Activation of EP4 receptors contributes to prostaglandin E 2 -mediated stimulation of renal sensory nerves

【摘要】  Induction of cyclooxygenase-2 (COX-2) in the renal pelvic wall increases prostaglandin E 2 (PGE 2 ) leading to stimulation of cAMP production, which results in substance P (SP) release and activation of renal mechanosensory nerves. The subtype of PGE receptors involved, EP2 and/or EP4, was studied by immunohistochemistry and renal pelvic administration of agonists and antagonists of EP2 and EP4 receptors. EP4 receptor-like immunoreactivity (LI) was colocalized with calcitonin gene-related peptide (CGRP)-LI in dorsal root ganglia (DRGs) at Th 9 -L 1 and in nerve terminals in the renal pelvic wall. Th 9 -L 1 DRG neurons also contained EP3 receptor-LI and COX-2-LI, each of which was colocalized with CGRP-LI in some neurons. No renal pelvic nerves contained EP3 receptor-LI and only very few nerves COX-2-LI. The EP1/EP2 receptor antagonist AH-6809 (20 µM) had no effect on SP release produced by PGE 2 (0.14 µM) from an isolated rat renal pelvic wall preparation. However, the EP4 receptor antagonist L-161,982 (10 µM) blocked the SP release produced by the EP2/EP4 receptor agonist butaprost (10 µM) 12 ± 2 vs. 2 ± 1 and PGE 2, 9 ± 1 vs. 1 ± 0 pg/min. The SP release by butaprost and PGE 2 was similarly blocked by the EP4 receptor antagonist AH-23848 (30 µM). In anesthetized rats, the afferent renal nerve activity (ARNA) responses to butaprost 700 ± 100 and PGE 2 ·780 ± 100%·s (area under the curve of ARNA vs. time) were unaffected by renal pelvic perfusion with AH-6809. However, 1 µM L-161,982 and 10 µM AH-23848 blocked the ARNA responses to butaprost by 94 ± 5 and 78 ± 10%, respectively, and to PGE 2 by 74 ± 16 and 74 ± 11%, respectively. L-161,982 also blocked the ARNA response to increasing renal pelvic pressure 10 mmHg, 85 ± 5%. In conclusion, PGE 2 increases renal pelvic release of SP and ARNA by activating EP4 receptors on renal sensory nerve fibers.

EP3 receptors; cyclooxygenase-2; substance P; butaprost; L-161,982

【关键词】  Activation receptors contributes prostaglandin mediated stimulation

PROSTAGLANDIN E 2 (PGE 2 ) is the major product of cyclooxygenase (COX)-induced metabolism of arachidonic acid in the kidney ( 3 ) and plays a critical role for normal renal function by its effects on renal microvasculature and urinary water and sodium excretion. In addition to its direct effects on tubular sodium and water reabsorption, our studies indicate that PGE 2 also modulates urinary sodium excretion by its effects on afferent renal nerves ( 25, 27, 29 ).

The majority of the afferent renal nerves containing substance P and calcitonin gene-related peptide (CGRP) are located in the renal pelvic wall ( 28, 33, 54 ). These nerves are activated by increases in renal pelvic pressure of a magnitude, 3 mmHg ( 26, 30 ), seen during moderate volume expansion. The increase in afferent renal nerve activity (ARNA) produced by the increased renal pelvic pressure leads to a reflex decrease in efferent renal sympathetic nerve activity (ERSNA) and a diuresis and natriuresis, i.e., a renorenal reflex response ( 31 ).

Among the various mechanisms activated by stretching the renal pelvic wall is induction of COX-2 leading to increased renal pelvic synthesis of PGE 2 ( 25, 27, 29 ). PGE 2 increases the release of substance P via activation of the cAMP-protein kinase A transduction pathway ( 25 ). Substance P activates the afferent renal nerves by stimulating neurokinin-1 receptors in the renal pelvic area ( 32 ). Regarding the role of CGRP, our studies suggest that CGRP potentiates the effect of substance P by retarding the metabolism of released substance P ( 17 ).

COX-2 mRNA is expressed in the renal pelvic wall ( 27 ) but it is not known whether COX-2 is present in or adjacent to the sensory nerves in the pelvic wall. Also, there is currently little evidence for COX-2 in dorsal root ganglia (DRG) in normal rats ( 9, 50, 51 ). However, it is well established that COX-2 mRNA and protein are present in areas in the brain and spinal cord involved in processing and integration of nociceptive visceral and sensory input ( 4, 52 ). Therefore, we studied whether COX-2 is localized in the Th 9 -L 1 DRGs and in the afferent nerves in the renal pelvic wall using immunohistochemistry. The DRGs at Th 9 -L 1 contain the majority of the cell bodies of the afferent renal nerves ( 7, 14, 54 ).

PGE receptors have been classified into four general subtypes, EP1, EP2, EP3, and EP4 based on cloning and pharmacological interventions ( 2, 41 ). Stimulation of EP1 receptors leads to activation of protein kinase C and increases in intracellular calcium. EP2 and EP4 receptors are coupled through the G s protein to increase cAMP. EP3 receptors have multiple splice variants. Although activation of these variants can lead to increases in intracellular calcium and increases or decreases in cAMP, the major effect of EP3 activation is a decrease in cAMP. The important role for activation of cAMP in the PGE 2 -mediated release of substance P and activation of renal mechanosensory nerves ( 25 ) suggests that PGE 2 exerts its effects by stimulating EP2 and/or EP4 receptors in the renal pelvic area. These findings together with the expression of EP4 mRNA in the renal pelvic wall ( 6 ) led us to examine whether EP4 receptors are located on or close to the sensory nerves in the pelvic wall and in Th 9 -L 1 DRG neurons using immunohistochemistry.

In parallel functional studies, we examined whether activation of EP2 and/or EP4 receptors contributed to the PGE 2 -mediated activation of renal sensory nerves. Because EP3 receptors are widely distributed in the central nervous system involved in the processing of peripheral sensory information, including DRGs ( 40 ), and in the renal medulla ( 5, 20, 47 ), we also searched for EP3 receptors in close conjunction to the nerve fibers in the renal pelvic wall.

METHODS

The experimental protocols were approved by the Institutional Animal Care and Use Committee and performed according to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health.

The study was performed in male Sprague-Dawley rats, weighing 188-454 g (mean 287 ± 5 g), anesthetized with pentobarbital sodium (0.2 mmol/kg ip) and fed a normal-sodium diet.

Immunohistochemical Procedures

The immunohistochemical procedures for kidney and DRG tissue have been previously described in detail ( 15, 28 ). In brief, male Sprague-Dawley rats anesthetized with pentobarbital sodium (0.2 mmol/kg ip) were perfused transcardially with fixative containing 4% wt/vol paraformaldehyde and 0.2% wt/vol picric acid in 0.1 M phosphate-buffered NaCl. The kidneys and Th 9 -L 1 DRG were quickly dissected, placed first in fixative, and then stored in 10% sucrose at 4°C. DRGs, embedded in O.C.T. compound (Tissue-Tek, Sakura Finetek, Torrence, CA), and kidneys, frozen in CO 2, were cut at 14 µm with a cryostat (Microm, Heidelberg, Germany) and thaw-mounted onto chromium potassium sulfate/gelatin-coated slides.

EP4 and EP3 receptors and COX-2. Sections were processed for tyramide signal amplification (TSA) immunohistochemistry (1, TSA Plus, PerkinElmer Life and Analytical Sciences, Boston, MA). The tissues were incubated overnight with primary antiserum for the EP4 human receptor ( 39 ) (rabbit; 1:6,000), the EP3 rat receptor ( 40 ) (rabbit; kidney, 1:400; DRGs, 1:100), or murine COX-2 (rabbit; kidney, 1:1,000; DRGs, 1:2,400; Cayman Chemical, Ann Arbor, MI). The following day, horseradish peroxidase-conjugated swine antirabbit IgG (1:200; DAKO, Copenhagen, Denmark) was applied followed by biotinylated tyramine. The reactions were detected with streptavidin conjugated with fluorescein. The specificity of the antisera was tested by preincubation of the primary antisera with an excess amount of the fusion protein used as the immunogen, the concentrations being 50, 250, and 10 µg/ml for the immunogens of EP4 and EP3 receptors and COX-2, respectively.

CGRP, tyrosine hydroxylase, and -smooth muscle actin. After completion of the protocol for TSA for detection of EP4 and EP3 receptors or COX-2, tissue sections were further processed by the indirect immunofluorescence technique ( 11 ). The tissue sections were incubated overnight with primary antiserum for CGRP ( 28 ) (mouse; 1:400; Drs. J. H. Walsh and H. C. Wong), tyrosine hydroxylase (TH; mouse; 1:400; Incstar, Stillwater, MN), or -smooth muscle (SM) actin (mouse; 1:400, Sigma, St. Louis, MO). The tissue-bound antibodies were detected by Rhodamine-Red-X-conjugated donkey anti-mouse antibody (1:80, Jackson Immuno Research, West Grove, PA).

The sections were examined in a Nikon Eclipse E600 fluorescence microscope (Tokyo, Japan) and in a Radiance Plus confocal laser-scanning system (Bio-Rad, Hemel Hemstead, UK) installed on a Nikon Eclipse E600 fluorescence microscope. Digital images were acquired with Nikon DXN 1200 digital still camera or the confocal system and optimized for image resolution, brightness, and contrast and color images were merged using Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA).

In Vitro Studies

The procedures for stimulating the release of substance P from an isolated rat renal pelvic wall preparation have been previously described in detail ( 21 ). In short, renal pelvises were placed in wells containing 400 µl of HEPES/indomethacin buffer ( 21 ). Indomethacin was included in the incubation buffer to minimize the influence of endogenous PGE 2 on substance P release.

The experiment was started following a 2-h equilibration period. All experiments consisted of four 5-min control, one 5-min experimental, and four 5-min recovery periods. The incubation medium, aspirated every 5 min, was placed in siliconized vials and stored at -80°C for later analysis of substance P.

Effects of an EP1/EP2 receptor antagonist on PGE 2 -mediated substance P release. One group ( n = 8) was studied. Throughout the experiment, the ipsilateral pelvis was incubated in HEPES/indomethacin buffer containing the EP1/EP2 receptor antagonist AH-6809 (20 µM) ( 53 ) and the contralateral pelvis in HEPES/indomethacin buffer containing vehicle (0.15 M NaCl). During the experimental period, PGE 2 (0.14 µM) was added to the incubation baths of both pelvises.

Effects of EP4 receptor antagonists on substance P release. Five groups were studied. In the first ( n = 14) and second groups ( n = 8), the ipsilateral pelvis was incubated in HEPES/indomethacin buffer containing the EP4 receptor antagonist L-161,982 (10 µM) ( 34 ) and the contralateral pelvis in buffer containing vehicle (0.15 M NaCl) throughout the experiment. During the experimental period, PGE 2 (0.14 µM) was added to the incubation baths of both pelvises in the first group and the EP2/EP4 agonist butaprost (10 µM) ( 16 ) in the second group. In the third group ( n = 8), the ipsilateral pelvis was incubated in L-161,983 (10 µM), the inactive enantiomer of L-161,982, and the contralateral pelvis in vehicle throughout the experiment. PGE 2 (0.14 µM) was added to both pelvises during the experimental period. The experimental protocols in the fourth ( n = 8) and fifth groups ( n = 6) were similar to those in the first two groups, except that the ipsilateral pelvis was incubated with the EP4 receptor antagonist AH-23848 (30 µM) ( 10 ) throughout the experiment.

In Vivo Studies

After induction of anesthesia, an intravenous infusion of pentobarbital sodium (0.04 mmol·kg -1 ·h -1 ) at 50 µl/min into the femoral vein was started and continued throughout the course of the experiment. Arterial pressure was recorded from a catheter in the femoral artery. The procedures for stimulating and recording ARNA have been previously described in detail ( 22 - 32 ). In brief, the left kidney was approached by a flank incision, a PE-10 catheter was placed in the right ureter for collection of urine, and a PE-60 catheter was placed in the left ureter with its tip in the renal pelvis. The left renal pelvis was perfused, via a PE-10 catheter placed inside the PE-60 catheter, throughout the experiment at 20 µl/min with vehicle or various renal perfusates described below. In two groups of rats, renal pelvic pressure was increased by elevating the fluid filled catheter above the level of the kidney. ARNA was recorded from the peripheral portion of the cut end of one renal nerve branch. ARNA was integrated over 1-s intervals, the unit of measure being microvolts per second per 1 s. Postmortem renal nerve activity was subtracted from all values of renal nerve activity. ARNA was expressed in percentage of its baseline value during the control period (22-32).

Experimental Protocol

Effects of an EP1/EP2 receptor antagonist on the ARNA responses to PGE 2 and butaprost. One group ( n = 7) was studied. The experiment was divided into two parts with a 10-min interval. Each part consisted of two 10-min control, 5-min experimental, and 10-min recovery periods. PGE 2 (0.14 µM) and butaprost (10 µM) were added to the renal pelvic perfusate during the two experimental periods in random order. The renal pelvis was perfused throughout the experiment, during the first part with vehicle (0.15 M NaCl), and during the second part with AH-6809 (20 µM).

Effects of EP4 receptor antagonists on the ARNA responses to PGE 2 and butaprost. Three groups were studied. The experimental protocols in the three groups were similar to that described above except the renal pelvis was perfused with AH-23848 (10 µM; n = 8), L-161,982 (1 µM; n = 8), or vehicle ( n = 7) during the second part of the experiments. Thus the last group served as time control.

Effects of an EP4 receptor antagonist on the ARNA responses to increased renal pelvic pressure. Two groups were studied. In the first group, n = 8, the experiment was divided into three parts separated by a 10-min interval. A 10-min control, 5-min experimental, and 10-min recovery period was performed during each part. The renal pelvis was perfused during the first part with vehicle, the second part with L-161,982 (5 µM), and the third part with vehicle. In the second group ( n = 8), the experiment was divided into two parts, each part consisting of a 10-min control, 5-min experimental, and 10-min recovery period. The renal pelvis was perfused with vehicle during the first part and L-161,983 (5 µM) during the second part. Renal pelvic pressure was increased 10 mmHg during each of the experimental periods in the two groups.

Drugs. L-161,982 and L-161,983 were gifts from Merck Frosst Canada (Center for Therapeutic Research, Kirkland, Quebec, Canada) and AH-23848 from GlaxoSmithKline Research and Development (Research Triangle Park, NJ). Substance P antibody (IHC 7451) was acquired from Penninsula Laboratories (San Carlos, CA) and PGE 2 and butaprost from Cayman Chemicals. All other agents were from Sigma unless otherwise stated. Indomethacin was dissolved together with Na 2 CO 3 (2:1 weight ratio) in HEPES buffer. Butaprost, methyl acetate solution evaporated, was dissolved in DMSO and further diluted in the various incubation buffers (in vitro studies) or 0.15 M NaCl (in vivo studies), final DMSO concentration being 0.1%. All other agents were dissolved in the various incubation buffers (in vitro studies) or 0.15 M NaCl (in vivo studies).

Analytic Procedures

Right urinary sodium excretion, measured in two groups, was expressed per gram kidney weight. Urinary sodium concentrations were determined with a flame photometer.

Substance P in the incubation medium was measured by ELISA, as previously described in detail ( 21 - 29 ).

Statistical Analysis

In vitro, the release of substance P during the experimental period was compared with that during the control and recovery periods using Friedman 2-way analysis of variance and shortcut analysis of variance. The Wilcoxon matched-pairs signed-rank test was used to compare the increases in substance P release from ipsilateral and contralateral renal pelvises. In vivo, the ARNA responses to PGE 2, butaprost, and renal pelvic pressure were calculated as the area under the curve (AUC) of ARNA vs. time, where ARNA was expressed as percentage of its baseline value during the bracketing control and recovery periods. Friedman 2-way analysis of variance and shortcut analysis of variance were used to determine the effects of the various treatments on the ARNA responses within each rat. A significance level of 5% was chosen. Data in text and figures are expressed as means ± SE ( 45, 48 ).

RESULTS

Immunohistochemistry

Localization of EP4 receptors in renal tissue and DRG. Many neuronal cell bodies in Th 9 DRGs were labeled with the antibody to EP4 receptors ( Fig. 1 a ). The EP4 receptor-LI was blocked by adsorption with the peptide ( Fig. 1 b ). Double-labeling experiments showed that some of the neuronal cell bodies that were EP4 receptor-immunoreactive (ir) also contained CGRP-like immunoreactivity (LI) ( Fig. 1, c - e ). A similar distribution of EP4 receptor- and CGRP-LI was found in all Th 9 -L 1 DRGs studied. Furthermore, strong labeling with the EP4 receptor antibody was observed in nerve fibers in the renal pelvic wall ( Fig. 1 g ). This staining was also blocked by adsorption with the peptide ( Fig. 1 h ). Double-labeling showed that the EP4 receptor-ir nerves in the pelvic wall also contained CGRP-LI ( Fig. 1, f, i, j ). Higher magnification of a thin nerve bundle in the renal pelvic wall revealed EP4 receptor-LI in and adjacent to CGRP-ir nerve fibers ( Fig. 1 f ). Likewise, in thicker nerve bundles in the renal pelvic area, EP4 receptor-LI was found in CGRP-ir nerve fibers ( Fig. 2, a - c ). However, there were also EP4 receptor-ir nerve fibers that did not contain CGRP-LI. Double-labeling kidney sections with antibodies to the EP4 receptors and TH, a marker for sympathetic nerves, showed some nerve bundles in the renal pelvic wall containing both receptor and enzyme but also single-labeled fibers ( Fig. 2, d - f ).

Fig. 1. a : Immunofluorescence labeling for EP4 receptors shows many positive neuronal cell bodies in T 9 dorsal root ganglia (DRG), x 120 magnification. In this slide, 40% of the cell bodies contain EP4-LI. Double-labeling this ganglion with antibodies to EP4 receptors ( c ) and calcitonin gene-related peptide (CGRP; d ) shows that 40% of the EP4-ir cells also contain CGRP-LI (arrows), x 240 magnification ( e ). Immunofluorescence double-labeling of renal tissue for EP4 receptors ( g ) and CGRP ( i ) shows colocalization in virtually all renal pelvic nerve terminals (arrows), x 240 magnification ( i ) and x 720 magnification ( f ). Preadsorption with the peptide blocks the EP4 receptor labeling both in DRG ( b ) and pelvic nerves in ( h ).

Fig. 2. Immunofluorescence double-labeling of renal tissue for EP4 receptors ( a ) and CGRP ( b ) and for EP4 receptors ( d ) and tyrosine hydroxylase (TH; e ), x 1,240 magnification. In small nerve bundles in the renal pelvic area ( a - c ) and in the renal pelvic wall ( d - f ), EP4 receptor-LI is present in nerve fibers on and/or close to CGRP-ir axons ( c ) and on and/or close to TH-ir axons ( f ).

The EP4 receptor antibody also labeled thin nerve fibers along arterioles close to glomeruli, among vasa recta bundles, and in arterial walls and veins throughout the kidney (data not shown). EP4 receptor-LI was also found in tissue surrounding the arteries in the renal pelvic area. Most of the EP4 receptor-ir nerve bundles and nerve terminals in nonpelvic renal tissue were also TH-ir. Furthermore, EP4 receptor staining was also found in the apical membrane in cortical tubular structures adjacent to the glomeruli. All EP4 receptor staining was prevented by preadsorption with the peptide used for immunization.

Localization of EP3 receptors in renal tissue and DRG. In agreement with previous studies ( 40 ), EP3 receptor-LI was found in DRGs ( Fig. 3 a ). Furthermore, our studies showed that a small portion of the neuronal cell bodies in Th 9 -L 1 DRGs contained both EP3 receptor- and CGRP-LI ( Fig. 3, a - c ). Therefore, we examined whether renal pelvic sensory nerve fibers contained EP3 receptor-LI. However, no EP3 receptor-LI was found in the CGRP-ir nerve fibers in the pelvic wall ( Fig. 3, d - f ). Also, EP3 receptor-LI was not found on TH-ir nerve fibers in the renal tissue (data not shown). Instead, at the renal pelvic tip, strong labeling with the EP3 receptor antibody was observed in fibers that were also labeled with an -SM actin antibody, a marker for smooth muscle fibers ( Figs. 3, g - i, and 4 a ). In agreement with in situ hybridization studies ( 5, 47 ), there was strong labeling with the EP3 receptor antibody in macula densa cells (data not shown) and tubular structures in the inner stripe of the outer medulla and papilla ( Fig. 4, c and e ). The EP3 receptor antibody also labeled the apical/brush-border membrane in proximal tubules ( Fig. 4 f ). The EP3 receptor staining of the muscle fibers in the pelvic tip, the tubular structures in the inner stripe of outer medulla, and the proximal tubular apical/brush border were blocked by adsorption with the peptide ( Fig. 4, b and d ).

Fig. 3. Immunofluorescence double-labeling for EP3 receptors ( a ) and CGRP ( b ) in T 11 DRG shows colocalization in a small portion of the neuronal cell bodies (arrows, c ). In this slide, <20% of the EP3-ir neurons contain CGRP-LI (arrows, c ). Double-labeling of renal tissue for EP3 receptors ( d ) and CGRP ( e ) shows lack of colocalization in the renal pelvic wall ( f ). Double-labeling for EP3 receptors ( g ) and -smooth muscle (SM) actin ( h ) shows colocalization in muscle fibers at the tip of the renal pelvic wall (yellow arrows; i ), x 240 magnification. EP3 receptor-LI was also found on the membrane of fat cells (*, d ).

Fig. 4. Immunofluorescence labeling of renal tissue shows EP3 receptor-LI in muscle fibers in the pelvic wall, x 240 magnification ( a ), in tubular structures in the inner stripe of the outer medulla (IS), x 48 magnification ( c ), in inner medulla (IM), x 240 magnification ( e ), and on the apical/brush-border membrane in proximal tubules, x 1,440 magnification ( f ). The EP3 receptor labeling in renal pelvic wall and IS is blocked by adsorption with the peptide ( b and d ).

Localization of COX-2 in renal tissue and DRG. Because PGE 2 is known only to act in the vicinity of its production, we examined whether COX-2 is distributed in DRGs and in or close to the renal sensory nerves. Numerous neuronal cell bodies in Th 9 -L 1 DRGs were labeled with the antibody to COX-2 ( Fig. 5 a ). Double-labeling showed many more neuronal cell bodies labeled with COX-2 antibody ( Fig. 5 a ) than the CGRP antibody ( Fig. 5 b ). However, many CGRP-ir cell bodies also contained COX-2. Blocking the COX-2 antibody with the immunogenic peptide abolished the labeling ( Fig. 5 c ).

Fig. 5. Immunofluorescence double-labeling of T 9 DRG shows many COX-2-ir ( a ) and fewer CGRP-ir ( b ) neuronal cell bodies. In these DRG sections, 40% of the COX-2-ir cell bodies also contain CGRP-LI (arrows, a and b ). Preadsorption with the peptide blocks the COX-2 labeling, x 120 magnification ( c ). In the kidney, there is COX-2-LI in the uroepithelium ( d ). Only a few CGRP-ir nerve fibers also contain COX-2-LI, x 240 magnification (arrows, d and e ). A nerve bundle in the renal pelvic wall contains COX-2-LI ( f ). Labeling an adjacent section for EP4 receptor ( g ) suggests presence of COX-2- and EP4 receptor-LI in the same nerve bundle in the renal pelvic wall, x 1,440 magnification.

In agreement with previous studies ( 19 ), COX-2-LI was localized in the macula densa cells and renal medullary interstitial cells (not shown). COX-2-LI was also observed in the uroepithelium ( Fig. 5 d ). Only very few sensory nerve fibers in the renal pelvic wall were labeled with the COX-2 antibody ( Fig. 5, d and e ). However, COX-2-LI was found in some nerve bundles along the renal pelvic wall ( Fig. 5 f ). In adjacent sections, the same nerve bundle also contained EP4 receptor-LI ( Fig. 5 g ).

In Vitro Studies

Effects of an EP1/EP2 receptor antagonist on PGE 2 -mediated substance P release. Because PGE 2 increases the renal pelvic release of substance P by stimulating cAMP production in the renal pelvic wall ( 25 ), we tested whether AH-6809, an EP receptor antagonist with equal affinity for EP1 and EP2 receptors and much greater affinity for EP2 than EP4 receptors ( 2, 53 ), would alter the substance P release produced by PGE 2. However, our data show that the increase in renal pelvic release of substance P produced by PGE 2 was unaltered by the presence of AH-6809 in the incubation bath ( Fig. 6 ).

Fig. 6. Effects of PGE 2 (0.14 µM) on the release of substance P from the isolated renal pelvic wall in the absence (vehicle) and presence of the EP1/EP2 receptor antagonist AH-6809 (20 µM). ** P < 0.01 vs. control and recovery periods.

Effects of EP4 receptor antagonists on substance P release. Butaprost, an agonist with a higher affinity for EP2 than EP4 receptors ( 2, 10, 41 ), activates renal sensory nerves by increasing cAMP activity, similar to PGE 2 ( 25 ). However, the concentration 4 µM ( 25 ), suggesting that this effect may not be related only to activation of EP2 but also EP4 receptors. This hypothesis was tested by examining the effects of two EP4 receptor antagonists with different molecular structures. As shown in Fig. 7, incubating the renal pelvises with the selective EP4 receptor antagonist L-161,982 abolished the increases in renal pelvic substance P release produced by either PGE 2 or butaprost. Likewise, the EP4 receptor antagonist AH-23848 blocked the renal pelvic release of substance P produced by PGE 2 and butaprost ( Table 1 ). Importantly, the PGE 2 -mediated release of substance P was not affected by L-161,983, the inactive enantiomer of L-161,982 ( Table 2 ).

Fig. 7. Effects of PGE 2 (0.14 µM; A ) and butaprost (10 µM; B ) on the release of substance P from the isolated renal pelvic wall in the absence (vehicle) and presence of the EP4 receptor antagonist L-161,982 (10 µM). ** P < 0.01 vs. control and recovery periods. P < 0.01 vs. increase in substance P release produced by PGE 2 /butaprost in the presence of L-161,982.

Table 1. Effects of the EP4 receptor antagonist AH-23848 (30 µM) on the renal pelvic release of substance P produced by PGE 2 (0.14 µM) and butaprost (10 µM) in an isolated renal pelvic wall preparation

Table 2. Effects of L-161,983 (10 µ M) the inactive enantiomer of L-161,982 on the PGE 2 -mediated release of substance P in an isolated renal pelvic wall preparation

In Vivo Studies

Effects of an EP1/EP2 receptor antagonist on the ARNA responses to PGE 2 and butaprost. Renal pelvic administration of PGE 2 and butaprost results in increases in ARNA that are of a similar magnitude and blocked by inhibiting adenylyl cyclase ( 25 ). PGE 2 (0.14 µM) and butaprost (10 µM) produced an increase in ARNA that was of a similar magnitude ( Fig. 8 ) and duration, 46 ± 8 and 34 ± 3 s, respectively. There were no significant differences among the increases in ARNA produced by PGE 2 and butaprost before and during renal pelvic perfusion with AH-6809. Arterial pressure (111 ± 3 mmHg) and heart rate (349 ± 15 beats/min) were unaltered by PGE 2, butaprost, and AH-6809.

Fig. 8. Effects of renal pelvic administration of PGE 2 (0.14 µM; A ) and butaprost (10 µM; B ) on ipsilateral afferent renal nerve activity (ARNA) in the presence of renal pelvic perfusion with vehicle and the EP1/EP2 receptor antagonist AH-6809 (20 µM). ** P < 0.01 vs. 0.

Effects of EP4 receptor antagonists on the ARNA responses to PGE 2 and butaprost. Because our in vivo studies suggested that activation of EP2 receptors did not contribute to the ARNA responses to PGE 2 and butaprost and our in vitro studies suggested that PGE 2 and butaprost increased substance P release by activation of EP4 receptors, we examined whether the ARNA responses to renal pelvic administration of PGE 2 and butaprost were blocked by renal pelvic perfusion with the EP4 receptor antagonists L-161,982 and/or AH-23848. As shown in Fig. 9 and Table 3, renal pelvic perfusion with either L-161,982 or AH-23848 produced marked blockade of the ARNA responses to both PGE 2 and butaprost. Neither L-161,982 nor AH-23848 altered arterial pressure (110 ± 3 and 109 ± 1 mmHg) or heart rate (353 ± 11 and 345 ± 13 beats/min) in the two groups, respectively. Time control experiments showed that repeated administration of PGE 2 and butaprost resulted in reproducible increases in ARNA ( Table 3 ).

Fig. 9. Effects of renal pelvic administration of PGE 2 (0.14 µM; A ) and butaprost (10 µM; B ) on ipsilateral ARNA in the presence of renal pelvic perfusion with vehicle and the EP4 receptor antagonist L-161,982 (1 µM). ** P < 0.01 vs. 0. P < 0.01 vs. ARNA responses to PGE 2 and butaprost during renal pelvic perfusion with vehicle.

Table 3. Effects of renal pelvic perfusion with the EP4 receptor antagonist AH-23848 (10 µ M) on the ARNA responses to renal pelvic administration with PGE 2 (0.14 µ M) and butaprost (10 µ M) and effects of repeated administration of PGE 2 and butaprost on ARNA in the presence of renal pelvic perfusion with vehicle

Effects of an EP4 receptor antagonist on the ARNA responses to increased renal pelvic pressure. Because the increase in ARNA produced by increased renal pelvic pressure involves increased renal PGE 2 syntheses and stimulation of cAMP production, and blocking renal EP4 receptors reduces the ARNA response to PGE 2, we examined whether renal pelvic perfusion with the EP4 receptor antagonist L-161,982 would alter the ARNA responses to increasing renal pelvic pressure. Elevating renal pelvic pressure 9.8 ± 0.2 mmHg increased ipsilateral ARNA ( Fig. 10 ) and contralateral urinary sodium excretion, 27 ± 10% ( P < 0.02) from 1.2 µmol·min -1 ·g -1. Renal pelvic perfusion with L-161,982 produced a reversible reduction of the ARNA and contralateral natriuretic responses, the latter being 14 ± 9% (not significant) and 38 ± 10% ( P < 0.02) during and after L-161,982, respectively. Arterial pressure (101 ± 2 mmHg) and heart rate (333 ± 21 beats/min) remained unaltered throughout the experiment. Subsequent studies examining the effects of L-161,983, the inactive enantiomer of L-161,982, showed that elevating renal pelvic pressure 10.2 ± 0.2 mmHg increased ipsilateral ARNA 4,640 ± 280 %·s (AUC) before and 5,220 ± 320 %·s (both P < 0.01) during renal pelvic perfusion with L-161,983. Likewise, L-161,983 had no effect on the contralateral natriuretic responses to increased renal pelvic pressure 19 ± 6% ( P < 0.02) from 1.7 ± 0.6 µmol·min -1 ·g -1 before and 32 ± 6% ( P < 0.01) from 2.2 ± 0.5 µmol·min -1 ·g -1 during perfusion with the inactive enantiomer.

Fig. 10. Effects of increasing renal pelvic pressure 9.8 ± 0.2 mmHg on ipsilateral ARNA in the presence of renal pelvic perfusion with vehicle and the EP4 receptor antagonist L-161,982 (5 µM). ** P < 0.01 vs. 0. P < 0.01 vs. ARNA responses to increased renal pelvic pressure during renal pelvic perfusion with vehicle.

DISCUSSION

The results of these studies show numerous EP4 receptor-ir thin nerve fibers in the renal pelvic wall. The majority of these nerve fibers also contained CGRP-LI. Because these two markers were also colocalized in Th 9 -L 1 DRG neurons, these findings suggest that EP4 receptor-LI is present in the majority of the sensory nerves in the renal pelvic wall. Studies using an isolated renal pelvic wall preparation showed that the increases in substance P release produced by PGE 2 and butaprost were blocked by the EP4 receptor antagonists AH-23484 and L-161,982 but not by AH-6809, an EP1/EP2 receptor antagonist. Likewise, our in vivo studies showed that the ARNA responses to renal pelvic administration of PGE 2 and butaprost were blocked by renal pelvic perfusion with AH-23484 and L-161,982 but not by AH-6809. Importantly, renal pelvic perfusion with L-161,982 also blocked the ARNA response to increased renal pelvic pressure. Taken together, our studies suggest that PGE 2 activates mechanosensory nerves by stimulating EP4 receptors located on or adjacent to the sensory nerve fibers in the renal pelvic wall.

EP4 Receptors in DRG and Neural and Nonneural Renal Tissue

Due to our previous studies showing that PGE 2 increases substance P release and activates renal mechanosensory nerves via stimulation of cAMP production ( 25 ), we reasoned that the EP receptor subtype involved was either of the EP2 or EP4 subtypes ( 10 ). Preliminary studies using an antibody raised against the human-EP2 receptor ( 39 ) failed to label any structures in the rat kidney. Although we cannot exclude that the human EP2 receptor antibody does not recognize rat EP2 receptors, a likely explanation may also be the very low expression of EP2 receptors in normal rat kidneys ( 20 ). On the other hand, EP4 receptors are more widespread throughout the body, including the kidney ( 6, 20, 41, 43 ). EP4 receptors have been found in hypothalamus and lower brain stem ( 41 ) and PGE 2 acting via EP4 receptors has an excitatory effect on parasympathetic preganglionic spinal neurons innervating the pelvic visceral organs ( 38 ). Of particular importance for the current studies are the findings showing that EP4 mRNA is expressed in DRGs ( 41 ). PGE 2 -mediated activation of EP4 receptors in cultured DRGs increases cAMP activity ( 46 ). In agreement with these findings, our present studies show EP4 receptor-LI in Th 9 -L 1 DRGs. Furthermore, the current data show that DRG neurons as well as renal pelvic nerve terminals contain both EP4 receptor- and CGRP-LI, suggesting that EP4 receptors are present in peripheral renal sensory nerves. These EP4 receptor-ir nerve fibers were distributed in the uroepithelium and in the muscle layer of the renal pelvic wall. Whether these EP4 receptors are derived from the DRGs and/or from a local synthesis, suggested from the demonstrated strong EP4 mRNA signal in the uroepithelium ( 6 ), is currently not known.

EP4 receptor-LI was also found in thin nerve fibers along glomerular arterioles, among vasa recta bundles, and in vessel walls throughout the kidney. Because the majority of these fibers also contained TH-LI, these findings suggest the presence of EP4 receptors on sympathetic renal nerves. The EP4-receptor labeling of apical membrane of cortical structures adjacent to glomeruli in the current study is consistent with the marked expression of EP4 receptor mRNA in rat distal tubules ( 20 ).

The labeling of nerve fibers in DRG and renal tissue was specific to the EP4 receptor antibody in the sense that it was abolished by preadsorption with the immunogenic peptide (see also Ref. 39 ).

Previous immunohistochemistry studies in human unfixed renal tissue have shown EP4 receptor-LI in arterial muscle wall ( 39, 49 ). This was not observed in the present study. The reason for this apparent discrepancy is not known but could be related to different treatment of the tissue, unfixed vs. fixed, and species studied, human vs. rat tissue.

EP3 Receptors in Renal Nonneural and Neural Tissue and DRG

Because of the considerable evidence for EP3 receptors in the areas of the central nervous system involved in the processing of sensory input, including nucleus tractus solitarii, laminae I and II of the dorsal spinal horn, nodose ganglia, and DRG ( 40 ) and the role of EP3c receptors in the PGE 2 -mediated activation of cAMP in DRG ( 46 ), we reasoned that EP3 receptors may also be present on peripheral renal sensory nerves. Our initial studies confirmed the presence of EP3 receptors in a small number of neuronal cell bodies in Th 9 -L 1 DRGs ( 40 ). Some of these neurons also contained CGRP-LI. However, we could not detect EP3-receptor-LI in any nerve fibers throughout the kidney. The lack of EP3 receptor labeling of peripheral renal nerve fibers was most likely not due to our antibody not recognizing the EP3 receptors because we found strong labeling with the EP3 receptor antibody in the tubular structures in the cortex, inner stripe of the outer medulla, and inner medulla/papilla. This is in agreement with previous in situ hybridization studies and reverse transcription-PCR on microdissected tubules showing abundant EP3 receptor mRNA in thick ascending limb and collecting ducts ( 5, 47 ). Our current findings showing EP3 receptor-LI in the "atypical" smooth muscle fibers in the pelvic tip described by Gosling and Dixon ( 18 ) suggest that activation of these EP3 receptors may contribute to the pelvic contractions produced by PGE 2 ( 5, 35 ). The lack of EP3 receptor staining in renal nerve terminals together with the presence of EP3 receptor-ir neuronal cell bodies in TH 9 -L 1 DRGs suggests that these EP3 receptor-ir neurons project to other organs than the kidney, are localized specifically in the cell body and the central endings of the sensory nerves ( 40 ), or centrifugally transported at such low levels that they cannot be detected with our methodology.

COX-2 in Renal Nonneural and Neural Tissue and DRG

COX-2 is constitutively expressed and widely distributed in the central nervous system ( 4 ) including the superficial dorsal horn of the spinal cord ( 52 ). However, several studies have reported lack of COX-2 labeling in DRGs in normal rats ( 9, 50, 51 ). In contrast, the current study shows intense COX-2 labeling of neuronal cell bodies in Th 9 -L 1 DRGs in normal rats. The labeling was specific to the COX-2 antibody in the sense that it was abolished by preadsorption with the immunogenic peptide. The apparent differences in the results between the current and previous studies may be explained by the DRGs studied. Whereas the current study concerned Th 9 -L 1 DRGs, previous studies have focused on more caudal lumbar DRGs ( 9, 51 ). The current study further showed that many of the COX-2-ir neurons in the DRGs also contained CGRP-LI.

The marked inhibition of the ARNA response to increased renal pelvic pressure produced by renal pelvic administration of COX-2 inhibitors ( 27 ) suggests that COX-2 in the renal pelvic wall contributes importantly to the activation of renal pelvic mechanosensory nerves. Due to its rapid metabolism, the actions of PGE 2 on its receptors should occur in the vicinity of its site of synthesis. Interestingly, COX-2 has been found to be colocalized with EP4 receptors in the vasculature in human kidneys ( 49 ). Thus we speculated that COX-2 may be located in or close to the renal pelvic sensory nerves. However, the current study showed only very few thin COX-2-ir nerve fibers in the pelvic wall. This relative absence of COX-2-LI despite its presence in many CGRP-LI-containing neurons in Th 9 -L 1 DRGs and in nerve bundles along the renal pelvic wall may be explained by the sensitivity of our immunohistochemistry being too low to detect the enzyme in the sensory nerve terminals, as discussed above. Whereas the presence of COX-2- and EP4 receptor-LI in nerve bundles along the renal pelvic wall suggests PG synthesis in or in close vicinity to the renal nerve fibers, the strong COX-2 mRNA signal in the uroepithelium and renal pelvic muscle layer ( 27 ) suggests that a large portion of PGE 2 synthesis occurs in the tissue surrounding the renal sensory nerves.

However, our findings do not exclude the possibility that PGE 2 involved in the activation of renal pelvic sensory nerves may, at least in part, be derived from COX-1 present in or close to the these sensory nerves. COX-1 is present in renal tissue ( 8, 49 ). Although there is currently little anatomic evidence for COX-1 in renal pelvic tissue, our previous studies showing that the nonselective COX inhibitor indomethacin produced a more marked inhibition of renal sensory nerve activation than selective COX-2 inhibitors ( 27 ) may suggest that induction of both COX-1 and COX-2 contributes to the PGE 2 -mediated stimulation of renal sensory nerves.

Role of EP2 and EP4 Receptors in the Activation of Renal Sensory Nerves

Butaprost, a selective EP2 receptor agonist at nanomolar concentrations, displays affinity for the EP4 receptors at micromolar concentrations ( 13, 16 ). Because our previous studies showed that the renal sensory nerves were activated by butaprost at 10 µM but not 4 µM ( 25 ), we hypothesized that PGE 2 (and butaprost) activates renal pelvic sensory nerves by stimulating EP4 and not EP2 receptors. Examining the effects of various EP receptor antagonists on the responses to activation of renal sensory nerves both in vitro and in vivo confirmed our hypothesis. The EP1/EP2 receptor antagonist AH-6809 ( 53 ) failed to attenuate the PGE 2 -mediated increase in substance P release from the isolated renal pelvises or the increases in ARNA produced by either PGE 2 or butaprost. On the other hand, the increases in substance P release and ARNA produced by PGE 2 and butaprost in vitro and in vivo, respectively, were abolished by AH-23848, a selective but relatively weak EP4 receptor antagonist ( 10 ). Importantly, similar results were obtained with a more potent selective EP4 receptor antagonist of a different molecular structure, L-161,982 ( 34 ).

The PGE 2 -mediated substance P release is a crucial mechanism in the activation of renal mechanosensory nerves ( 25, 27, 29 ). Therefore, we also examined whether the increase in ARNA produced by elevated renal pelvic pressure is modulated by an EP4 receptor antagonist. Indeed, renal pelvic perfusion with L-161,982 produced a reversible blockade of the ipsilateral ARNA and contralateral natriuretic responses to increases in renal pelvic pressure. Taken together, our functional data showing a role for EP4 receptors in the PGE 2 -mediated activation of renal pelvic nerves support our immunohistochemical findings of EP4 receptor-LI on these nerve fibers.

Activation of EP4 receptors contributes to the PGE 2 -mediated increase in cAMP from cultured mouse juxtaglomerular (JG) granular cells ( 20 ) and the PGE 2 -mediated increase in renin release ( 44 ). Although EP4 receptor mRNA has been demonstrated in cultured JG granular cells ( 20 ), EP4 receptor-LI was not detected in these cells in the current study. The lack of EP4 receptor-ir JG cells may be related to the levels of EP 4 receptors being too low in whole kidney sections from rats fed normal-sodium diet ( 20 ) to be detected with our methodology.

The functional role of the colocalization of EP4 receptor and TH-LI in renal nerve fibers is currently not known. We like to speculate that activation of the presynaptic EP4 receptors on sympathetic renal nerve fibers contributes to the PGE 2 -mediated renal vascular effects. There is considerable evidence for PGE 2 reducing norepinephrine release in both central and peripheral neural tissue by activating presynaptic EP receptors ( 36 ). However, pharmacological studies would indicate a role for EP3 receptors in the PGE 2 -mediated reduction of stimulated norepinephrine release (e.g., Ref. 42 ). Our studies failed to show EP3 receptor-LI on TH-LI or CGRP-LI containing nerve fibers in the kidney. Although we cannot exclude the possibility that EP3 receptors located on thin nerve fibers in the kidney were not detected by the EP3 receptor antibody applied, the strong labeling of non-neural renal tissue by the EP3 receptor antibody would argue against this hypothesis. The possible functional role of presynaptic EP4 receptors in modulating norepinephrine release from renal sympathetic nerve fibers awaits further study.

Preliminary experiments examined the effects of the EP1/EP3 receptor agonist sulprostone ( 2, 5, 41 ) on the activation of the renal nerves. However, the results were inconsistent. Whereas renal pelvic perfusion with 0.2 µM sulprostone produced a small reduction (35 ± 8%, n = 9) of the ARNA response to increasing renal pelvic pressure in vivo, sulprostone (0.2 or 1.0 µM) failed to alter baseline substance P release, from 4.30 ± 0.5 to 4.4 ± 0.8 pg/min ( n = 8), from the isolated renal pelvic wall preparation. This was in marked contrast to the effects of PGE 2 (0.14 µM), which increased baseline substance P release from the contralateral pelvis from 4.9 ± 0.6 to 14.1 ± 1.5 pg/min ( P < 0.01). Further in vitro studies showed that sulprostone also did not alter the PGE 2 -mediated substance P release from the isolated renal pelvic wall. In agreement with our studies are studies in cultured DRGs, which failed to show an effect of sulprostone on PGE 2 -mediated cAMP activity ( 43 ). The lack of effects of sulprostone may be related to sulprostone being a nonselective agonist of EP1 receptors and EP3a, EP3b, and EP3c receptors. On the other hand, the data may suggest relative absence of EP3 receptors modulating renal pelvic sensory nerves as suggested by our immunohistochemical studies.

Physiological Significance of the Renorenal Reflexes

The responsiveness of the afferent renal nerves is enhanced by a high- and suppressed by a low-sodium diet, suggesting that this reflex mechanism contributes to total body sodium and fluid volume balance by assisting in the excretion of sodium and water ( 24 ). This hypothesis was confirmed by our previous studies in dorsal rhizotomized rats. Interrupting the afferent renal nerve input to the spinal cord at Th 9 -L 1 results in salt-sensitive hypertension ( 23 ). Thus during a high-sodium intake, interruption of the afferent limb of the renorenal reflexes results in the development of increased arterial pressure, presumably to facilitate natriuresis and establishment of sodium balance. In view of the renorenal reflexes being impaired in rats fed fatty acid-deficient diet ( 30 ), it is interesting that these rats become hypertensive when placed on a high-sodium diet ( 12 ). Also, selective inhibition of renal medullary COX-2 activity results in salt-sensitive hypertension ( 37 ). Furthermore, the renorenal reflexes are impaired in spontaneously hypertensive rats ( 22 ) and rats with congestive heart failure ( 26 ), suggesting that the decreased responsiveness of the renal sensory nerves may contribute to the increased ERSNA and sodium retention in these pathological conditions.

In summary, the present study shows EP4 receptor-LI in CGRP-ir nerves in the renal pelvic wall and Th 9 -L 1 DRGs, suggesting the presence of this subtype of PGE receptors on renal pelvic sensory nerves. These findings are supported by our functional studies showing that the increases in substance P release and ARNA produced by PGE 2 were blocked by selective EP4 receptor antagonists but not by an EP2 receptor antagonist. Also, the EP4 receptor antagonist blocked the increases in ARNA produced by elevated renal pelvic pressure. Our immunohistochemical studies further showed the presence of COX-2-LI in the vicinity of EP4 receptor-ir nerves in the renal pelvic area suggesting the synthesis of PGE 2 close to EP4 receptors. On the other hand, there was no evidence for EP3 receptor-LI on or close to renal pelvic nerve fibers. Taken together, our data suggest that PGE 2 activates renal pelvic mechanosensory nerve fibers by stimulating EP4 receptors located on or in the vicinity of the renal pelvic sensory nerve fibers.

GRANTS

This work was supported by grants from the Department of Veterans Affairs, National Heart, Lung, and Blood Institute, RO1-HL-66068, and Specialized Center of Research Grants HL-55006, American Heart Association Grant-In-Aid 0150024N, the Swedish Medical Research Council (04-2887), the Marianne and Marcus Wallenberg and the Knut and Alice Wallenberg Foundations, an EU Grant (LSHM-CT-2004-503474), an unrestricted Bristol-Myers Squibbs Neuroscience Grant, and the Japan Society for the Promotion of Science (No. 3814).

ACKNOWLEDGMENTS

We are grateful for the generous supply of L-161,982 and L-161,983 from Dr. R. N. Young (Merck Frosst Canada, Center for Therapeutic Research, Kirkland, Quebec, Canada), AH-23848 from Dr. D. P. Brooks (GlaxoSmithKline Research and Development, King of Prussia, PA), and CGRP antisera from Drs. J. H. Walsh and H. C. Wong (The Center for Ulcer Research and Education of the Veterans Affairs/University of California Gastroenteric Biology Center, Los Angeles, CA) (antibody/RIA Core Grant #DK-41301).

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作者单位：1 Departments of Internal Medicine and Pharmacology, Department of Veterans Affairs Medical Center and University of Iowa Carver College of Medicine, Iowa City, Iowa 52242; 2 Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-850 Japan; 3 De