Termination of prostaglandin (PG) signaling has been proposed to involve carrier-mediated uptake across the plasma membrane followed by cytoplasmic oxidation. Here, we tested this hypothesis directly by coexpressing the PG uptake carrier prostaglandin transporter (PGT) in various cell types with and without human PG 15 dehydrogenase (PG15DH). In HeLa cells, which express neither PGT nor PG15DH, exogenously added PGE2 or PGF2␣ were rapidly oxidized to the 13,14-dihydro,15-keto metabolites only when PGT and PG15DH were coexpressed, directly confirming the two-step hypothesis. Cells expressing PG15DH that were broken open formed more PG metabolites than cells in which the PGs could gain access to PG15DH only via PGT. Similar results were obtained using the human prostate cancer cell line LNCaP, in which endogenous PG15DH is induced after exposure to dihydrotestosterone. Because PGT in vivo is expressed in renal collecting duct epithelia, we also expressed PGT in Madin-Darby canine kidney cells grown on filters, where it mediated both the active uptake of PGE2 across the apical membrane and the transepithelial transport of PGE2 to the basolateral compartment. When PG15DH was coexpressed with PGT in these epithelial monolayers, about half of the PGE2 taken up apically was oxidized to 13,14-dihydro,15-keto-PGE2, which in turn exited the cells nondirectionally into both the apical and basolateral compartments. Our data represent reconstitution of the longstanding model of PG metabolism consisting of sequential carrier-mediated PG uptake, cytoplasmic oxidation, and diffusional efflux of the PG metabolite.

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The PG transporter (PGT) is expressed in subapical vesicles in the kidney collecting duct. To gain insight into the possible function of the PGT in this tubule segment, we tagged rat PGT with green fluorescent protein at the COOH terminus and generated stable PGT-expressing Madin-Darby canine kidney cell lines. When grown on permeable filters, green fluorescent protein-PGT was expressed predominantly at the apical membrane. Although the basal-to-apical transepithelial flux of [(3)H]PGE(2) was little changed by PGT expression, the apical-to-basolateral flux was increased 100-fold compared with wild-type cells. Analysis of driving forces revealed that this flux represents PGT-mediated active transepithelial PGE(2) transport. We propose that endogenous PGT is exocytically inserted into the collecting duct apical membrane, where it could control the concentration of luminal PGs.

During water deprivation, prostaglandin E(2) (PGE(2)), formed by renal medullary interstitial cells (RMICs), feedback inhibits the actions of antidiuretic hormone. Interstitial PGE(2) concentrations represent the net of both PGE(2) synthesis by cyclooxygenase (COX) and PGE(2) uptake by carriers such as PGT. We used cultured RMICs to examine the effects of hyperosmolarity on both PG synthesis and PG uptake in the same RMIC. RMICs expressed endogenous PGT as assessed by mRNA and immunoblotting. RMICs rapidly took up [(3)H]PGE(2) to a level 5- to 10-fold above background and with a characteristic time-dependent "overshoot." Inhibitory constants (K(i)) for various PGs and PGT inhibitors were similar between RMICs and the cloned rat PGT. Increasing extracellular hyperosmolarity to the range of 335-485 mosM increased the net release of PGE(2) by RMICs, an effect that was concentration dependent, maximal by 24 h, reversible, and associated with increased expression of COX-2. Over the same time period, there was decreased cell-surface activity of PGT due to internalization of the transporter. With continued exposure to hyperosmolarity over 7-10 days, PGE(2) release remained elevated, COX-2 returned to baseline, and PGT-mediated uptake became markedly reduced. Our findings suggest that hyperosmolarity induces coordinated changes in COX-2-mediated PGE(2) synthesis and PGT-mediated PGE(2) uptake in RMICs.

Prostaglandins mediate autacrine and paracrine signaling over short distances. We used the renal collecting duct as a model system to test the hypothesis that local control of prostaglandin signaling is achieved by expressing inactivation in the same cell as synthesis. Immunocytochemical studies demonstrated that renal collecting ducts in situ express the prostaglandin (PG) synthesis enzyme, cyclooxygenase-1 (COX-1), as well as both components of prostaglandin metabolic inactivation, i.e. the prostaglandin uptake carrier prostaglandin transporter (PGT) and the enzyme 15-hydroxyprostaglandin dehydrogenase. We characterized this system further using the collecting duct cell line Madin-Darby canine kidney (MDCK), which retains COX-2 and prostaglandin dehydrogenase expression but which has lost PGT expression. When we reintroduced PGT, it was correctly sorted to the apical membrane where it altered the sidedness of prostaglandin E2 (PGE2) release, a process we call "vectorial release via sided reuptake." Importantly, although COX-2 and prostaglandin dehydrogenase are expressed in the same MDCK cell, they must be compartmentalized because even in the presence of excess dehydrogenase newly synthesized PGE2 is released largely un-oxidized. However, when PGE2 undergoes first release and then PGT-mediated reuptake, significant oxidation takes place, suggesting that PGT imports PGE2 into the prostaglandin dehydrogenase compartment. Our data are consistent with a new model that offers significant new mechanisms for the fine control of eicosanoid signaling.Prostaglandins (PGs) 1 represent an extreme example of context-dependent autacrine or paracrine signaling. A single type of prostaglandin, PGE2, can activate any of four receptor subtypes (EP 1 , EP 2 , EP 3 , and EP 4 ) so as to mediate changes in physiological function as diverse as gastric acid secretion, body temperature, intraocular pressure, blood pressure, and airway reactivity (1-3).Even within a single organ, such as the kidney, PGE2 has diverse effects including afferent arteriolar vasodilatation, reduction of NaCl resorption by the thick ascending limb of Henle, vasodilatation of medullary vasa rectae, and inhibition of osmotic water flow in the cortical collecting duct (2). On a smaller scale, the renal collecting expresses luminal EP 4 receptors, activation of which increases Na ϩ reabsorption and increases water reabsorption, and basolateral EP 1 receptors, activation of which signals the opposite effects (4, 5). Clearly, to achieve the requisite fidelity in PGE2 signaling, rapid inactivation must occur.PG inactivation involves active uptake into the cell followed by cytoplasmic oxidation (6). Our laboratory identified the prostaglandin transporter PGT (Slc21a2; oatp2A1) (7), which is the lead candidate for the uptake step. Targeted deletion of mouse PGT results in death at post-natal day 1, most likely the result of an inability to inactivate circulating PGE2. 2 The enzyme 15-hydroxyprostaglandin dehydrogenase (PGDH) has been extensively characterized by ot...