Departments of 1 Medicine and 2 Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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PGT is a broadly expressed transporter of
prostaglandins (PGs) and thromboxane that is energetically poised to
take up prostanoids across the plasma membrane. To gain insight into
the function of PGT, we generated mouse monoclonal antibody 20 against
a portion of putative extracellular loop 5 of rat PGT. Immunoblots of
endogenous PGT in rat kidney revealed a 65-kDa protein in a zonal
pattern corresponding to PG synthesis rates (papilla medulla > cortex). Immunocytochemically, PGT in rat kidneys was
expressed in glomerular endothelial and mesangial cells, arteriolar
endothelial and muscularis cells, principal cells of the collecting
duct, medullary interstitial cells, medullary vasa rectae endothelia,
and papillary surface epithelium. Proximal tubules, which are known to
take up and metabolize PGs, were negative. Immunoblotting and
immunocytochemistry revealed that rat platelets also express abundant
PGT. Coexpression of the PG synthesis apparatus (cyclooxygenase) and
PGT by the same cell suggests that prostanoids may undergo release and reuptake.
carrier proteins; biological transport; molecular cloning
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INTRODUCTION |
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PROSTAGLANDINS (PGS) AND THROMBOXANES (Txs) play fundamental roles in context-dependent autocrine and paracrine signaling. In the kidney, for example, depending on the site of release and the receptors activated, PGE2 vasodilates or vasoconstricts blood vessels, stimulates renin release, and modulates Na, Cl, and water transport (24).
The kidney exemplifies the principle that prostanoid synthesis and degradation are compartmentalized into separate cell types and tissue zones. Regionally, the highest rates of renal PG synthesis occur in the papilla. Renal cell types that synthesize PGs and/or express cyclooxygenases (COXs) include glomerular mesangial cells and endothelia, collecting ducts, and medullary interstitial cells (24). In contrast, renal oxidation of PGs occurs in the cortex and juxtamedullary regions (24, 32), primarily by means of the proximal straight tubule, which actively secretes both native and oxidized PGs (13, 16).
Our laboratory recently identified a rat cDNA encoding PGT, the first known PG transporter. When expressed heterologously in cultured cells or Xenopus laevis oocytes, PGT mediates the uptake of PGE2 and TxB2, among other eicosanoids (17, 21). The broad expression pattern of PGT mRNA in rats, humans, and mice (17, 21, 28) has suggested a possible physiological role in the release of newly synthesized prostanoids and/or PG uptake before intracellular oxidation (32).
To further explore the physiological role of this transporter, we have immunolocalized PGT in rat kidneys and have also sought evidence for PGT expression in rat platelets, which synthesize and release TxA2 (1).
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MATERIALS AND METHODS |
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Generation of monoclonal antibody 20 against rat PGT. We PCR-amplified a portion of the rat PGT (rPGT) cDNA corresponding to deduced amino acids 430-505 on putative exofacial loop 5 and cloned it into the vector pGEX to generate an rPGT-glutathione S-transferase (GST) fusion protein. Mice were immunized with the purified fusion protein, myeloma fusions were performed by using the standard Kohler-Milstein approach (20), and hybridomas were screened by ELISA by using the fusion peptide vs. GST alone. Of several monoclonal antibodies generated, mouse monoclonal antibody 20 (MAb20) of the class IgG1 was expanded for use.
Transient expression of PGT cDNAs and determination of tracer PGE2 uptake. HeLa cells, grown to 70-80% confluence on 35-mm dishes, were infected with recombinant vaccinia virus vTF7-3 (10 plaque-forming units/cell) as described previously (17). Cells were then transfected with 10 µg of full-length rPGT. The transfection medium was removed after 3 h, and the cells were incubated for an additional 20-26 h at 37°C in culture media (DMEM with 10% fetal bovine serum and antibiotics).
Tracer PG uptakes were performed as previously described (17). Briefly, after the cell monolayers were washed twice with Waymouth solution, timed uptake was begun by changing to Waymouth solution containing [3H]PGE2 (171 Ci/mmol; DuPont-New England Nuclear, Boston, MA) at a final concentration of 0.1 µCi/ml (0.6 nM PGE2). Uptake was terminated by aspirating the uptake medium and washing the cell monolayers twice with ice-cold Waymouth solution containing 5% bovine serum albumin and twice with ice-cold unaltered Waymouth solution. Cells were scraped and suspended in 1 ml of Waymouth solution, which was then added to 10 ml of scintillation fluid for determination of radioactive counts by liquid scintillation spectrophotometry. In separate experiments, water or in vitro transcribed and capped rPGT cRNA was injected into X. laevis oocytes (50 ng mRNA/oocyte), and uptake of [3H]PGE2 was determined 3 days after injection to confirm expression (11).Immunocytochemistry and immunoblotting of heterologously expressed rPGT. For immunocytochemistry, HeLa cells seeded onto glass coverslips were transiently transfected with rPGT as described in Transient expression of PGT cDNAs and determination of tracer PGE2 uptake and were then fixed in 2% paraformaldehyde for 60 min. After being blocked with 5% goat serum, cells were incubated with MAb20 as the supernatant at 4°C overnight, followed by incubation with FITC-coupled goat anti-mouse IgG. Fluorescence was visualized with a Bio-Rad MRC 600 laser scanning confocal microscope.
For immunoblotting, 10 water-injected control X. laevis oocytes and 10 rPGT-expressing oocytes (confirmed by tracer PGE2 uptake as described in Transient expression of PGT cDNAs and determination of tracer PGE2 uptake) were extracted with Triton X-100 (0.5%, 10 min, 4°C) and centrifuged at 12,000 g for 10 min. Equivalent aliquots of supernatant were subjected to SDS-PAGE electrophoresis. Proteins were electrophoretically transferred to nitrocellulose membranes, blocked in 5% nonfat milk, and immunoblotted with MAb20. In separate experiments, rPGT was transiently expressed in HeLa cells as described in Transient expression of PGT cDNAs and determination of tracer PGE2 uptake, and the cells were scraped into protease inhibitors and subjected to SDS-PAGE, followed by transfer and immunoblotting with MAb20. Primary antibody was added as the undiluted hybridoma supernatant; the secondary antibody was horseradish peroxidase-coupled rabbit anti-mouse IgG (Cappel, Westchester, PA). Bands were visualized by enhanced chemiluminescence (Amersham).Immunocytochemistry and immunoblotting of endogenous rPGT in rat kidney. Kidney immunocytochemistry was performed in one of three ways. In each case, male Sprague-Dawley rats were anesthetized with Nembutal.
In some experiments, rat kidneys were perfusion fixed. After the kidneys were perfused free of blood with heparinized saline, they were perfused with 2% paraformaldehyde for 2-5 min at 27°C, cut into coronal sections, and immersion fixed overnight in 2% paraformaldehyde at 4°C. Vibratome sections (50 µm) were cut and incubated in PBS for 1-2 days at 4°C with daily changes, then blocked in 5-10% goat serum, incubated in MAb20 (as hybridoma supernatant) overnight at 4°C, washed, and incubated in FITC-coupled goat anti-mouse IgG. For ultrastructural kidney localization, vibratome sections generated as described above were blocked for 2 h in 5% nonfat milk, incubated overnight in MAb20 and then for 2 h in goat anti-mouse IgG coupled to 20-nm gold (EY Laboratories), and washed, after which the gold was silver enhanced for 5-15 min (Vector Laboratories, Burlingame, CA). In separate experiments, unfixed rat kidneys were frozen, and 5-µm cryosections were cut onto glass slides, fixed with 0.5% paraformaldehyde for 30 min at 27°C, and washed and labeled as above. Sections were visualized by confocal microscopy. Carbonic anhydrase type II was visualized in rat kidneys with a rabbit polyclonal antibody (7, 9), followed by visualization with a rhodamine-coupled goat anti-rabbit IgG. COX-1 was visualized by using a mouse monoclonal antibody of the IgG2b class (Oxford Biomedical Research) followed by a type-specific rhodamine-coupled goat anti-mouse secondary antibody (Fisher, Hampton, NH). In this case, MAb20 was visualized by using an FITC-coupled goat anti-mouse IgG1-specific antibody (Vector Laboratories). COX-2 was visualized by using an anti-sheep goat polyclonal antibody (Cayman Chemical, Ann Arbor, MI). Von Willibrand factor (vWF) was detected by using a rabbit anti-human vWF antibody (Dako, Carpinteria, CA) followed by a lissamine rhodamine-coupled goat anti-rabbit immunoglobulin. Synaptopodin was detected by using a rabbit polyclonal antibody that was courtesy of Dr. Peter Mundel (27). For immunoblots of endogenous rPGT in kidney, rats were anesthetized, their kidneys were perfused with heparin and homogenization buffer (0.32 M sucrose, 5 mM Tris · HCl, 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin), removed, and cut into coronal slices, which were further cut into cortical, medullary, and papillary zones. Pooled samples were pulverized by using a liquid nitrogen mortar and pestle and scraped into homogenization buffer, and 20 µg protein/lane were subjected to SDS-PAGE electrophoresis and immunoblotting as described in Immunocytochemistry and immunoblotting of heterologously expressed rPGT for heterologous expression of PGT in HeLa cells. Band densities were quantified from radiographs by scanning and measuring pixel intensities with Adobe Photoshop.Immunoblotting and immunocytochemistry of rPGT in rat platelets. Blood was harvested from the abdominal vena cava of anesthetized rats, dripped into a polypropylene tube containing 0.372 ml of 10% EDTA, and centrifuged at 200 g for 15 min at 21°C, and the platelet-rich plasma (PRP) was pipetted off. Column purification of PRP was performed by applying PRP to a running Sepharose 2B column at 21°C as described previously (29, 33).
For platelet immunocytochemistry, washed platelets (~4 ×105/µl) were equilibrated at 37°C, and aliquots were incubated with or without thrombin (2.5 U/ml) for 1 min, pelleted by centrifugation (2,000 g), and resuspended in 50 µl wash buffer. They were then dispersed in liquefied 10% gelatin. After solidification on ice, the gelatin blocks were embedded in OCT and stored at ![]() |
RESULTS |
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Validation of anti-rPGT antibody.
We first set out to validate mouse MAb20 by showing that it can
recognize the rPGT holoprotein immunocytochemically in fixed cells and
also when PGT is subjected to immunoblotting. We used HeLa cells and
X. laevis oocytes because our laboratory has previously shown that these cell types have a low level of background PG transport
and also express heterologous PGT well (11, 17). HeLa
cells were transiently transfected with full-length cDNAs encoding
rPGT. After expression of the transporter at the plasma membrane was
confirmed by tracer PGE2 uptake (data not shown) (17), the cells were examined immunocytochemically. Mouse
MAb20 recognized rPGT at the plasma membrane (Fig.
1A, arrows) as well as in an
intracellular compartment that probably represents the Golgi apparatus.
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Immunological characterization of PGT in rat
kidneys.
On the basis of the hypothesis that insights into PGT function could be
gained by knowing the cell types in which it is expressed, rat kidneys
were sectioned into zones and probed by immunoblotting. Figure
2A shows that MAb20 recognizes
an immunoreactive band of 65 kDa in rat kidney with the zonal
distribution cortex (C) < medulla (M) papilla (P).
Quantitation on two separate blots showed that the PGT band revealed
5.6 average densitometry units of cortex, 8.1 of medulla, and 9.7 of
papilla. Adsorption of MAb20 with the immunogen abrogated
immunoreactivity (data not shown).
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Immunological characterization of PGT in rat
platelets.
The results in rat kidney suggest that PGT expression is confined to
cell types that synthesize and release PGs. To test this hypothesis
further, we turned to rat platelets, a readily available cell that
synthesizes Tx. Figure 5A,
top, shows that rat platelets embedded in gelatin express strong
PGT expression by fluorescence immunocytochemistry. This labeling is
abolished when the PGT-GST fusion peptide is mixed with mAb20 (Fig.
5A, bottom). Addition of thrombin before gelatin
embedding causes platelet aggregation (Fig. 5A,
right). It is unclear whether the apparent increase in
fluorescence intensity after thrombin addition (Fig. 5A,
top) can be ascribed to either aggregation or unmasking of
PGT epitope(s), or both.
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DISCUSSION |
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We have generated a monoclonal antibody (MAb20) against a putative
extracytoplasmic loop of rPGT. We validated the ability of the antibody
to recognize the intact transporter by using heterologous expression in
cultured cells and X. laevis oocytes. When used to probe for
endogenous PGT expression in kidneys and platelets, this antibody
revealed that the pattern of PGT expression by kidney zones
(papilla medulla > cortex) and in distinct kidney cell types correlates with known PG synthesis, including glomerular endothelial and mesangial cells, cortical arteriolar endothelium and
muscularis, collecting ducts, and medullary interstitial cells and vasa
rectae. Further immunological studies indicated that PGT is strongly
expressed in rat platelets. In contrast, the proximal straight tubule,
which is known to take up, oxidize, and secrete PGs in a process of
metabolic clearance, does not express PGT.
Topper et al. (34) reported that antisera against the COOH terminus of human PGT labeled endothelia, but not other structures, in the human kidney, results which vary from the present data. In addition to recognizing PGT in endothelia, our anti-rat mAb20 also recognizes immunoreactivity in glomerular mesangial cells, arteriolar muscularis layers, collecting ducts, medullary interstitial cells, and papillary surface epithelia. Differences in fixation technique may account for the variation in labeling between our work and that of Topper et al., because we found glomerular and cortical arteriolar labeling to be strongest in cryosections, whereas tubular and interstitial cell labeling was best obtained with perfusion fixation. In addition, species differences in expression may play a role. Finally, the human PGT gene structure suggests that multiple protein isoforms might arise by means of alternative splicing (23). Thus it is possible that different domains of the transporter are expressed in different tissues and might be recognized variably by antisera against differing protein domains.
Although PGT is expressed at the plasma membrane of medullary vasa recta cells, in collecting duct and medullary interstitial cells it is expressed in cytoplasmic vesicles. Nonetheless, we hypothesize that PGT can be recruited to the plasma membrane by means of exocytic-endocytic cycling, as is the case with many other plasma membrane transporters, such as sodium channels and glucose transporters (6, 8). In support of this hypothesis, we have shown in companion experiments that green fluorescence protein-tagged rPGT is sorted in Madin-Darby canine kidney cells to the apical plasma membrane, where it mediates active uptake and transepithelial PG resorption (12). Further experiments will be required to delineate the stimuli, if any, that could induce exocytic insertion into the plasma membrane in collecting duct and medullary interstitial cells.
The glomerular labeling for PGT shows a possible nuclear or perinuclear labeling pattern. Although this is most likely due to a shift of antigen during fixation (26), we cannot exclude the possibility that PGT in glomeruli is expressed in a different cellular compartment than that in tubules, endothelia, and interstitial cells. If this is the case, it could indicate multiple functions for the transporter, depending on cell type.
On the basis of its broad expression, we previously postulated that PGT might play a role in the metabolic clearance and/or release of PGs (32). The present results help us to better consider these possibilities.
The lack of PGT expression in the proximal tubule argues strongly against a role in the known metabolic clearance of PGs by this nephron segment (32). Presumably, uptake of PGs by the proximal tubule is mediated by any of several organic anion transporters identified recently, some of which have been shown to be expressed at the proximal tubule basolateral membrane (35).
The present data show that PGT expression is limited to cells that synthesize and release PGs. Indeed, we have shown in recent preliminary studies that COX-2 and PGT are coordinately induced by serum in Swiss 3T3 cells (22). Because PGs efflux readily from cells by simple diffusion (11) and because PGT is coupled to glycolysis to be energetically poised for PG uptake (10), it seems unlikely that PGT directly mediates the release of newly synthesized prostanoids.
Instead, we speculate that PGT at the plasma membrane mediates PG
reuptake. Such a model might be similar to that of synaptic signaling,
in which the secreted neurotransmitter is taken up by the cell that
released it and also by other cell types. In the brain,
-aminobutyric acid is cleared from the synaptic cleft by specific,
high-affinity Na- and Cl-dependent transporters located on both
presynaptic terminals and surrounding glial cells (5). In
the retina, glutamate is released by photoreceptor cells and is taken
up by glutamate transporters expressed on both photoreceptor cells and
Muller cells (36).
In the case of PGs, we propose that efflux would occur by unregulated simple diffusion (11). In the presence of an outwardly directed lactate gradient from aerobic glycolysis [for example, in collecting ducts (2, 15)], PGT would be poised to take PGs back into the cytoplasm. PG reuptake might 1) control net release either as a negative-feedback loop when PG synthesis is stimulated or in quiescent cells to minimize "leak" of basal PGs or 2) signal intracellular events using pathways separate from cell-surface prostanoid receptors, e.g., by binding to prostanoid receptors expressed on the nuclear envelope (3, 4) or to peroxisome proliferator activator receptors in the nucleus (14, 19). Testing of these models will require further experiments.
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ACKNOWLEDGEMENTS |
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We thank Dr. Matthew Scharff and Susan Buhl for invaluable assistance with the monoclonal antibody, Dr. Peter Mundel for the antibodies to synaptopodin, Dr. Wendy Cammer for the antibodies to carbonic anhydrase, and the Einstein Analytical Imaging Facility for use of the Bio-Rad MRC 600 laser scanning confocal microscope and electron microscope sectioning and viewing.
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FOOTNOTES |
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A portion of this work was supported by grants to V. L. Schuster from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-49688) and the American Heart Association (New York City affiliate).
Address for reprint requests and other correspondence: V. L. Schuster, Renal Div., Ullmann Bldg. Rm. 615, 1300 Morris Park Ave; Bronx, NY 10461 (E-mail: schuster{at}aecom.yu.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.
10.1152/ajprenal.00152.2001
Received 15 May 2001; accepted in final form 27 December 2001.
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