Apelin and Acetylcholine Activate Distinct Nitric Oxide Signaling Pathways to Cause Endothelium‐Dependent Relaxation of Isolated Coronary Arteries

Abstract

The peptide, apelin, is expressed in fat cells, endothelial cells, and CNS neurons. Increasing evidence (e.g. inotropic and vasomotor effects) supports a role for apelin in the regulation of the cardiovascular system. Apelin is reported to cause vasodilation in peripheral arteries but the underlying molecular mechanisms are not well understood. In the present study, we investigated the role of nitric oxide (NO) in apelin‐induced relaxation of isolated rat coronary arteries, and compared it with the classic endothelium‐dependent vasodilator, acetylcholine. Receptors for apelin (APJ receptors) were expressed in coronary arteries, as determined by Western blot and RT‐PCR analysis (n=3). Immunofluorescence staining and confocal microscopy demonstrated the presence of APJ receptors on endothelial and smooth muscle cells in the coronary arterial wall. In freshly isolated coronary endothelial cells, apelin (10−6 M) caused an increase in DAF‐2 fluorescence that was abolished by nitro‐l‐arginine (3 × 10−5 M; NLA) and F13A (10−7 M), an APJ receptor antagonist, consistent with an increase in NO production. In coronary arterial rings contracted with 5‐HT (10−7 M), apelin caused endothelium‐dependent relaxations (pD2=6.90 ± 0.1; Emax=45 ± 6%; n=9) that were abolished in the presence of NLA, F13A, or iberiotoxin (10−7 M; IBTx), as well as in rings contracted with a depolarizing concentration of K+ (40 mM). Neither ODQ (10−5 M) nor DT‐2 (10−6 M), a protein kinase G (PKG) inhibitor, had any effect on apelin‐induced relaxations, and apelin (10−6 M) itself had no effect on intracellular cGMP accumulation in coronary arteries (0.09 ± 0.03 vs 0.05 ± 0.01 pmol/mg; n=4; p>0.05). By contrast, endothelium‐dependent relaxations to acetylcholine (pD2=6.89 ± 0.1; Emax=97 ± 2%; n=8) were inhibited by NLA, ODQ, and DT‐2. Acetylcholine (10−6 M) increased DAF‐2 fluorescence in isolated endothelial cells, and increased cGMP accumulation (0.08 ± 0.04 vs 0.26 ± 0.02 pmol/mg; n=4; p<0.05) in coronary arteries in an NLA‐ and ODQ‐sensitive manner. Patch clamp studies in freshly isolated coronary smooth muscle cells demonstrated that the NO donor, DEA NONOate (10−6 M) caused an increase in large conductance, calcium‐activated K (BKCa) current (peak current density (+80mV) = 53.02 ± 10.4 vs. 90.42 ± 8.6 pA/pF without and with DEA NONOate, respectively; n=6; p<0.05), that was inhibited by IBTx (peak current density = 33.8 ± 5.3 pA/pF; n=6; p<0.05 vs DEA NONOate alone) but not by ODQ (peak current density = 90.2 ± 7.9 pA/pF; n=6; p>0.05 vs DEA NONOate alone). Taken together, the data demonstrate that, in coronary arteries, endothelium‐derived NO causes arterial relaxation via two distinct signaling pathways: 1) which is activated by apelin, is sensitive to changes in membrane potential and requires activation of BKCa channels; this pathway is independent of cGMP formation and may be due to direct activation of BKCa channels by NO; and 2) which is activated by acetylcholine, is cGMP‐dependent and is mediated by activation of PKG. Differential regulation of distinct NO‐signaling pathways may provide opportunities for developing novel therapeutic strategies for the treatment of the coronary arterial dysfunction that often occurs in cardiovascular disease.Support or Funding InformationSupported by a grant from the NIH National Heart, Lung and Blood Institute (HL124338)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.