Inorganic phosphate and lactate potentiate the pressor response to acidic stimuli in rats

Abstract

H+, lactate and inorganic phosphate (Pi) are produced by contracting skeletal muscles to evoke, in part, the metabolic component of the exercise pressor reflex. Because of their disparate dissociation constants (i.e., pKa), the contribution of each acid to the muscle metaboreflex is unclear. This lack of information prompted us to determine the reflex pressor responses of injecting acidic saline, lactate (24mM) and Pi (86mM) at various pHs (from 2.66 through 7.5), alone or in combination, into the arterial supply of hindlimb skeletal muscle of decerebrated rats. In particular, we were prompted to test the hypothesis that the pressor response to an injection of a combination of lactate and phosphate at an acidic pH evoked a greater pressor response than did injection of either phosphate or lactate alone at the same pH as was used to inject the combined stimulus. We found that injecting acidic saline produced a pressor response only at a pH of 2.66 (7±4 mmHg), an effect that was potentiated when the solution contained lactate (50±20 mmHg). At a pH of 6.0, however, this effect was lost. At a pH of 6.0 only the injection of Pi produced a significant pressor response (23±12 mmHg). A large potentiating effect was found when lactate was added to the Pi solution (39±18 mmHg), an effect that was lost at a pH > 7.0. Our findings lead to the conclusion that the pressor response to injection of acidic solutions was driven by H+ ions, and that Pi and lactate functioned as sensitizing agents.

Introduction

The exercise pressor reflex is evoked by metabolic and mechanical stimuli arising in contracting skeletal muscles and functions to improve blood flow and oxygen delivery (Kaufman et al., 1983; O’Leary et al., 1999; Amann et al., 2011). Among the different metabolites produced by the contracting muscle, acidosis arising from anaerobic glycolysis is considered to be a major contributor (Rotto et al., 1989; Fadel et al., 2003; Tsuchimochi et al., 2011). For example, the exercise pressor reflex is blunted in myophosphorylase deficient patients, a pathophysiological condition in which muscle pH does not change during static exercise due to the absence of the enzymes that convert glycogen to glucose (Fadel et al., 2003). In contrast, the exercise pressor reflex is exaggerated in patients with peripheral artery disease, a condition in which muscle acidosis is presumably exacerbated due to the arterial supply of the exercising muscles being partially occluded (Baccelli et al., 1999; Muller et al., 2012).

Despite the important information gained from these studies, the exact acidic stimulus responsible for evoking the metabolic component of the exercise pressor reflex remains unclear. For example, injecting lactic acid into the arterial supply of the cats’ hindlimb evoked a pressor response that was greater than that evoked by injecting hydrochloric acid at the same pH (Rotto et al., 1989). This result suggests that the accumulation of lactic acid plays a key role in evoking the metabolic component of the exercise pressor reflex. Nevertheless, the pH of this lactic acid solution injected into the arterial supply of the hindlimb by Rotto et al. (1989) was not physiological (i.e., < 3.0). Consequently, the possibility exists that the pressor responses to the exogenous injection of putative metabolic by-products of contraction may not be representative of what is happening during normal exercise. Sinoway et al. (1994), for example, found that injecting 50mM lactic acid at a pH of 6.0 increased blood pressure by only few mmHg in cats.

The lack of responsiveness to lactic acid injection at a more physiological pH might arise from the fact that the pKa of lactic acid (i.e. the pH at which lactic acid is half dissociated) is 3.86. Lactic acid is therefore present in its dissociated form, lactate, at a pH > 6.0. Despite the fact that lactate facilitates the opening of acid-sensing ion channels (Immke & McCleskey, 2001; 2003), another acid needs to be considered for being responsible for evoking the acidosis-induced metaboreflex. Inorganic phosphate released from cross-bridge cycle and ATP hydrolysis might be a good candidate.

Being that the pKa of inorganic phosphate (${\text{H}}_2{\text{PO}}_4^{-}$/${\text{HPO}}_4^{2-}$⁻) is 6.8, inorganic phosphate are predominantly present in its monoprotonated form (${\text{HPO}}_4^{2-}$⁻) at a neutral pH and in its diprotonated form (${\text{H}}_2{\text{PO}}_4^{-}$) at a more acidic pH (e.g. 6.0). Several experiments showed that the diprotonated form of phosphate produced a significant pressor response when injected into the arterial supply of cats or rats hindlimb muscles (Sinoway et al., 1994; Gao et al., 2006) These results suggest that inorganic phosphate might be a better compound to evoke the metabolic component of the exercise pressor reflex than is lactate. However, the true contribution of each compound might be more complex. Indeed, the blunted pressor response to static exercise in myophosphorylase deficient patients occurred despite accumulation of inorganic phosphate (Fadel et al., 2003). Consequently, it is difficult to conclude on the stimulus responsible for the pressor response evoked by muscle acidosis because the contribution of each compound in evoking the metabolic component of the exercise pressor reflex has never been isolated from each other. In addition, it is possible that inorganic phosphate interacts with lactate to potentiate the exercise pressor reflex but data regarding that hypothesis are lacking.

Therefore, this lack of understanding prompted us to determine the isolated and combined contributions of hydrogen ions, inorganic phosphate or lactate to evoke reflex pressor responses in rats. We hypothesized that 1) injecting acidic saline evoked a pressor response only at a very acidic pH; 2) injecting lactate or inorganic phosphate evoked a greater pressor response than did acidic saline injection when the pH of the three solution was the same; 3) injecting inorganic phosphate evoked a greater pressor response than did injecting lactate when the pH of the solution was the same; 4) the pressor responses evoked by injections of lactate or inorganic phosphate decreased when the pH of the injected solutions increased; 5) injecting a solution of lactate combined with inorganic phosphate evoked a pressor response that is potentiated compared to the pressor response evoked by injecting lactate or inorganic phosphate alone.

Methods

Ethical approval

The Institutional Care and Use Committee of the Pennsylvania State University College of Medicine approved all of the procedures (PRAMS200946708). The authors understand and conformed to the ethical guidelines of the journal for animal use in research.

Animal characteristics, wellness and sample size

Experiments were conducted at constant room air temperature (21 °C) on 24 male Sprague-Dawley rats (Charles River) weighing 350–550 g.

Rats were housed within the central animal facility of the Pennsylvania State University - College of Medicine, with access to food and water ad libitum and exposure to a 50/50 light/dark cycle. All attempts were made to minimize animal discomfort and pain.

Surgical procedures

Each rat was anesthetized initially by inhalation of 4 % isoflurane in oxygen. The surgical procedure was initiated only when the corneal reflex was abolished by the anesthesia and when pinching the hind paw did not produce a withdrawal reflex. The rat’s trachea was cannulated and its lungs were mechanically ventilated (683, Harvard apparatus Inc., Holliston, MA, USA). The concentration of isoflurane ventilating the lungs was reduced to 2 % for the rest of the surgery. The left, right common carotid and right jugular vein were cannulated with RenaPulse™ High Fidelity Pressure Tubing (RPT040, Braintree Scientific Inc., Braintree, MA, USA) to record arterial blood pressure (P23XL, Gould-Statham Instruments Inc., Los Angeles CA, USA) and to draw arterial blood samples. The left superficial epigastric artery, which is a side branch of the femoral artery, was cannulated (SUBL-140, Braintree Scientific Inc., Braintree, MA, USA). A snare (2.0 silk suture) was placed around the femoral artery and vein ~0.5–1 cm upstream of the superficial epigastric artery. Finally, the muscular branch of the femoral artery and saphenous arteries were ligated (4.0 silk suture) to increase the probability that the injectate accessed the triceps surae circulation.

All injections described below were made into the superficial epigastric artery and were flushed with 100 μL of saline. During these experiments, the rat was paralyzed by intravenous injection of pancuronium bromide (0.2 mg ∙mL⁻¹; 0.2 ml). The snare placed around the femoral artery and vein, when tightened partially trapped injected solutions in the hindlimb circulation. The snare was released two minutes after the injection was completed. At least 10 minutes separated every injection.

The rat was decerebrated by sectioning the brain less than 1 mm rostral to the superior colliculus (Dobson & Harris, 2012). The isoflurane was then discontinued and the lungs ventilated with room air. Blood pH (7.35–7.45), arterial ${\text{P}}_{{\text{O}}_2}$ (100 – 150 mmHg), ${\text{P}}_{{\text{CO}}_2}$ (35 – 40 mmHg) and [HC${\text{O}}_3^{-}$] (22–26 mmol∙L⁻¹) were kept within physiological range. Body temperature was maintained around 37 °C using a heating lamp. At the end of the experiment, the decerebrated rats were euthanized by intravenous injection of a supersaturated KCl solution.

Experimental procedures

To determine the effect of injecting H+, lactate, or inorganic phosphate (${\text{H}}_2{\text{PO}}_4^{-}$/${\text{HPO}}_4^{2-}$⁻) alone or in combination on arterial blood pressure and heart rate, we performed five experiments. The concentration of lactic acid/lactate or ${\text{H}}_2{\text{PO}}_4^{-}$/${\text{HPO}}_4^{2-}$⁻ (i.e. inorganic phosphate) depending on the pH of the solutions is presented Table 1.

Table 1.

Concentrations of the different acid/base couple depending on the pH of the solution

	Acid/base couple
pH	Lactic acid/lactate	${\text{H}}_2{\text{PO}}_4^{-}/{\text{HPO}}_4^{2-}$
2.66	22.4/1.6	-
4.5	4.0/20.0	-
5.0	1.4/22.6	-
6.0	0.2/23.8	74.2/11.8
6.5	0.05/23.95	57.3/28.7
7.0	0.02/23.98	33.3/52.7
7.5	0.01/23.99	14.3/71.7

Concentrations have been calculated using the Henderson–Hasselbalch equation. We assumed that the sum of the concentrations of the compounds was equal to 24mM and 86mM and we assumed a pKa of 3.86 and 6.8 for lactic acid and inorganic phosphate, respectively.

1. We compared the pressor and cardioaccelerator responses to injection acidic saline or 24 mM lactate at two different pHs, namely 2.66 and 6.0. In addition we compared these responses to injection of acidic saline and 86 mM inorganic phosphate at pH of 6.0. We made no attempt to inject inorganic phosphate at a pH of 2.66 because it is unlikely that these conditions happen during exercise.

2. We determined the role played by pH in the pressor and cardioaccelerator responses to injection of 24 mM lactate four different pHs, namely 2.66, 4.5, 5.0, or 6.0. We made no attempt to inject lactate under mild pH condition (> 6.0) because no significant pressor response was found at a pH of 6.0.

3. We determined the role played by pH in the pressor and cardioaccelerator responses to injection of 86mM inorganic phosphate at four different pHs, namely 6.0, 6.5, 7.0, and 7.5.

4. We determined the pressor and cardioaccelerator responses to injections of equal volumes (i.e., 100 μL) of 24 mM lactate at pH of 2.66 or 6.0, 86 mM inorganic phosphate at a pH of 6.0 or 86mM inorganic phosphate combined with 24mM lactate at a pH of 6.0.

5. We determined the role played by pH in the pressor and cardioaccelerator responses to injection of 86mM inorganic phosphate combined with 24 mM lactate at four different pHs, namely 6.0, 6.5, 7.0, and 7.5.

The order of injections was randomized. In an attempt to limit animal usage, two experiments were sometimes performed on a single rat. When two experiments were performed on the same rat, the second experiment started once all the injections of the first experiment were completed. Experiment 1 and 2 were performed on the same rat on four occasions. Experiment 2 and 4 were performed on the same rats on six occasions. Experiment 3 and 4 were performed on the same rat on three occasions. Experiment 3 and 5 were performed on the same rat on five occasions. Experiment 4 and 5 were performed on the same rat on two occasions.

We made the assumption that the concentration of lactate (24 mM) and inorganic phosphate (86mM) injected into the superficial epigastric artery would be diluted in the triceps surae muscles, whose volume was approximately ~3 mL. If 100 % of the injectate accessed the triceps surae muscles, the concentration of inorganic phosphate at the muscle level would be ~3 mM, whereas concentration of lactate at the muscle level would be ~1 mM, both of which are lower than their respective concentrations measured during exhausting exercise using muscle biopsies or ³¹P-MRS (Sahlin et al., 1976; Bangsbo et al., 1993; Broxterman et al., 2017).

To verify that injections into the superficial epigastric artery accessed the triceps surae muscles, we injected 0.2 mL of Evans Blue dye into the superficial epigastric artery in each rat tested. We considered that the solution entered into the triceps surae muscles when the belly of the triceps surae muscles was stained blue. If the color of the triceps surae muscles did not change, we excluded the data from the study.

Drug preparation

Acidic saline at a pH of either 2.66 or 6.0 were made by adding HCl to 0.9 % saline or 10 mM HEPES, respectively. The lactate solutions were made by dissolving lactic acid (SigmaAldrich) in 0.9 % saline. The pH of 24 mM lactic acid solution was 2.66. The pH of the solution was increased to 4.5, 5.0 and 6.0 by adding NaOH. The solution of inorganic phosphate was made by dissolving equimolar concentrations of Na₂HPO₄ with NaH₂PO₄ in 10 mM HEPES. When lactate was combined with inorganic phosphate, crystals of lactic acid were dissolved directly in the solution of inorganic phosphate. The pH of the solution of inorganic phosphate with or without lactate was adjusted to 6.0, 6.5, 7.0 or 7.5 by adding HCl or NaOH. The concentrations of the different acid/base couple based on the pH of the solution are presented Table 1. Because lactate is predominant at pH above 3.86, which is its pK, the term “lactic acid solution” will later be used to designate solutions at a pH of 2.66 and the term “lactate solution” will be used to designate solutions at a pH greater than 4.5.

Data Analysis

The arterial blood pressure signal was amplified using Gould Universal amplifier (Gould-Statham Instruments Inc., CA, USA), displayed and recorded at 1 kHz using an A/D converter (Micro1401 mkII, Cambridge Electronic Design Limited, Cambridge, England) and its associated commercially available software (Spike2, 7.20, RRID: SCR_000903, Cambridge Electronic Design Limited, Cambridge, England). Heart rate was determined beat-by-beat from the arterial pressure pulse signal and expressed as beats per minute. For each compound injected, the peak increase in blood pressure was calculated. When no discernable pressor response was visually detectable, the peak pressor response was determined during the 30s that followed the injection.

Statistical analysis

Data are presented as the mean ± SD. Using a Kolmogorov-Smirnoff test, we verified that our samples respected a normal distribution. For each experiment, the pre- to post-injection change in peak pressor response and peak heart rate was evaluated using two-way repeated measures ANOVA. The difference in the pressor and heart rate response to the compounds injected was evaluated using one-way repeated measures ANOVA. The level of significance was set at P < 0.05. When statistical difference was found, post-hoc multiple-ccomparisons were performed using Tukey’s honestly significant difference test.. Effect size was calculated using partial η-squared (η²). An η² for effect size was considered as small, medium or large when η² was close to 0.02, 0.13 or 0.26, respectively (Cohen, 1977). When individual data are not presented, effect size was also calculated using 95% confidence intervals (Curran-Everett, 2009). Confidence intervals are presented as the lower and upper limit of the interval that should, if this experiment is repeated, contain 95% of the time the true value of the treated effect (Curran-Everett, 2009). Statistical analyses were conducted using Statistica 8.0 (RRID: SCR_014213, StatSoft Inc., Tulsa, OK, US).

Results

Experiment 1

At a pH of 2.66, injection of acidic saline (0.1 mL) or lactic acid (24 mM, 0.1 mL) into the arterial supply of the hindlimb evoked significant pressor and cardioaccelerator responses. The pressor response evoked by injection of lactic acid was greater than the pressor response evoked by acidic saline. In contrast, at a pH of 6.0, injection of acidic saline or lactic acid did not evoke significant pressor and cardioaccelerator responses whereas injection of inorganic phosphate did (86 mM, 0.1 mL, Tables 2–3, Fig. 1).

Table 2.

Pressor response to intraarterial injection of acidic saline, lactate or inorganic phosphate.

Experiment	Stimulus	pH	Baseline	Peak	95% CI	P value
1 (n = 6)	Saline	2.66	111 ± 12	119 ± 13	[3 12]	P = 0.04
		6.0	109 ± 18	111 ± 16	[−1 6]	P = 0.99
	Lactic acid	2.66	113 ± 13	163 ± 21	[28 71]	P < 0.001
		6.0	115 ± 15	118 ± 14	[1 7]	P = 0.99
	Pi	6.0	110 ± 15	138 ± 27	[11 45]	P < 0.001
2 (n = 10)	Lactic acid	2.66	100 ± 24	152 ± 26	[39 66]	P < 0.001
		4.5	92 ± 22	103 ± 21	[8 14]	P = 0.014
		5.0	94 ± 28	99 ± 28	[3 7]	P = 0.66
		6.0	101 ± 21	105 ± 20	[3 4]	P = 0.94
3 (n = 8)	Pi	6.0	110 ± 17	139 ± 29	[16 43]	P < 0.001
		6.5	103 ± 15	120 ± 24	[7 28]	P < 0.001
		7.0	106 ± 22	115 ± 29	[2 17]	P = 0.053
		7.5	105 ± 20	110 ± 20	[2 8]	P = 0.60
4 (n = 13)	Lactic acid	2.66	100 ± 18	145 ± 28	[34 57]	P < 0.001
		6.0	98 ± 22	101 ± 21	[2 5]	P = 0.96
	Pi	6.0	100 ± 17	123 ± 18	[17 30]	P < 0.001
	Pi + Lactic acid	6.0	96 ± 21	135 ± 25	[28 50]	P < 0.001
5 (n = 8)	Pi + Lactic acid	6.0	111 ± 22	150 ± 32	[16 61]	P < 0.001
		6.5	101 ± 26	124 ± 24	[9 37]	P = 0.004
		7.0	108 ± 28	117 ± 30	[4 14]	P = 0.57
		7.5	103 ± 24	109 ± 25	[3 9]	P = 0.94

Results are presented as mean ± SD. 95% confidence interval is presented as the lower and upper boundary of the interval containing the true value of the effect of stimulus on mean arterial pressure. Mean arterial pressure is expressed in mmHg. P values were calculated using post hoc Tukey’s HSD. A significant ANOVA interaction effect was found for every experiment (P < 0.05, η² > 0.36). 95% CI, 95% confidence interval

Table 3.

Cardioaccelerator response to intraarterial injection of acidic saline, lactic acid or inorganic phosphate.

Experiment	Stimulus	pH	Baseline	Peak	95% CI	P value
1 (n = 6)	Saline	2.66	382 ± 46	385 ± 65	[1 4]	P = 0.43
		6.0	380 ± 69	382 ± 69	[0 3]	P = 0.89
	Lactic acid	2.66	388 ± 63	396 ± 64	[0 2]	P < 0.001
		6.0	377 ± 72	378 ± 72	[0 2]	P = 0.97
	Pi	6.0	379 ± 73	385 ± 75	[3 10]	P < 0.001
2 (n = 10)	Lactic acid	2.66	358 ± 50	364 ± 52	[3 10]	P < 0.001
		4.5	356 ± 40	359 ± 40	[2 4]	P = 0.004
		5.0	349 ± 51	351 ± 51	[1 3]	P = 0.16
		6.0	361 ± 69	362 ± 69	[1 2]	P = 0.72
3 (n = 8)	Pi	6.0	350 ± 73	354 ± 73	[1 7]	P = 0.002
		6.5	363 ± 67	365 ± 67	[1 3]	P = 0.024
		7.0	365 ± 62	367 ± 63	[0 3]	P = 0.31
		7.5	363 ± 65	365 ± 64	[0 3]	P = 0.32
4 (n = 13)	Lactic acid	2.66	377 ± 79	384 ± 81	[4 10]	P < 0.001
		6.0	364 ± 82	366 ± 81	[1 2]	P = 0.86
	Pi	6.0	364 ± 78	367 ± 77	[1 5]	P = 0.016
	Pi + Lactic acid	6.0	362 ± 66	368 ± 67	[4 8]	P < 0.001
5 (n = 8)	Pi + Lactic acid	6.0	366 ± 68	370 ± 68	[2 6]	P < 0.001
		6.5	374 ± 61	377 ± 60	[1 4]	P = 0.003
		7.0	375 ± 55	378 ± 54	[0 2]	P = 0.005
		7.5	376 ± 57	378 ± 56	[0 2]	P = 0.44

Results are presented as mean ± SD. 95% confidence interval is presented as the lower and upper boundary of the interval containing the true value of the effect of stimulus on heart rate. Heart rate is expressed in beats.min⁻¹. P values were calculated using post hoc Tukey’s HSD. A significant ANOVA interaction effect was found for every experiment (P < 0.05, η² > 0.36). 95% CI, 95% confidence interval

Figure 1. The pressor responses to injections of acidic saline, lactate and inorganic phosphate solutions into the arterial circulation of the hindlimb.

Data are presented as individual (black dots, n = 6) and group mean (grey bars) for the peak increase in mean arterial blood pressure (MAP) resulting from an injection of acidic saline (pH 6.0 or 2.66), lactate (LA, 24mM, pH 6.0 or 2.66) or inorganic phosphate (Pi, 86mM, pH 6.0) into the superficial epigastric artery. *, P < 0.05 compared to LA at pH of 2.66; ***, P < 0.001 compared to LA at pH of 2.66; †, P < 0.05 compared to Pi at a pH of 6.0; ††, P < 0.01 compared to Pi at a pH of 6.0.

Experiment 2 and 3

Similar to experiment 1, injection of lactic acid at a pH of 2.66 or inorganic phosphate at a pH of 6.0 evoked significant pressor and cardioaccelerator responses (Table 2–3). When the pH of lactic acid or inorganic phosphate solutions was increased, the pressor responses evoked by their injection significantly decreased until not differing from baseline when the pH was greater than 5.0 or 7.0, respectively (Fig. 2 and 3).

Figure 2. Increasing the pH of the solution decreased the pressor response to injections of lactate solutions into the arterial circulation of the hindlimb.

Data are presented as individual (black dots) and group mean (grey bars) for the peak increase in mean arterial blood pressure (MAP) resulting from an injection of lactate (24mM, n = 10) at different pH. ***, P < 0.001 compared to lactate at a pH of 2.66.

Figure 3. Increasing the pH of the solution decreased the pressor response to injections of inorganic phosphate solutions into the arterial circulation of the hindlimb.

Data are presented as individual (black dots) and group mean (grey bars) for the peak increase in mean arterial blood pressure (MAP) resulting from an injection of inorganic phosphate (Pi, 86mM, n = 8) at different pH. *, P < 0.05 compared to Pi at a pH of 6.0; ***, P < 0.001 compared to Pi at a pH of 6.0; †, P < 0.05 compared to Pi at a pH of 6.5.

Experiment 4

Injecting inorganic phosphate combined with lactate at a pH of 6.0 (0.1 mL) evoked a significant pressor and cardioaccelerator response (Table 2–3) that was greater than the pressor response evoked by lactate or inorganic phosphate injected alone at the same pH (6.0). Moreover, the pressor response to injection of a combined lactate and inorganic phosphate solution at a pH of 6.0 was significantly greater than the arithmetic sum of the pressor responses to injection of lactate or inorganic phosphate at a pH of 6.0. No difference was found between the pressor response of the combined injection of inorganic phosphate and lactate at a pH of 6.0 and the pressor response to lactic acid injected alone at a pH of 2.66 (Fig. 4).

Figure 4. Inorganic phosphate combined with lactate potentiated the pressor response to injection of inorganic phosphate or lactate alone.

Data are presented as individual (black dots, n = 13) and group mean (grey bars) for the peak increase in mean arterial blood pressure (MAP) resulting from an injection of lactate (LA, 24mM, pH 2.66 or 6.0), inorganic phosphate (Pi, 86mM, pH 6.0) or lactate combined with inorganic phosphate (Pi/LA, pH 6.0). Sum (LA + Pi), artithmetic sum of the peak pressor responses evoked by separated injections of LA and Pi; ***, P < 0.001 compared to lactate at a pH of 2.66; †, P < 0.05 compared to Pi at a pH of 6.0; ††, P < 0.01 compared to Pi at a pH of 6.0; †††, P < 0.001 compared to Pi at a pH of 6.0; ##, P < 0.01 compared to lactate combined with Pi at a pH of 6.0.

Experiment 5

Similar to experiment 2 and 3, when the pH of a solution containing inorganic phosphate with lactate was increased, the pressor response to injection decreased until being not different from baseline at a pH greater than 7.0 (Fig. 5, Table 2–3). Quantitatively, the pressor response of inorganic phosphate combined with lactate was comparable to the pressor response to inorganic phosphate alone at a pH greater than 7.0 (Fig. 6).

Figure 5. Increasing the pH of the solution decreased the pressor response to injections of lactate combined with inorganic phosphate solutions into the arterial circulation of the hindlimb.

Data are presented as individual (black dots) and group mean (grey bars) for the peak increase in mean arterial blood pressure (MAP) resulting from an injection of lactate (24mM) combined with inorganic phosphate (Pi, 86mM; n = 7) at different pH. **, P < 0.001 compared to lactate combined with Pi at a pH of 6.0; ***, P < 0.001 compared to lactate combined with Pi at a pH of 6.0.

Figure 6. Summary of the averaged pressor responses measured during the different experiments.

Data are presented as mean ± SE for the peak increase collected during experiment 1, 2, 3 and 5. For all injections, we injected 100μL of solutions. The concentration of lactate solutions was 24mM and the concentration of Pi was 86mM. Pi, inorganic phosphate; MAP, mean arterial pressure.

Discussion

We found that injection of inorganic phosphate combined with lactate at an acidic pH evoked a pressor response that was potentiated compared to the pressor response evoked by each metabolite injected individually. It is important to state that the pH and volume of solutions used in this comparison were identical to each other. The potentiating effect was dependent on the pH of the solution injected into the arterial supply of the hindlimb. In addition, our data showed that inorganic phosphate had a larger potentiating effect than did lactate.

The fact that an injection of inorganic phosphate combined with lactate at an acidic pH produced a greater pressor response than the pressor response of each ion injected individually is the first in vivo evidence showing that a combination of metabolites is more potent than a single metabolite to increase reflexively blood pressure. These results echoed previous works on in vitro or ex vivo single afferent recording showing that within the physiological range afferent neurons respond to a combination of several metabolites but not to one (Light et al., 2008; Jankowski et al., 2013). The pressor response evoked by the combined metabolites being greater than the arithmetic sum of the pressor response evoked by the compounds injected separately demonstrate that the effect was synergistic and not additive.

Our data suggest that neither inorganic phosphate nor lactate by themselves can act as direct agonists of afferent receptors involved in the metabolic component of the exercise pressor reflex. Indeed, the injection of inorganic phosphate or lactate at a pH greater than 7.0 or 6.0, respectively, did not evoke a significant increase in blood pressure (Fig. 2–3). These results support findings showing that extremely high, non-physiologic, concentrations of inorganic phosphate or lactate at neutral pH did not activate group III-IV afferent fibers (Kniffki et al., 1978; Rotto & Kaufman, 1988; Hoheisel et al., 2004). In contrast, decreasing the pH (i.e. increasing the concentration of H⁺) of a saline solution from 6.0 to 2.66 evoked a pressor response that was modest but significant. These results suggest that the agonist compound to the afferent receptors was H⁺ ions, and that inorganic phosphate and lactate ions acted as potentiating agents. Our experiments do not provide mechanistic evidence regarding how inorganic phosphate and lactate act synergistically on afferent receptors to potentiate the resulting pressor response, but inorganic phosphate and lactate together could sensitize acid-sensing ion channels to pH by acting as chelating agents that remove Ca²⁺ ions blocking the access of H+ ions to the channel (Immke & McCleskey, 2001; 2003).

Given that the pressor response to an injection of inorganic phosphate at a pH of 6.0 evoked a greater pressor response than did injection of lactate at the same pH, our result suggest that inorganic phosphate is more efficient than lactate to sensitize the afferent neurons to pH. We are not aware of any evidence explaining the greater efficacy of inorganic phospahte to evoke a greater pressor response than lactate, but it is possible that ${\text{H}}_2{\text{PO}}_4^{-}$ and ${\text{HPO}}_4^{2-}$ (i.e. the diprotonated or monoprotonated form of inorganic phosphate ions) are more efficient as chelating agents than is lactate, ${\text{HPO}}_4^{2-}$ having the possibility to bind one Ca²⁺ ion, whereas two lactate ions are needed to bind one Ca²⁺ ion.

Similar to the pressor response to injections of lactate at different pH, the pressor response to an injection of inorganic phosphate combined with lactate decreased as the pH of the solution increased (Fig. 5). Because at a pH greater than 7.0, the pressor response to an injection of inorganic phosphate alone or combined with lactate was not significantly different than baseline, our results suggest that the potentiating effect of lactate and inorganic phosphate was lost. In addition, our results suggest that a combination of inorganic phosphate and lactate are not sufficient to evoke the metabolic component of the exercise pressor reflex at a pH greater than 7.0. This result is key because the pH of the interstitial space at rest is ~7.4 (Street et al., 2001). Therefore, early in the exercise when inorganic phosphate and lactate concentrations increase greatly but pH change is limited (Karlsson & Saltin, 1970; Broxterman et al., 2017), other mechanisms or metabolites need to compensate for the lack of effectiveness of inorganic phosphate and lactate to produce a pressor response. This indirectly supports recent results from our laboratory showing that the mechanisms responsible for evoking the exercise pressor reflex are redundant and arise from a wide variety of receptors (Stone et al., 2015). Among the different mechanisms possible, activation of P2X receptors due to ATP release early in the exercise appear to be a good candidate (Hanna & Kaufman, 2004; Hayes et al., 2008).

Methodological consideration and limits

Several methodological considerations must be taken into account when considering our findings. First, the concentrations of inorganic phosphate injected was ~3.5 times greater than the concentration of lactate. This difference raises the possibility that the greater efficacy of inorganic phosphate to produce a pressor response at a pH of 6.0 might due to the presence of greater amount of sensitizing agents. However, Sinoway et al. (1994) found in cats that injecting 50 mM of inorganic phosphate produced a greater pressor response than 50 mM of lactate at a pH of 6.0.

Second, we calculated the concentrations of the compound injected assuming that the injectate concentration at the interstitial space level would have been diluted in the hindlimb muscles. For example, Gao et al. (2007) found that infusing Ringer solution at a pH ranging from 6.5 to 4.5 into the arterial supply of the triceps surae muscles changed the interstitial pH from 7.5 to 7.2 and 6.6, respectively. However, one might argue that the concentration at the muscle level remained non-physiologic despite this diluting effect. This cannot be verified and measuring the change in concentrations at the interstitial space level would not have been possible using microdialysis technique because the compounds were injected as a bolus. Nevertheless, whether the concentrations at the interstitial space level were physiologically representative or not, changing the concentration of the compounds would affect the magnitude of the pressor response, but not the way the compounds interact with each other. The conclusion that a combination of inorganic phosphate and lactate at a pH of 6.0 produced a greater pressor response than the pressor response of each metabolite injected individually remained therefore valid.

Finally, if inorganic phosphate and lactate are among the metabolites which concentrations increase the most during intense exercise, they are not the only metabolites produced by the contracting muscles that could sensitize the afferent arm of the exercise pressor reflex. For example, findings showed that bradykinin, arachidonic acid by-products, and ATP produce a pressor response when injected into the arterial supply of the muscles (Stebbins & Longhurst, 1985; Koba et al., 2011; Stone et al., 2014). Further experiments are therefore warranted to determine if together these secondary by-products can interact with other metabolites and potentiate the exercise pressor reflex.

Perspective and significance

The present experiment demonstrated that a combination of metabolites injected into the arterial supply of the triceps surae muscles evoked a greater pressor response than did of each metabolite injected individually. This pressor response is driven by the concentration of protons while inorganic phosphate and lactate provide a potentiating effect, the former being the more potent than the latter. Further studies, using for example patch-clamp experiments, are warranted to better understand the synergistic mechanisms by which lactate and inorganic phosphate sensitize the afferent receptors.

New findings.

What is the central question of this study?

What is the contribution of the main acidic compounds accumulated during contractions, namely H+, lactic acid and inorganic phosphate to evoke the metabolic component of the exercise pressor reflex?

What is the main finding and its importance?

We found that the pressor response to acidic stimuli is driven by the concentration of hydrogen ions and that lactate and inorganic phosphate acted as potentiating agents.