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When contracting muscles are freely perfused, the acid-sensing ion channel 3 (ASIC3) on group IV afferents plays a minor role in evoking the exercise pressor reflex. We recently showed in isolated dorsal root ganglion neurons innervating the gastrocnemius muscles that two mu opioid receptor agonists, namely endomorphin 2 and oxycodone, potentiated the sustained inward ASIC3 current evoked by acidic solutions. This in vitro finding prompted us to determine whether endomorphin 2 and oxycodone, when infused into the arterial supply of freely perfused contracting hindlimb muscles, potentiated the exercise pressor reflex. We found that infusion of endomorphin 2 and naloxone in decerebrated rats potentiated the pressor responses to contraction of the triceps surae muscles. The endomorphin 2-induced potentiation of the pressor responses to contraction was prevented by infusion of APETx2, an ASIC3 antagonist. Specifically, the peak pressor response to contraction averaged 19.3 ± 5.6 mmHg for control ( n = 10), 27.2 ± 8.1 mmHg after naloxone and endomorphin 2 infusion ( n = 10), and 20 ± 8 mmHg after APETx2 and endomorphin 2 infusion ( n = 10). Infusion of endomorphin 2 and naloxone did not potentiate the pressor responses to contraction in ASIC3 knockout rats ( n = 6). Partly similar findings were observed when oxycodone was substituted for endomorphin 2. Oxycodone infusion significantly increased the exercise pressor reflex over its control level, but subsequent APETx2 infusion failed to restore the increase to its control level ( n = 9). The peak pressor response averaged 23.1 ± 8.6 mmHg for control ( n = 9), 33.2 ± 11 mmHg after naloxone and oxycodone were infused ( n = 9), and 27 ± 8.6 mmHg after APETx2 and oxycodone were infused ( n = 9). Our data suggest that after opioid receptor blockade, ASIC3 stimulation by the endogenous mu opioid, endomorphin 2, potentiated the exercise pressor reflex. NEW & NOTEWORTHY This paper provides the first in vivo evidence that endomorphin 2, an endogenous opioid peptide, can paradoxically increase the magnitude of the exercise pressor reflex by an ASIC3-dependent mechanism even when the contracting muscles are freely perfused.

The exercise pressor reflex is initiated by the contraction-induced activation of group III and IV muscle afferents. The reflex is manifested by increases in arterial blood pressure and cardiac output, which, in turn, are generated by increases in the sympathetic outflow to the heart and vasculature and decreases in the vagal outflow to the heart. In previous experiments, we used a pharmacological approach to assess the role played by the acid-sensing ion channel 3 (ASIC3) on group III and IV afferents in evoking the exercise pressor reflex. In the present experiments, we used an alternative approach, namely functional knockout (KO) of the ASIC3 gene, to confirm and extend our previous finding that pharmacological blockade of the ASIC3 had only a small impact on the expression of the exercise pressor reflex when the arterial supply to the contracting hindlimb muscles of rats was patent. Using this alternative approach, we compared the magnitude of the exercise pressor reflex evoked in ASIC3 KO rats with that evoked in their wild-type (WT) counterparts. We found both WT and ASIC3 KO rats displayed similar pressor responses to static contraction (WT, n = 10, +12 ± 2 mmHg; KO, n = 9, +11 ± 2 mmHg) and calcaneal tendon stretch (WT, n = 9, +13 ± 2 mmHg; KO, n = 7, +11 ± 2 mmHg). Likewise, both WT and ASIC3 KO displayed similar pressor responses to intra-arterial injection of 12 mM lactic acid (WT, n = 9, +14 ± 3 mmHg; KO, n = 8, +18 ± 5 mmHg), 24 mM lactic acid (WT, n = 9,+24 ± 2 mmHg; KO, n = 8, +20 ± 5 mmHg), capsaicin (WT, n = 9,+27 ± 5 mmHg; KO, n = 10, +29 ± 5 mmHg), and diprotonated phosphate ([Formula: see text]; WT, n = 6,+22 ± 3 mmHg; KO, n = 6, +32 ± 6 mmHg). We conclude that redundant receptors are responsible for evoking the pressor reflexes arising from group III and IV afferents.

Mechanical and metabolic stimuli arising from contracting muscles evoke the exercise pressor reflex. This reflex is greater in a rat model of simulated peripheral arterial disease in which a femoral artery is chronically ligated than it is in rats with freely perfused femoral arteries. The role played by the mechanically sensitive component of the exaggerated exercise pressor reflex in ligated rats is unknown. We tested the hypothesis that the mechano-gated channel inhibitor GsMTx4, a relatively selective inhibitor of mechano-gated Piezo channels, reduces the exercise pressor reflex in decerebrate rats with ligated femoral arteries. Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the pressor response to Achilles tendon stretch (a purely mechanical stimulus) but had no effect on the pressor responses to intra-arterial injection of α,β-methylene ATP or lactic acid (purely metabolic stimuli). Moreover, injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced both the integrated pressor area (control 535 ± 21, GsMTx4 218 ± 24 mmHg·s; P < 0.01), peak pressor (control 29 ± 2, GsMTx4 14 ± 3 mmHg; P < 0.01), and renal sympathetic nerve responses to electrically induced intermittent hindlimb muscle contraction (a mixed mechanical and metabolic stimulus). The reduction of the integrated pressor area during contraction caused by GsMTx4 was greater in rats with ligated femoral arteries than it was in rats with freely perfused femoral arteries. We conclude that the mechanically sensitive component of the reflex contributes to the exaggerated exercise pressor reflex during intermittent hindlimb muscle contractions in rats with ligated femoral arteries., (Copyright © 2016 the American Physiological Society.)

Key points Mechanical and metabolic stimuli from contracting muscles evoke reflex increases in blood pressure, heart rate and sympathetic nerve activity. Little is known, however, about the nature of the mechano-gated channels on the thin fibre muscle afferents that contribute to evoke this reflex, termed the exercise pressor reflex., We determined the effect of GsMTx4, an inhibitor of mechano-gated Piezo channels, on the exercise pressor reflex evoked by intermittent contraction of the triceps surae muscles in decerebrated, unanaesthetized rats., GsMTx4 reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to intermittent contraction but did not reduce the pressor responses to femoral arterial injection of compounds that stimulate the metabolically-sensitive thin fibre muscle afferents., Expression levels of Piezo2 channels were greater than Piezo1 channels in rat dorsal root ganglia., Our findings suggest that mechanically-sensitive Piezo proteins contribute to the generation of the mechanical component of the exercise pressor reflex in rats., Abstract Mechanical and metabolic stimuli within contracting skeletal muscles evoke reflex autonomic and cardiovascular adjustments. In cats and rats, gadolinium has been used to investigate the role played by the mechanical component of this reflex, termed the exercise pressor reflex. Gadolinium, however, has poor selectivity for mechano-gated channels and exerts multiple off-target effects. We tested the hypothesis that GsMTX4, a more selective mechano-gated channel inhibitor than gadolinium and a particularly potent inhibitor of mechano-gated Piezo channels, reduced the exercise pressor reflex in decerebrate rats. Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 5, GsMTx4: 12 ± 5 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to tendon stretch, a purely mechanical stimulus, but had no effect on the pressor responses to intra-arterial injection of α,β-methylene ATP or lactic acid. Moreover, injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 2, GsMTx4: 14 ± 3 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to electrically-induced intermittent hindlimb muscle contractions. By contrast, injection of 10 μg of GsMTx4 into the jugular vein had no effect on the pressor, cardioaccelerator, or renal sympathetic nerve responses to contraction. Quantitative RT-PCR and western blot analyses indicated that both Piezo1 and Piezo2 channel isoforms were natively expressed in rat dorsal root ganglia tissue. We conclude that GsMTx4 reduced the exercise pressor reflex in decerebrate rats and that the reduction was attributable, at least in part, to its effect on mechano-gated Piezo channels. [ABSTRACT FROM AUTHOR]