Cross-sensitization of histamine-independent itch in mouse primary sensory neurons

Abstract

Overexpression of pruritogens and their precursors may contribute to the sensitization of histamine-dependent and -independent itch-signaling pathways in chronic itch. We presently investigated self- and cross-sensitization of scratching behavior elicited by various pruritogens, and their effects on primary sensory neurons. The MrgprC11 agonist BAM8-22 exhibited self- and reciprocal cross-sensitization of scratching evoked by the protease-activated receptor-2 (PAR-2) agonist SLIGRL. The MrgprA3 agonist chloroquine unidirectionally cross-sensitized BAM8-22-evoked scratching. Histamine unidirectionally cross-sensitized scratching evoked by chloroquine and BAM8-22. SLIGRL unidirectionally cross-sensitized scratching evoked by chloroquine. Dorsal root ganglion (DRG) cells responded to various combinations of pruritogens and algogens. Neither chloroquine, BAM8-22 nor histamine had any effect on responses of DRG cell responses to subsequently applied pruritogens, implying that their behavioral self- and cross-sensitization effects are mediated indirectly. SLIGRL unilaterally cross-sensitized responses of DRG cells to chloroquine and BAM8-22, consistent with the behavioral data. These results indicate that unidirectional cross-sensitization of histamine-independent itch-signaling pathways might occur at a peripheral site through PAR-2. PAR-2 expressed in pruriceptive nerve endings is a potential target to reduce sensitization associated with chronic itch.

Fig. 1

Time course of self- and cross-sensitization of pruritogen-evoked scratching behavior. A. Mean number of scratch bouts/5-min after chloroquine alone (none; ○) or when preceded by chloroquine (●) or BAM8–22 (▲).Error bars: SEM. n=6–24/group. B. As in A; chloroquine preceded by histamine (■) or PAR-2 agonist (solid diamonds). *, significantly different from chloroquine alone (p < 0.05, two-way repeated-measures ANOVA, Student-Newman-Keuls test, F (47, 336) =3.57). C. Histamine alone (○) or preceded by chloroquine (●) or BAM8–22 (▲). Note the different scale for the y-axis. D. BAM8–22 alone (none; ○) or preceded by chloroquine (●) or BAM8–22 (▲). *, significantly different from BAM8–22 alone (p < 0.0001, F (47, 336) =10). E. BAM8–22 preceded by histamine (■) or PAR-2 agonist (solid diamonds). *, significantly different from first trial of BAM8–22 (p < 0.0001, , F (47, 336) =10). F. PAR-2 agonist alone (none; ○) or when preceded by chloroquine (●) or BAM8–22 (▲). *, significantly different from PAR-2 agonist alone (p < 0.05, F (23, 168) =4.95). Note the different scale for the y-axis.

Fig. 2

Summary of self- and cross-sensitization of pruritogen-evoked scratching behavior. A. Mean number of scratch bouts/40 min elicited by chloroquine alone (none; open bar) and when preceded by chloroquine (black bar), BAM8–22 (dark gray bar), histamine (light gray bar) or PAR-2 agonist (hatched bar). Error bars: SEM. *, significantly different from first trial of chloroquine(p < 0.05, One-way ANOVA, Bonferroni post-test, n=6–24/group). B. As in A for histamine. Note the different scale for the y-axis here and in C, D. C. As in A for BAM8–22. *, significantly different from BAM8–22 alone (p < 0.05, n=6/group). D. As in A for PAR-2 agonist. *, significantly different from first trial of PAR-2 agonist (p < 0.05, n=6/group).

Fig. 3

Pruritogen activation of DRG cells. A: Graph plots 340/380 nm ratio as a function of time. Black bars indicate time of application of each indicated chemical. One cell (blue trace; encircled in blue in B-F) responded to CQ twice followed by His. Other cells (orange, black and magenta traces; encircled in white in B-F) responded to His, but not CQ. All cells responded high K+ (HiK). B-F: Fluorescence images of DRG cells. Scale bar in B is 50 µm and applies to B-F. B: Before application. C: post first application of chloroquine (CQ; 300 µM). D: post second application of CQ. E: post- histamine (His; 100 µM). F: post application of high potassium solution (K+).

Fig. 4

Summary of self- and cross-sensitization of pruritogen-evoked responses in DRG cells. A: Peak ratio was normalized to baseline. Bar graph plots mean increase in peak response from baseline of DRG cells elicited by chloroquine when it was applied first (open bar) and when it was applied 3 min after a prior application of chloroquine (black bar), BAM8–22 (dark gray bar), histamine (light gray bar) or the PAR-2 agonist (dotted bar). Error bars: SEM. *, significantly different from chloroquine alone (none, p < 0.05, One-way ANOVA, Bonferroni post-test, n=46–189/group). B: As in A for histamine. (n= 45–173/group). Error bars: SEM. C: As in A for BAM8–22. *, significantly different from BAM8–22 alone (p < 0.05, One-way ANOVA, Bonferroni post-test, n=13–87/group). Note the different scale for the y-axis here and in D. D: As in A for the PAR-2 agonist (n=13–24/group).

Fig. 5

Proportion of DRG cells responsive to various pruritogens. Each pie chart shows the percentages of DRG cells that responded to either or both of the first two pruritogens tested in a given experimental sequence. A: Chloroquine (CQ; 300 µM) followed by BAM8–22 (BAM; 10 µM). + indicates that the cell responded and − indicates lack of response. The number in each slice is the number of DRG cells in the corresponding group (percentage in parentheses). B: Chloroquine (CQ) followed by histamine (His; 100 µM). C: CQ followed by PAR-2 agonist (PAR2; 100 µM). D: As in BAM8–22 (BAM; 10 µM) followed by histamine (His; 100 µM). E: BAM followed by PAR-2 agonist (PAR2; 100 µM).

Fig. 6

Proportion of DRG cells responsive to various pruritogens and algogens. Each pie chart shows the percentages of DRG cells that responded to either or both of pruritogens and algogens tested in a given experimental sequence. A, B, C and D: Chloroquine (CQ; 300 µM), BAM8–22 (BAM; 10 µM), histamine (His; 100 µM) or PAR-2 agonist (PAR2; 100 µM) followed by capsaicin (CAP; 1 µM), respectively. + indicates that the cell responded and – indicates lack of response. The number in each slice is the number of DRG cells in the corresponding group (percentage in parentheses). E, F, G and H: As in A for CQ, BAM, His or PAR2 followed by allyl isothiocyanate (AITC; 100 µM), respectively.