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

Abstract

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 Х medulla Ͼ 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 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, PGE 2 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 PGE 2 and TxB 2 , 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 TxA 2 (1). MATERIALS AND METHODS 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 im...

Prostaglandin Signaling in the Renal Collecting Duct

“…COX-1 was expressed in a perinuclear pattern consistent with its known localization to the nuclear envelope (21). PGT was expressed at the apical membrane as reported previously by our laboratory (10). PGDH was expressed throughout the cytoplasm of cortical collecting duct cells.…”

“…B, immunolocalization of endogenous PGT in rat kidney cortical collecting ducts using a mouse monoclonal antibody. Arrows indicate localization at or near the apical plasma membrane (10). C, immunolocalization of endogenous PGDH in rat kidney cortex.…”

“…However, whereas collecting ducts express PGT in subapical vesicles or at the apical plasma membrane (10,32), MDCK cells have lost this expression. Similarly, PGT expression is lost from endothelial cells when cultured, but expression is regained when shear stress is applied (32)(33)(34)(35).…”

“…For PGT, sections were incubated overnight at 4°C with mouse monoclonal antibody #20 as straight hybridoma supernatatant (10). For PGDH and cyclooxygenase-1 (COX-1), sections were incubated overnight at 4°C with a rabbit antiserum to PGDH (catalog number 160615, 1:100) or with rabbit anti-mouse COX-1 (catalog number * This work was supported by National Institutes of Health Grants R01DK049688 and P50DK064236 (to V. L. S.).…”

Prostaglandin transporter expression in mouse brain during development and in response to hypoxia

“…Second, we used in our experiments microsomal preparations from four different brain regions (cortex, hippocampus, cerebellum and brainstem/diencephalon) and demonstrated that the cortex has the highest expression of PGT in the mouse brain. We would like to note that PGT expression may be highly coordinated with the synthesis and release of prostaglandins since Bao et al (2002) have shown that PGT expression is limited to cells that synthesize and release prostaglandins. The same investigators have also shown that cyclooxygenase-2 (COX-2) -an inducible enzyme which catalyses PG formation in the brain, as well as PGT, are coordinately induced in Swiss 3T3 cells in-vitro (Schuster, 1998).…”