u-0126 and Acidosis

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a proton in exchange for extracellular sodium. Renal tissues are subject to metabolic and respiratory acidosis, and acidosis has been shown to acutely activate NHE1 activity in other cell types. We examined if NHE1 is activated by acute acidosis in HEK293 and Madin-Darby canine kidney (MDCK) cells. Acute sustained intracellular acidosis (SIA) activated NHE1 in both cell types. We expressed wild-type and mutant NHE1 cDNAs in MDCK cells. All the cDNAs had a L163F/G174S mutation, which conferred a 100-fold resistance to EMD87580, an NHE1-specific inhibitor. We assayed exogenous NHE1 activity while inhibiting endogenous activity with EMD87580 and while inhibiting the NHE3 isoform of the Na⁺/H⁺ exchanger using the isoform-specific inhibitor S3226. We examined the activation and phosphorylation of the wild-type and mutant NHE1 proteins in response to SIA. In MDCK cells we demonstrated that the amino acids Ser⁷⁷¹, Ser⁷⁷⁶, Thr⁷⁷⁹, and Ser⁷⁸⁵ are important for NHE1 phosphorylation and activation after acute SIA. SIA activated ERK-dependent pathways in MDCK cells, and this was blocked by treatment with the MEK inhibitor U0126. Treatment with U0126 also blocked activation of NHE1 by SIA. These results suggest that acute acidosis activates NHE1 in mammalian kidney cells and that in MDCK cells this activation occurs through an ERK-dependent pathway affecting phosphorylation of a distinct set of amino acids in the cytosolic regulatory tail of NHE1.

Topics: Acidosis; Amino Acids; Animals; Blotting, Western; Butadienes; Cation Transport Proteins; Cell Line; Cells, Cultured; Dogs; Electrophoresis, Polyacrylamide Gel; Guanidines; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Immunoprecipitation; Kidney; MAP Kinase Signaling System; Methacrylates; Mutagenesis, Site-Directed; Nitriles; Phosphorylation; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Sulfones; Transfection

In vitro it has been shown that the functional activity of P-glycoprotein (Pgp), an important drug transporter responsible for multidrug resistance, can be strongly increased by extracellular acidosis. Here mitogen-activated protein kinases (MAPK) (p38, ERK1/2) seem to play an important role for signal transduction. However, it is unclear whether these effects are also relevant in vivo.. With the newly developed PET tracer Schiff base-based (68)Ga-MFL6.MZ the functional Pgp activity was visualized under acidic conditions and during inhibition of MAPKs non-invasively by means of microPET in rat tumours. Tumours were acidified either by inspiratory hypoxia (8% O(2)) or by injection of lactic acid. Inhibitors of the MAPK were injected intratumourally.. With increasing tumour volume the tumour pH changed from 7.0 to 6.7 and simultaneously the Pgp activity increased almost linearly. When the tumour was acidified by direct lactic acid injection the PET tracer uptake was reduced by 20% indicating a higher transport rate out of the cells. Changing the inspiratory O(2) fraction to 8% dynamically led to a reduction of extracellular pH and in parallel to a decrease of tracer concentration. While inhibition of the p38 pathway reduced the Pgp transport rate, inhibition of ERK1/2 had practically no impact.. An acidic extracellular environment significantly stimulates the Pgp activity. The p38 MAPK pathway plays an important role for Pgp regulation in vivo, whereas ERK1/2 is of minor importance. From these results new strategies for overcoming multidrug resistance (e.g. reducing tumour acidosis, inhibition of p38) may be developed.

Topics: Acidosis; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Butadienes; Cell Line, Tumor; Extracellular Space; Gallium Radioisotopes; Hydrogen-Ion Concentration; Male; MAP Kinase Signaling System; Nitriles; Pharmaceutical Preparations; Positron-Emission Tomography; Protein Kinase Inhibitors; Radioactive Tracers; Rats

This study aimed to identify the signaling pathway for the proposed link between phosphodiesterase-5A (PDE5A) inhibition and decreased cardiac Na(+)/H(+) exchanger (NHE-1) activity.. NHE-1 activity was assessed in rat isolated papillary muscles by the Na(+)-dependent initial pH(i) recovery from a sustained acidosis (ammonium prepulse). ERK1/2, p90RSK and NHE-1 phosphorylation state during acidosis was determined.. PDE5A inhibition (1 μmol/L sildenafil, SIL) did not modify basal pH(i) but significantly blunted pH(i) recovery after sustained acidosis. Although preventing ERK1/2- p90RSK signaling pathway (10 μmol/L U0126) mimicked SIL effect, SIL did not blunt the acidosis-mediated increase in kinases activation. SIL+U0126 did not show additive effect on NHE-1 activity. Then, we hypothesized that SIL could be activating phophasatases (PP1 and/or PP2A) to directly dephosphorylate NHE-1 despite preserved ERK1/2-p90RSK activation. Non-specific phosphatases inhibition (1 μmol/L okadaic acid) canceled SIL effect on pH(i) recovery from acidosis. Same result was observed by inhibiting PP2A either with a lower dose of okadaic acid (1 nmol/L) or, more specifically, with 100 μmol/L endothall. Consistently, NHE-1 phosphorylation at Ser703 increased after acidosis, SIL prevented this effect and PP2A inhibition (endothall) reverted SIL effect.. We suggest that PDE5A inhibitors decrease NHE-1 phosphorylation and activity through a mechanism that involves PP2A activation.

Topics: Acidosis; Animals; Butadienes; Cyclic Nucleotide Phosphodiesterases, Type 5; Dicarboxylic Acids; Hydrogen-Ion Concentration; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Okadaic Acid; Papillary Muscles; Phosphodiesterase Inhibitors; Phosphorylation; Piperazines; Protein Phosphatase 1; Protein Phosphatase 2; Purines; Rats; Ribosomal Protein S6 Kinases, 90-kDa; Sildenafil Citrate; Sodium-Hydrogen Exchangers; Sulfones

Activity of the Na+/H+ exchanger (NHE) isoform 1 (NHE1) is increased by intracellular acidosis through the interaction of intracellular H+ with an allosteric modifier site in the transport domain. Additional regulation is achieved via kinase-mediated modulation of the NHE1 regulatory domain. To determine if intracellular acidosis stimulates NHE1 activity solely by the allosteric mechanism, we subjected cultured neonatal rat ventricular myocytes (NRVM) with native NHE1 expression to intracellular acidosis (pHi approximately 6.6) for up to 6 min by transient exposure to NH4Cl and its washout in the presence of NHE inhibition (by zero [Na+]o or the NHE1 inhibitor cariporide) in HCO3- -free medium. After the desired duration of acidosis, NHE was reactivated (by reintroduction of [Na+]o or removal of cariporide), and the rate of recovery of pHi (dpHi/dt) was measured as the index of NHE activity. Regardless of the method used when intracellular acidosis was sustained for > or =3 min, subsequent NHE activity was significantly increased (>4-fold). Similar NHE stimulatory effects of sustained acidosis were observed in adult rat ventricular myocytes and COS-7 cells. Sustained (3 min) intracellular acidosis activated several NHE1 kinases in NRVM, in an in-gel kinase assay using as substrate a glutathione S-transferase fusion protein of the NHE1 regulatory domain. Detailed investigation of ERK and its downstream effector p90RSK, two putative NHE1 kinases, revealed time-dependent activation of both by intracellular acidosis in NRVM. Furthermore, inhibition of MEK1/2 by pretreatment of NRVM with two structurally distinct inhibitors, PD98059 (30 microM) or UO126 (3 microM), inhibited the activation of ERK and p90RSK and abolished the stimulation of NHE activity by sustained (3 min) intracellular acidosis. Our data show that not only the extent but also the duration of intracellular acidosis regulates NHE1 activity and suggest that the stimulatory effect of sustained intracellular acidosis occurs through a novel mechanism mediated by activation of the ERK pathway.

Topics: Acidosis; Animals; Butadienes; Cell Membrane; Cells, Cultured; Chlorocebus aethiops; COS Cells; Enzyme Inhibitors; Flavonoids; Mitogen-Activated Protein Kinases; Nitriles; Rats; Rats, Wistar; Ribosomal Protein S6 Kinases, 90-kDa; Sodium-Hydrogen Exchangers