Apelin Does Not Impair Coronary Artery Relaxation Mediated by Nitric Oxide-Induced Activation of BKCa Channels

Abstract

Apelin-APJ receptor signaling regulates vascular tone in cerebral and peripheral arteries. We recently reported that apelin inhibits BK_Ca channel function in cerebral arteries, resulting in impaired endothelium-dependent relaxations. In contrast, apelin causes endothelium-dependent relaxation of coronary arteries. However, the effects of apelin on BK_Ca channel function in coronary arterial myocytes have not yet been explored. We hypothesized that apelin-APJ receptor signaling does not have an inhibitory effect on coronary arterial BK_Ca channels and hence does not alter nitric oxide (NO)-dependent relaxation of coronary arteries. Patch clamp recording was used to measure whole cell K⁺ currents in freshly isolated coronary smooth muscle cells. Apelin had no effect on the increases in current density in response to membrane depolarization or to NS1619 (a BK_Ca channel opener). Moreover, apelin did not inhibit NO/cGMP-dependent relaxations that required activation of BK_Ca channels in isolated coronary arteries. Apelin-APJ receptor signaling caused a marked increase in intracellular Ca²⁺ levels in coronary arterial smooth muscle cells, but failed to activate PI3-kinase to increase phosphorylation of Akt protein. Collectively, these data provide mechanistic evidence that apelin has no inhibitory effects on BK_Ca channel function in coronary arteries. The lack of inhibitory effect on BK_Ca channels makes it unlikely that activation of APJ receptors in coronary arteries would adversely affect coronary flow by creating a vasoconstrictive environment. It can be expected that apelin or other APJ receptor agonists in development will not interfere with the vasodilator effects of endogenous BK_Ca channel openers.

Keywords: BKCa channels; apelin; coronary artery; nitric oxide; vasorelaxation.

FIGURE 1

Effect of apelin on whole cell K⁺ currents in coronary arterial myocytes. Whole cell K⁺ currents were recorded in freshly isolated myocytes in response to successive voltage pulses of 800 ms duration, increasing in 10 mV increments from −60 to +80 mV with or without apelin (10⁻⁷ M) or apelin plus IBTx (10^-7 M). (A) Representative traces showing the current recordings from smooth muscle cell in the absence and presence of apelin alone or apelin plus IBTx. (B) Summary I–V curves of whole cell K⁺ currents at baseline (control) and after treatment with apelin (10⁻⁷ M) alone or in the presence of IBTx (10⁻⁷ M). Dotted line represents zero current level (at 0 mV). Values are presented as the mean ± S. E. M. (n = 6). *p < 0.05 vs. control current density.

FIGURE 2

Effect of apelin on NS1619-induced BK_Ca currents in coronary arterial myocytes. Whole cell BK_Ca currents induced in response to NS1619 (10^-5 M) were recorded in freshly isolated coronary arterial myocytes. Representative traces showing the current recordings from smooth muscle cells after treatment with (A) NS1619 (10⁻⁵ M, 2 min) alone or (B) in the presence of IBTx (10⁻⁷ M, 5 min) or (C) apelin (10⁻⁷ M, 5 min). (D–F) Summary I–V curves of BK_Ca currents at baseline and after treatment with (D) NS1619 (10⁻⁵ M) alone or in the presence of (E) IBTx (10⁻⁷ M) or (F) apelin (10⁻⁷ M). Dotted line represents zero current level (at 0 mV). Values are presented as the mean ± S.E.M. (n = 6–7). *p < 0.05 vs. control current density.

FIGURE 3

Effect of apelin and iberiotoxin on NO-cGMP dependent vasorelaxation of coronary arteries. Log concentration-response curves in producing relaxation of coronary arteries for DEA NONOate (A,B), acetylcholine (C,D) or 8-Bromo-cGMP (E,F) in the absence and presence of IBTx (10⁻⁷ M) (A, C, E) or apelin (10⁻⁷ M) (B, D, F). Values are presented as the mean ± S.E.M. (n = 4–6). *p < 0.05 vs. control (in the absence of inhibitor).

FIGURE 4

Effect of apelin-APJ signaling on coronary intracellular Ca²⁺ levels and PI3-kinase activity. Representative images of freshly isolated coronary arterial smooth muscle cells loaded with Fluo-four AM (5 µM) (A) before and (B) after exposure to apelin (10⁻⁷ M). (C) Bar graph summarizing changes in fluorescence (F/F0) in response in apelin alone (10⁻⁷ M) or in the presence of F13A (10⁻⁷ M); 60 mM K⁺ was used as a positive control for the experiments (n = 3). (D) Representative blots showing changes in phosphorylation of Akt (p-Akt) or Akt (total) protein expression in a time-dependent manner following treatment with apelin (10⁻⁷ M). (E) Summary data showing effect of apelin on PI3-kinase activity expressed as a ratio of p-Akt/Akt protein levels (n = 3). Values are presented as the mean ± S.E.M. *p < 0.05 vs. apelin alone; **p < 0.05 vs. control.