BNP facilitates NMB-encoded histaminergic itch via NPRC-NMBR crosstalk

Abstract

Histamine-dependent and -independent itch is conveyed by parallel peripheral neural pathways that express gastrin-releasing peptide (GRP) and neuromedin B (NMB), respectively, to the spinal cord of mice. B-type natriuretic peptide (BNP) has been proposed to transmit both types of itch via its receptor NPRA encoded by Npr1. However, BNP also binds to its cognate receptor, NPRC encoded by Npr3 with equal potency. Moreover, natriuretic peptides (NP) signal through the G_i-couped inhibitory cGMP pathway that is supposed to inhibit neuronal activity, raising the question of how BNP may transmit itch information. Here, we report that Npr3 expression in laminae I-II of the dorsal horn partially overlaps with NMB receptor (NMBR) that transmits histaminergic itch via G_q-couped PLCβ-Ca²⁺ signaling pathway. Functional studies indicate that NPRC is required for itch evoked by histamine but not chloroquine (CQ), a nonhistaminergic pruritogen. Importantly, BNP significantly facilitates scratching behaviors mediated by NMB, but not GRP. Consistently, BNP evoked Ca²⁺ responses in NMBR/NPRC HEK 293 cells and NMBR/NPRC dorsal horn neurons. These results reveal a previously unknown mechanism by which BNP facilitates NMB-encoded itch through a novel NPRC-NMBR cross-signaling in mice. Our studies uncover distinct modes of action for neuropeptides in transmission and modulation of itch in mice.

Keywords: BNP; GRP; NPRA; NPRC; itch; mouse; neuroscience; spinal cord.

Figure 1.. Expression of Npr1, 2,and 3 and other molecular markers in the spinal cord.

(A) Diagram shows crosstalk between NPs and NP receptors. BNP can bind NPRA and NPRC. (B) BNP dose-dependently evoked scratching behaviors 60 min after i.t. injection. n = 6. *p < 0.05, **p < 0.01, one-way ANOVA followed by Tukey’s test. (C) Time-course of scratching behaviors induced by different doses of BNP shows a delayed onset of scratching responses. (D, F, H, J, L, N) Images of double RNAscope ISH showing that the overlapping expression of Npr1 (green) with Grpr (red) (D), Nmbr (F), of Npr3 (green) with Nmbr (red) (H), Npr3 (red) with Vglut2 (green) (J), Vgat (green) (L), or Npr1 (green) (N) in laminae I-II of the dorsal horn. Dashed white lines divide laminae I-II from III. White boxes are shown at higher magnification in the right panel. Arrows indicate double-positive neurons. E, G, I, K, M, O, Venn diagrams showing the overlap between Npr1 and Grpr (E), Nmbr (G), between Npr3 and Nmbr (I), Vglut2 (K), Vgat (M) or Npr1 (O). n = 10–15 sections from 3 mice. Scale bar, 20 µm in D – N.

Figure 1—figure supplement 1.. Failure of ANP and CNP in facilitating histamine itch.

(A) ANP 1 ~ 20 µg, (equivalent to 6–120 µM, i.t.) failed to induce robust scratching behaviors in mice. n = 6. (B) Only BNP (30 µM, i.t.) facilitated histamine itch. Note that neither ANP (60 µM, i.t.) nor CNP (60 µM, i.t.) exhibited facilitatory effect. n = 6–7. *p < 0.05, ***p < 0.001, one-way ANOVA followed by Dunnett’s test. Values are presented as mean ± SEM.

Figure 1—figure supplement 2.. Normal innervation of primary afferents in Npr1 KO mice and WT mice.

(A-D) Comparable expression of CGRP (red) and IB4 staining (green), TRPV1, GRP, and SP in the superficial dorsal horn of WT and Npr1 KO mice. Scale bar, 50 µm. (E) Images of double RNAscope ISH showing that Npr1 (green) is partially co-expressed with Grp (red) in the dorsal horn. Arrows indicate double-positive neurons. Scale bar, 20 µm. (F) Venn diagram showing partial overlapping of Npr1 and Grp expression. (G) Scratching behaviors elicited by i.t. BNP (150 µM) were significantly enhanced by isoflurane. (H) Time course of i.t. NMB (1 nmol) and GRP (1 nmol) evoked scratching behavior. n = 6. **p < 0.01, ***p < 0.001, two-way ANOVA followed by Bonferroni’s test. Values are presented as mean ± SEM. Scale bar, 50 µm in A-D, 20 µm in E.

Figure 2.. NPRA and NPRC are involved in acute itch.

(A) Npr1 KO mice and their WT littermates showed comparable scratching behaviors in response to GRP (0.05 nmol, i.t.) and NMB (0.5 nmol, i.t.). n = 6–8. (B) Npr1 KO mice showed significantly reduced scratching behavior elicited by histamine (200 µg, i.d.) and CQ (200 µg, i.d.). n = 9–11. *p < 0.05, **p < 0.01, unpaired t test. (C, D) Mice treated with Npr1 siRNA showed significantly reduced scratching responses to histamine (C), CQ (D), wherea mice treated with Npr3 siRNA displayed deficits only in histamine (C) but not CQ itch (D). n = 6–7. *p < 0.05, **p < 0.01, one-way ANOVA followed by Dunnett’s test. (E, F) Real-time PCR confirmed the reduced Npr1-3 expression by Npr1, Npr2, and Npr3 siRNA knockdown in the spinal cord (E) and DRG (F). n = 4. **p < 0.01, one-way ANOVA followed by Dunnett’s test. Values are presented as mean ± SEM.

Figure 3.. BNP facilitates histamine itch.

(A) Pre-injection of BNP (30 µM, i.t.) for 1 min significantly enhanced scratching behavior evoked by i.d. injection of histamine (Hist.) (100 µg). n = 6. (B) Scratching behavior evoked by i.d. injection of CQ (50 µg, i.d.) was significantly enhanced by pre-injection of BNP for 1 min. n = 6. (C, D) Co-injection of 1 µg BNP (30 µM, i.t.) facilitated scratching behaviors evoked by NMB (0.05 nmol, i.t.) (C) but not GRP (0.01 nmol) (D). n = 6. (E) Pre-injection of 1 µg BNP (30 µM, i.t.) for 1 min significantly enhanced scratching behavior evoked by i.d. injection of histamine (100 µg) in Grpr KO mice. n = 8. (F, G) Pre-injection of NMB (0.05 nmol, i.t.) had no effect on scratching behavior induced by histamine (F) or CQ (G). Note that NMB barely evoked scratching bouts. n = 6. (H), NPRC agonist ANP-4–23 (6 nmol, i.t.) facilitates NMB (0.005 nmol, i.t.) induced scratching behavior. n = 5–9. (I), Histamine (25 µg, i.d.)-induced scratching behavior facilitated by BNP (30 µM, i.t.) was attenuated with AP 811 (10 µM, i.t.) or U 73122 (13.5 nmol, i.t.) treatment. n = 6–11. (I–K) Double RNAScope ISH images (J and L) and Venn diagrams (K and M) showing 60% of Nppb neurons co-express Nmb (J and K), but little Grp in DRGs (L and M). Values are presented as mean ± SEM, *p < 0.05, **p < 0.01, unpaired t test in (A–E), one-way ANOVA in (F and G). Scale bar, 20 µm in J, 50 µm in L.

Figure 4.. Potentiation of NMB-evoked calcium and scratching responses by BNP requires G_i-G_q crosstalk between NPRC-NMBR.

(A) A diagram showing the procedure for calcium imaging on dissociated spinal cord dorsal horn neurons. (B) Sample traces showing that co-application of BNP and NMB at low doses evoked Ca²⁺ transients in WT dorsal horn neurons (n = 8 neurons from 33 NMBR neurons analyzed, n = 10 pups). These neurons responded to both BNP/NMB at the low doses responded to NMB at 20 nM robustly, indicating that they are healthy neurons. (C) No dorsal horn neurons responded to NMB (20 nM) isolated from the spinal cord of Nmbr KO mice (n = 2 mice), whereas they responded to KCI, indicating that they were healthy neurons. (D) Co-application of BNP (1 µM) with subthreshold of NMB (1 pM) evoked robust calcium response in HEK 293 cells co-expressing NMBR, which was significantly attenuated by Npr3 siRNA treatment. (E) Calcium transients induced by BNP and NMB were attenuated by pre-incubation of PTX (200 ng/ml), gallein or AP 811 (0.1 µM) for 30 min. n = 6 slides per group with at least 50 cells imaged on each slide. (F) Quantification of calcium concentration ([Ca2+]i) of E. (G) I.t. gallein (20 nmol) significantly reduced scratching behavior evoked by histamine (25 µg, i.d.) facilitated with BNP (30 µM, i.t.). Values are presented as mean ± SEM, n = 6–10. *p < 0.05, ***p < 0.001, one-way ANOVA followed by Tukey’s test.

Figure 4—figure supplement 1.. real-time RT-PCR detected endogenous expression of Npr1, Npr2, and Npr3 in HEK 293 cells.

Figure 5.. BNP-sap ablates spinal cord neurons expressing Npr1 and Npr3.

(A-F) RNAscope ISH images (A and C) and quantified data (F) showing that BNP-sap ablated Npr1+ (A), Npr3+ (C), Grp+ (D), and Nmbr+ (E) neurons (red) in the dorsal horn of the spinal cord, while Npr2+ (B) neurons (red) were not affected. n = 4. (G) Incubation of BNP (10 µM) for 30 min caused internalization of Npr1-mCh and Npr3-mCh in HEK 293 cells transfected with NMBR cDNA as indicated by arrows. No internalization of Npr2-mCh was observed. Scale bar, 20 µm. mCh: mCherry. (H, I) Scratching behaviors induced by histamine (H), but not CQ (I) were significantly reduced in BNP-sap treated mice. n = 7–8. Values are presented as mean ± SEM. *p < 0.05, **p < 0.01, unpaired t test. Scale bar, 50 µm in A–F, 10 µm in G.

Figure 6.. SST evoked both pain and itch responses in mice.

(A) Pre-injection of morphine (10 mg/kg, i.p.) for 30 min attenuated scratching behaviors induced by i.t. injection of SST (5 nmol). n = 6 mice per group. Sal, saline; Mor, morphine. (B, C) SST (5 nmol, i.t.)(B) and OCT (C) -evoked scratching behaviors were significantly reduced in bombesin-saporin-treated mice comparing with control mice that were treated with blank saporin. n = 5–6 mice per group. Ctrl, control; BB-sap, bombesin-saporin. (D) Raster plot of scratching behavior induced by light stimulation of skin in Sst-ChR2 and Sst-cre mice. (E) Number of scratches in 5 min induced by 3 s – 1, 5, 10, or 20 Hz light stimulation of nape skin in Sst-ChR2 and Sst-cre mice. n = 8–10 mice. ns – not significant, one-way ANOVA with Tukey post hoc. (F) IHC images of Sst-ChR2/Sst co-expression in DRG of Sst-ChR2 mice (Left). Arrowheads indicate co-expression. Scale bar, 10 µm. Venn diagram showing overlapping expression of Sst-ChR2 and Sst in DRG neurons (Right). (G) IHC images of Sst-ChR2/CGRP/IB4 (left), Sst-ChR2/NF-H (middle), and Sst-ChR2/TRPV1 (right) in DRG of Sst-ChR2 mice. Arrowheads indicate co-expression. (H) IHC image of Sst-ChR2/βIII-Tubulin in hairy nape skin. Dashed line marks epidermal/dermal boundary. Arrowheads indicate ChR2 expression in lanceolate endings of hair follicles. Values are presented as mean ± SEM. *p < 0.05, **p < 0.01, unpaired t test. Scale bars, 10 µm in F, 100 µm in G and H.

Figure 6—figure supplement 1.. BNP-NPRA signaling is dispensable for nonhistaminergic itch and neuropathic itch.

(A) Npr1 KO mice and WT littermates showed comparable spontaneous scratching behaviors in the dry skin model. n = 6, p = 0.1283, F1,50 = 2.392, repeated measures Two-way ANOVA. (B) Real-time RT PCR showing significantly reduced levels of Grp, Nmb, Nppb, Sst, and Tac1 in DRGs of dry skin mice relative to WT mice. n = 4. (C), RNA scope ISH images showing that Nppb and Sst were largely co-expressed in WT DRG neurons. Nppb and Sst signals were dramatically reduced in DRGs of BRAF^Nav1.8 mice. (D) Venn diagram showing overlapping expression of Nppb and Sst. (E) Quantified data of RNA scope showing that the numbers of Nppb neurons and Sst neurons were significantly reduced in the DRGs of BRAF^Nav1.8 mice. n = 4. Values are presented as mean ± SEM. **p < 0.01, ***p < 0.001, unpaired t test. Scale bars, 50 µm.

Figure 7.. Schematics for the BNP-NPRC facilitated signaling pathway and distinct neuropeptide pathways for histamine-dependent and -independent itch.

(A) A schematic showing a model for NMBR-NPRC cross-signaling facilitated by BNP via the NMB-NMBR pathway. In response to histamine, NMB and BNP are released from primary afferents to activate NMBR and NPRC concurrently. Activation of NMBR by NMB at a low concentration may prime PLCβ signaling, whereas activation of NPRC by BNP stimulates Gai signaling, which in turn stimulates PLCβ to activate downstream Ca2+ signaling. (B) A hypothetic model depicting the respective roles of neuropeptides and glutamate in itch transmission. CQ itch is mediated in part by GRP-GRPR signaling independent of glutamatergic transmission. In contrast, histamine itch is mediated by NMB-NMBR signaling from primary afferents to NMBR neurons and by glutamatergic transmission from NMBR neurons to GRPR neurons. BNP facilitates NMB-NMBR signaling via NPRC independent of GRP-GRPR signaling but dependent on GRPR neurons. Glu: glutamate; GRP: gastrin-releasing peptide; BNP: B-type natriuretic peptide; NMB: neuromedin B.

Figure 7—figure supplement 1.. A hypothetic model depicting the role of BNP, NMB, and SST in facilitation of itch and disinhibition of pain, respectively.

In response to histamine injection, NMB is released from primary afferents to activate NMBR neurons, while BNP is released to activate NPRC to facilitate NMBR signaling in NMBR neurons. Note that NMB and BNP do not have to be released from the same sensory neurons since NMB is also expressed in non-BNP neurons that may also innervate NMBR/NPRC neurons. During itch transmission, SST is not released. However, in response to certain types of noxious stimuli, SST may be released due to more intense firing of primary afferents to inhibit SST2R neurons, contributing to nociceptive transmission as a result of disinhibition.