Mechanisms of itch evoked by β-alanine

Abstract

β-Alanine, a popular supplement for muscle building, induces itch and tingling after consumption, but the underlying molecular and neural mechanisms are obscure. Here we show that, in mice, β-alanine elicited itch-associated behavior that requires MrgprD, a G-protein-coupled receptor expressed by a subpopulation of primary sensory neurons. These neurons exclusively innervate the skin, respond to β-alanine, heat, and mechanical noxious stimuli but do not respond to histamine. In humans, intradermally injected β-alanine induced itch but neither wheal nor flare, suggesting that the itch was not mediated by histamine. Thus, the primary sensory neurons responsive to β-alanine are likely part of a histamine-independent itch neural circuit and a target for treating clinical itch that is unrelieved by anti-histamines.

Figure 1.

Intradermal injection of β-alanine evokes itch in humans. A, Time course of the mean perceived intensity of itch and nociceptive sensations evoked by intradermal injection of 90 μg (100 mm) of β-alanine or l-alanine. The mean ratings of itch, pricking/stinging, and burning obtained from 11 subjects are plotted for successive intervals of 30 s after injection. Beginning with the peak rating of each sensory quality, the SEM is plotted every 5 min (error bars). The positions of three verbal descriptors (vertical scale on the right) are shown in correspondence with the ratings of mean perceived intensity (y-axes). B, Time course of the mean perceived intensity of itch and nociceptive sensations evoked by different doses of β-alanine and l-alanine in the same subjects.

Figure 2.

MrgprD is required for β-alanine-induced scratching in mice. A, Cheek injection of β-alanine induced site-directed scratching in WT but not MrgprD^−/− mice. β-alanine induced significant scratching in WT mice during the first 15 min after injection in a dose-dependent manner (50 mm, n = 6; 20 mm, n = 5; 10 mm, n = 5). In contrast, few scratching bouts were detected in MrgprD^−/− mice after β-alanine cheek injection (n = 5). l-alanine, the proteinogenic α-amino acid, did not induce a significant itch response (WT, n = 6; KO, n = 8). B, Orally administering β-alanine induced itch in WT but not MrgprD^−/− (KO) mice. The total number of scratching bouts during the 40 min after β-alanine feeding (40 mg/kg body weight) was significantly higher in WT mice than in MrgprD^−/− mice (n = 5 per genotype; *WT vs KO). All error bars represent SEM. *p < 0.05, **p < 0.01; Student's t test.

Figure 3.

The response of DRG neurons to β-alanine is MrgprD-dependent. A, Approximately 12% of WT DRG neurons responded to β-alanine (β-ala; 1 mm) with increased [Ca²⁺]_i, whereas MrgprD^−/− DRG neurons did not respond (number of neurons analyzed: WT, n = 282; KO, n = 212, n = 3 mice per genotype). The response to histamine was not impaired in MrgprD^−/− neurons. The percentage of MrgprD^−/− neurons responding to histamine (100 μm) was similar to that of WT neurons (>300 neurons analyzed, 3–4 mice per genotype). Error bars represent SEM. B, Representative traces of DRG neurons from MrgprD^GFP/+ mice in calcium imaging assays. β-alanine only activated GFP⁺ neurons as monitored by increased [Ca²⁺]_i with calcium imaging. β-alanine-sensitive neurons (black, green, and blue traces; n = 91) did not respond to histamine (His; 100 μm) or CQ (1 mm), and histamine- and CQ-sensitive neurons (yellow, red, and orange traces) did not respond to β-alanine (>200 neurons from 3 mice). C, β-alanine (BA; 1 mm) induced action potentials in DRG neurons. In WT DRG, β-alanine-sensitive neurons (as determined by calcium imaging, n = 8) fired action potentials upon repeated β-alanine treatment. In contrast, none of the neurons tested from MrgprD^−/− mice showed any response to the drug (n = 7).

Figure 4.

β-alanine-sensitive neurons are C-mechanoheat nociceptive and do not respond to histamine. A, Bright-field image of a neuronal recording (arrow) with an extracellular electrode (outlined with dashed blue lines). B, Fluorescent microscopy revealed the expression of GFP (i.e., MrgprD⁺) in this neuron. C, Location of the cutaneous receptive field (red dot) of this neuron on the hairy skin of the hindpaw and conduction velocity (0.64 m/s, lower trace) obtained with electrical stimulation (arrow) of the receptive field. D, Responses of this neuron to a 60 mN force via a 200 μm diameter probe applied to the receptive field (1 s) with original extracellular recording trace (Ie) and action potentials (AP) indicated below. E, Response to heat stimulation (38–51°C, 5 s) indicates that this is a CMH nociceptive neuron. F, Responses of the above CMH neuron to the intradermal injection of vehicle (VEH), histamine (HIS), and β-alanine (BAL).