Ras triggers acidosis-induced activation of the extracellular-signal-regulated kinase pathway in cardiac myocytes.
In cardiac myocytes, sustained (3 min) intracellular acidosis activates the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway and, through this pathway, increases sarcolemmal NHE (Na+/H+ exchanger) activity [Haworth, McCann, Snabaitis, Roberts and Avkiran (2003) J. Biol. Chem. 278, 31676-31684]. In the present study, we aimed to determine the time-dependence, pH-dependence and upstream signalling mechanisms of acidosis-induced ERK1/2 activation in ARVM (adult rat ventricular myocytes). Cultured ARVM were subjected to intracellular acidosis for up to 20 min by exposure to NH4Cl, followed by washout with a bicarbonate-free Tyrode solution containing the NHE1 inhibitor cariporide. After the desired duration of intracellular acidosis, the phosphorylation status of ERK1/2 and its downstream effector p90(RSK) (90 kDa ribosomal S6 kinase) were determined by Western blotting. This revealed a time-dependent transient phosphorylation of both ERK1/2 and p90(RSK) by intracellular acidosis (intracellular pH approximately 6.6), with maximum activation occurring at 3 min and a return to basal levels by 20 min. When the degree of intracellular acidosis was varied from approximately 6.8 to approximately 6.5, maximum ERK1/2 phosphorylation was observed at an intracellular pH of 6.64. Inhibition of MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK kinase 1/2) by pre-treatment of ARVM with U0126 or adenoviral expression of dominant-negative D208A-MEK1 protein prevented the phosphorylation of ERK1/2 by sustained intracellular acidosis, as did inhibition of Raf-1 with GW 5074 or ZM 336372. Interference with Ras signalling by the adenoviral expression of dominant-negative N17-Ras protein or with FPT III (farnesyl protein transferase inhibitor III) also prevented acidosis-induced ERK1/2 phosphorylation, whereas inhibiting G-protein signalling [by adenoviral expression of RGS4 or Lsc, the RGS domain of p115 RhoGEF (guanine nucleotide-exchange factor)] or protein kinase C (with bisindolylmaleimide I) had no effect. Our data show that, in ARVM, sustained intracellular acidosis activates ERK1/2 through proximal activation of the classical Ras/Raf/MEK pathway.
Haworth
Robert S
RS
Cardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, UK. robert.haworth@kcl.ac.uk
Dashnyam
Semjidmaa
S
Avkiran
Metin
M
D000138:Acidosis / Q000201:enzymology*; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007552:Isotonic Solutions / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000201:enzymology*; D063065:Organophosphonates / Q000494:pharmacology; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D015398:Signal Transduction; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors; D013450:Sulfones / Q000494:pharmacology
C436083:2-(2-oxo-2-((3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy)aminoethyl)phosphonic acid, (2,2-dimethyl-1-oxopropoxy)methyl ester sodium; D027682:Cation Transport Proteins; D006146:Guanidines; D007552:Isotonic Solutions; D063065:Organophosphonates; D011993:Recombinant Fusion Proteins; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; C037692:Tyrode's solution; D000643:Ammonium Chloride; C093373:cariporide; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)