Ras triggers acidosis-induced activation of the extracellular-signal-regulated kinase pathway in cardiac myocytes.

Abstract

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.

• Authors: Robert S Haworth, Semjidmaa Dashnyam, Metin Avkiran
• Journal: The Biochemical journal (Biochem J) Vol. 399 Issue 3 Pg. 493-501 (Nov 01 2006) ISSN: 1470-8728 [Electronic] England
• PMID: 16831126 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• 2-(2-oxo-2-((3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy)aminoethyl)phosphonic acid, (2,2-dimethyl-1-oxopropoxy)methyl ester sodium
• Cation Transport Proteins
• Guanidines
• Isotonic Solutions
• Organophosphonates
• Recombinant Fusion Proteins
• SLC9A1 protein, human
• Sodium-Hydrogen Exchanger 1
• Sodium-Hydrogen Exchangers
• Sulfones
• Tyrode's solution
• Ammonium Chloride
• cariporide
• Ribosomal Protein S6 Kinases, 90-kDa
• Mitogen-Activated Protein Kinase 1
• Mitogen-Activated Protein Kinase 3
• HRAS protein, human
• Proto-Oncogene Proteins p21(ras)

Topics

• Acidosis (enzymology)
• Ammonium Chloride (pharmacology)
• Animals
• Cation Transport Proteins (antagonists & inhibitors)
• Cells, Cultured (drug effects, enzymology)
• Enzyme Activation (drug effects)
• Guanidines (pharmacology)
• Hydrogen-Ion Concentration
• Isotonic Solutions (pharmacology)
• Mitogen-Activated Protein Kinase 1 (metabolism)
• Mitogen-Activated Protein Kinase 3 (metabolism)
• Myocytes, Cardiac (enzymology)
• Organophosphonates (pharmacology)
• Proto-Oncogene Proteins p21(ras) (genetics, physiology)
• Rats
• Recombinant Fusion Proteins (physiology)
• Ribosomal Protein S6 Kinases, 90-kDa (metabolism)
• Signal Transduction
• Sodium-Hydrogen Exchanger 1
• Sodium-Hydrogen Exchangers (antagonists & inhibitors)
• Sulfones (pharmacology)