Molecular basis for angiotensin II-induced increase of chloride/bicarbonate exchange in the myocardium.
Plasma membrane anion exchangers (AEs) regulate myocardial intracellular pH (pH(i)) by Na(+)-independent Cl(-)/HCO(3)(-) exchange. Angiotensin II (Ang II) activates protein kinase C (PKC) and increases anion exchange activity in the myocardium. Elevated anion exchange activity has been proposed to contribute to the development of cardiac hypertrophy. Our Northern blots showed that adult rat heart expresses AE1, AE2, AE3fl, and AE3c. Activity of each AE isoform was individually measured by following changes of pH(i), associated with bicarbonate transport, in transfected HEK293 cells. Exposure to the PKC activator, PMA (150 nmol/L), increased the transport activity of only the AE3fl isoform by 50+/-11% (P<0.05, n=6), consistent with the increase observed in intact myocardium. Cotransfection of HEK293 cells with AE3fl and AT1(a)-Ang II receptors conferred sensitivity of anion transport to Ang II (500 nmol/L), increasing the transport activity by 39+/-3% (P<0.05, n=4). PKC inhibition by chelerythrine (10 micromol/L) blocked the PMA effect. To identify the PKC-responsive site, 7 consensus PKC phosphorylation sites of AE3fl were individually mutated to alanine. Mutation of serine 67 of AE3 prevented the PMA-induced increase of anion transport activity. Inhibition of MEK1/2 by PD98059 (50 micromol/L) did not affect the response of AE3fl to Ang II, indicating that PKC directly phosphorylates AE3fl. We conclude that following Ang II stimulation of cells, PKCepsilon phosphorylates serine 67 of the AE3 cytoplasmic domain, inducing the Ang II-induced increase in anion transport observed in the hypertrophic myocardium.
Alvarez
B V
BV
Department of Physiology, Canadian Institutes of Health Research (CIHR) Group in Molecular Biology of Membrane Proteins, University of Alberta, Edmonton, Canada.
Fujinaga
J
J
Casey
J R
JR
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D017920:Antiporters / Q000235:genetics / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D017136:Ion Transport / Q000187:drug effects*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051744:Protein Kinase C-epsilon; D051381:Rats; D017207:Rats, Sprague-Dawley; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D017920:Antiporters; D001639:Bicarbonates; D002712:Chlorides; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D020033:Protein Isoforms; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; C489237:SLC4A3 protein, human; C489238:Slc4a3 protein, rat; D000804:Angiotensin II; C497257:Prkce protein, rat; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate