Regulation of mOAT-mediated Organic Anion Transport by Okadaic Acid and Protein Kinase C in LLC-PK1 Cells

Abstract

Organic anion transporters in the kidney proximal tubule play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites, thereby preventing their potentially toxic effects within the body. We have previously cloned a cDNA encoding an organic anion transporter from mouse kidney (mOAT) (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478; Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519 -1524). In the present study, we assessed the potential for regulation of this transporter by heterologous expression of mOAT in the pig proximal tubule-like cell line, LLC-PK 1 . We report here that both protein phosphatase (PP1/PP2A) inhibitor, okadaic acid, and protein kinase C (PKC) activators down-regulate mOAT-mediated transport of para-aminohippuric acid (PAH), a prototypic organic anion, in a time-and concentrationdependent manner. However their mechanisms of action for this down-regulation are distinct. Okadaic acid modulated PAH transport, at least in part, through phosphorylation/dephosphorylation of mOAT; phosphoamino acid analysis indicated this phosphorylation occurs on serine. In contrast, PKC activation induced a decrease in the maximum transport velocity (V max ) of PAH transport without direct phosphorylation of the transporter protein. Together these results provide the first demonstration that regulation of organic anion transport by mOAT is likely to be tightly controlled directly and indirectly by phosphatase PP1/PP2A and PKC. Our results also suggest that kinases other than PKC are involved in this process.Renal organic anion transport plays a vital role in the elimination of a wide variety of potentially toxic and negatively charged waste products of metabolism, drugs, environmental pollutants, and their metabolites from the body. The transport mechanisms responsible for this elimination have been extensively studied (3-5). Based on these studies, it has been suggested that the transport of organic anions is a complex process involving distinctly different proteins at the apical and basolateral membranes of the proximal tubule cells. Organic anions are transported across the basolateral membrane into the cell in exchange for intracellular dicarboxylates, which are subsequently returned into the cell via a sodium-dependent dicarboxylate transporter. Once inside the cell, organic anions are subject to intracellular binding and sequestration within vesicular structures. Finally, luminal exit is thought to occur by anion exchange and/or facilitated diffusion (3-5).We (1, 2) and others (6 -11) have recently cloned the organic anion transporter cDNA from kidneys of multiple species. Using computer modeling based on hydropathy analysis, the predicted proteins share several common features, including 12 putative membrane-spanning segments, a cluster of potential glycosylation sites located in the first extracellular...

Organic anion transporters: discovery, pharmacology, regulation and roles in pathophysiology

“…Therefore, these data clearly indicated that PKC activation can result in downregulation of basolateral organic anion transport. Several studies supported this observation, the first of which focused on regulation of mOat1 stably expressed in a porcine renal proximal tubule cell line, LLC-PK 1 [101]. It was demonstrated that PKC activation decreased V max and reduced mOat1-mediated transport of PAH, although direct phosphorylation of mOat1 by PKC was not observed.…”

“…Finally, serine/threonine phosphatases have been shown to increase p-aminohippurate (PAH) uptake by an undetermined mechanism. Thus far, the relevance of OAT regulation by direct phosphorylation of the transporter is virtually unknown mOat1 function, and this was indeed because of direct phosphorylation of the mOat1 protein [101]. Others demonstrated that activation of PKC decreased PAH secretion in an opossum kidney cell line as a result of reduction in both renal basolateral uptake and apical efflux [102].…”

Co-localization and interaction of organic anion transporter 1 with caveolin-2 in rat kidney

“…The presence of these conserved phosphorylation sites suggested that OAT might be subject to phosphorylation-induced functional regulation. You and co-workers (You et al, 2000) have shown the involvement of reversible phosphorylation in regulating the function of mOAT1. Okadaic acid, a protein phosphatase inhibitor, significantly increased the level of mOAT1 phosphorylation and it enhanced the phophorylation in a dose-dependent and time-dependent manner that was closely correlated to the decreased PAH transport.…”

Short-Term Regulation of Basolateral Organic Anion Uptake in Proximal Tubular OK cells

“…Inhibition of the net secretory transport of organic anions by bradykinin and phenylephrine via PKC was shown in isolated perfused rabbit proximal tubules (23). Recently, You et al (20) showed that PKC inhibits murine OAT without direct phosphorylation of the transport protein itself.…”

“…Inhibition of basolateral organic anion transport by stimulation of protein kinase C (PKC) was reported in isolated tubules of killifish (18). rOAT3 from rat is also inhibitable by PKC (19), and mOAT from mouse is regulated by PKC and phosphatases (20). The basolateral exchanger of organic anions and dicarboxylates in isolated proximal tubules of rabbit kidney was shown to be regulated by Ca 2ϩ /calmodulin-dependent protein kinase II, thyroxin kinase, phosphatidylinositol-3-kinase, and mitogen-activated protein kinases (MAPK) (21).…”