Molecular Basis for Angiotensin II-Induced Increase of Chloride/Bicarbonate Exchange in the Myocardium

Abstract

Abstract-Plasma membrane anion exchangers (AEs) regulate myocardial intracellular pH (pH i ) by Na ϩ -independent Cl Ϫ /HCO 3 Ϫ 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 50Ϯ11% (PϽ0.05, nϭ6), 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 39Ϯ3% (PϽ0.05, nϭ4). PKC inhibition by chelerythrine (10 mol/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 mol/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, PKC⑀ phosphorylates serine 67 of the AE3 cytoplasmic domain, inducing the Ang II-induced increase in anion transport observed in the hypertrophic myocardium. Key Words: hypertrophy Ⅲ anion exchange Ⅲ pH regulation Ⅲ angiotensin II Ⅲ protein kinase C I ntracellular pH (pH i ) regulates excitation-contraction coupling in cardiac cells through ionic conductances, 1 metabolic pathways, 2 Ca 2ϩ homeostasis, 3 contractility, 4 and electrical conduction. 5 Four well-characterized membrane ion transporters regulate cardiomyocyte pH i . Acid loads activate Na ϩ /H ϩ exchange (NHE) and Na ϩ /HCO 3 Ϫ symport (NBC), 6,7 whereas after an alkaline load, Na ϩ -independent Cl Ϫ /HCO 3 Ϫ exchangers (AEs) 8 and a second dual acid-loading mechanism 9 are activated. Anion exchange proteins, which facilitate the reversible electroneutral exchange of Cl Ϫ for HCO 3 Ϫ across the plasma membrane, regulate pH i , intracellular chloride concentration, bicarbonate metabolism and cell volume. The AE family comprises 3 members: AE1, AE2, and AE3. 10 Heart and retina coexpress 2 different isoforms of AE3, AE3 full length (AE3fl, the most abundant AE protein expressed in the brain) and AE3 cardiac (AE3c), which result from alternative promoter usage. 11,12 Rat AE3fl is 1227 amino acids long and AE3c contains 1034 amino acids. The C-terminal 957 amino acids of both polypeptides are identical, but the AE3c protein contains a unique N-terminal sequence of 73 amino acids, which replaces the unique first 270 amino acids of the AE3fl form. 11 A truncated form of AE1 was recently characterized in rat ventricular my...

Sasanquasaponin promotes cellular chloride efflux and elicits cardioprotection via the PKCε pathway

“…A previous study by our group showed that AE3 is closely linked with the inhibitory effects of SQS on sI/R-induced elevation of [Cl -] i (9). Of note, Alvarez et al (29) showed that AE3 is the PKC-sensitive anion exchange protein of the heart, and that the PKCε-dependent phosphorylation of serine 67 on AE3 can cause an increase in anion transport. Therefore, it may be speculated that SQS inhibits sI/R-induced elevation of [Cl -] i and induces cardioprotection through activation of the PKCε pathway and consequent PKCε-dependent phosphorylation of serine 67 on AE3.…”

Influence of Na + -Independent Cl − -HCO 3 − Exchange on the Slow Force Response to Myocardial Stretch

“…On one hand, it has been shown by some authors of the present study that exogenous applied Ang II induces AE3 activation by PKC-⑀-dependent phosphorylation of the AE3fl isoform on Ser67. 41 Myocardial stretch probably shares the same pathway. On the other hand, Moor and Fliegel 42 demonstrated that ET induces NHE-1 stimulation through the 42/44 mitogen-activated protein kinase and p90, which are both known as downstream effectors from PKC.…”

“…Perhaps the AE3 isoforms, and particularly the AE3fl, are the ones linked to PKC-dependent activation. In addition to be PKC-⑀-dependent phosphorylated by Ang II, 41 the AE3fl isoform is upregulated in the hypertrophied myocardium of the SHR where pH i is compensated in spite of the enhanced NHE-1 activity. 17…”

Plasma membrane Cl-/HCO3-exchangers: Structure, mechanism and physiology

“…54 Involvement of AE3 in cardiac hypertrophy is supported by the observation that it is the only family member that can be activated by protein kinase C (PKC), which is the kinase implicated in hypertrophic pathways. 55 AE3 is also expressed in the retina, where it may regulate pH and HCO 3 -levels since the retina is the most energy-utilizing tissue of the body, with an associated high HCO 3 -load. In the retina there is differential expression of AE3c and AE3fl in the Müller glial cell or horizontal cell layers, and there is also differential expression at different developmental stages.…”