Abstract 12634: Amplified P2x 3 Pathway Activity in IB4 Positive Muscle DRG Neurons and Exercise Pressor Reflex Regulation in Hindlimb Ischemia-Reperfusion

Abstract

Background: Hindlimb ischemia-reperfusion (IR) represents one of the most prominent pathological conditions observed in peripheral artery disease (PAD). Arterial blood pressure response (BP) during exercise, partly driven by the exaggerated exercise pressor reflex (EPR), is associated with an elevated risk of cardiovascular events in PAD patients. However, the precise underlying mechanisms leading to this exaggerated BP response are poorly elucidated. The purinergic P2X 3 signaling pathway, which plays a key role in modifying the EPR, was the focus of the present study. Objectives: To examine the regulatory role of P2X 3 on the EPR in a rat model of hindlimb IR, which was induced by 6 hours’ ischemia followed by 18 hours’ reperfusion (referred to as IR rats). Results: We found that, in IR rats, there were: 1) upregulation of P2X 3 protein expression in the L4-6 dorsal root ganglion (DRG) (1.48±0.16 in IR rats/n=8 vs. 1.00±0.41 in sham rats/n=9; P<0.05), which was localized in IB4 positive DRG neurons by the method of immunohistochemistry, 2) amplified P2X currents in isolated muscle DRG neurons (P2X transient currents: 1281.64±776.74 pA in IR rats/n=25, vs. 717.91±556.2 pA in sham rats/n=9, P<0.05; and sustain currents: 466.94±168.68 pA in IR rats/n=11, vs. 271.49±200.90 pA in sham rats/n=8, P<0.05); 3) amplification of P2X-mediated BP response (29±8 mmHg in IR rats/n=12 vs. 21±4 mmHg in sham rats/n=7; P<0.05); and 4) alleviation of the exaggerated BP response induced by static muscle contraction (18±8 mmHg in IR +A317491/n=8, vs. 29±11 mmHg in IR/n=7, P<0.05) and P2X-mediated BP response by α, β-me-ATP injection (15±4 mmHg in IR+A317491 /n=6 vs. 23±7 mmHg in IR/n=4, P<0.05) following the inhibition of muscle afferents’ P2X 3 receptor by A-317491. We further verified that both A-317491 and siRNA knockdown of P2X 3 significantly decreased the activity of P2X currents in the isolated muscle DRG neurons (all P<0.05 vs. their respective controls). Conclusion: With the above evidence from multiple approaches, we concluded that the P2X 3 signaling pathway activity is amplified following hindlimb IR via the enhancing expression and activity of P2X 3 receptors in IB4 positive muscle DRG afferents. This contributes to the exacerbated EPR responses in PAD.