Short-Term Regulation of Basolateral Organic Anion Uptake in Proximal Tubular Opossum Kidney Cells: Prostaglandin E2 Acts via Receptor-Mediated Activation of Protein Kinase A

Abstract

ABSTRACT. It was shown previously that EGF induces release of the important prostanoid prostaglandin E2 (PGE2) in proximal tubular opossum kidney (OK) cells and PGE2 then stimulates initial basolateral uptake of organic anions (OA) dose dependently. PGE2 is a receptor agonist and a known substrate for the basolateral exchanger mediating OA uptake (OAT1 and/or OAT3). This study investigated the mechanism of short-term PGE2 action on initial basolateral OA uptake in OK cells. PGE2 stimulation of OA uptake was abolished by selective inhibition of adenylate cyclase (by MDL-12, 330A) or protein kinase A (PKA; by H89). PGE2 stimulation of OA uptake persisted after preloading the cells with glutarate and was still abolished by inhibition of PKA. Selective activation of adenylate cyclase by forskolin led to identical results. These data contradicted the hypothesis that PGE2 action on OA uptake is due to its action as a counter ion. Therefore, we tested whether the PGE2 receptors (EP1 to 4) are involved in stimulation of OA uptake in OK cells by PGE2. Because of their intracellular signaling profile, EP1 and EP3 were not taken into account as possible receptors for mediation of PGE2-induced OA uptake. With the use of selective agonists (11-deoxy PGE1 and butaprost), EP4 was pharmacologically identified as the receptor responsible for PGE2-mediated stimulation of OA uptake. By reverse transcription–PCR, cloning, and subsequent sequencing, a homologue fragment to EP4 was identified in OK cells. EGF-induced stimulation of basolateral organic anion uptake was abolished by inhibition of adenylate cyclase or PKA. This indicates that EGF action is mediated by generation of PGE2. The following model is proposed: PGE2 generated in the cells does not act as a counter ion but activates adenylate cyclase. This is mediated by a homologue of EP4 receptor. cAMP then activates PKA, which stimulates initial basolateral uptake of OA in OK cells by a not-yet-known mechanism. PGE2 is an organic anion, a potential stimulator of organic anion excretion, and an important mediator of inflammation all at once. Thus, the mechanism presented here may contribute to a limitation of inflammatory events in the kidney cortex interstitium.