Activation of cAMP-dependent signaling induces oxidative modification of the cardiac Na+-K+ pump and inhibits its activity.
Cellular signaling can inhibit the membrane Na(+)-K(+) pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the beta(1) subunit of the pump alpha/beta heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump, and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47(phox) and p22(phox), required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na(+)-K(+) pump beta(1) subunit and decreased its co-immunoprecipitation with the alpha(1) subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the alpha(1)/beta(1) subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive electrogenic Na(+)-K(+) pump current (arising from the 3:2 Na(+):K(+) exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, the inhibitory peptide for the epsilon-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMP- and PKC-dependent inhibition of the cardiac Na(+)-K(+) pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pump beta(1) subunit.
White
Caroline N
CN
North Shore Heart Research Group, Kolling Institute, University of Sydney, Sydney 2065, Australia.
Liu
Chia-Chi
CC
Garcia
Alvaro
A
Hamilton
Elisha J
EJ
Chia
Karin K M
KK
Figtree
Gemma A
GA
Rasmussen
Helge H
HH
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000201:enzymology*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010084:Oxidation-Reduction; D051744:Protein Kinase C-epsilon / Q000378:metabolism; D011817:Rabbits; D015290:Second Messenger Systems; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D005576:Colforsin; D000242:Cyclic AMP; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D017868:Cyclic AMP-Dependent Protein Kinases; D051744:Protein Kinase C-epsilon; D010738:Type C Phospholipases; D000262:Adenylyl Cyclases; D000254:Sodium-Potassium-Exchanging ATPase; D005978:Glutathione