Increased NHE1 expression is associated with serum deprivation-induced differentiation in immortalized rat proximal tubule cells.
We studied the proton secretion mechanisms involved with pHi regulation in immortalized rat proximal tubule cells (IRPTC), a SV40-immortalized cell line derived from rat proximal tubule, and characterized the effects of serum deprivation on them. Using pHi measurements with the fluorescent probe BCECF, we demonstrated that the IRPTC express both Na+/H+ exchanger and H+-ATPase, but only NHE1 is modulated by serum deprivation. In these cells, 24 h of serum starvation increased pHi from 7.08+/-0.008 (n=34) to 7.18+/-0.018 (n=33) as well as the pH recovery rate from intracellular acidification with NH4Cl from 0.29+/-0.022 pH U/min (n=14) to 0.50+/-0.024 pH U/min (n=14), without modifying their buffering capacity. These effects were followed by several modifications in morphological features, indicating an increase in differentiation status. The altered activity of NHE1 was consistent with an increase of both transcription and translation of the antiporter, as the utilization of actinomycin D and cycloheximide significantly inhibited the upregulation of NHE1 induced by serum withdrawal. Inhibition of tyrosine phosphorylation by genistein blocked the serum deprivation-dependent activation of NHE. Moreover, the pharmacological inhibition of MEK1/2, the upstream activator of ERK1/2 by UO-126, significantly inhibited the stimulatory effect of serum starvation on Na+/H+ exchanger activity, whereas the putative p38 MAPK inhibitor SB-203580 failed to cause any effect on pHi recovery rates. Our findings indicate that during IRPTC differentiation by serum deprivation, there was a net enhancement of NHE1 activity. This upregulation of NHE by serum removal was consistent with an increase of RNA and protein synthesis of the exchanger, which depends on tyrosine kinase phosphorylation and ERK pathway activation.
Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1524, 05508-900 São Paulo, SP, Brazil.
Luciene Regina
LR
Carraro-Lacroix
Marco Antonio
MA
Ramirez
Telma M T
TM
Zorn
Nancy Amaral
NA
Rebouças
Gerhard
G
Malnic
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D005786:Gene Expression Regulation; D019833:Genistein / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000032:analysis / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000032:analysis / Q000502:physiology; D014176:Protein Biosynthesis / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000378:metabolism; D051381:Rats; D044967:Serum / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology; D015854:Up-Regulation
C452850:Slc9a1 protein, rat; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D003609:Dactinomycin; D003513:Cycloheximide; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D020930:MAP Kinase Kinase Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D006180:Proton-Translocating ATPases