Roles for substance P and gastrin-releasing peptide as neurotransmitters released by primary afferent pruriceptors

Abstract

Recent studies support roles for neurokinin-1 (NK-1) and gastrin-releasing peptide (GRP) receptor-expressing spinal neurons in itch. We presently investigated expression of substance P (SP) and GRP in pruritogen-responsive primary sensory neurons and roles for these neuropeptides in itch signaling. Responses of dorsal root ganglion (DRG) cells to various pruritogens were observed by calcium imaging. DRG cells were then processed for SP, GRP, and isolectin B-4 (IB4; a marker for nonpeptidergic neurons) immunofluorescence. Of pruritogen-responsive DRG cells, 11.8-26.8%, 21.8-40.0%, and 21.4-26.8% were immunopositive for SP, GRP, and IB4, respectively. In behavioral studies, both systemic and intrathecal administration of a NK-1 receptor antagonist significantly attenuated scratching evoked by chloroquine and a protease-activated receptor 2 agonist, SLIGRL, but not histamine, bovine adrenal medulla peptide 8-22 (BAM8-22), or serotonin. Systemic or intrathecal administration of a GRP receptor antagonist attenuated scratching evoked by chloroquine and SLIGRL but not BAM8-22 or histamine. The GRP receptor antagonist enhanced scratching evoked by serotonin. These results indicate that SP and GRP expressed in primary sensory neurons are partially involved as neurotransmitters in histamine-independent itch signaling from the skin to the spinal cord.

Fig. 1.

Prutirogen-responsive dorsal root ganglion (DRG) cells double-labeled for substance P (SP), gastrin-releasing peptide (GRP), or isolectin B-4 (IB4) immunoreactivity. A: fluorscence microscopic image of calcium response following application of chloroquine (CQ; encircled DRG cell). Scale bar applies to A and B. B: same cell in A double-labeled for SP. C: example of DRG cells labeled for SP. D: example of DRG cells labeled for GRP. E: example of DRG cells labeled for IB4. Scale bar applies to C–E. F: summary of percentages of CQ-responsive DRG cells (assessed by calcium imaging) that were double labeled for SP (black bar), GRP (open bar), or IB4 (striped bar; n = 14–83/group). G: bovine adrenal medulla peptide 8–22 [BAM8-22 (BAM)]-sensitive cells (n = 15–44/group). H: histamine (His)-sensitive cells (n = 11–87/group). I: protease-activated receptor 2 agonist SLIGRL-NH2-sensitive cells (n = 10–29/group).

Fig. 2.

Time course of pruritogen-elicited scratching. A: mean number of scratch bouts/5 min over the 30-min period following intradermal (id) pruritogen injection preceded by systemic injection of vehicle (saline; open circles), neurokinin-1 (NK-1) receptor antagonist L-733060 (10 mg/kg; filled circles), or GRP receptor antagonist RC-3095 (0.3 mg/kg; filled triangles). Error bars: SE; n = 6/group. B: as in A for BAM8-22. C: as in A for histamine. D: as in A for SLIGRL-NH2. E: as in A for 5-HT. *Significantly different from vehicle group; P < 0.05, post hoc Student-Newman-Keuls test, following 2-way repeated-measures ANOVA; F(10, 97) = 7.29; n = 6/group.

Fig. 3.

Summary of systemic effects of NK-1 and GRP receptor antagonist on pruritogen-elicited scratching. A: bar graph plots, from left to right, the mean number of scratch bouts/30 min elicited by id microinjection of CQ, 30 min after prior systemic injection of vehicle (Veh; saline; open bar), NK-1 antagonist L-733060 (10 mg/kg; filled bar), or GRP antagonist RC-3095 (0.3 mg/kg; striped bar). Error bars: SE. *Significantly different from vehicle group; P < 0.05, 1-way ANOVA, Bonferroni post-test, n = 6/group. B: as in A for BAM8-22. C: as in A for histamine. D: as in A for SLIGRL-NH2. *Significantly different from vehicle group; P < 0.05, 1-way ANOVA, Bonferroni post-test, n = 6/group. E: as in A for 5-HT. *Significantly different from vehicle group; P < 0.05, 1-way ANOVA, Bonferroni post-test, n = 6/group.

Fig. 4.

Summary of effects of of NK-1 and GRP receptor antagonists on pruritogen-elicited scratching. Antagonists were injected intrathecally in A–D and id (Id) in E. A: bar graph plots, from left to right, the mean number of scratch bouts/30 min elicited by id microinjection of CQ, 30 min after prior intrathecal injection of vehicle (saline; open bar), NK-1 antagonist L-733060 (22.7 nmol/5 μl; filled bar), or GRP antagonist RC-3095 (0.3 nmol/5 μl; striped bar). Error bars: SE. *Significantly different from vehicle group; P < 0.05, 1-way ANOVA, Bonferroni post-test, n = 6/group. B: as in A for BAM8-22. C: as in A for histamine. D: as in A for SLIGRL-NH2. *Significantly different from vehicle group; P < 0.05, 1-way ANOVA, Bonferroni post-test, n = 6/group. E: a mixture of either vehicle and 5-HT (Id Veh + 5-HT; saline; 10 μl; open bar) or 5-HT and GRP receptor antagonist RC-3095 (Id GRP + 5-HT; 0.6 nmol/10 μl, striped bar) was injected id. *Significantly different from vehicle group; P < 0.05, paired t-test, n = 6/group.