Article Figures & Data
Figures
- Fig. 1.
Gender-related differences in NK2 receptor–mediated contractility. Contractile responses of female (n = 11) and male (n = 10 to 11) human colonic circular smooth muscle strips to (A) the endogenous NK2 receptor ligand NKA and (B) the selective NK2 receptor agonist LMN-NKA. Data are expressed as mean tension (grams) ± S.E.M. Significance by two-way ANOVA is indicated.
- Fig. 2.
Gender-specific distinctions in the potency of the selective NK2 receptor antagonist ibodutant. The effect of different concentrations of ibodutant on contractile responses induced by the endogenous NK2 receptor ligand NKA (left panel) and the selective NK2 receptor agonist LMN-NKA (right panel) in female (A and B) and male (C and D) human colonic circular smooth muscle strips. Data are expressed as a tension (grams), and curves are presented as a percentage of the mean maximum response for each concentration of ibodutant. n = 9–11 for each curve, and closed circles indicate means ± S.E.M.
- Fig. 3.
The Schild plots generated to calculate pA2 values for ibodutant against the endogenous NK2 receptor ligand NKA (left panel) and the selective NK2 receptor agonist LMN-NKA (right panel) in female (A and B) and male (C and D) human colonic circular muscle strips (n = 11).
- Fig. 4.
Gender-related differences in the neuronal release of tachykinin peptides. (A and B) Basal release of NKA and SP from female (n = 14 and 10, respectively) and male (n = 15 and nine, respectively) human colonic smooth muscle strips. Horizontal lines denote medians. Significance by Mann-Whitney test is indicated, where *P = 0.0279. (C and D) The release of NKA and SP in response to increasing frequencies of EFS. Data are expressed as a percentage of the basal amount released and are presented as means ± S.E.M. One-sample t test analysis of EFS-induced tachykinin release compared with basal level is indicated by *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
- Fig. 5.
Western blot and densitometry analysis of NK2 receptor protein expression in human colonic sigmoid smooth muscle. Results are expressed as a percentage of the loading control GAPDH. (A) NK2 receptor and GAPDH protein blots with specific bands corresponding to the expected molecular weight of ∼45 and ∼36 kDa, respectively. (B) NK2 receptor protein expression in female (n = 25) and male (n = 15) samples. Horizontal lines denote medians. (C) NK2 receptor protein expression in samples from patients younger (closed circles) or older (open circles) than 50 years of age. (D) Spearman correlation analysis of NK2 receptor expression and age in females (r = 0.5023, *P = 0.0105) and males (r = 0.127, P = 0.6499).
- Fig. 6.
Fluorescent immunoreactivity of the NK2 receptor (left panel) in the smooth muscle layers of the human sigmoid colon. The cell markers used (middle panel) are α-SMA (B, n = 2), the interstitial cells of Cajal marker c-kit (E and H, n = 2), and the neuronal marker β-tubulin (K, n = 6). The merged images (right panel) contain the nuclear marker DAPI (blue). Staining is present on myenteric ganglia (mg), circular muscle (cm), and longitudinal (lm) muscle. Scale bar, 20 µm. Representative images were taken from female specimens aged 62 years old (A–C and J–L) and 46 years old (G–I) and a male specimen aged 49 years old (D–F).
- Fig. 7.
Fluorescent staining of the NK2 receptor (left panel) within the myenteric ganglia of the human sigmoid colon. The cell markers used (middle panel) are the nerve cell body marker HuC/D (B, n = 2), the neuronal marker β-tubulin (E, n = 6), and the glial cell markers S100 (H, n = 6) and GFAP (K, n = 2). The merged images (right panel) contain the nuclear marker DAPI (blue). Staining is present on myenteric ganglia (mg), circular muscle (cm), and longitudinal (lm) muscle. Scale bar, 20 µm. Representative images were taken from a female specimen aged 62 years old (A–F) and a male specimen aged 49 years old (G–L).
- Fig. 8.
Gender-related variations in NK2 receptor–mediated second messenger signaling pathways. Contractile responses of female (red) and male (blue) human colonic circular smooth muscle strips to NKA (left panel) and LMN-NKA (right panel) at 0.3 μM, either alone (dashed lines) or in the presence of the PLC inhibitor U73122 (A and B), the inositol IP3 receptor inhibitor 2-APB (C and D), the PKC inhibitor GF109203X (E and F), or the Rho kinase inhibitor Y-27632 dihydrochloride (G and H) at 3, 30, 100, and 300 μM. Data are expressed as a percentage of the contractile response before pharmacological inhibition and are presented as means ± S.E.M. (n = 5–11 for each curve). Significance by two-way ANOVA is indicated by a single symbol for P < 0.05, two for P < 0.01, three for P < 0.001, and four for P < 0.0001 (* for before vs. after inhibition within each gender; † for females vs. males).
- Fig. 9.
Gene expression of the Gαq family of G proteins (A–C) and the PLCβ isoenzymes (D–G) in female (n = 26 to 27) and male (n = 25–27) human colonic smooth muscle. Results are normalized to GAPDH expression and presented as fold change relative to the inter-run calibrator. Horizontal lines denote medians.
Tables
- TABLE 1
Summary of Schild plot results for ibodutant against NKA and the selective NK2 receptor agonist LMN-NKA in human colonic circular smooth muscle
pA2 values and slopes were generated from Schild plot and expressed as means ± S.E.M. All slopes were not significantly different from 1, and there was no difference between the slopes. Values in parentheses are the mean apparent pKB of individual ibodutant concentrations calculated using the equation pKB = log (dose ratio − 1) − log [B], where [B] is the antagonist concentration (Arunlakshana and Schild, 1959; Kenakin, 2009).
Parameters Ibodutant against NKA Ibodutant against LMN-NKA Female Male Female Male pA2 ± S.E.M. (apparent pKB) 8.36 ± 0.38* (8.22 ± 0.17)** 7.26 ± 0.21 (7.10 ± 0.14) 8.14 ± 0.36 (7.99 ± 0.19) 8.33 ± 0.22* (8.11 ± 0.08)** Schild slope ± S.E.M. 0.76 ± 0.14 0.81 ± 0.055 0.72 ± 0.17 0.90 ± 0.12 Slope different from 1 No No No No P = 0.24 P = 0.077 P = 0.24 P = 0.51 ↵* P < 0.05; **P < 0.01, compared with male against NKA (one-way ANOVA with Bonferroni’s test).
Data Supplement
- Supplemental Data -
Supplementary Table 1 - Forward (FP) and reverse (RP) primers of human target and housekeeping genes used in real-time PCR studies. Primer melting temperatures and amplification efficiency scores are shown.
Supplementary Figure 1 - Contractile responses of human colonic circular muscle to A) the endogenous NK2 receptor ligand neurokinin A (NKA), B) the selective NK2 receptor agonist [Lys5,MeLeu9, Nle10]NKA(4-10) (LMN-NKA) and C) the selective NK1 receptor agonist [Pro9] substance P ([Pro9] SP).
Supplementary Figure 2 - The effect of atropine on the contractile responses of human colonic circular muscle to A) the endogenous NK2 receptor ligand neurokinin A (NKA) and B) the selective NK2 receptor agonist [Lys5
,MeLeu9, Nle10]NKA(4-10) (LMN-NKA).
- Supplemental Data -
In this issue
Tachykinin NK2 Receptors in Human Colonic Smooth Muscle
