Proteinase-Activated Receptor-2 Sensitivity of Amplified TRPA1 Activity in Skeletal Muscle Afferent Nerves and Exercise Pressor Reflex in Rats with Femoral Artery Occlusion

Abstract

Background/aims: Limb ischemia occurs in peripheral artery disease (PAD). Sympathetic nerve activity (SNA) that regulates blood flow directed to the ischemic limb is exaggerated during exercise in this disease, and transient receptor potential channel A1 (TRPA1) in thin-fiber muscle afferents contributes to the amplified sympathetic response. The purpose of the present study was to determine the role of proteinase-activated receptor-2 (PAR2) in regulating abnormal TRPA1 function and the TRPA1-mediated sympathetic component of the exercise pressor reflex.

Methods: A rat model of femoral artery ligation was employed to study PAD. Dorsal root ganglion (DRG) tissues were obtained to examine the protein levels of PAR2 using western blot analysis. Current responses induced by activation of TRPA1 in skeletal muscle DRG neurons were characterized using whole-cell patch clamp methods. The blood pressure response to static exercise (i.e., muscle contraction) and stimulation of TRPA1 was also examined after a blockade of PAR2.

Results: The expression of PAR2 was amplified in DRG neurons of the occluded limb, and PAR2 activation with SL-NH2 (a PAR2 agonist) increased the amplitude of TRPA1 currents to a greater degree in DRG neurons of the occluded limb. Moreover, FSLLRY-NH2 (a PAR antagonist) injected into the arterial blood supply of the hindlimb muscles significantly attenuated the pressor response to muscle contraction and TRPA1 stimulation in rats with occluded limbs.

Conclusions: The PAR2 signal in muscle sensory nerves contributes to the amplified exercise pressor reflex via TRPA1 mechanisms in rats with femoral artery ligation. These findings provide a pathophysiological basis for autonomic responses during exercise activity in PAD, which may potentially aid in the development of therapeutic approaches for improvement of blood flow in this disease.

Keywords: Hindlimb ischemia; Muscle afferent nerve; PAR2; Peripheral arterial disease; TRPA1.

Fig. 1

A, Original band and averaged data: protein expression levels of PAR2 were amplified in DRG tissues of occluded limbs compared with those of control limbs. *P<0.05 vs. control limbs. N=5 in each group. B, Original traces: the effects of prior application of 100 µM of SL-NH2 on AITC-induced currents in DRG neurons of a control limb and an occluded limb. C, Averaged data: SL-NH2 amplified the amplitude of AITC-activated currents in muscle DRG neurons. *P<0.05 compared with AITC alone. D, The percentage of augmented amplitude of AITC-induced currents by SL-NH2 appeared to be greater in DRG neurons of occluded limbs than that in DRG neurons of control limbs. *P<0.05 vs. control. N=10–12 in each group.

Fig. 2

A, Blocking PLC pathways attenuated amplified TRPA1 currents in DRG neurons induced by activation of PAR2. However, blocking PKC pathways failed to antagonize amplified currents induced by AITC in DRG neurons. Top panel: original traces; and bottom panel: average data. ET-18-OCH3 (ET, 20 µM): PLC inhibitor; and GF109203X (GF, 20 µM): PKC inhibitor. *P<0.05 vs. AITC and SL-NH2+ET+AITC. N=10–12 in each group. B, PIP2 (10 µM) added to the internal patch solution attenuated amplified TRPA1 currents in DRG neurons induced by activation of PAR2. PIP2: phosphatidylinositol-4,5-bisphosphate. *P<0.05 vs. AITC alone. There were no significant differences in AITC-induced currents for AITC vs. SL-NH2+AITC with PIP2. N=10–12 in each group.

Fig. 3

Pressor response to muscle contraction in control rats (n=8) and occluded rats (n=8) after blocking PAR2 using FSLLRY-NH2. There were no significant differences in MAP response evoked by muscle contraction in control rats after FSLLRY-NH2. However, FSLLRY-NH2 attenuated MAP response in occluded rats. *P<0.05 vs. saline control. There were no significant differences in developed muscle tension among groups.

Fig. 4

Sympathetic and pressor responses to activation of TRPA1 by arterial injection of AITC (20 µg/kg) with prior blockade of PAR2 using FSLLRY-NH2 in control rats and occluded rats. Note that a higher dose of FSLLRY-NH2 was observed to attenuate sympathetic and pressor responses evoked by AITC in control rats. *P<0.05 vs. saline control. n=10 in control rats and n=12 in occluded rats.