Phorbol ester stimulation of RasGRP1 regulates the sodium-chloride cotransporter by a PKC-independent pathway.
The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of one of the most effective classes of antihypertensive medications, thiazide diuretics. We developed a cell model system to assess NCC function in a mammalian cell line that natively expresses NCC, the mouse DCT (mDCT) cell line. We used this system to study the complex regulation of NCC by the phorbol ester (PE) 12-O-tetradecanoylphorbol-13-acetate (TPA), a diacylglycerol (DAG) analog. It has generally been thought that PEs mediate their effects on transporters through the activation of PKC. However, there are at least five other DAG/PE targets. Here we describe how one of those alternate targets of DAG/PE effects, Ras guanyl-releasing protein 1 (RasGRP1), mediates the PE-induced suppression of function and the surface expression of NCC. Functional assessment of NCC by using thiazide-sensitive (22)Na(+) uptakes revealed that TPA completely suppresses NCC function. Biotinylation experiments demonstrated that this result was primarily because of decreased surface expression of NCC. Although inhibitors of PKC had no effect on this suppression, MAPK inhibitors completely prevented the TPA effect. RasGRP1 activates the MAPK pathway through activation of the small G protein Ras. Gene silencing of RasGRP1 prevented the PE-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of RasGRP1 in mediating the PE-induced suppression of NCC activity through the stimulation of the MAPK pathway.
Ko
Benjamin
B
Department of Medicine, University of Chicago, Chicago, IL 60637, USA.
Joshi
Leena M
LM
Cooke
Leslie L
LL
Vazquez
Norma
N
Musch
Mark W
MW
Hebert
Steven C
SC
Gamba
Gerardo
G
Hoover
Robert S
RS
D000042:Absorption / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D007686:Kidney Tubules, Distal / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D011493:Protein Kinase C / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology; D049992:Sodium Chloride Symporters / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014982:Xenopus laevis
D020662:Guanine Nucleotide Exchange Factors; C112278:Rasgrp1 protein, mouse; D049993:Sodium Chloride Symporter Inhibitors; D049992:Sodium Chloride Symporters; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate