Prostaglandin transporter PGT is expressed in cell types that synthesize and release prostanoids

Yi Bao1, Michael L. Pucci1, Brenda S. Chan1, Run Lu1, Shigekazu Ito1, and Victor L. Schuster1,2

Departments of 1 Medicine and 2 Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 congruent  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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 -80°C. Thereafter, 5-µm cryosections were cut, thawed onto glass coverslips, and fixed in 0.5% paraformaldyde for 1 h. After an overnight rinsing in PBS at 4°C, the sections were exposed to MAb20, followed by exposure to an FITC-coupled secondary antibody.

For immunoblots of endogenous PGT in rat platelets, protein lysates from washed rat platelets were separated by SDS-PAGE, transferred to nitrocellulose membranes, probed with MAb20 followed by a horseradish peroxidase-coupled secondary antibody, and visualized using chemiluminescence.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Validation of monoclonal antibody 20 (MAb20) with heterologously expressed rat prostaglandin transporter PGT (rPGT). A: fluorescence immunocytochemistry of rPGT transiently expressed in HeLa cells and labeled with MAb20. PGT is expressed in an intracellular compartment (probably Golgi) and at the plasma membrane (arrows). B: expression of rPGT in Xenopus laevis oocytes. Triton-soluble fractions were separated by SDS-PAGE and immunoblotted with MAb20. Lanes 1 and 3: oocytes were injected with rPGT cRNA. Lanes 2 and 4: water-injected oocytes. Lanes 3 and 4: MAb20 was preadsorbed with the immunizing fusion protein. C: expression of rPGT in HeLa cells. Lane 1, transfection with vector control; lane 2, transfection with rPGT cDNA.

X. laevis oocytes were injected with water or cRNA encoding rPGT, and the membranes were extracted with Triton X-100, subjected to SDS-PAGE under nonreducing conditions, and immunoblotted with MAb20 (Fig. 1B). The strong immunoreactive band at 68 kDa (Fig. 1B, lane 1), corresponding to the predicted PGT molecular mass of 68 kDa, was not seen in control (water-injected) oocytes (Fig. 1B, lanes 2 and 4), or when the hybridoma supernatant was preabsorbed with the fusion peptide (Fig. 1B, lanes 3 and 4).

Separately, HeLa cell monolayers transiently transfected with rPGT were immunoblotted with MAb20 under both reducing and nonreducing conditions. Similar to oocytes, mAb20 recognized a 65-kDa immunoreactive band (Fig. 1C, lane 2) that was not present after transfection with the vector control (Fig. 1C, lane 1). Immunoreactivity was abrogated by including the immunogen peptide or when the reducing agent dithiothreitol was used (data not shown). The latter finding is consistent with our modeling of the immunogenic region, a region rich in cysteine residues, as being extracytoplasmic and thus structurally defined by means of disulfide bonds (32).

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) congruent  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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Fig. 2.   Expression of endogenous PGT in rat kidney zones. A: rat kidney was divided by hand into cortex (C), medulla (M), and papilla (P), and these zones were subjected to SDS-PAGE and immunoblotting with MAb20. B: 50-µm coronal cryosections of kidney were cut and labeled with MAb20 hybridoma supernatant (left) or with mAb20 supernatant that had been preadsorbed with the immunogen (right). Detection was by the peroxidase method.

We next probed rat kidney immunocytochemically for PGT expression by using several complementary methods. Perfusion fixation gives better overall tissue preservation than cryosectioning, because the tissue is well penetrated by fixative and sectioning with a vibratome leaves a substantial core of central material undamaged by the knife. However, because the fixative concentration is highest in the vascular lumen, this method can result in false-negative vascular immunolocalization, especially when monoclonal antibodies are used, if that epitope happens to be easily denatured by fixation. Therefore, we used a combination of perfusion fixation for light and ultrastructural labeling, and we also used light-level cryosectioning to capture all possible PGT immunoreactivity.

Figure 2B shows a whole-kidney "zonal" approach similar to that taken above with the immunoblot. A 50-µm cryosection of whole rat kidney was labeled immunocytochemically with MAb20. As with the immunoblot, this low-power view reveals that the kidney cortex has relatively sparse overall PGT protein labeling, whereas the medulla and papilla demonstrate more pronounced, and roughly equivalent, PGT labeling intensities.

Figure 3 shows MAb20-labeled rat renal vascular structures in cryosectioned tissue (Fig. 3, A-H). Figure 3A shows MAb20-labeled glomeruli in a possible nuclear or perinuclear labeling pattern. Some glomerular regions were positive for the endothelial marker vWF (Fig. 3B). Double labeling revealed that these regions coexpress PGT and vWF (Fig. 3C), indicating that PGT is expressed in glomerular endothelial cells.


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Fig. 3.   Expression of endogenous rPGT in vascular structures. A-H: confocal sections of 1- to 2-µm optical thickness from kidney cryosections. I: section from perfusion-fixed kidney. A-C: PGT in glomerular endothelial cells. Labeling is for PGT (A), von Willibrand factor (vWF; expressed by endothelia; B), and the merging of A and B (C). C: yellow-labeled cells represent endothelial cells expressing both PGT and vWF. D-F: lack of PGT expression in glomerular podocytes. Labeling is for PGT (D), synaptopodin (expressed by podocytes; E), and the merging of images D and E (F). F: lack of yellow-labeled cells indicates that PGT is not expressed in podocytes. G and H: low-power view of rat kidney cortex. Arrows, glomeruli fed by arterioles; *, an isolated arteriole. Labeling for PGT alone (G) and double-labeling for vWF (endothelia; H) reveal that PGT is expressed in arteriolar endothelia (yellow label in the 2 arterioles leading into the glomeruli and in the arteriole at bottom left) and in the muscularis layer (green label outside of the yellow regions in the arterioles). I: transmission electron micrograph of rat kidney labeled for PGT by the preembedding immunogold method. An outer medullary vasa recta endothelial cell body is shown with gold particles in the cytoplasm (presumably vesicular) and at the plasma membrane (arrows).

Glomerular cells devoid of vWF expression were also labeled with MAb20 (Fig. 3C, green); these could represent either mesangial cells or podocytes. Glomerular podocytes selectively express synaptopodin (27). Double labeling for PGT with MAb20 (Fig. 3D) and a rabbit polyclonal anti-synaptopodin antibody (Fig. 3E) revealed that podocytes do not express PGT (Fig. 3F). Therefore, by elimination, the cells in Fig. 3C that were positive for PGT but negative for vWF appear to be mesangial cells.

PGT expression was also observed in adjacent renal cortical arterioles (Fig. 3G). Double labeling for vWF demonstrates PGT expression in both the endothelial layer (coexpression with vWF) and the muscularis layer (Fig. 3H). PGT could be visualized ultrastructurally in medullary vasa rectae cell bodies in the cytoplasm and at the plasma membrane (Fig. 3I).

Perfusion-fixation methods also revealed PGT expression in nonvascular structures. Figure 4A shows a representative cortical collecting duct in which PGT is expressed in what appear to be subapical vesicles in some, but not all, cells. In contrast to the case with vasa rectae, ultrastructural examination revealed no expression at the plasma membrane in collecting duct cells (data not shown). The PGT-positive cells are principal cells, as shown by double labeling for PGT and the intercalated cell-specific enzyme carbonic anhydrase II (Fig. 4B) (18, 31). Indeed, PGT is expressed in cytoplasmic vesicles along the collecting duct, including the outer medullary and papillary segments (Fig. 4C). The papillary surface epithelium also expressed a strong pattern of vesicular PGT (not shown). Medullary interstitial cells of the initial portion of the inner medulla also express PGT in cytoplasmic vesicles. Double labeling revealed that these cells express PG endoperoxide H synthase-1 (Fig. 4D).


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Fig. 4.   Expression of PGT in nonvascular structures of rat kidney. Confocal sections of 1- to 2-µm optical thickness from kidney vibratome sections. A: cortical collecting duct. PGT is expressed in subapical vesicles in some, but not all, cells. B: cortical collecting duct, double-labeled for PGT (immunogold labeling method, in which the gold particles are pseudocolored green) and carbonic anhydrase II (red). Arrows, PGT in subapical locations in principal cells; *, intercalated cells rich in carbonic anhydrase. C: papillary collecting ducts. Green label is PGT in cytoplasmic vesicles. Red label is rhodamine-tagged lectin. D: medullary interstitial cells double-labeled for PGT (green) and cyclooxygenase-2 (red). Cyclooxygenase-2 label (COX-2) is at the nuclear membrane, whereas PGT appears to be asymmetrically expressed in vesicles at one end of the cells.

Immunocytochemistry using anti-COX-2 antisera revealed weak labeling of both macula densa and medullary interstitial cells, as expected in rats on a controlled-sodium diet (37). There was no colocalization between PGT and COX-2 in the macula densa, whereas PGT did colocalize with COX-2 in interstitial cells in a pattern indistinguishable from that of COX-1 (data not shown).

The proximal tubules of rat kidney were negative in all of these studies. We also used other fixation regimens, such as periodate-lysine-paraformaldehyde (25) and "antigen retrieval" with detergents (30); none of these methods revealed labeling of the proximal tubule (data not shown).

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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Fig. 5.   Expression of PGT in rat platelets. A: fluorescence immunocytochemistry of rat platelets embedded in gelatin. Platelets were not exposed to thrombin (left) or were induced to aggregate by exposure to thrombin (2.5 U/ml) for 1 min (right). Bottom: MAb20 was preadsorbed with the immunizing peptide. B: immunoblot of endogenous PGT in rat platelets (plts) compared with recombinant rPGT heterologously expressed in HeLa cells (left). Single immunoreactive band is recognized by MAb20 in both lanes. Right: MAb20 was preadsorbed with the immunogen, i.e., the PGT-glutathione S-transferase fusion protein.

On immunoblots of rat platelets, MAb20 recognized a band of 65 kDa that comigrates with rPGT heterologously expressed in HeLa cells (68 kDa), which was adsorbed out almost completely with the GST-PGT fusion peptide (Fig. 5B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 congruent  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, gamma -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Von Bruchhausen, F, and Walter U (Editors). Platelets and Their Factors. New York: Springer, 1997.

2.   Bagnasco, S, Good D, Balaban R, and Burg M. Lactate production in isolated segments of the rat nephron. Am J Physiol Renal Fluid Electrolyte Physiol 248: F522-F526, 1985[ISI][Medline].

3.   Bhattacharya, M, Peri K, Ribeiro-da-Silva A, Almazan G, Shichi H, Hou X, Varma DR, and Chemtob S. Localization of functional prostaglandin E2 receptors ep3 and ep4 in the nuclear envelope. J Biol Chem 274: 15719-15724, 1999[Abstract/Free Full Text].

4.   Bhattacharya, M, Peri KG, Almazan G, Ribeiro-da-Silva A, Shichi H, Durocher Y, Abramovitz M, Hou X, Varma DR, and Chemtob S. Nuclear localization of prostaglandin E2 receptors. Proc Natl Acad Sci USA 95: 15792-15797, 1998[Abstract/Free Full Text].

5.   Borden, LA. GABA transporter heterogeneity: pharmacology and cellular localization. Neurochem Int 29: 335-356, 1996[ISI][Medline].

6.   Bradbury, NA, and Bridges RJ. Role of membrane trafficking in plasma membrane solute transport. Am J Physiol Cell Physiol 267: C1-C24, 1994[Abstract/Free Full Text].

7.   Brion, LP, Cammer W, Satlin LM, Suarez C, Zavilowitz BJ, and Schuster VL. Expression of carbonic anhydrase IV in carbonic anhydrase II-deficient mice. Am J Physiol Renal Physiol 273: F234-F245, 1997[Abstract/Free Full Text].

8.   Brown, D. Membrane recycling and epithelial cell function. Am J Physiol Renal Fluid Electrolyte Physiol 256: F1-F12, 1989[Abstract/Free Full Text].

9.   Cammer, W, and Zhang H. Comparison of immunocytochemical staining of astrocytes, oligodendrocytes, and myelinated fibers in the brains of carbonic anhydrase II-deficient mice and normal littermates. J Neuroimmunol 34: 81-86, 1991[ISI][Medline].

10.   Chan, BS, Endo S, Kanai N, and Schuster VL. Identification of lactate as a driving force for prostanoid transport by prostaglandin transporter PGT. Am J Physiol Renal Physiol 282: F1097-F1102, 2002[Abstract/Free Full Text].

11.   Chan, BS, Satriano JA, Pucci ML, and Schuster VL. Mechanism of prostaglandin E2 transport across the plasma membrane of HeLa cells and Xenopus oocytes expressing the prostaglandin transporter "PGT." J Biol Chem 273: 6689-6697, 1998[Abstract/Free Full Text].

12.   Endo, S, Nomura T, Chan BS, Lu R, Pucci ML, Bao Y, and Schuster VL. Expression of the prostaglandin transporter PGT in MDCK cell monolayers: polarized apical localization and induction of active prostaglandin transport. Am J Physiol Renal Physiol 282: F618-F622, 2002[Abstract/Free Full Text].

13.   Farman, N, Pradelles P, and Bonvalet JP. PGE2, PGF2-alpha , 6-keto-PGF1-alpha , and TxB2 synthesis along the rabbit nephron. Am J Physiol Renal Fluid Electrolyte Physiol 252: F53-F59, 1987[Abstract/Free Full Text].

14.   Forman, BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, and Evans RM. 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell 83: 803-812, 1995[ISI][Medline].

15.   Guder, WG, Wagner S, and Wirthensohn G. Metabolic fuels along the nephron: pathways and intracellular mechanisms of interaction. Kidney Int 29: 41-45, 1986[ISI][Medline].

16.   Irish, JM. Secretion of prostaglandin E2 by rabbit proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 237: F268-F273, 1979[ISI][Medline].

17.   Kanai, N, Lu R, Satriano JA, Bao Y, Wolkoff AW, and Schuster VL. Identification and characterization of a prostaglandin transporter. Science 268: 866-869, 1995[ISI][Medline].

18.   Kim, J, Tisher CC, Linser PJ, and Madsen KM. Ultrastructural localization of carbonic anhydrase II in subpopulations of intercalated cells of the rat kidney. J Am Soc Nephrol 1: 245-256, 1990[Abstract].

19.   Kliewer, SA, Lenhard JM, Willson TM, Patel I, Morris DC, and Lehmann JM. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell 83: 813-819, 1995[ISI][Medline].

20.   Kohler, G, and Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497, 1975[ISI][Medline].

21.   Lu, R, Kanai N, Bao Y, and Schuster VL. Cloning, in vitro expression, and tissue distribution of a human prostaglandin transporter cDNA (hPGT). J Clin Invest 98: 1142-1149, 1996[Abstract/Free Full Text].

22.   Lu, R, Pucci ML, Nomura T, and Schuster VL. Characterization and regulation of the prostaglandin transporter PGT in the mouse Swiss 3t3 fibroblast cell line. J Am Soc Nephrol 12: A3035, 2001.

23.   Lu, R, and Schuster VL. Molecular cloning of the gene for the human prostaglandin transporter hPGT: gene organization, promoter activity, and chromosomal localization. Biochem Biophys Res Commun 246: 805-812, 1998[ISI][Medline].

24.   Mattix, HJ, and Badr KF. Arachidonic acid metabolites and the kidney. In: The Kidney, edited by Brenner BM.. Philadelphia, PA: Saunders, 2000, p. 756-792.

25.   McLean, IW, and Nakane PF. Periodate lysine paraformaldehyde fixative: a new fixative for immunoelectron microscopy. J Histochem Cytochem 22: 1077-1083, 1974[ISI][Medline].

26.   Melan, MA, and Sluder G. Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy. J Cell Sci 101: 731-743, 1992[Abstract].

27.   Mundel, P, Heid HW, Mundel TM, Kruger M, Reiser J, and Kriz W. Synaptopodin: an actin-associated protein in telencephalic dendrites and renal podocytes. J Cell Biol 139: 193-204, 1997[Abstract/Free Full Text].

28.   Pucci, ML, Bao Y, Chan B, Itoh S, Lu R, Copeland NG, Gilbert DJ, and Schuster VL. Cloning of mouse prostaglandin transporter PGT cDNA: species-specific substrate affinities. Am J Physiol Regulatory Integrative Comp Physiol 277: R734-R741, 1999[Abstract/Free Full Text].

29.   Reverter, JC, Beguin S, Kessels H, Kumar R, Hemker HC, and Coller BS. Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis." J Clin Invest 98: 863-874, 1996[Abstract/Free Full Text].

30.   Sabolic, I, Herak-Kramberger CM, Breton S, and Brown D. Na/K-ATPase in intercalated cells along the rat nephron revealed by antigen retrieval. J Am Soc Nephrol 10: 913-922, 1999[Abstract/Free Full Text].

31.   Schuster, VL. Function and regulation of collecting duct intercalated cells. Annu Rev Physiol 55: 267-288, 1993[ISI][Medline].

32.   Schuster, VL. Molecular mechanisms of prostaglandin transport. Annu Rev Physiol 60: 221-242, 1998[ISI][Medline].

33.   Scudder, LE, Kalomiris EL, and Coller BS. Preparation and functional characterization of monoclonal antibodies against glycoprotein Ib. Methods Enzymol 215: 295-311, 1992[ISI][Medline].

34.   Topper, JN, Cai J, Stavrakis G, Anderson KR, Woolf EA, Sampson BA, Schoen FJ, Falb D, and Gimbrone MA, Jr. Human prostaglandin transporter gene (hPGT) is regulated by fluid mechanical stimuli in cultured endothelial cells and expressed in vascular endothelium in vivo. Circulation 98: 2396-2403, 1998[Abstract/Free Full Text].

35.   Van Aubel, RA, Masereeuw R, and Russel FG. Molecular pharmacology of renal organic anion transporters. Am J Physiol Renal Physiol 279: F216-F232, 2000[Abstract/Free Full Text].

36.   Wu, SM. Synaptic transmission in the outer retina. Annu Rev Physiol 56: 141-168, 1994[ISI][Medline].

37.   Yang, T, Singh I, Pham H, Sun D, Smart A, Schnermann JB, and Briggs JP. Regulation of cyclooxygenase expression in the kidney by dietary salt intake. Am J Physiol Renal Physiol 274: F481-F489, 1998[Abstract/Free Full Text].


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