Role of diprotonated phosphate in evoking muscle reflex responses in cats and humans

Lactic acid and H⁺ evoke muscle reflexes that raise sympathetic nerve activity. Whether these substances are direct afferent stimulants or markers for the acidification of other substances is unknown. Diprotonated phosphate (H₂PO₄/^-), a possible mediator of fatigue, increases as the cell acidifies and phosphate is produced. Its role in evoking muscle reflexes is unknown. We used ³¹P-nuclear magnetic resonance to measure forearm muscle H⁺ and H₂PO₄/^- and microneurography to measure muscle sympathetic nerve activity (MSNA, peroneal nerve) during a handgrip protocol designed to dissociate H⁺ from H₂PO₄/^-. Ischemic handgrip (50% maximal voluntary contraction x 2 min) was followed by a 1-min rest period during which the muscle was freely perfused. This was followed by a second bout of ischemic handgrip and a 5- min recovery. In seven of eight subjects, MSNA correlated with H₂PO₄/^-, whereas it correlated with pH in only one subject. To determine whether muscle reflex responses are evoked by H⁺, lactic acid, monoprotonated phosphate (HPO₄/^2-), or H₂PO₄/^-, we injected H⁺, lactate, H₂PO₄/^- [all 50 mM in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffered to pH 6], and HPO₄/^2- (50 mM, pH 7.5 in 10 mM HEPES) into the arterial supply of the triceps surae of the cat (n = 9) as we measured mean arterial blood pressure (MAP). H₂PO₄/^- increased MAP more than HPO₄/^2-, H⁺, or lactate (27.1 ± 3.7 vs. 5.0 ± 1.3, 4.6 ± 3.1, and 7.7 ± 3.2 rise in mmHg). This effect was a reflex, since H₂PO₄/^- did not raise MAP after the sciatic nerve was cut. Thus H₂PO₄/^- is a major stimulant of muscle afferents and in large part determines the degree of sympathoexcitation during exercise.