Fig 1: Representative microphotographs of the lung of non-COVID-19 (A) and of SARS-CoV-2-positive samples (B-D). Hematoxylin-eosin stained sections (A and B) show clear histological changes caused by SARS-CoV-2 infection, including proliferation of type II pneumocytes (arrows in B). Immunofluorescence for RAMP1 shows the distribution of this receptor component in the bronchiolar epithelium (C) and the hyperplastic type II pneumocytes (D) in SARS-CoV-2 samples. Scale bar for A and B = 200 µm. Scale bar for C and D = 50 µm.
Fig 2: Schwann cell RAMP1 mediates PMA evoked by CGRP.a Representative real-time PCR plot and cumulative data for GAPDH, S100, CLR and RAMP1 mRNA in HSCs (n = 3 independent experiments) and MSCs from trigeminal or sciatic nerve (MSCs from sciatic nerve n = 3 independent experiments; MSCs from trigeminal nerve, n = 4 independent experiments). b Representative images of DAPI and immunoreactive S100, RAMP1 and CLR in human and mouse cutaneous nerve bundles (scale, 10 µm human, 50 µm mouse) (n = 3 subjects). c PMA induced by CGRP (1.5 nmol) or vehicle in male and female Plp1-CreERT+/Ramp1fl/fl and Control mice treated with periorbital 4-OHT (n = 8 mice per group). d Representative images and colocalization value (Rcoloc) of S100 and RAMP1 in periorbital nerve and sciatic nerve trunks from Plp1-CreERT+;Ramp1fl/fl and Control mice (scale, 20 µm) (n = 4 replicates). TN (trigeminal nerve), SN (sciatic nerve). e PMA induced by intraperitoneal (i.p.) CGRP (0.1 mg/kg) or vehicle in male and female C57BL/6 J mice (n = 8 mice per group). f PMA and (g) paw mechanical allodynia induced by intraperitoneal (i.p.) CGRP (0.1 mg/kg) or vehicle in male Plp1-CreERT+/Ramp1fl/fl and Control mice (n = 8 mice per group) treated with periorbital 4-OHT. Mean±SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Veh, Control-Veh, and TN-Control, §§§P < 0.001. vs. Control-CGRP. 2-way (c, e, f, g) or 1-way (d) ANOVA, Bonferroni correction. Source data are provided as a Source Data file.
Fig 3: DIPMA-MK-3207 nanoparticles target endosomal CLR/RAMP1 signaling and provide superior relief from CGRP-evoked PMA.a pH-responsive DIPMA-MK-3207. b Transmission electron micrograph image of DIPMA-MK-3207 (Scale: 0.1 µm), from two different nanoparticle preparations (3 images captured per sample). c Physicochemical properties of DIPMA-MK-3207 and DIPMA-Ø. d Uptake of DIPMA-Cy5 into HSCs expressing EEA1-GFP. Cells were preincubated with DIPMA-Cy5 (40–60 ng/ml) for 30 min and were then incubated with TAMRA-CGRP (100 nM) for 30 min. Arrows denote accumulation of TAMRA-CGRP in early endosomes containing DIPMA-Cy5. Representative images from n = 5 independent experiments (Scale: 10 µm). e–g Effects of DIPMA-MK-3207, MK-3207, DIPMA-Ø or vehicle on CGRP- (100 nM) stimulated cAMP formation in HEK-rCLR/RAMP1 cells. e Time course and f, g integrated response (AUC) before (1st phase) and after (2nd phase) washing to remove extracellular CGRP (n = 6 independent experiments). h Concentration-response curves of the inhibition by DIPMA-MK-3207 or free MK-3207 on the Ca2+ response to CGRP in HSCs (DIPMA-MK-3207: -9M, n = 145; -8M, n = 361; -7M, n = 213: -6.5 M, n = 150; or free MK-3207: -8M, n = 83; -6M, n = 106; -5M, n = 87; -4M, n = 127: -3M, n = 127 cells). i PMA, expressed as AUC, after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in C57BL/6 J male mice pre-treated (0.5 h) with DIPMA-MK-3207, MK-3207 (0.1, 0.3, 1 pmol), DIPMA-Ø or vehicle (n = 8 mice per group). Mean±SEM. ***P < 0.001 vs. DIPMA-Ø/Veh, ###P < 0.001 vs. MK-3207 0.3 pmol and MK-3207 1 pmol. f, g, i 1-way ANOVA, Bonferroni correction. Source data are provided as a Source Data file.
Fig 4: Functional CLR/RAMP1 is expressed by HSCs and undergoes clathrin- and dynamin-mediated endocytosis, which underlies nociception.a, b Effects of graded concentrations of CGRP on cAMP formation (n = 5 independent experiments). c, d Effects of graded concentrations of olcegepant on CGRP (100 nM)-evoked cAMP formation (n = 4 independent experiments). e Pharmacological targets. f Representative images of HSCs expressing Rab5a-GFP at 30 min after incubation with TAMRA-CGRP (100 nM). Arrows denote colocalization of TAMRA-CGRP and Rab5a-GFP. Arrowheads denote retention of a weak TAMRA-CGRP signal at the plasma membrane. Cells were preincubated with vehicle, Dyngo-4a (Dy4), Pitstop 2 (PS2), inactive analogs (PS2 and Dy4 inact) (all 30 µM) or sucrose (0.45 M) (n = 4 independent experiments). Scale, 10 µm. g Quantification of localization of TAMRA-CGRP in endosomes (data represent n = 949 veh 0 min, n = 1209 veh 30 min, n = 1111 Dy4 30 min, n = 1016 Dy4 inact 30 min, n = 1700 PS2 30 min, n = 714 PS2 inact 30 min, n = 1896 sucrose) and (h) quantification of the number of TAMRA-CGRP+ve endosomes (data represent n = 5 veh 0 min, n = 7 veh 30 min, n = 7 Dy4 30 min, n = 5 Dy4 inact 30 min, n = 7 PS2 30 min, n = 5 PS2 inact 30 min, n = 5 sucrose). i, j PMA induced by periorbital CGRP (1.5 nmol) or vehicle in C57BL/6 J male mice pretreated (0.5 h) with PS2, Dy4, PS2 or Dy4 inact (all 500 pmol) (n = 8 mice per group). k, l PMA induced by periorbital capsaicin (CPS, 50 pmol) or vehicle in C57BL/6 J male mice pretreated (0.5 h) with PS2, Dy4, PS2 or Dy4 inact (all 500 pmol) (n = 8 mice per group). Mean±SEM. ***P < 0.001 vs. Veh 0 min, and Veh/Veh; §§P < 0.01, §§§P < 0.001 vs. Veh 30 min, PS2 30 min, Dy4 30 min, CGRP/PS2 inact, CGRP/Dy4 inact, CPS/PS2 inact, CPS/Dy4 inact. 1-way (g, h) or 2-way (i–l) ANOVA, Bonferroni correction. Source data are provided as a Source Data file.
Fig 5: CGRP leads to Ga protein activation and ßARR2 recruitment at the plasma membrane and in early endosomes in HEK-hCLR/RAMP1 cells and HSCs-hCLR/RAMP1.Endosomal signaling generates sustained formation of cAMP in HSCs. (a–d) CGRP (100 nM) increased EbBRET between Rluc8-mGas, Rluc8-mGasq, Rluc8-mGasi, and Rluc2-ßARR2 with RGFP-CAAX (a, b) and tdRGFP-Rab5a (c, d) in HEK-hCLR/RAMP1 cells. a, c time course. b, d area under curve (AUC) (a, b n = 10 mGas, n = 8 mGasq, n = 7 mGasi, n = 7 ßARR2, n = 9 veh; c, d n = 8 mGas, n = 8 mGasq, n = 8 mGasi, n = 6 ßARR2, n = 9 veh). e–h CGRP (100 nM) increased EbBRET between Rluc8-mGas, Rluc8-mGasq, Rluc8-mGasi, and Rluc2-ßARR2 with RGFP-CAAX (e, f) and tdRGFP-Rab5a (g, h) in HSC-hCLR/RAMP1 cells. e, g time course. f, h AUC (e, f n = 7 mGas, n = 9 mGasq, n = 5 mGasi, n = 6 ßARR2, n = 9 veh; g, h n = 7 mGas, n = 7 mGasq, n = 7 mGasi, n = 5 ßARR2, n = 7 veh). (i) Hypertonic sucrose (0.45 M) inhibited CGRP (100 nM)-stimulated EbBRET between hCLR-Rluc8 and tdRGFP-Rab5a in HEK-hCLR/RAMP1 cells (n = 5 independent experiments). (j, k) Hypertonic sucrose (0.45 M) inhibited CGRP (100 nM)-stimulated EbBRET between Rluc8-mGas, Rluc8-mGasq, Rluc8-mGasi and Rluc2-ßARR2 with tdRGFP-Rab5a in HEK-hCLR/RAMP1 cells (j, k n = 10 independent experiments). (l, m) Sucrose (0.45 M) inhibited CGRP (100 nM)-stimulated EbBRET between Rluc8-mGas, Rluc8-mGasq, Rluc8-mGasi, and Rluc2-ßARR2 with tdRGFP-Rab5a in HSC-hCLR/RAMP1 cells. (n = 8 independent experiments). (n, o) Sucrose (0.45 M) inhibited CGRP (100 nM)-stimulated formation of cAMP in HSCs. n time course. o, AUC (n = 5 independent experiments). Mean±SEM.. *P < 0.05, **P < 0.01, ***P < 0.001, vs. Veh. 1-way ANOVA, Dunnett’s correction (b, d, f, h) or parametric unpaired t test (i–m, o). Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-RAMP1 antibody [EPR10867]