Regulation of sarcolemmal Na(+)/H(+) exchange by hydrogen peroxide in adult rat ventricular myocytes.
OBJECTIVE
To characterise the effects of exogenous H(2)O(2) on sarcolemmal Na(+)/H(+) exchanger (NHE) activity and determine the roles of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC) in observed effects.


METHODS
Sarcolemmal H(+) efflux rate (J(H)) was determined by microepifluorescence at a pH(i) of 6.70 in adult rat ventricular myocytes, after two consecutive acid pulses in HCO(3)(-)-free medium; before the second pulse, cells (n=7-10/group) were exposed to H(2)O(2) or vehicle and the change in J(H) (DeltaJ(H)) was used to quantify the change in NHE activity. ERK and p38 MAPK activities were determined by immunoblotting with phosphospecific antibodies.


RESULTS
Relative to control, DeltaJ(H) was increased by a 10-min exposure to 100, but not 1 or 10 microM H(2)O(2) (1000 microM was not tolerated); 3 or 6 min exposure to 100 microM H(2)O(2) was without effect. ERK and p38 MAPK activities were both increased by 100 microM H(2)O(2) (peak at 6 min); the ERK kinase inhibitor PD98059 (10 microM), but not the p38 MAPK inhibitor SB203580 (1 microM), inhibited the H(2)O(2)-induced increase in DeltaJ(H). H(2)O(2)-induced ERK activation was inhibited not only by PD98059 (10 microM), but also by the non-selective tyrosine kinase inhibitor genistein (3-100 microM), the EGF receptor kinase inhibitor AG1478 (3-300 nM) and the Src family kinase inhibitor PP2 (0.1-10 microM). The PKC inhibitors GF109203X (0.3-10 microM) and chelerythrine (1-30 microM) were without effect on ERK activation, although the former abolished the H(2)O(2)-induced increase in DeltaJ(H).


CONCLUSIONS
Our data demonstrate that, in adult rat ventricular myocytes, (i) hydrogen peroxide stimulates sarcolemmal NHE activity, (ii) this response requires activation of ERK and PKC, but not p38 MAPK, (iii) ERK activation occurs through tyrosine kinase-mediated, but PKC-independent, mechanisms
Snabaitis
Andrew K
AK
Centre for Cardiovascular Biology and Medicine, King's College London, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK.
Hearse
David J
DJ
Avkiran
Metin
M
D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D005419:Flavonoids / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D015151:Immunoblotting / Q000379:methods; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010940:Plant Proteins / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D011799:Quinazolines; D051381:Rats; D017208:Rats, Wistar; D012508:Sarcolemma / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D020032:Tyrphostins / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D007211:Indoles; D008301:Maleimides; D010940:Plant Proteins; D011725:Pyridines; D011799:Quinazolines; D017923:Sodium-Hydrogen Exchangers; D020032:Tyrphostins; C083694:phloem protein-2, Physcomitrella patens; C101044:RTKI cpd; D006861:Hydrogen Peroxide; D019833:Genistein; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D019061:src-Family Kinases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C070515:bisindolylmaleimide I; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one