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 p90RSK (90 kDa ribosomal S6 kinase) were determined by Western blotting. This revealed a time-dependent transient phosphorylation of both ERK1/2 and p90RSK by intracellular acidosis (intracellular pH ∼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 ∼6.8 to ∼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.
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Research Article|
October 13 2006
Ras triggers acidosis-induced activation of the extracellular-signal-regulated kinase pathway in cardiac myocytes
Robert S. Haworth;
Robert S. Haworth
1
1Cardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, U.K.
1To whom correspondence should be addressed (email robert.haworth@kcl.ac.uk).
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Semjidmaa Dashnyam;
Semjidmaa Dashnyam
1Cardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, U.K.
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Metin Avkiran
Metin Avkiran
1Cardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, U.K.
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Publisher: Portland Press Ltd
Received:
October 05 2005
Revision Received:
June 07 2006
Accepted:
July 10 2006
Accepted Manuscript online:
July 10 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 399 (3): 493–501.
Article history
Received:
October 05 2005
Revision Received:
June 07 2006
Accepted:
July 10 2006
Accepted Manuscript online:
July 10 2006
Citation
Robert S. Haworth, Semjidmaa Dashnyam, Metin Avkiran; Ras triggers acidosis-induced activation of the extracellular-signal-regulated kinase pathway in cardiac myocytes. Biochem J 1 November 2006; 399 (3): 493–501. doi: https://doi.org/10.1042/BJ20051628
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