Blockade of acid sensing ion channels attenuates the augmented exercise pressor reflex in rats with chronic femoral artery occlusion

Abstract

We found previously that static contraction of the hindlimb muscles of rats whose femoral artery was ligated evoked a larger reflex pressor response (i.e. exercise pressor reflex) than did static contraction of the contralateral hindlimb muscles which were freely perfused. Ligating a femoral artery in rats results in blood flow patterns to the muscles that are remarkably similar to those displayed by humans with peripheral artery disease. Using decerebrated rats, we tested the hypothesis that the augmented exercise pressor reflex in rats with a ligated femoral artery is attenuated by blockade of the acid sensing ion channel (ASIC) 3. We found that femoral arterial injection of either amiloride (5 and 50 μg kg(-1)) or APETx2 (100 μg kg(-1)) markedly attenuated the reflex in rats with a ligated femoral artery. In contrast, these ASIC antagonists had only modest effects on the reflex in rats with freely perfused hindlimbs. Tests of specificity of the two antagonists revealed that the low dose of amiloride and APETx2 greatly attenuated the pressor response to lactic acid, an ASIC agonist, but did not attenuate the pressor response to capsaicin, a TRPV1 agonist. In contrast, the high dose of amiloride attenuated the pressor responses to lactic acid, but also attenuated the pressor response to capsaicin. We conclude that ASIC3 on thin fibre muscle afferents plays an important role in evoking the exercise pressor reflex in rats with a compromised arterial blood supply to the working muscles.

Figure 1. Effects of two doses of amiloride (5 and 50 μg kg⁻¹), injected into the femoral artery, on the peak pressor and cardioaccelerator responses to femoral arterial injection of lactic acid (A) and capsaicin (B) in rats with either freely perfused femoral arteries or femoral arteries that were ligated 72 h before the start of the experiment

Control represents the peak response before injection of amiloride. In this and subsequent figures probability levels calculated by a one-way ANOVA are given above the bars, which represent the mean increase in either mean arterial pressure (MAP) or heart rate (HR). Vertical brackets above each bar represent standard errors of the mean. Asterisks (*) represent that the peak increase was significantly greater (P < 0.05) than its corresponding average baseline level, which is given along with its standard error of the mean below each bar. Daggers (†) represent that the pressor responses to lactic acid and capsaicin are significantly greater (P < 0.05) in the ligated rats than they are in the freely perfused rats. Horizontal brackets connect means that are significantly different from each other.

Figure 2. Effects of two doses of amiloride (5 and 50 μg kg⁻¹), injected into the femoral artery, on the peak pressor and cardioaccelerator responses to static contraction (A) and tendon stretch (B) in rats with either freely perfused femoral arteries or femoral arteries that were ligated 72 h before the start of the experiment

Control represents the peak response before femoral artery injection of amiloride.

Figure 3. Effects of two doses of amiloride (5 and 50 μg kg⁻¹), injected into the femoral artery, on the time course of the pressor and cardioaccelerator responses to static contraction in rats with either freely perfused hindlimbs (A) or femoral arteries that were ligated 72 h before the start of the experiment (B)

The responses to contraction were averaged across rats starting at 2 s after the start of contraction and then at each 5 s time point until the manoeuvre ended. Open circles represent the time course of the responses before amiloride was given. Filled circles represent the time course of the responses after 5 μg kg⁻¹ of amiloride was given; likewise the open triangles represent the responses after 50 μg kg⁻¹ of amiloride was given. Daggers (†) represent time points that were significantly less (P < 0.05) than their corresponding time point in control. Horizontal bars identify time points that were significantly greater (P < 0.05) than their corresponding baseline level, which in this case is no change (i.e. 0).

Figure 4. Effects of two doses of amiloride (5 and 50 μg kg⁻¹), injected into the femoral artery, on the time course of the pressor and cardioaccelerator responses to tendon stretch in decerebrated rats with either freely perfused hindlimbs or femoral arteries that were ligated 72 h before the start of the experiment

The responses to stretch were averaged across rats starting at two seconds after the start of stretch and then at each 5 s time point until the manoeuvre ended. See Fig. 3 for explanation of symbols.

Figure 5. Examples of the effects of amiloride (5 μg kg⁻¹; i.a.; A) and APETx2 (100 μg kg⁻¹; i.a.; B) on the cardiovascular responses to static contraction in ligated rats

Note that the white line in the arterial pressure trace is the mean pressure.

Figure 6. Effects of two doses of amiloride (5 and 50 μg kg⁻¹), injected intravenously, on the peak pressor and cardioaccelerator responses to static contraction (A) and to tendon stretch (B) in decerebrated rats with either freely perfused femoral arteries or femoral arteries that were ligated 72 h before the start of the experiment

Daggers (†) represent that the pressor response to contraction is significantly greater (P < 0.05) in the ligated rats than it is in the freely perfused rats. See Fig. 1 for an explanation of other symbols.

Figure 7. Effect of APETx2 (100 μg kg⁻¹), injected into the femoral artery, on the peak pressor and cardioaccelerator responses to femoral arterial injection of lactic acid (A) and capsaicin (B) in rats with either freely perfused femoral arteries or femoral arteries that were ligated 72 h before the start of the experiment

Control represents the peak response before injection of APETx2. Daggers (†) represent that the pressor responses to lactic acid and capsaicin are significantly greater (P < 0.05) in the ligated rats than they are in the freely perfused rats. Horizontal brackets connect means that are significantly different from each other. See Fig. 1 for an explanation of other symbols.

Figure 8. Effect of APETx2 (100 μg kg⁻¹), injected into the femoral artery, on the peak pressor and cardioaccelerator responses to static contraction (A) and tendon stretch (B) in rats with either freely perfused femoral arteries or femoral arteries that were ligated 72 h before the start of the experiment

Control represents the peak response before femoral artery injection of APETx2. Dagger (†) represents that the pressor response to contraction is significantly greater (P < 0.05) in the ligated rats than it is in the freely perfused rats. See Fig. 1 for an explanation of other symbols.

Figure 9. Effect of APETx2 (100 μg kg⁻¹), injected into the femoral artery, on the time course of the pressor and cardioaccelerator responses to static contraction in rats with either freely perfused hindlimbs (A) or femoral arteries that were ligated 72 h before the start of the experiment (B)

The responses to contraction were averaged across rats starting at 2 s after the start of contraction and then at each 5 s time point until the manoeuvre ended. Daggers (†) represent time points that were significantly less (P < 0.05) than their corresponding time point in control. See Fig. 3 for explanation of other symbols.

Figure 10. Effect of APETx2 (100 μg kg⁻¹), injected into the femoral artery, on the time course of the pressor and cardioaccelerator responses to tendon stretch in decerebrated rats with either freely perfused hindlimbs or femoral arteries that were ligated 72 h before the start of the experiment

The responses to stretch were averaged across rats starting at 2 s after the start of stretch and then at each 5 s time point until the manoeuvre ended. Daggers (†) represent time points that were significantly less (P < 0.05) than their corresponding time point in control. See Fig. 3 for explanation of other symbols.