Regulation of mOAT-mediated Organic Anion Transport by Okadaic Acid and Protein Kinase C in LLC-PK1Cells

In order to investigate the regulation of canalicular organic-anion transport, we used a hepatocyte transport assay in which canalicular secretion of a model organic anion, dinitrophenyl-glutathione (GS-DNP), was measured in the presence of stimulators and inhibitors of the Ca2+/protein kinase C (PKC) second-messenger system and of the cyclic AMP (cAMP) second-messenger system. Vasopressin (24 nM) and the phorbol ester phorbol 12-myristate 13-acetate (1 microgram/ml), both stimulators of PKC, stimulated GS-DNP efflux by 65 +/- 36% and 55 +/- 28% respectively, whereas staurosporine (10 microM), an inhibitor of PKC, inhibited efflux by 53 +/- 13%. Glucagon and forskolin, both stimulators of the cAMP second-messenger system, as well as the cAMP analogue dibutyryl cAMP and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, did not significantly influence the GS-DNP efflux. It can be concluded that canalicular organic-anion transport in hepatocytes is either directly or indirectly regulated by PKC.

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Regulation by protein kinase C of organic anion transport driven by rat organic anion transporter 3 (rOAT3)

Life Sciences ◽

10.1016/s0024-3205(00)00694-9 ◽

2000 ◽

Vol 67 (9) ◽

pp. 1087-1093 ◽

Cited By ~ 47

Author(s):

Michio Takeda ◽

Takashi Sekine ◽

Hitoshi Endou

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Organic Anion ◽

Anion Transport ◽

Organic Anion Transporter ◽

Organic Anion Transport ◽

Kinase C ◽

Anion Transporter ◽

Organic Anion Transporter 3

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Protein kinase C regulation of organic anion transport in renal proximal tubule

AJP Renal Physiology ◽

10.1152/ajprenal.1998.274.1.f156 ◽

1998 ◽

Vol 274 (1) ◽

pp. F156-F164 ◽

Cited By ~ 15

Author(s):

David S. Miller

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Ester ◽

Kinase Inhibitors ◽

Organic Anion ◽

Anion Transport ◽

Protein Kinase Inhibitors ◽

Organic Anion Transport ◽

Kinase C ◽

Tissue Fluorescence

Fluorescence microscopy and digital image analysis were used to examine the role of protein kinase C (PKC) in the control of organic anion (fluorescein, FL) transport in killifish renal proximal tubules. Phorbol ester (1–100 nM) reduced cellular and luminal accumulation of FL, and protein kinase inhibitors [staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, 10–1,000 nM] increased cellular and luminal accumulation. Phorbol ester effects were blocked by staurosporine. The increases in tissue fluorescence caused by staurosporine were blocked by p-aminohippurate, indicating that they represent increased FL transport on the organic anion system. Neither phorbol ester nor staurosporine had any effects on the cell-to-lumen transport of a fluorescent organic anion that was generated intracellularly from a nonfluorescent, uncharged precursor. Finally, studies with a fluorescent PKC inhibitor showed that phorbol ester caused PKC translocation from cytoplasm to the plasma membrane. Together, these findings indicate that renal organic anion transport is negatively correlated with PKC activity and that PKC directly or indirectly controls the basolateral step in transport.

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Nedd4-2 but not Nedd4-1 is critical for protein kinase C-regulated ubiquitination, expression, and transport activity of human organic anion transporter 1

AJP Renal Physiology ◽

10.1152/ajprenal.00522.2015 ◽

2016 ◽

Vol 310 (9) ◽

pp. F821-F831 ◽

Cited By ~ 11

Author(s):

Da Xu ◽

Haoxun Wang ◽

Qiang Zhang ◽

Guofeng You

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Surface ◽

Organic Anion ◽

The Body ◽

Organic Anion Transporter ◽

Transport Activity ◽

Kinase C ◽

Anion Transporter ◽

In Cells

Human organic anion transporter 1 (hOAT1) expressed at the membrane of the kidney proximal tubule cells mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and antiinflammatories. Therefore, understanding the regulation of hOAT1 will provide significant insights into kidney function and dysfunction. We previously established that hOAT1 transport activity is inhibited by activation of protein kinase C (PKC) through accelerating hOAT1 internalization from cell surface into intracellular endosomes and subsequent degradation. We further established that PKC-induced hOAT1 ubiquitination is an important step preceding hOAT1 internalization. In the current study, we identified two closely related E3 ubiquitin ligases, neural precursor cell expressed, developmentally downregulated 4-1 and 4-2 (Nedd4-1 and Nedd4-2), as important regulators for hOAT1: overexpression of Nedd4-1 or Nedd4-2 enhanced hOAT1 ubiquitination, reduced the hOAT1 amount at the cell surface, and suppressed hOAT1 transport activity. In further exploring the relationship among PKC, Nedd4-1, and Nedd4-2, we discovered that PKC-dependent changes in hOAT1 ubiquitination, expression, and transport activity were significantly blocked in cells transfected with the ligase-dead mutant of Nedd4-2 (Nedd4-2/C821A) or with Nedd4-2-specific siRNA to knockdown endogenous Nedd4-2 but not in cells transfected with the ligase-dead mutant of Nedd4-1 (Nedd4-1/C867S) or with Nedd4-1-specific siRNA to knockdown endogenous Nedd4-1. In conclusion, this is the first demonstration that both Nedd4-1 and Nedd4-2 are important regulators for hOAT1 ubiquitination, expression, and function. Yet they play distinct roles, as Nedd4-2 but not Nedd4-1 is a critical mediator for PKC-regulated hOAT1 ubiquitination, expression, and transport activity.

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Cloning of the human kidney PAH transporter: narrow substrate specificity and regulation by protein kinase C

AJP Renal Physiology ◽

10.1152/ajprenal.1999.276.2.f295 ◽

1999 ◽

Vol 276 (2) ◽

pp. F295-F303 ◽

Cited By ~ 38

Author(s):

Run Lu ◽

Brenda S. Chan ◽

Victor L. Schuster

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Substrate Specificity ◽

Proximal Tubule ◽

Organic Anion ◽

Human Kidney ◽

Time Dependent ◽

Organic Substrates ◽

Chromosome 11 ◽

Kinase C

Conserved from fish to mammals, renal proximal tubule organic anion secretion plays an important role in drug and xenobiotic elimination. Studies with the model substrate p-aminohippurate (PAH) have suggested that a basolateral PAH/α-ketoglutarate exchanger imports diverse organic substrates into the proximal tubule prior to apical secretion. cDNAs encoding PAH transporters have been cloned recently from rat and flounder. Here we report the cloning of a highly similar human PAH transporter (hPAHT) from human kidney. By Northern blot analysis and EST database searching, hPAHT mRNA was detected in kidney and brain. PCR-based monochromosomal somatic cell hybrid mapping placed the hPAHT gene on chromosome 11. When expressed transiently in vitro, hPAHT catalyzed time-dependent and saturable [3H]PAH uptake ( K m of ∼5 μM). Preincubation with unlabeled α-ketoglutaric or with glutaric acid stimulated tracer PAH uptake, and preincubation with unlabeled PAH stimulated tracer α-ketoglutarate uptake, results that are consistent with PAH/α-ketoglutarate exchange. Several structurally diverse organic anions cis-inhibited PAH uptake. Like rat OAT1 organic anion transporter, hPAHT was inhibited by furosemide, indomethacin, probenecid, and α-ketoglutarate. Unlike OAT1, hPAHT was not inhibited by prostaglandins or methotrexate (MTX). Moreover, tracer PGE2 and MTX were not transported, indicating that the substrate specificity for transport by hPAHT is not broad. PAH uptake was inhibited by phorbol 12-myristate 13-acetate (PMA) in a dose- and time-dependent fashion, but not by the inactive 4α-phorbol-12,13 didecanoate. PMA-induced inhibition was blocked by staurosporine. Thus the protein kinase C-mediated inhibition of basolateral organic anion entry previously reported in intact tubules is likely due, at least in part, to direct modulation of the PAH/α-ketoglutarate exchanger.

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