Femoral artery occlusion increases expression of ASIC3 in dorsal root ganglion neurons

Abstract

Acid-sensing ion channels (ASICs) in sensory nerves are responsive to increases in the levels of protons in the extracellular medium. Prior studies suggest that the muscle metabolite, lactic acid, plays a role in reflex sympathetic and cardiovascular responses via stimulation of thin muscle afferent nerves. Also, femoral artery occlusion augments the reflex sympathetic nerve response in rats. ASIC3 is a main subtype to appear in sensory nerves in mediating the response induced by increases in protons in the interstitial space of contracting muscles. Thus, in this article, we hypothesized that femoral occlusion increases the expression of ASIC3 in primary afferent neurons innervating muscles, and this contributes to the exaggerated reflex sympathetic responses. Femoral occlusion/vascular insufficiency of the hindlimb muscles was induced by the femoral artery ligation in rats. First, Western blot analysis shows that 24-72 h of femoral artery ligation significantly increased the expression of ASIC3 protein in dorsal root ganglion (optical density, 1.0 ± 0.07 in control vs. 1.65 ± 0.1 after 24 h of occlusion, P < 0.05; n = 6 in each group). There were no significant differences for increases in ASIC3 24 and 72 h postocclusion. Second, experiments using fluorescent immunohistochemistry and retrograde-labeling technique show that a greater percentage of ASIC3 staining neurons are localized in muscle-innervating dorsal root ganglion neurons after the arterial occlusion (78 ± 3% in 24 h post occlusion vs. 59 ± 5% in control, P < 0.05; n = 6 in each group). Third, the reflex responses in renal sympathetic nerve and arterial blood pressure induced by the stimulation of ASIC were examined after an injection of lactic acid into the arterial blood supply of hindlimb muscles of control rats and ligated rats. The results demonstrate that the sympathetic and pressor responses to lactic acid were significantly augmented after femoral occlusion compared with those in the control group. The data of this study suggest that enhanced ASIC3 expression in muscle afferent nerves contributes to the exaggerated reflex sympathetic and pressor responses to lactic acid as seen in arterial occlusion.

Fig. 1.

Effects of femoral artery occlusion on acid-sensing ion channel-3 (ASIC3) expression in dorsal root ganglion (DRG) neurons at different time courses. Western blot assay was employed to examine ASIC3 proteins in L4–L6 DRG at time points of 6, 24 and 72 h following femoral artery occlusion. The ligation was performed on the one hindlimb. The sham-operated procedure was performed on the contralateral limb of the same rats, and this served as control. A: representative bands of ASIC3 expression. Bands of β-actin are used as control for an equal protein loading. B: average data. The optical density is expressed in arbitrary units normalized against a control sample. Data in histograms represent means ± SE; n = 3–6 in each group. *P < 0.05 compared with control (0 h).

Fig. 2.

Upregulation of ASIC3 induced by femoral artery occlusion is localized in the lumbar level of DRG. ASIC3 proteins were examined in L4–6 and cervical DRG neurons of occluded limb and control limb using Western blot analysis. Femoral artery ligation (24 h) was performed. The sham-operated procedures were performed on contralateral limb as control, 24 h lumbar (L) DRG, 24 h ipsilateral cervical (IC) DRG, and 24 h contralateral cervical (CC) DRG. A: representative Western blots. B: densitometrical analysis was used to show average data; n = 6 for each group. The optical density is expressed in arbitrary units normalized against a control sample. *P < 0.05 compared with control (0 h).

Fig. 3.

Localization of ASIC3 in muscle-innervating DRG neurons. Fluorescence immunohistochemistry was employed to examine double labeling of ASIC3 and Fluoro-Gold (FG) in DRG neurons of control limb and occluded limb. FG was injected into the hindlimb muscles a week before DRGs were dissected. A: representative photomicrographs. Arrows indicate cells positive for FG and ASIC3. Scale bar = 50 μM. B: histograms show that percentage of double-labeling neurons against total FG-positive cells is greater in DRG neurons' femoral artery occlusion (n = 6) than that in control (n = 6). *P < 0.05 compared with control.

Fig. 4.

Changes in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in response to stimulation of ASIC3 receptors with arterial injection of lactic acid. Lactic acid (1–4 μmol/kg body wt) was injected into arterial blood supply of hindlimb muscles of control rats (n = 8) and rats with femoral artery occlusion (n = 6). A: typical recordings of RSNA and arterial pressure (AP) responses. Arrows indicate a start of injections. B: average data of MAP and RSNA. Note that baseline MAP is 123 ± 8 mmHg in control rats and 115 ± 10 mmHg in occluded rats (P < 0.05 vs. control). Values are means ± SE. *P < 0.05 compared with sham-operated control.