Abstract 527: Apelin-13 Inhibits Large-Conductance Ca2+-Activated K+ Channels in Cerebral Artery Smooth Muscle Cells via a PI3-Kinase-Dependent Mechanism

Abstract

Apelin-13 has been reported to act directly on APJ receptors in vascular smooth muscle (VSM) to induce vasocontraction. However, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca2+-activated K+ (BKca) channels in VSM cells are critical regulators of membrane potential and vascular tone. Thus, in the present study, we examined the effect of apelin-13 on BKca channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001μM-1μM) caused dose-dependent inhibition of BKca in VSM cells. Apelin-13 (0.1μM) significantly decreased the BKca current density from 71.25±8.14 pA/pF to 44.52±7.10 pA/pF (n=14 cells, P<0.05). This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which apelin-13 (0.1μM) decreased the open-state probability (NPo) of BKca channels from 0.0239 ±.00408 to 0.0148 ± 0.00267 (n=9 cells, P<0.05) in freshly isolated VSM cells. However, in inside-out patches, application of apelin-13 (0.1μM) into the pipette solution did not alter the NPo of BKca channels (NPo of BKca: 0.72±0.06 and 0.79±0.08 before and after apelin-13 administration, n=10, p>0.05), suggesting that the inhibitory effect of apelin-13 on BKca is not mediated by its direct action on BKca. In whole cell patches, pretreatment of VSM cells with LY-294002 (10 μM), a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BKca current density by 52%. Next, we examined the effect of apelin-13 on PI3- kinase activity in rat cerebral arteries using western blot analysis. Treatment with apelin-13 (0.1μM) significantly increased the ratio of phosphorylated-Akt/total Akt by 40%, indicating that apelin-13 significantly increases PI3-kinase activity. In summary, apelin-13 inhibits BKca channel current density via a PI3-kinase-dependent signaling pathway, which may contribute to its regulatory action in the control of vascular tone.