AJP - Renal AJP: Gastrointestinal and Liver Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 289: F768-F776, 2005. First published May 24, 2005; doi:10.1152/ajprenal.00177.2005
0363-6127/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/4/F768    most recent
00177.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, R.
Right arrow Articles by Kishore, B. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, R.
Right arrow Articles by Kishore, B. K.

Chronic dDAVP infusion in rats decreases the expression of P2Y2 receptor in inner medulla and P2Y2 receptor-mediated PGE2 release by IMCD

Rujia Sun,1,2,3 R. Lance Miller,4 Andrew C. Hemmert,1,3 Ping Zhang,4 Huihui Shi,1,3 Raoul D. Nelson,4 and Bellamkonda K. Kishore1,2,3

Departments of 1Medicine, 2Physiology, and 4Pediatrics, University of Utah Health Sciences Center, and 3Nephrology Research, Veterans Affairs Salt Lake City Health Care System, Salt Lake City, Utah

Submitted 25 April 2005 ; accepted in final form 18 May 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of P2Y2 receptor (P2Y2-R) in inner medullary collecting duct (IMCD) of rat decreases AVP-induced water flow and releases PGE2. We observed that dehydration of rats decreases the expression of P2Y2 receptor in inner medulla (IM) and P2Y2-R-mediated PGE2 release by IMCD. Because circulating vasopressin (AVP) levels are increased in dehydrated condition, we examined whether chronic infusion of desmopressin (dDAVP) has a similar effect on the expression and activity of P2Y2-R. Groups of rats were infused with saline or dDAVP (5 or 20 ng/h sc, 5 or 6 days) via osmotic minipumps and euthanized. Urine volume, osmolality, and PGE2 metabolite content were determined. AQP2- and P2Y2- and V2-R mRNA and/or protein in IM were quantified by real-time RT-PCR and immunoblotting, respectively. P2Y2-R-mediated PGE2 release by freshly prepared IMCD was assayed using ATP{gamma}S as a ligand. Chronic dDAVP infusion resulted in low-output of concentrated urine and significantly increased the AQP2 protein abundance in IM. On the contrary, dDAVP infusion at 5 or 20 ng/h significantly decreased P2Y2-R protein abundance (~40% of saline-treated group). In parallel, the relative expression of P2Y2-R vs. AQP2- or V2-R mRNA was significantly decreased. Furthermore, the P2Y2-R-mediated PGE2 release by IMCD was significantly decreased in rats infused 20 ng/h but not 5 ng/h of dDAVP. Urinary PGE2 metabolite excretion, however, did not change with dDAVP infusion. In conclusion, chronic dDAVP infusion decreases the expression and activity of P2Y2-R in IM. This may be due to a direct effect of dDAVP or dDAVP-induced increase in medullary tonicity.

arginine vasopressin; V2 receptor; prostanoids; nucleotides; desmopressin; inner medullary collecting duct; prostaglandin E2

THE RENAL COLLECTING DUCT system, which expresses vasopressin-regulated AQP2 water channel, is the site of regulated water transport. Under the influence of AVP, it accounts for the absorption of 15–20% of filtered water that determines the concentration of the final voided urine. AVP, acting through its V2 receptor, a G protein-coupled receptor, in the collecting duct principal cells, activates membrane-bound adenylyl cyclase to produce cAMP as a second messenger. The cellular effects of cAMP are believed to be connected to the activation of protein kinase A, which phosphorylates various key proteins. AVP has both short- and long-term effects on the collecting duct water permeability. The short-term effect, which occurs within a time frame of few to several minutes, involves the translocation of AQP2 water channels from a pool of subapical vesicles to the plasma membrane (21). The apical membrane is the rate-limiting barrier for the transepithelial water movement. The long-term effect, which occurs over a time span of several hours to days, involves an increase in the absolute amount of AQP2 mRNA and protein. Water deprivation (14, 21) and vasopressin stimulation (4, 15, 28) both increase AQP2 expression. These long-term effects of AVP are also apparently mediated by cAMP. cAMP is capable of stimulating AQP2 gene transcription by acting through cAMP-responsive element (CRE) and AP1 sites in the AQP2 promoter (10, 17, 31). cAMP activation of APQ2 gene likely occurs by phosphorylation of CRE binding protein and the ability of phosphorylated CRE binding protein to activate AQP2 gene transcription via binding to CRE sites in the AQP2 promoter. Furthermore, the fact that levels of cAMP and AQP2 mRNA are low in the mice with constitutively active cAMP-phosphodiesterase suggests that expression of AQP2 in vivo is regulated by cAMP (7, 9).

Apart from AVP, a variety of autocrine and paracrine agents, such as PGE2, endothelin, and extracellular nucleotides (ATP/UTP), regulate the collecting duct water permeability. Similar to AVP, these agents bind to specific G protein-coupled receptors but mediate their effects through phosphoinositide signaling pathway. Experimental studies have shown that these local mediators potentially decrease the AVP-stimulated water flow in the collecting duct (8, 11, 16, 20, 24, 25). Thus, in the collecting duct, cAMP and phosphoinositide systems are mutually opposing signaling pathways (27). Despite this, our understanding of the potential in vivo interactions of these nonvasopressin regulators with vasopressin and vice versa is not as advanced as the molecular physiology and cell biology of AVP action on the collecting duct (23).

Our research on the purinergic regulation of medullary collecting duct function over the past few years unraveled the potential roles of extracellular nucleotide-stimulated P2Y2 receptor. P2Y2 receptor is a G protein-coupled receptor with an agonist potency order of UTP ≥ ATP > ATP{gamma}S >> 2-MeS-ATP. Pharmacological and molecular approaches localized P2Y2 receptor in the medullary collecting duct of rat (6, 12). Using an approach of isolated microperfused medullary collecting duct of rat, we showed that agonist activation of P2Y2 receptor results in a decrease in the AVP-stimulated osmotic water permeability (11). Subsequently, we showed that agonist activation of P2Y2 receptor in the medullary collecting duct preparations causes production and release of PGE2 (29). More recently, we demonstrated that the expression of P2Y2 receptor in the inner medulla is altered depending on the hydration status of the animal. We showed that P2Y2 receptor mRNA expression and protein abundance are significantly elevated in hydrated rats compared with the dehydrated rats (14). In parallel, we documented that the altered expression of P2Y2 receptor in hydrated and dehydrated rats is associated with corresponding changes in the P2Y2 receptor-stimulated ex vivo PGE2 release by the inner medullary collecting duct (IMCD). We observed that IMCD from hydrated rats show significantly higher levels of purinergic-stimulated PGE2 release compared with the IMCD of normal rats (26). Interestingly, this effect is blunted in dehydrated rats. Thus our studies demonstrate that, in IMCD, activation of purinergic signaling drives the production of PGE2, and this driving capacity is accentuated in hydrated state and blunted in dehydrated condition. PGE2 affects transport of water, salt, and urea by IMCD (8, 11, 20, 24, 25). Hence, our observations have profound physiological significance.

Because dehydrated rats exhibit an increase in circulating vasopressin (AVP) levels as well as medullary tonicity, we hypothesized that chronically elevated levels of vasopressin should also have similar effects on the expression and activity of P2Y2 receptor. Hence, in this study, we examined whether chronic infusion of dDAVP (desmopressin), a V2-receptor-specific analog of vasopressin, to rats decreases P2Y2 receptor in inner medulla and P2Y2-receptor-mediated PGE2 release by the IMCD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. The animal experiments were carried out according to the protocols approved by the Institutional Animal Care and Use Committees of the University of Utah and Veterans Affairs Salt Lake City Health Care System. Specific pathogen-free male Sprague-Dawley rats (Harlan, Indianapolis, IN) were housed in the Veterinary Medical Unit of the Veterans Affairs Salt Lake City Health Care System. This facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (International) and approved by the US Department of Agriculture and the Office of Laboratory Animal Welfare of the Public Health Service. The rats were housed two or three per cage in pathogen-free state, fed ad libitum a commercial rodent diet, and had free access to drinking water. The rats were acclimated to the housing conditions for ~1 wk before experiments were conducted. The rats weighed 309 ± 3 g (mean ± SE) at the time of euthanasia.

Chronic infusion of dDAVP to rats. Rats were subcutaneously infused with saline or with one of the two different doses of dDAVP as described previously (15). Briefly, groups of rats (n = 9 per group) were anesthetized with isofluorane (3–5%) using rodent anesthetic machine. Under aseptic conditions, osmotic minipumps (model 2002; flow rate of 0.5 µl/h; Alzet, Palo Alto, CA) preloaded with either saline (0.9% NaCl, USP grade) or dDAVP (catalog no. V-1005; Sigma, St. Louis, MO) were implanted subcutaneously on the back of the napes between the two shoulder blades. dDAVP was dissolved in sterile saline at two different concentrations, so as to deliver either 5 or 20 ng of dDAVP per hour. Skin wounds were closed by surgical clips. Rats had free access to standard rodent chow and drinking water during the experimental period. dDAVP-infused rats and their respective saline-infused rats were euthanized after 6 (5 ng/h series) or 5 (20 ng/h series) days of infusion.

Collection and analysis of urine samples. Twenty-four hour urine samples were collected from all rats before the implantation of minipumps and just before euthanasia by placing them in individual metabolic cages. After collection, volumes of 24-h urine samples were recorded. Aliquots of urine samples were centrifuged to remove suspended particles, and the osmolalitiy of the supernatants were measured by vapor pressure method (model 5100; Wescor, Logan, UT). PGE2 metabolite in the urine of three rats in each group was assayed with the PGE metabolite EIA kit (catalog No. 514531; Cayman Chemical, Ann Arbor, MI). PGE2 in urine is not stable and is rapidly converted to its 13,14-dihydro-15-keto metabolite. The PGE metabolite assay kit supplied by the Cayman Chemical converts all of the immediate PGE2 metabolites in the urine samples to a single, stable derivative that could be easily quantified by EIA. Urine samples were processed for the assay according to the manufacturer's instructions and as described in detail previously (26). The measured amounts of PGE2 metabolite in the urine samples were normalized to the 24-h urine output and expressed as picograms of PGE2 metabolite per 24 h.

Euthanasia and collection of tissue samples. At the end of the experimental period, animals were euthanized by pentobarbital sodium overdose. Both kidneys were quickly removed and chilled in ice-cold PBS. Inner medullae were dissected on ice. Inner medullae from six rats in each group were flash frozen in liquid nitrogen and stored at –85°C until analyzed for RNA and protein. Inner medullae from the other three rats in each group were transferred to physiological solution and kept on ice for the preparation of fractions enriched in medullary collecting ducts. These were processed immediately for the determination of P2Y2 receptor-stimulated ex vivo PGE2 release as described below.

Preparations of tissue samples and Western analysis. Tissue samples were prepared and immunoblotted by the methods described earlier (12–14). Briefly, whole tissue homogenates were prepared by homogenizing one inner medulla from each rat in a homogenizing buffer containing protease inhibitors. Protein concentrations of the homogenates were determined by bicinchoninic acid protein reagent (Pierce Endogen) and then solubilized in Laemmli sample buffer at 60°C for 20 min. Semiquantitative immunoblotting approach was used to assess alterations in protein abundances as described previously (12–14). Equality of protein loading was checked by running loading gels and staining the separated proteins by Coomassie blue (Gel Code Blue; Pierce Endogen). The membranes were probed with affinity-purified polyclonal antibodies to P2Y2 receptor (L246) or AQP2 (GN-762). Generation, purification, and characterization of these antibodies were described previously (12, 13). Sites of antigen-antibody reaction were visualized by chemiluminescence (SuperSignal substrate; Pierce Endogen) and captured on light-sensitive imaging film (Kodak X-Omat AR or Kodak Biomax ML). Films showing optimal gray scale were digitized by photographic imaging with a high-pixel digital camera. Relative band densities were quantified using Un-Scan-It software (Silk Scientific, Orem, UT). Densitometry results are expressed as volume integrated values expressed as percentage of the mean values in saline-infused rats (100%).

Quantitative real-time RT-PCR assays. Quantitative real-time RT-PCR was carried out as described previously (14, 18). Total RNA from one inner medulla per rat was isolated using the Trizol method (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The extracted RNA was further purified using RNeasy mini columns with DNase I treatment (Qiagen, Valencia, CA) to remove traces of genomic DNA present in the samples.

cDNA was made by reverse transcribing 2.5 µg of total RNA using oligo(dT) priming and superscript reverse transcriptase II (Invitrogen) according to manufacturer's instructions. cDNA was quantified using Smart Cycler II system (Cepheid, Sunnyvale, CA) and SYBR green for detection. Each PCR reaction contained the following final concentrations: 1x buffer (20 mM Tris·HCl, pH 8.3), 50 mM KCl, 3 mM MgCl2, 0.3 µM forward primer, 0.3 µM reverse primer, 1x additive reagent (0.2 mg/ml BSA, 150 mM trehlose, 0.2% Tween 20), 0.25x SYBR green, 1.5 U platinum Taq polymerase, 200 µM each dNTPs, 1 µl DNA, and H2O to bring the final volume to 25 µl. Table 1 shows the nucleotide sequences of the primer pairs used for the amplification of AQP2 water channel and P2Y2 and V2 receptors. The cDNA was amplified according to the following steps: 1) 95°C for 2 min, 2) 95°C for 12 s, 3) 58–62°C for 15 s (optimized for each primer pair), 4) 72°C for 20 s, and 5) 85°C for 6 s to detect SYBR green (nonspecific products melt at <85°C, therefore, are not detected). Steps 2–5 were repeated for an additional 39 cycles; at the end of the last cycle, temperature was increased from 60 to 95°C (0.2°C/s) to produce a melt curve.


View this table:
[in this window]
[in a new window]
  		Table 1. Nucleotide sequences of the primer pairs used in PCR

 
Standard curves were generated using serial dilutions of plasmid templates at the following concentrations: 107, 106, 105, 104, 103, and 102 copies for reaction. PCR reactions were considered valid only if the amplification was linear, i.e., r2 ≥ 0.98, and amplification efficiency was ≥95%. Plasmid templates for quantitative real-time RT-PCR were made by cloning PCR products into the pGEMT plasmid vector (Promega, Madison, WI). Gene copy number per nanogram of DNA was calculated using the size of PCR product plus vector base pairs. Optical density of plasmid template at 260 nm was used to measure concentration of DNA.

Preparation of fractions enriched in IMCD. Fractions enriched in IMCD were prepared from the inner medullae as described previously (12, 26, 29). Briefly, freshly obtained inner medullae were digested with collagenase and hyaluronidase in an oxygenated physiological buffer until a uniform digest of tubular fragments was obtained. Collecting duct fragments in the digested material were separated from the noncollecting duct elements of inner medulla (thin limbs and vasa recta) by repeated low-speed centrifugations and washings. The final fraction enriched in collecting ducts was suspended in physiological buffer and used for the determination of ATP{gamma}S-stimulated PGE2 release.

Incubations of IMCD preparations with ATP{gamma}S and assay of PGE2. Fractions enriched in IMCD were incubated with or without the addition of ATP{gamma}S to a final concentration of 50 µM as described previously (26, 29). Incubations were carried out for 20 min at 37°C. Reactions were terminated by adding chilled incubation buffer. Tubular fragments were pelleted by centrifugation. PGE2 content in the supernatants was determined using PGE2 EIA kit-monoclonal (catalog No. 514010; Cayman Chemical) according to the instructions of the manufacturer and as described in detail previously (26, 29). The pellets were delipidated, and the protein content of each incubation was measured by Coomassie Plus protein assay reagent kit (Pierce Endogen, Rockford, IL) as described previously (26, 29). The concentrations of PGE2 in the incubations were normalized with the corresponding protein content and expressed as nanograms of PGE2 released per milligram of protein.

Statistical analyses. Values are expressed as mean ± SE. Differences between the means of two groups were analyzed by unpaired t-test. Differences between the means of more than two groups were analyzed by ANOVA followed by assessment of differences by Tukey-Kramer's multiple comparison test. P values <0.05 were considered significant. The statistical analyses were performed using GraphPad InStat version 3.0 software (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Urine parameters in saline- or 5 ng/h dDAVP-infused rats. Figure 1 shows the urine output and osmolalities in rats infused with saline or 5 ng/h dDAVP subcutaneously for 6 days. As expected, chronic dDAVP infusion significantly decreased urine output and increased urine osmolalities. The pretreatment mean values of urine output and osmolalities between the saline- or dDAVP-infused groups were not significantly different (9.2 ± 1.0 vs. 10.1 ± 1.4 ml/24 h for urine volume and 1,690 ± 93 vs. 1,746 ± 48 mosmol/kgH2O for osmolality). Furthermore, there were no significant differences between these two groups with respect to pre- and posttreatment body weights. Hence, the observed differences between these two groups at the end of treatment period are attributable to the effect of chronic dDAVP infusion.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1. Urine output and osmolality in saline- or 5 ng/h dDAVP-infused rats. A: 24-h urine volumes. B: urine osmolalities. Values are expressed as means ± SE. The level of statistical significance and the number of animals studied (N) are shown above the bars. dDAVP, desmopressin.

 
AQP2 water channel protein abundance in saline- or 5 ng/h dDAVP-infused rats. Figure 2 shows the protein abundance of AQP2 water channel in inner medullas of rats infused with saline or 5 ng/h dDAVP subcutaneously for 6 days, as assessed by semiquantitative immunoblotting. AQP2 immunoblots typically show two bands. The 29-kDa band corresponds to unglycosylated form, whereas the smear from 35 to 50 kDa represents various glycosylated forms of the protein (22). As shown in Fig. 2A, chronic dDAVP infusion, as expected, increased the abundances of both 29-kDa and 35- to 50-kDa protein bands of AQP2 water channel. Figure 2B shows the mean densities of these bands in the saline- or dDAVP-infused rats plotted as percentages of mean values in the saline-infused rats. dDAVP infusion caused about six- and fourfold increases in the mean densities of 29-kDa and 35- to 50-kDa protein bands, respectively.



View larger version (46K):
[in this window]
[in a new window]
  		Fig. 2. Protein abundance of AQP2 water channel in inner medulla of saline- or 5 ng/h dDAVP-infused rats, as determined by semiquantitative immunoblotting. A: representative profile of AQP2 immunoblot as captured on X-ray film. Blots were generated by loading equal amounts of whole tissue proteins from 1 rat per each lane. Blots were probed with primary antibody to AQP2 protein. B: mean densities of 29- and 35- to 50-kDa AQP2 protein bands in saline- and dDAVP-infused groups plotted as percentage of the mean values in saline group (means ± SE). The level of statistical significance and the number of animals examined are shown above the bars.

 
P2Y2 receptor protein abundance in saline- or 5 ng/h dDAVP-infused rats. Figure 3 shows the protein abundance of P2Y2 receptor in inner medullas of rats infused with saline or 5 ng/h dDAVP, as assessed by semiquantitative immunoblotting. As characterized and documented by us previously, our P2Y2 receptor antibody consistently shows two sets of distinct immunoreactive bands in kidney and lung (12, 14). Both sets of bands appear to be specific, as they were ablated by preadsorption of the antibody with the immunizing peptide (12). The lower set of bands migrating at 47 kDa corresponds to glycosylated forms of the receptor protein. The exact nature of the upper set of bands migrating at ~105 kDa is not known. It may represent a dimer of the receptor protein, or a covalent complex of the receptor with another membrane and/or receptor-associated protein. It may also represent an alternatively processed form of the receptor. As shown in Fig. 3A, the intensities of the 47-kDa bands were reduced in dDAVP-infused rats. Densitometry showed that the mean density of the 47-kDa band in dDAVP-infused rats was reduced to ~40% compared with the saline-infused group.



View larger version (47K):
[in this window]
[in a new window]
  		Fig. 3. Protein abundance of P2Y2 receptor (R) in inner medulla of saline- or 5 ng/h dDAVP-infused rats, as determined by semiquantitative immunoblotting. A: representative profile of P2Y2 receptor immunoblot as captured on X-ray film. Blots were generated by loading equal amounts of whole tissue proteins from 1 rat per each lane. Blots were probed with primary antibody to P2Y2 receptor protein. B: mean densities of 47-kDa P2Y2 receptor protein bands in saline- and dDAVP-infused groups plotted as percentage of the mean values in saline group (means ± SE). The level of statistical significance and the number of animals examined are shown above the bars.

 
Relative expression of AQP2, P2Y2, and V2 receptor mRNA in saline- or 5 ng/h dDAVP-infused rats. As documented above, Western analysis showed that the protein abundance of AQP2 water channel increased, whereas the protein abundance of P2Y2 receptor was decreased, in dDAVP-infused rats. To examine whether the observed changes in protein abundances of these molecules were also associated with similar changes in the mRNA expression, we preformed real-time RT-PCR analysis on RNA extracted from the inner medullas of saline- or dDAVP-infused rats. Because chronic dDAVP infusion may alter the expression of standard housekeeping genes, such as GAPDH and {beta}-actin (3), and the validity of other housekeeping genes under our experimental conditions is not known, we directly compared the relative expression of AQP2, P2Y2, and V2 receptors with each other. Because the cDNAs from a single reverse-transcription reaction tube were used to amplify target sequences of these molecules, such direct comparisons were made possible. Figure 4A shows the relative expression of APQ2 mRNA copies per copy of P2Y2 receptor mRNA, whereas Fig. 4B shows the relative expression of V2 receptor mRNA copies per copy of P2Y2 receptor mRNA. As shown in Fig. 4, chronic dDAVP infusion significantly increased the ratios of AQP2/P2Y2 and V2/P2Y2 by 168 and 73%, respectively, compared with the saline-infused rats.



View larger version (36K):
[in this window]
[in a new window]
  		Fig. 4. Relative expression of AQP2-, P2Y2-, and V2-R mRNA in the inner medulla of saline- or 5 ng/h dDAVP-infused rats, as determined by quantitative real-time RT-PCR. A: ratio of AQP2-R to P2Y2-R mRNA expression. B: ratio of V2-R to P2Y2-R mRNA expression. Results (means ± SE) are computed as copies of AQP2 or V2 receptor mRNA per copy of P2Y2-R mRNA. The level of statistical significance and the number of animals examined are shown above the bars.

 
Urine parameters in saline- or 20 ng/h DDAVP-infused rats. Figure 5 shows the urine output and osmolalities in rats infused with saline or 20 ng/h dDAVP subcutaneously for 5 days. Chronic dDAVP infusion significantly decreased urine output and increased urine osmolalities. The pretreatment mean values of urine output and osmolalities between the saline- or dDAVP-infused groups were not significantly different (11.0 ± 0.7 vs. 12.5 ± 1.0 ml/24 h for urine volume and 1,840 ± 107 vs. 1,865 ± 160 mosmol/kgH2O for osmolality). There were no significant differences between these two groups with respect to pre- and posttreatment body weights.



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 5. Urine output and osmolality in saline- or 20 ng/h dDAVP-infused rats. A: 24-h urine volumes. B: urine osmolalities. Values are expressed as means ± SE. The level of statistical significance and the number of animals studied are shown above the bars.

 
AQP2 water channel protein abundance in saline- or 20 ng/h dDAVP-infused rats. Figure 6 shows the protein abundance of AQP2 water channel in inner medullas of rats infused with saline or 20 ng/h dDAVP subcutaneously for 5 days, as assessed by semiquantitative immunoblotting. As shown in Fig. 6A, chronic dDAVP infusion increased the abundances of both 29-kDa and 35- to 50-kDa protein bands of AQP2 water channel. Figure 6B shows the mean densities of these bands in the saline- or dDAVP-infused rats plotted as percent of mean values in the saline-infused rats. dDAVP infusion caused ~3.5- and 10-fold increases in the mean densities of 29-kDa and 35- to 50-kDa protein bands, respectively.



View larger version (47K):
[in this window]
[in a new window]
  		Fig. 6. Protein abundance of AQP2 water channel in inner medulla of saline- or 20 ng/h dDAVP-infused rats, as determined by semiquantitative immunoblotting. A: representative profile of AQP2 immunoblot as captured on X-ray film. Blots were generated by loading equal amounts of whole tissue proteins from 1 rat per each lane. Blots were probed with primary antibody to AQP2 protein. B: mean densities of 29- and 35- to 50-kDa AQP2 protein bands in saline- and dDAVP-infused groups plotted as percentage of the mean values in saline group (means ± SE). The level of statistical significance and the number of animals examined are shown above the bars.

 
P2Y2 receptor protein abundance in saline- or 20 ng/h dDAVP-infused rats. Figure 7 shows the protein abundance of P2Y2 receptor in inner medullas of rats infused with saline or 20 ng/h dDAVP, as assessed by semiquantitative immunoblotting. As shown in Fig. 7A, the intensities of the 105-kDa bands were reduced in dDAVP-infused rats. Densitometry showed that the mean density of the 105-kDa band in dDAVP-infused rats was significantly reduced to 39% compared with the saline-infused group. Furthermore, in contrast to the 5 ng/h dDAVP-infused series, here we observe that the 105-kDa bands were more prominent than the 47-kDa bands. Earlier, our group (12) documented that 105 kDa was more prominent in lung compared with the kidney medulla. Over the past few years, we (unpublished observations) also observed variations in the relative abundances of these two sets of bands in different batches of rats.



View larger version (48K):
[in this window]
[in a new window]
  		Fig. 7. Protein abundance of P2Y2 receptor in inner medulla of saline- or 20 ng/h dDAVP-infused rats, as determined by semiquantitative immunoblotting. A: representative profile of P2Y2 receptor immunoblot as captured on X-ray film. Blots were generated by loading equal amounts of whole tissue proteins from 1 rat per each lane. Blots were probed with primary antibody to P2Y2 receptor protein. B: mean densities of 105-kDa P2Y2 receptor protein bands in saline- and dDAVP-infused groups plotted as percentage of the mean values in saline group (means ± SE). The level of statistical significance and the number of animals examined are shown above the bars.

 
P2Y2 receptor-stimulated PGE2 release by IMCD preparations in saline- or dDAVP (5 or 20 ng/h)-infused rats. Fractions enriched in IMCD were prepared from inner medullas of saline- or 5 or 20 ng/h dDAVP-infused rats and challenged with 50 µM ATP{gamma}S at 37°C for 20 min. Amounts of PGE2 released into the incubation medium were determined and normalized to the protein contents of the incubations. Figure 8 shows the PGE2 released by the IMCD under basal (vehicle) or stimulated (50 µM ATP{gamma}S) conditions. As expected, stimulation of IMCD with ATP{gamma}S in saline-infused rats significantly increased PGE2 release. The basal and ATP{gamma}S-stimulated PGE2 released by IMCD from 5 ng/h dDAVP-infused rats were not different from the corresponding values in the saline-infused groups. However, infusion of 20 ng/h dDAVP significantly decreased the stimulated PGE2 release compared with saline- or 5 ng/h dDAVP-infused rats. Furthermore, the basal PGE2 release (vehicle) in 20 ng/h dDAVP-infused group was numerically less compared with the basal PGE2 release in the saline-infused group (72% of saline-infused group). The difference between these two was not statistically significant when analyzed by ANOVA. However, a direct comparison of basal PGE2 release in the saline- or 20 ng/h dDAVP-infused rats by unpaired t-test gave a P value of <0.003.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 8. P2Y2 receptor-stimulated PGE2 release by inner medullary collecting duct (IMCD) preparations in saline- or dDAVP (5 or 20 ng/h)-infused rats. Fractions enriched in IMCD were freshly prepared from pooled inner medulla in each group (3 rats/group). Aliquots of IMCD fractions, after warming up to 37°C for 5 min, were incubated in the presence of incubation buffer alone (vehicle) or 50 µM ATP{gamma}S for 20 min. PGE2 concentrations in the supernatants were assayed by EIA and normalized to the protein contents of the incubations. Incubations in each group were performed in quadruplicate (n = 4). Data (means ± SE) are shown as percentage of the mean values in vehicle alone incubations in the saline-infused group. *Significantly different from the respective vehicle alone values (P < 0.001 for saline group and P < 0.01 for dDAVP 5 ng/h group). **P < 0.05 vs. respective vehicle values and P < 0.01 vs. 50 µM ATP{gamma}S incubations in the saline or dDAVP 5 ng/h groups.

 
Urinary excretion of PGE2 metabolite in saline- or dDAVP (5 or 20 ng/h)-infused rats. As shown in Fig. 9, chronic dDAVP infusion did not alter urinary excretion of PGE2 even at the higher dose of dDAVP.



View larger version (51K):
[in this window]
[in a new window]
  		Fig. 9. Urinary excretion of PGE2 metabolite in saline- or dDAVP (5 or 20 ng/h)-infused rats. Twenty-four hour urine samples were collected and processed for the assay of PGE2 metabolite as described under MATERIALS AND METHODS. From volume of urine collected, urinary PGE2 metabolite excreted over 24-h periods was calculated and expressed as ng/24 h. Data shown are means ± SE for 3 rats in each group. Means of the 3 groups are not significantly different from each other.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this communication, we documented two significant findings. First, we demonstrated that the expression of P2Y2 receptor in inner medulla is significantly decreased by chronic dDAVP infusion. Second, we showed that P2Y2 receptor-mediated ex vivo PGE2 release by IMCD is significantly decreased by chronic dDAVP infusion at a higher dose. These findings constitute the first molecular evidence of long-term effects of higher circulating vasopressin levels on P2Y2 receptor expression and activity. These findings may have physiological significance in the regulation of medullary collecting duct function.

AVP, a potent circulating hormone, acts through cAMP second messenger system and increases the water permeability of the collecting duct by regulating the expression and apical membrane targeting of AQP2 (4, 22, 28). In contrast to vasopressin, whose release is regulated by systemic and central mechanisms, the autocrine or paracrine agents that act through the phosphoinositide signaling pathway in the collecting duct, such as endothelin, PGE2 and extracellular nucleotides, are locally produced, regulated, and metabolized. It was known that in the medullary collecting duct cAMP and phosphoinositide signaling pathways are mutually opposing (27). Earlier studies documented that, in acute experiments, these autocrine or paracrine agents inhibited AVP-stimulated water flow in the medullary collecting duct (8, 11, 16, 20, 24, 25). Some of these studies also documented the putative cross-talk points between the vasopressin system and the phosphoinositide signaling, through which these autocrine or paracrine agents act. However, to the best of our knowledge no studies are available in the literature documenting the chronic effect of elevated circulating vasopressin levels on the expression and activity of a G protein-coupled receptor involved in the activation phosphoinositide signaling. In this context, our findings that chronic dDAVP infusion decreases the expression of P2Y2 receptor in the inner medulla and P2Y2 receptor-mediated PGE2 release by IMCD are perhaps the first of its kind. Our observations also provide the molecular basis for the interaction of vasopressin with nonvasopressin mediators of medullary collecting duct function, in this case purinergic system.

In our previous studies, we observed that dehydration of rats by water deprivation for 48 h resulted in a significant decrease in the expression of P2Y2 receptor protein and mRNA in the inner medulla, associated with a significant decrease in P2Y2 receptor-mediated ex vivo PGE2 release by IMCD (14, 26). Dehydration is associated with increased circulating levels of AVP and AVP-induced increase in medullary interstitial tonicity. Hence, we hypothesized that chronic dDAVP infusion to rats also should result in similar changes in the expression and activity of P2Y2 receptor as seen under dehydrated conditions. Indeed, our results presented here document that chronic dDAVP infusion resulted in similar alterations in the expression and activity of P2Y2 receptor as seen in the dehydration model. The large elevation in the relative expression of AQP2 vs. P2Y2 receptor mRNA following chronic dDAVP infusion compared with the dehydration model (14) may be mostly due to a large increase in AQP2 expression in dDAVP-infused rats, which is more dramatic compared with dehydration model (14). Furthermore, a relative increase in the expression of V2 receptor vs. P2Y2 receptor as documented here may tilt the balance in favor of the cAMP signaling pathway.

We also observed that chronic dDAVP infusion at higher (20 ng/h) dose, but not at lower (5 ng/h) dose, resulted in significant decrease in P2Y2 receptor-mediated ex vivo PGE2 release by IMCD preparations. These observations suggest that decreased expression of P2Y2 receptor may not necessarily be associated with decreased ability of this receptor to stimulate PGE2 release. It is possible that these two may be independently regulated. Thus chronic dDAVP infusion, depending on the dose administered, may have a dual effect on the P2Y2 receptor expression and activity. In this context, the effects of dehydration and chronic dDAVP infusion on the P2Y2 receptor-mediated PGE2 release apparently differ. In the dehydration model, we showed that the basal levels of PGE2 release by IMCD were low and P2Y2 receptor stimulation did not result in an increase in the PGE2 release (26). However, it should be noted that dehydrated rats, unlike the dDAVP-infused rats, were water deprived. So, dehydrated rats, unlike the dDAVP-infused rats, have to conserve body water. This may be the reason for very low urine flow in the dehydrated rats compared with dDAVP-infused rats. The mean urine output in the dehydrated rats (14, 26) was about one-third of the mean values in dDAVP-infused rats. The urine osmolality in dehydrated rat was also higher (mean values of ~3,500 or ~4,000 mosmol/kgH2O) compared with the dDAVP-infused rats (mean values of ~2,800 or ~3,100 mosmol/kgH2O) (14, 26). Thus the necessity to conserve maximum amounts of water in dehydrated rats, or a lack of it in the dDAVP-infused rats, may play a role in the differential regulation of P2Y2 receptor expression and activity in these two conditions. Furthermore, dehydration induces an increase in AVP, which has both V1-receptor-mediated pressor and V2-receptor-mediated antidiuretic effects. dDAVP, on the other hand, has only V2-receptor-mediated antidiuretic effect. Previous studies demonstrated that AVP and dDAVP differ in their ability to stimulate PGE2 production in rat renal medullary tubular and interstitial cells (1, 19, 30). In view of these, more systematic studies, which are beyond the scope of this communication, are needed to examine and establish these possible mechanisms.

The medullary collecting duct accounts for ~66% of the total PGE2 synthesized in the kidney (2), and urinary prostaglandins probably reflect renal synthesis (5). Despite the observation of significantly decreased P2Y2 receptor-mediated PGE2 release by the IMCD, the urinary excretion of PGE2 metabolite in 20 ng/h dDAVP-infused rats did not change. This observation is in contrast to our earlier finding that dehydrated rats had very low urinary PGE2 excretion. This difference between dehydrated and dDAVP-infused rats may be due to the significant difference in the urine flow rates in these two groups, which is again related to the necessity or lack of it to conserver body water under these conditions as explained above.

Finally, future studies are needed to address whether the decreased expression and activity of P2Y2 receptor seen in dehydrated and dDAVP-infused rats are due to a direct effect of vasopressin/dDAVP or to the increased medullary tonicity induced by these agents in vivo. Pending such studies, at this stage, our present and previous studies clearly establish the in vivo and in vitro interactions among vasopressin and purinergic and prostanoid systems. These studies also exemplify the complex nature of these interactions, which may influence the medullary collecting duct function in health and disease conditions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-61183 (B. K. Kishore) and DK-53990 (R. D. Nelson) and the resources and facilities at the Veterans Administration Salt Lake City Health Care System, Salt Lake City, Utah.


   ACKNOWLEDGMENTS
 
The authors thank Drs. Mark Knepper and Donald Kohan for critical reading of the manuscript.

Parts of this work were presented at the 37th Annual Meeting of the American Society of Nephrology, October–November 2004, St. Louis, MO, and appeared in abstract form in the proceedings of that meeting.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. K. Kishore, Nephrology Research (151M), VA Salt Lake City Health Care System, 500 Foothill Dr., Salt Lake City, UT 84148 (e-mail: BK.Kishore{at}hsc.utah.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Beck TR, Hassid A, and Dunn MJ. The effect of arginine vasopressin and its analogs on the synthesis of prostaglandin E2 by rat renal medullary interstitial cells in culture. J Pharmacol Exp Ther 215: 15–19, 1980.[Abstract/Free Full Text]
  2. Bonvalet JP, Pradelles P, and Farman N. Segmental synthesis and actions of prostaglandins along the nephron. Am J Physiol Renal Fluid Electrolyte Physiol 253: F377–F387, 1987.[Abstract/Free Full Text]
  3. Brooks HL, Ageloff A, Kwon TH, Brandt W, Terris JM, Seth A, Michea L, Nielsen S, Fenton R, and Knepper MA. cDNA array identification of genes regulated in rat renal medulla in response to vasopressin infusion. Am J Physiol Renal Physiol 284: F218–F228, 2003.[Abstract/Free Full Text]
  4. DiGiovanni SR, Nielsen S, Christensen EI, and Knepper MA. Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat. Proc Natl Acad Sci USA 91: 8994–8988, 1994.[Abstract/Free Full Text]
  5. Dunn MJ and Hood VL. Prostaglandins and the kidney. Am J Physiol Renal Fluid Electrolyte Physiol 22: F169–F184, 1977.
  6. Ecelbarger CA, Maeda Y, Gibson CC, and Knepper MA. Extracellular ATP increases intracellular calcium in rat terminal collecting duct via a nucleotide receptor. Am J Physiol Renal Fluid Electrolyte Physiol 267: F998–F1006, 1994.[Abstract/Free Full Text]
  7. Frokiær J, Marples D, Valtin H, Morris JF, Knepper MA, and Nielsen S. Low aquaporin-2 levels in DI+/+ severe mice with constitutively high cAMP-phosphodiesterase activity. Am J Physiol Renal Physiol 276: F179–F190, 1999.[Abstract/Free Full Text]
  8. Han JS, Maeda Y, Ecelbarger CA, and Knepper MA. Vasopressin-independent regulation of collecting duct water permeability. Am J Physiol Renal Fluid Electrolyte Physiol 266: F139–F146, 1994.[Abstract/Free Full Text]
  9. Homma S, Gapstur SM, Coffey A, Valtin H, and Dousal TP. Role of cAMP-phosphodiesterase isoenzymes in pathogenesis of murine nephrogenic diabetes insipidus. Am J Physiol Renal Fluid Electrolyte Physiol 261: F345–F353, 1991.[Abstract/Free Full Text]
  10. Hozawa S, Holtzman EJ, and Ausiello DA. cAMP motifs regulating transcription in the aquaporin-2 gene. Am J Physiol Cell Physiol 270: C1695–C1702, 1996.[Abstract/Free Full Text]
  11. Kishore BK, Chou CL, and Knepper MA. Extracellular nucleotide receptor inhibits AVP-stimulated water permeability in inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 269: F863–F869, 1995.[Abstract/Free Full Text]
  12. Kishore BK, Ginns SM, Krane CM, Nielsen S, and Knepper MA. Cellular localization of P2Y2 receptor in rat renal medulla and lung. Am J Physiol Renal Physiol 278: F43–F51, 2000.[Abstract/Free Full Text]
  13. Kishore BK, Krane CM, Di Iulio D, Menon AG, and Cacini W. Expression of renal aquaporins 1, 2, and 3 in a rat model of cisplatin-induced polyuria. Kidney Int 58: 701–711, 2000.[CrossRef][Web of Science][Medline]
  14. Kishore BK, Krane CM, Miller RL, Shi H, Zhang P, Hemmert A, Sun R, and Nelson RD. P2Y2 receptor mRNA and protein expression is altered in inner medullae of hydrated and dehydrated rats: relevance to AVP-independent regulation of IMCD function. Am J Physiol Renal Physiol 288: F1164–F1172, 2005.[Abstract/Free Full Text]
  15. Kishore BK, Terris JM, and Knepper MA. Quantitation of aquaporin-2 abundance in microdissected collecting ducts: axial distribution and control by AVP. Am J Physiol Renal Fluid Electrolyte Physiol 271: F61–F70, 1996.
  16. Kohan DE and Hughe K. Autocrine role of endothelin in rat IMCD: inhibition of AVP-induced cAMP accumulation. Am J Physiol Renal Fluid Electrolyte Physiol 265: F126–F129, 1993.[Abstract/Free Full Text]
  17. Matsumura Y, Uchida S, Rai T, Sasaki S, and Marumo F. Transcriptional regulation of aquaporin-2 water channel gene by cAMP. J Am Soc Nephrol 8: 861–867, 1997.[Abstract]
  18. Miller RL, Zhang P, Smith M, Beaulieu V, Paunescu TG, Brown D, Breton S, and Nelson RD. V-ATPase subunit promoter drives expression of EGFP in intercalated cells of kidney, clear cells of epididymis and airway cells of lung in transgenic mice. Am J Physiol Cell Physiol 288: C1134–C1144, 2005.[Abstract/Free Full Text]
  19. Moses AM, Scheinman SJ, and Schroeder ET. Antidiuretic and PGE2 responses to AVP and dDAVP in subjects with central and nephrogenic diabetes insipidus. Am J Physiol Renal Fluid Electrolyte Physiol 248: F354–F359, 1985.[Abstract/Free Full Text]
  20. Nadler SP, Zimpelmann JA, and Hebert RL. PGE2 inhibits water permeability at a post-cAMP site in rat terminal inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 262: F229–F235, 1992.[Abstract/Free Full Text]
  21. Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, and Knepper MA. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc Natl Acad Sci USA 92: 1013–1017, 1995.[Abstract/Free Full Text]
  22. Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, and Harris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci USA 90: 11663–11667, 1993.[Abstract/Free Full Text]
  23. Nielsen S, Kwon TH, Christensen BM, Promeneur D, Frokiær J, and Marples D. Physiology and pathophysiology of renal aquaporins. J Am Soc Nephrol 10: 647–663, 1999.[Abstract/Free Full Text]
  24. Roman RJ and Lechene C. Prostaglandin E2 and F2{alpha} reduced urea reabsorption from the rat collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 241: F53–F60, 1981.[Abstract/Free Full Text]
  25. Rouch AJ and Kudo LH. Role of PGE2 in {alpha}2-induced inhibition of AVP- and cAMP-stimulated H2O, Na+, and urea transport in rat IMCD. Am J Physiol Renal Physiol 279: F294–F301, 2000.[Abstract/Free Full Text]
  26. Sun R, Carlson NG, Hemmert AC, and Kishore BK. P2Y2 receptor-mediated release of prostaglandin E2 by IMCD is altered in hydrated and dehydrated rats: relevance to AVP-independent regulation of IMCD function. Am J Physiol Renal Physiol First published April 19, 2005; doi:10.1152/ajprenal.00050.2005.
  27. Teitelbaum I. Hormone signaling systems in inner medullary collecting ducts. Am J Physiol Renal Fluid Electrolyte Physiol 263: F985–F990, 1992.[Abstract/Free Full Text]
  28. Terris J, Ecelbarger CA, Nielsen S, and Knepper MA. Long-term regulation of four renal aquaporins in rats. Am J Physiol Renal Fluid Electrolyte Physiol 271: F414–F422, 1996.[Abstract/Free Full Text]
  29. Welch BD, Carlson NG, Shi H, Myatt L, and Kishore BK. P2Y2 receptor-stimulated release of prostaglandin E2 by rat inner medullary collecting duct preparations. Am J Physiol Renal Physiol 285: F711–F721, 2003.[Abstract/Free Full Text]
  30. Wuthrich RP and Vallotton MB. Prostaglandin E2 and cyclic AMP response to vasopressin in renal medullary tubular cells. Am J Physiol Renal Fluid Electrolyte Physiol 251: F499–F505, 1986.[Abstract/Free Full Text]
  31. Yasui M, Zelenina SM, Celsi G, and Aperia A. Adenylate cyclase-coupled vasopressin receptor activates AQP2 promoter via a dual effect on CRE and AP1 elements. Am J Physiol Renal Physiol 272: F442–F450, 1997.



This article has been cited by other articles:


Home page
 J. Am. Soc. Nephrol.Home page
S. S. P. Wildman, M. Boone, C. M. Peppiatt-Wildman, A. Contreras-Sanz, B. F. King, D. G. Shirley, P. M. T. Deen, and R. J. Unwin
Nucleotides Downregulate Aquaporin 2 via Activation of Apical P2 Receptors
J. Am. Soc. Nephrol., July 1, 2009; 20(7): 1480 - 1490.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
T. Rieg and V. Vallon
ATP and adenosine in the local regulation of water transport and homeostasis by the kidney
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2009; 296(2): R419 - R427.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
Y. Zhang, J. M. Sands, D. E. Kohan, R. D. Nelson, C. F. Martin, N. G. Carlson, C. D. Kamerath, Y. Ge, J. D. Klein, and B. K. Kishore
Potential role of purinergic signaling in urinary concentration in inner medulla: insights from P2Y2 receptor gene knockout mice
Am J Physiol Renal Physiol, December 1, 2008; 295(6): F1715 - F1724.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
T. Rieg, K. Pothula, J. Schroth, J. Satriano, H. Osswald, J. Schnermann, P. A. Insel, R. A. Bundey, and V. Vallon
Vasopressin regulation of inner medullary collecting ducts and compensatory changes in mice lacking adenosine A1 receptors
Am J Physiol Renal Physiol, March 1, 2008; 294(3): F638 - F644.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
V. Vallon
P2 receptors in the regulation of renal transport mechanisms
Am J Physiol Renal Physiol, January 1, 2008; 294(1): F10 - F27.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/4/F768    most recent
00177.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, R.
Right arrow Articles by Kishore, B. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, R.
Right arrow Articles by Kishore, B. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.