibility that P2Y 1 and P2X receptors are expressed in rat IMCD. Therefore, we focused our research efforts on understanding thecellular and molecular mechanisms of P2Y 2 receptor-mediated effectsin rat IMCD.

ATP S was the major agonist that we used to stimulate theP2Y 2 receptor because of its nonhydrolyzable nature, which prevents the formation of adenosine, which can interact with adenosine A 1 receptors in IMCD. We have also observed similar results using otherP2Y 2 agonists (data not shown here). In preliminary experimentsusing the same model of freshly prepared IMCD fractions where RIA was used forthe measurement of PGE 2, we observed that the agonists ATP and UTP(100 µM), but not the nonagonist ADP, stimulated comparable amounts ofPGE 2 release after 10 min of incubation at 37°C. Thosepreliminary studies also showed that carbachol, a muscarinic cholinergicagonist, used as a positive control for intracellular calcium rise, had a similar effect on the production of PGE 2 by IMCD (unpublished data).9 @9 ^; k9 U# s1 S' ~! }+ P' P2 s

The time course experiments clearly demonstrated that the P2Y 2 agonist-stimulated release of PGE 2 was significantly higher thanthe unstimulated basal release from IMCD. Concentration-response experimentsshowed that the PGE 2 release reaches a plateau at 25 µMATP S, with 50% of the increase at 10 µM. This finding is consistentwith the earlier observations by Ecelbarger et al.( 12 ), who showed that 10 µMATP increased intracellular calcium by 50% of that observed at 100 µM. In vitro microperfusion experiments conducted by us( 23 ) also showed that thedecrease in AVP-stimulated osmotic water permeability in the presence of 100µM ATP is not much different from that caused by 10 µM ATP, thusindicating that higher concentrations of agonists do not have any addedeffect.

Our experiments showed that the basal or unstimulated release ofPGE 2 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-specificinhibition. Although there is no evidence in the literature that IMCD cellsexpress COX-2 protein, a recent study using a combination of RT-PCR analysesof microdissected renal tubular segments and immunocytochemistry on tissuesections indicated that COX-2 mRNA, but not protein, was present in rat IMCD( 41 ). However, theseinvestigators could not exclude the possibility that the observed results forCOX-2 mRNA were due to small amounts of residual medullary interstitial cells that remained attached to the IMCD in their preparations( 41 ). Because medullaryinterstitial cells are known to express abundant amounts of COX-2 mRNA andprotein ( 17 ), even a few ofthese cells in the IMCD preparations could result in detectable amounts ofCOX-2 transcripts. The same could also be said for assays for COX-2 activitywith IMCD preparations, where small amounts of medullary interstitial cellscould yield detectable activity. Hence, the COX-2-dependent release ofPGE 2 that we observed under basal conditions was possibly due tosmall amounts of medullary interstitial cells that remained attached tightly to the IMCD preparations. Conversely, it may be due to a yet to be identifiedactivity of COX-2 in IMCD cells in our experimental conditions. This issueneeds further investigation, and we did not address these aspects in thisstudy.

Because the ATP S-stimulated PGE 2 release by IMCD wascompletely 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 P2Y 2 receptor-mediated release of PGE 2 in IMCD. On the other hand, ourstudies with differential COX inhibitors revealed that, unlike the basal orunstimulated production, the ATP S-stimulated PGE 2 release isdependent on COX-1, rather than COX-2, activity. These observations areconsistent with the documented expression of the P2Y 2 receptor andCOX-1 in IMCD cells ( 24, 41 ). In our immunocytochemicalstudies using a peptide-derived polyclonal antibody specific to theP2Y 2 receptor, we could not detect P2Y 2 receptor proteinin medullary interstitial cells( 24; unpublishedobservations). Hence, even if present in our IMCD preparations, theinterstitial cells may not respond to stimulation by ATP S./ y2 I! ~* a/ f, n; E: {; y5 _

The availability of arachidonic acid is the rate-limiting step in thesynthesis of prostanoids by COX in many tissues( 18 ). However, our attempts toexamine the effect of inhibition of cPLA 2 activity onATP S-stimulated PGE 2 release using the specific inhibitorAACOCF3 were not successful. This may be due to the difficulties in attainingeffective intracellular concentrations of AACOCF3 under our experimentalconditions. It is also possible that the release of arachidonic acid by IMCDafter P2Y 2 receptor activation is more complex and may likelyinvolve phospholipases other than cPLA 2. This aspect needs furtherinvestigation; hence, it was not probed further in the present series ofexperiments.: [" `( T% Z7 V& X6 G$ Y: Y' v# x

Finally, from our experiments where we could inhibit the release ofPGE 2 by the IMCD, it appears that the "release" is due to de novo synthesis, and not "secretion" of premade and stored PGE 2. The inhibitors that we used here are known to inhibit theactivities of COX and are not known to inhibit release or secretion ofPGE 2 from cells. Furthermore, available evidence shows that therelease of PGE 2 from cells, including collecting duct principalcells, is dependent on specific prostaglandin transporters (PGTs) in the cellmembranes ( 4 ). Several PGTs have been cloned and characterized. These are broadly expressed inCOX-positive cells and are coordinately regulated by COX. However, there issome evidence in Madin-Darby canine kidney cells that the PGTs areexocytotically inserted into the collecting duct apical membrane, where theycould control the concentration of luminal prostaglandins( 14 ). The various factors thatmay modulate the release of prostaglandins, such as the exocytotic insertionof PGTs into the cell membrane, are poorly understood. Further studies in thisarea of research will definitely shed new light in the near future on theparacrine regulation of collecting duct function.
' C+ [/ Y" a: T! \6 W
In conclusion, our study has important physiological significance withregard to the role of purinergic regulation of medullary collecting ductfunction beyond the direct modulation of AVP-stimulated water permeability.Because PGE 2 is known to affect the transport of water, salt, andurea in IMCD ( 29, 35, 36 ), the production andrelease of PGE 2 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-independentregulatory mechanisms that determine the overall function of IMCD in the renalconcentration mechanism.
8 q: s) E# J9 ~5 X
DISCLOSURES

This work was supported by National Institute of Diabetes and Digestive andKidney Diseases Grant DK-61183 (to B. K. Kishore) and the resources andfacilities at the Veterans Administration Salt Lake City Health CareSystem.
  W/ B3 R7 M+ q# X5 H) P
ACKNOWLEDGMENTS( l0 G3 n) i& E
, n6 @4 o2 s& E8 Z
The authors thank Drs. Mark Knepper and Donald Kohan for critical readingof the manuscript, Diane Brockman for helpful suggestions on technicalapproaches, and Arpana Mazumder for technical assistance.

This work was presented as a featured topic at the Experimental Biology2003 Meeting, April 2003, San Diego, CA.
      【参考文献】
Aitken H,Poyser NL, and Hollingsworth M. The effects of P2Y receptor agonists andadenosine on prostaglandin production by the guinea-pig uterus. BrJ Pharmacol 132:709-721, 2001.
# U8 v6 w3 Z+ h$ c" w$ N

Aksoy MO,Borenstein M, Li XX, and Kelsen SG. Eicosanoid production in rabbittracheal epithelium by adenine nucleotides: mediation by P2-purinoceptors. Am J Respir Cell Mol Biol 13:410-417, 1995.' r. Z& D0 P% L+ P9 Z" f

5 c' Z& g5 e( ?2 h1 q

Bailey MA,Hillman KA, and Unwin RJ. P2 receptors in the kidney. J AutonNerv Syst 81:264-270, 2000.% S0 k, m$ G7 ?/ L( s3 U( s4 h

. ]. e; o& h- G0 q* g4 }+ _
Bao Y, PucciML, Chan BS, Lu R, Ito S, and Schuster VL. Prostaglandin transporter PGTis expressed in cell types that synthesize and release prostanoids. Am J Physiol Renal Physiol 282:F1103-F1110, 2002.
, v. s8 V/ x; }9 E  o

Barnett J, ChowJ, Ives D, Chiou M, Mackenzie R, Osen E, Nguyen B, Tsing S, Bach C, Freire J,Chan H, Sigal E, and Ramesha C. Purification, characterization andselective inhibition of human prostaglandin G/H synthase 1 and 2 expressed inbacculovirus system. Biochim Biophys Acta 1209: 130-139,1994.: T4 R: j! s: D3 [& l- R! M9 D

8 Z+ f5 |" [0 D0 D( n) z# c; i
% j( o( i% B& ^: F
Bhattacharyya DK, Lecomte M, Dunn J, Morgans DJ, and Smith WL. Selective inhibition of prostaglandin endoperoxidase synthase-1(cyclooxygenase-1) by valerylsalicylic acid. Arch BiochemBiophys 317:19-24, 1995." i& @: A0 m! [& ]  W
" U  ?" f2 A$ X7 D8 J
3 L7 |) q3 {( F; [
  X- x* v' c1 ]7 X$ |2 i
Bonventre JV. The 85-kD cytosolic phospholipaseA 2 knockout mouse: a new tool for physiology and cell biology. J Am Soc Nephrol 10:404-412, 1999.
; V. e2 m) e9 @  ~% u$ H, z" j9 @
2 j8 M6 K: Q9 z) P- v0 r  a+ l6 }
1 `" i0 n1 M% L3 v
Carlson NG,Bacchi A, Rogers SW, and Gahring LC. Nicotine blocks TNF- -mediatedneuroprotection to NMDA by -bungarotoxin-sensitive pathway. J Neurobiol 35:29-36, 1998.* T4 L  o# E) O$ h: n

' l1 N6 n7 q8 A9 Z' i+ S9 H
9 }" ^- Y) S' ^  ?) Q4 e+ t
Carter TD,Hallam TJ, Cusack NJ, and Pearson JD. Regulation ofP2y-purinoceptor-mediated prostacyclin release from human endothelial cells bycytoplasmic calcium concentration. Br J Pharmacol 95: 1181-1190,1988.
6 ^% p! g6 j1 c. k

$ T1 S& c- {; g) h
Communi D,Janssens R, Suarez-Huerta N, Robaye B, and Boeynaems JM. Advances insignaling by extracellular nucleotides, the role and transduction mechanismsof P2Y receptors. Cell Signal 12: 351-360,2000.3 n* ?. F" t: e3 J$ r6 [9 D. r
/ N2 n) c, B- E8 W* u, F

Demolle D,Lagneau C, and Boeynaems JM. Stimulation of prostacyclin release fromaortic smooth muscle cells by purine and pyrimidine nucleotides. Eur J Pharmacol 155:339-343, 1988.$ [+ i4 ~: t9 ~: b2 O0 x
; x1 r$ e& M0 P5 l5 R

Ecelbarger CA,Maeda Y, Gibson CC, and Knepper MA. Extracellular ATP increasesintracellular calcium in rat terminal collecting duct via a nucleotidereceptor. Am J Physiol Renal Fluid Electrolyte Physiol 267: F998-F1006,1994.
, |; p! a7 k, S0 ~# t. J8 C" m: p2 i

Ecelbarger CA,Sands JM, Doran JJ, Cacini W, and Kishore BK. Expression of salt and ureatransporters in rat kidney during cisplatin-induced polyuria. Kidney Int 60:2274-2282, 2001., I! ?1 c, z& W7 _5 C, a& Z
9 B4 {5 ?' D+ D+ g+ o; w# l/ s+ R

4 M) M1 }6 X3 n0 h
Endo S, NomuraT, Chan BS, Lu R, Pucci ML, Bao Y, and Schuster VL. Expression of PGT inMDCK cell monolayers: polarized apical localization and induction of active PGtransport. Am J Physiol Renal Physiol 282: F618-F622,2002.3 J( e, f, O1 B
0 w& a; Q7 e8 }4 A/ Q: {) B+ o8 {
9 r/ |& n2 E( }  s, F. E! z. P
* h- Q  w! S, [5 |
Gaion RM andTrento M. The role of adrenergic, purinergic and opiate receptors in thecontrol of prostacyclin-induced contraction in the guinea-pig ileum. Arch Int Pharmacodyn Ther 271:33-44, 1984.' g* D/ Z6 R; X" R" D  h4 v) |
* d, P, u, J  q1 F+ k

Gebicke-Haerter PJ, Wurster S, Schobert A, and Hertting G. P2-purinoceptor-induced prostaglandin synthesis in primary rat astrocytecultures. Naunyn Schmiedebergs Arch Pharmacol 338: 704-707,1988.

: x! }* e4 L0 ~/ A/ f* z
Harris RC andBreyer MD. Physiological regulation of cyclooxygenase-2 in the kidney. Am J Physiol Renal Physiol 281:F1-F11, 2001.
) Q1 o( j1 ~) u! e! c% j2 ^' r

Hirabayashi T and Shimizu T. Localization and regulation of cytosolic phospholipaseA 2. Biochim Biophys Acta 1488: 124-138,2000.  `0 a; |% e/ s. e- [  Y: a
1 @, w8 u! Q- u' \0 x/ z1 R
' f7 }3 O3 p, ^/ a: Q; @( S

Inscho EW. Renal microvascular effects of P2 receptor stimulation. Clin ExpPharmacol Physiol 28:332-339, 2001.
. y) j* ~& X9 F/ ~

Ishimoto H,Nakahata N, Matsuoka I, and Nakanishi H. Effects of ATP onphosphoinositide hydrolysis and prostaglandin E 2 generation inrabbit astrocytes. J Pharm Pharamcol 49: 520-540,1997.2 v4 C$ M0 K- u" y9 w4 \: A

2 F( k0 f! P5 r2 X3 I. R# g
Jackson BA. Prostaglandin E 2 synthesis in the inner medullary collecting ductof the rat: implications for vasopressin-dependent cyclic AMP formation. J Cell Physiol 129:60-64, 1986.  k8 d7 \8 {. l5 O
, J7 A. q1 E# d
8 a+ ~9 D7 K2 J' i) j" S: u5 V+ C
. h# g# \; d3 I4 R( N" [
Kalgutkar AS,Crews BC, Rowlinson SW, Garner C, Seibert K, and Marnett LJ. Aspiring-likemolecules that covalently inactivate cyclooxygenase-2. Science 280:1268-1270, 1998.. f6 A6 x# U4 X" X5 d# k

8 {/ f$ Y/ C  f+ m# `7 }, T
Kishore BK,Chou CL, and Knepper MA. Extracellular nucleotide receptor inhibitsAVP-stimulated water permeability in inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 269: F863-F869,1995.6 a+ l2 F; \: P5 j# u0 N. w3 ^0 A
+ ]& r0 A! C; W2 C
' s3 X  `# w/ q/ E

Kishore BK,Ginns SM, Krane CM, Nielsen S, and Knepper MA. Cellular localization ofP2Y 2 purinoceptor in rat renal medulla and lung. Am JPhysiol Renal Physiol 278:F43-F51, 2000.* ^$ N1 i0 }4 W6 y

8 S. j: p) G, S+ g
Kishore BK,Krane CM, Di Iulio D, Menon AG, and Cacini W. Expression of renalaquaporins 1, 2, and 3 in a rat model of cisplatin-induced polyuria. Kidney Int 58:701-711, 2000.0 j, g& j6 t, W! w8 Q

- M! o! ]+ c# f0 l
: j- D. @1 B& y9 _
Kishore BK,Wade JB, Schorr K, Inoue T, Mandon B, and Knepper MA. Expression ofsynaptotagmin VIII in rat kidney. Am J Physiol RenalPhysiol 275:F131-F142, 1998.+ J/ G8 v( \! b+ R4 q) d; L  T
9 d/ {* e& J6 k. f- y  a4 J6 ]
$ G, R- l% }! g+ _1 u

Leipziger J. Control of epithelial transport via luminal P2receptors. Am J Physiol Renal Physiol 284: F419-F432,2003.# K' Z+ f# G. Q" @; z
  T6 l0 q3 _& q' o& T" B

Liu P, Lalor D,Bowser SS, Hayden JH, Wen M, and Hayashi J. Regulation of arachidonic acidrelease and prostaglandin E 2 production in thymic epithelial cellsby ATP S and transforming growth factor-. CellImmunol 188:81-88, 1998.* I+ `$ o/ l" V# j1 f+ r
' W6 Y* {, V5 g
8 D, _$ u' F% p8 b$ d

Nadler SP,Zimpelmann JA, and Hebert RL. PGE 2 inhibits water permeabilityat a post-cAMP site in rat terminal inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 262: F229-F235,1992.7 V, w8 K* d. y: C( G7 ^, \/ w

) p1 ?$ P0 H2 Z) x6 _4 S1 r
Neethling FA,Koscec M, Oriol R, Cooper DK, and Koren E. A reliable, rapid andinexpensive two-color fluorescence assay to monitor serum cytotoxicity inxenotransplantation. J Immunol Methods 222: 31-44,1999.
0 r; k  x! }/ L3 o( n5 Q+ ~

North RA. Molecular physiology of P2X receptor. Physiol Rev 82: 1013-1067,2002.! q& M1 i2 @: c

2 o5 m% B! f5 i# u% i& z
1 P- k& `3 V* S0 U8 A  I& Z1 b' L0 x
North RA andSuprenant A. Pharmacology of cloned P2X receptor. Annu RevPharmacol Toxicol 40:563-580, 2000.; K4 B/ |) v% {+ u$ m7 g& @3 I. }
% T4 I( M% e* d% X/ G; _

Pearson JD,Slakey LL, and Gordon JL. Stimulation of prostaglandin production throughpurinoceptors on cultured porcine endothelial cells. BiochemJ 214:273-276, 1983.5 }2 ]: W& u+ }
3 q, t5 h. |- L1 s) T- w

5 q4 l" O. R- Z9 p; Y- U
Pfeilschifter J, Thuring B, and Festa F. Extracellular ATPstimulates poly(inositol phospholipid) hydrolysis and eicosanoid synthesis inmouse peritoneal macrophages in culture. Eur J Biochem 186: 509-513,1989.

4 F/ W, y  E( o. v
9 Y* }. @/ e0 n
Roman RJ andLechene C. Prostaglandin E 2 and F 2 reducedurea reabsorption from the rat collecting duct. Am J Physiol RenalFluid Electrolyte Physiol 241:F53-F60, 1981.
( Y2 {% ?# J/ I! G6 n. v+ B/ L  ]# w

Rouch AJ andKudo LH. Role of PGE 2 in 2 -induced inhibitionof AVP- and cAMP-stimulated H 2 O, Na  , and urea transportin rat IMCD. Am J Physiol Renal Physiol 279: F294-F301,2000.
5 |! x$ T! W7 d, q" p  E0 m; ^

2 x$ s8 d8 L# m. w8 w
Schwiebert EM. ATP release mechanisms, ATP receptors andpurinergic signaling along the nephron. Clin Exp PharmacolPhysiol 28:340-350, 2001.

$ F; Q$ v) Q; D$ [% b: }6 x" c
4 I' J+ h9 Z, [
Schwiebert EM and Kishore BK. Extracellular nucleotide signaling along the renalepithelium. Am J Physiol Renal Physiol 280: F945-F963,2001.) _2 H! O7 ?$ e% m

7 j  r0 a( K) ~
" x! C! w7 g2 @
Takikawa R,Kurachi Y, Mashima S, and Sugimoto T. Adenosine-5'-triphosphate-induced sinus tachycardia mediated byprostaglandin synthesis via phospholipase C in the rabbit heart. Pflügers Arch 417:13-20, 1990.4 _8 q# R0 a: ?2 h
  y6 O, S! u. N+ Q( R

0 u( U7 a8 e# _
Teitelbaum I. Hormone signaling systems in inner medullarycollecting ducts. Am J Physiol Renal Fluid ElectrolytePhysiol 263:F985-F990, 1992.5 N, \% C9 c' D* P5 p
8 E5 \" v$ D$ ]! I0 ~; v, |

/ B6 i/ q: j  X9 O8 m+ C( B
Vitzthum H, AbtI, Einhellig S, and Kurtz A. Gene expression of prostanoid-forming enzymesalong the rat nephron. Kidney Int 62: 1570-1581,2002.) U1 t; g- \; ~- ^6 _
1 a* e) Z4 }$ W, G0 |" w# t, Z

Von Kugelgen I and Wetter A. Molecular pharmacology of P2Y-receptors. NaunynSchmiedebergs Arch Pharmacol 362:310-323, 2000.

6 q4 B3 P! R0 [3 U/ Q

Xing M,Firestein BL, Shen GH, and Insel PA. Dual role of protein kinase C in theregulation of cPLA 2 -mediated arachidonic acid release by P2ureceptors in MDCK-D1 cells: involvement of MAP kinase-dependent and-independent pathways. J Clin Invest 99: 805-814,1997.: r7 u, V  a  z7 ~
' V0 c+ m  W3 G$ N0 r$ ^
7 S& A7 B7 @+ q. e& Z. C
0 Y8 P  }8 j0 {* l& o3 f- M) G0 B
Zimmermann K,Reeh PW, and Averbeck B. ATP can enhance the proton-induced CGRP releasethrough P2Y receptors and secondary PGE 2 release in isolated ratdura mater. Pain 97:259-265, 2002.

收藏0 分享0 顶0 踩0