Effect of Intramuscular Protons, Lactate, and ATP on Muscle Hyperalgesia in Rats.

Chronic muscle pain is a significant health problem leading to disability[1]. Muscle fatigue can exacerbate muscle pain. Metabolites, including ATP, lactate, and protons, are released during fatiguing exercise and produce pain in humans. These substances directly activate purinergic (P2X) and acid sensing ion channels (ASICs) on muscle nociceptors, and when combined, produce a greater increase in neuron firing than when given alone. Whether the enhanced effect of combining protons, lactate, and ATP is the sum of individual effects (additive) or more than the sum of individual effects (synergistic) is unknown. Using a rat model of muscle nociceptive behavior, we tested each of these compounds individually over a range of physiologic and supra-physiologic concentrations. Further, we combined all three compounds in a series of dilutions and tested their effect on muscle nociceptive behavior. We also tested a non-hydrolyzable form of ATP (α,β-meATP) alone and in combination with lactate and acidic pH. Surprisingly, we found no dose-dependent effect on muscle nociceptive behavior for protons, lactate, or ATP when given alone. We similarly found no effect after application of each two-metabolite combination. Only pH 4 saline and α,β-meATP produced hyperalgesia when given alone. When all 3 substances were combined, however, ATP (2.4μm), lactate (10mM), and acidic pH (pH 6.0) produced an enhanced effect greater than the sum of the effects of the individual components, i.e. synergism. α,β me ATP (3nmol), on the other hand, showed no enhanced effects when combined with lactate (10mM) or acidic pH (pH 6.0), i.e. additive. These data suggest that combining fatigue metabolites in muscle produces a synergistic effect on muscle nociception

Comparison of Individual Components Versus Combined Solution on muscle withdrawal thresholds across a 4h time period.

<p>(A) Injection of protons, lactate, and ATP alone compared to the combined solution. Muscle withdrawal threshold was measured before and after intramuscular injection with pH 6 saline (n = 4), 10 mM lactate (n = 4), 2.4 μM ATP (n = 4), and all three in combination (n = 5). None of the individual components had an effect on muscle withdrawal threshold, but the combination produced significant decreases (Repeated measures ANOVA, F_3,13 = 54.568, p < 0.001, Tukey test, p < 0.05 for each compound alone). (B) Injection of protons, lactate, and α,β–meATP alone compared to the combined solution. Muscle withdrawal threshold was measured after intramuscular injection with pH 6 saline (n = 4), 10 mM lactate (n = 4), 3 nM α,β–meATP (n = 4), or all three in combination (n = 6). The 3 nM α,β–meATP and the combination solution produced similar decreases in muscle withdrawal threshold (Repeated-measures ANOVA F_3,14 = 5.373, p < 0.011, Tukey test p > 0.05 for 3 nM α,β–meATP compared to combination solutions, p < 0.05 the remaining) *, p < 0.05.</p

Doses Used in the Acidic pH, Lactate, αβ–meATP Isobole Dose Response Curve (Fig 2B).

<p>Doses Used in the Acidic pH, Lactate, αβ–meATP Isobole Dose Response Curve (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138576#pone.0138576.g002" target="_blank">Fig 2B</a>).</p

Comparison of Individual Components Versus Combined Solution on muscle withdrawal thresholds across a 4h time period.

<p>(A) Injection of protons, lactate, and ATP alone compared to the combined solution. Muscle withdrawal threshold was measured before and after intramuscular injection with pH 6 saline (n = 4), 10 mM lactate (n = 4), 2.4 μM ATP (n = 4), and all three in combination (n = 5). None of the individual components had an effect on muscle withdrawal threshold, but the combination produced significant decreases (Repeated measures ANOVA, F_3,13 = 54.568, p < 0.001, Tukey test, p < 0.05 for each compound alone). (B) Injection of protons, lactate, and α,β–meATP alone compared to the combined solution. Muscle withdrawal threshold was measured after intramuscular injection with pH 6 saline (n = 4), 10 mM lactate (n = 4), 3 nM α,β–meATP (n = 4), or all three in combination (n = 6). The 3 nM α,β–meATP and the combination solution produced similar decreases in muscle withdrawal threshold (Repeated-measures ANOVA F_3,14 = 5.373, p < 0.011, Tukey test p > 0.05 for 3 nM α,β–meATP compared to combination solutions, p < 0.05 the remaining) *, p < 0.05.</p

Combination of ATP (A) or αβ–meATP (B) with Lactate and Protons.

<p>Muscle withdrawal thresholds were assessed before and 30 min after intramuscular injection. (A) Protons, lactate, and ATP were given in combination across series dilutions. Combining protons, lactate, and ATP had a significant effect on muscle withdrawal (Repeated measures ANOVA F_5,27 = 17.986, p < 0.001), but only the most dilute combination (C4) was decreased relative to saline controls (Tukey test, p = 0.001). The positive control, intramuscular injection of 3% carrageenan shows a significant decrease 24h after injection (p<0.001). (B) Protons, lactate, and α,β–meATP were given in combination across a series of dilutions. None of combinations tested produced significant decreases in muscle withdrawal threshold (Repeated-measures ANOVA F_3,14 = 5.373, p < 0.011). *, p < 0.05.</p

Effects of paired solutions on muscle withdrawal thresholds across a 4h time period.

<p>Injection of the dual combinations of metabolites. Muscle withdrawal threshold were measured before and after each possible pairwise combination (2.4 μm ATP + pH 6, n = 4; 2.4 μm ATP + 10 mM lactate, n = 4; pH 6.0 + 10 mM lactate, n = 4). There was no significant difference for the paired combinations (repeated measures ANOVA, F_2,9 = 0.911, p = 0.436).</p

Effect of Intramuscular Injection of pH, lactate, ATP, and α,β–meATP on Muscle Withdrawal Threshold.

<p>Muscle withdrawal threshold was measured before and 30 minutes after injection of physiologic and supraphysiologic concentrations of pH, lactate, ATP, and α,β–meATP. (A) pH dose response curve. Rats were injected with normal saline adjusted to neutral pH (n = 6), pH 4.0 (n = 12), pH 4.5 (n = 6), pH 5.0 (n = 6), and pH 6.0 (n = 6). Of these doses, only pH 4.0 saline produced a significant decrease in muscle withdrawal threshold from baseline (Repeated measures ANOVA, F_4,31 = 16.761, p <0.001, Tukey test, p < 0.001). (B) Lactate dose response curve. Rats were injected with normal saline adjusted to pH 7.4 with lactate concentrations of 1.5 M (n = 6), 470 mM (n = 6), 150 mM (n = 6), 47 mM (n = 12), 15 mM (n = 12), 4.7 mM (n = 6), 1.5 mM (n = 6), and 470 μM (n = 6), saline control (n = 6). There was no significant change from baseline as compared to saline control (Repeated measures ANOVA, F_7,52 = 1.834, p = 0.100). (C) ATP dose response curve. Rats were injected with normal saline adjusted to pH 7.4 with ATP concentrations of 24 mM (n = 6), 7.6 mM (n = 6), 2.4 mM (n = 6), 760 μM (n = 12), 76 μM (n = 6), 760 nM (n = 6), 7.6 μM (n = 6), 760 nM (n = 6). ATP concentration had a significant effect on muscle withdrawal threshold (Repeated measures ANOVA, F_7,46 = 2.315, p = 0.041), but there was no significant difference between any dose and the saline control (Tukey test, p > 0.05 for all doses versus saline control). (D) α,β–me ATP dose response curve. Rats were injected with normal saline adjusted to pH 7.4 with α,β–meATP concentrations of 100 nM (n = 5), 30 nM (n = 5), 10 nM (n = 4), 3 nM (n = 4), 1 nM (n = 4), and saline control (n = 7). α,β–meATP significantly decreased muscle withdrawal threshold (Repeated measures ANOVA, F_5,23 = 6.949, p < 0.001), with the 100 nM and 30 nM doses being effective (Tukey test, p < 0.05 each dose). *, p < 0.05.</p