Gender-Related Differences of Tachykinin NK 2 Receptor Expression and Activity in Human Colonic Smooth Muscle s

The tachykinin NK 2 receptor plays a key role in gastrointes- tinal motor function. Enteric neurons release neurokinin A (NKA), which activates NK 2 receptors on gastrointestinal smooth muscle, leading to contraction and increased motility. In patients with diarrhea-predominant irritable bowel syndrome, the NK 2 receptor antagonist ibodutant had a greater therapeutic effect in females than males. The present study aimed to determine whether gender influences the expression and activity of NK 2 receptors in human colonic smooth muscle. In vitro functional studies were performed to examine the contractile responses of colonic muscle strips to NKA and the selective NK 2 receptor agonist [Lys 5 ,MeLeu 9 ,Nle 10 ]NKA(4-10). Contractions were also measured in the presence of ibodutant to determine its antag- onistic potency. The signal transduction pathways coupled to NK 2 receptor activation were investigated using second mes- senger inhibitors. Western blot and fluorescent immunohisto-chemistry were conducted to determine the protein expression and localization of NK 2 receptors. NK 2 receptor – mediated contractility was greater in females compared with males. When against NKA, ibodutant was more potent in females.


Introduction
Tachykinins are a family of peptides that regulate a broad range of biologic activities, including smooth muscle contraction, vasodilation, inflammation, and nociception. The major naturally occurring tachykinins are substance P (SP), neurokinin A (NKA), and neurokinin B, and they exhibit preferential affinity for tachykinin NK 1 , NK 2 , and NK 3 receptors, respectively (Nakamura et al., 2011). Tachykinin receptors primarily couple to the Ga q/11 class of G proteins, which activate phospholipase C (PLC), leading to the formation of inositol 1,4,5-triphosphate (IP 3 ) and the subsequent release of Ca 2+ from intracellular stores (Regoli et al., 1994;Mizuta et al., 2008). These receptors may also activate Ga s proteins, leading to increased cAMP levels (Nakajima et al., 1992;Regoli et al., 1994;Perdona et al., 2019).
In the gastrointestinal tract, SP and NKA are primarily released from myenteric and submucosal neurons and are involved in the modulation of motor and secretory events (Shimizu et al., 2008;Steinhoff et al., 2014). Among the three tachykinin receptor subtypes, NK 2 receptors have an important role in regulating human colonic motility. Numerous pharmacological studies using human colonic smooth muscle strips have revealed that the contractile effects of endogenous tachykinins are mediated by NK 2 receptors (Croci et al., 1998;Cao et al., 2000;Liu et al., 2002Liu et al., , 2009Nakamura et al., 2011;Tanaka et al., 2012). This is in line with the high mRNA https://doi.org/10. 1124/jpet.120.265967. s This article has supplemental material available at jpet.aspetjournals.org. expression levels of the tachykinin receptor 2 gene (encoding NK 2 receptors) in human colonic smooth muscle (Liu et al., 2011). Furthermore, NK 2 receptor activation has been shown to be responsible for the tachykinergic component of excitatory neurotransmission, and altered NK 2 receptor function has been implicated in colonic motility disorders, such as inflammatory bowel disease, diverticular disease, and slow transit constipation in both adults and children (Menzies et al., 2001;Mitolo-Chieppa et al., 2001;Liu et al., 2002;Stanton et al., 2003;Maselli et al., 2004;Auli et al., 2008;Burcher et al., 2008;Alvarez-Berdugo et al., 2015).
Given their key involvement in human colonic motor function, NK 2 receptors have been considered as possible drug targets for the development of effective therapies for diarrheapredominant irritable bowel syndrome (IBS) (Lecci et al., 2004;Corsetti et al., 2015;Szymaszkiewicz et al., 2019). Specifically, the NK 2 receptor antagonist ibodutant emerged as a promising candidate because of its high selectivity and potency for human NK 2 receptors, long duration of action, and good oral bioavailability (Cialdai et al., 2006;Meini et al., 2009;Santicioli et al., 2013). However, clinical trials uncovered a gender distinction in the effectiveness of ibodutant, whereby dose-dependent improvements in symptoms reached statistical significance over placebo in female patients with IBS only (Tack et al., 2017).
The gender difference observed in the therapeutic outcome of ibodutant in diarrhea-predominant IBS sparks an interest in the gender-specific variations surrounding colonic NK 2 receptors. Gender differences in the efficacy of ibodutant have also been reported in a guinea pig colitis model, in which colonic contractions induced by colorectal distension were inhibited by ibodutant at lower doses in females than those required in males (Bellucci et al., 2016). Previously, our findings revealed a gender-related distinction in the density of [ 125 I]NKA binding sites (corresponding to the number of NK 2 receptors) in human colonic smooth muscle, with a significantly higher binding capacity observed in males compared with females (Burcher et al., 2008). However, [ 125 I]NKA binding affinities and the expression levels of NK 2 receptor mRNA did not differ between the genders (Burcher et al., 2008).
Further investigation is required to explain the existence of gender distinctions in the efficacy of ibodutant and the abundance of NK 2 receptor binding sites. The present study aims to clarify these observations by uncovering any further gender-related differences in the expression and activity of NK 2 receptors in human colonic smooth muscle. Although NK 2 receptors primarily couple to the Ga q/11 -PLC-IP 3 -Ca 2+ transduction cascade to mediate smooth muscle contraction, such as in the bladder and airway (Mizuta et al., 2008;Der et al., 2019), this signal transduction does not appear to be the main pathway used by NK 2 receptors in human colonic smooth muscle (Warner et al., 2000;O'Riordan et al., 2001). Therefore, this study also aims to uncover the signal transduction pathways coupled to NK 2 receptor activation in the human colon.

Materials and Methods
Human Colon Specimens. Ascending and sigmoid colon segments were taken 10-20 cm away from the tumor site of patients with colorectal carcinoma undergoing surgical resection. Patients with obstructions or those who had received radiation therapy or chemotherapy were excluded from the study. The mean age of the specimens used was 58.5 years for females (age range: 31-85 years, n = 55) and 60.7 years for males (age range: 32-84, n = 43). There were no significant differences in the age of the female and male specimens used for the functional and expression studies. Specimens were collected from Hurstville Private, St George Private, and Prince of Wales Public and Private Hospitals. The research was performed in accordance with the Declaration of Helsinki and approved by the South Eastern Sydney Local Health District Human Research Ethics Committee, Sydney, NSW, Australia. The specimens were transported to the laboratory in ice-cold, carbogenated (95% O 2 and 5% CO 2 ) Krebs-Henseleit solution (NaCl 118 mM, KCl 4.7 mM, NaHCO 3 25 mM, KH 2 PO 4 1.2 mM, MgSO 4 1.2 mM, CaCl 2 2.5 mM, D-glucose 11.7 mM). Specimens were gently washed to remove fecal matter, and fat was trimmed. For the functional experiments, a portion of smooth muscle was dissected away from the other layers and stored in Krebs-Henseleit solution overnight at 4°C. The remaining tissue was processed for the expression studies as described below.
Functional Organ Bath Set-Up. Colonic smooth muscle strips (each approximately 3 Â 6 mm) were cut along the circular axis. Muscle strips were mounted under 1 g tension in 3.5-ml organ baths containing carbogenated Krebs-Henseleit solution at 37°C, as previously described (Liu et al., 2002). One end of each strip was fixed to a hook at the bottom of the bath, and the other end was connected to an isometric force transducer (Grass FTO3C, Warwick, RI). The change of muscle tension was recorded using the Polygraph computer program (E. Crawford, UNSW Sydney, NSW, Australia). Preparations were allowed to equilibrate for 30 minutes before exposure to antagonists and inhibitors and for 60 minutes before exposure to tachykinin agonists.
Contractile Responses to NK 2 Receptor Agents. During optimizing experimental conditions, it was shown that NKA-and [Lys 5 ,MeLeu 9 ,Nle 10 ]NKA(4-10) (LMN-NKA)-induced human colonic contractile responses remained unaffected in the presence of the NK 1 receptor antagonist CP9994 (100 nM) but were greatly reduced by the NK 2 receptor antagonist SR 48968 (100 nM) (Supplemental Fig. 1, A and B). Furthermore, the selective NK 1 receptor agonist [Pro 9 ] SP (up to 10 mM), was virtually ineffective in contracting human colonic smooth muscle (Supplemental Fig. 1C). These results suggest that the influence of NK 1 receptors is negligible. There was no evidence for the involvement of enteric neuron transmission, as atropine (1 mM) showed no effect to NKA and LMN-NKA (Supplemental Fig. 2). Thus, the subsequent experiments were conducted without the presence of an NK 1 antagonist nor atropine.
A single concentration-response curve to NKA or the selective NK 2 receptor agonist LMN-NKA (0.001-10 mM) was obtained from each strip, using 60-minute intervals to avoid desensitization. Washing occurred twice between each concentration of agonist. Acetylcholine (ACh, 10 mM) was used as an internal standard and applied at the start and end of each concentration-response curve, as per previously published protocols (Warner et al., 2000;Liu et al., 2002;Burcher et al., 2008). In parallel experiments, contractile responses were measured in the presence of the selective NK 2 receptor antagonist ibodutant (either 0.01, 0.1, or 1 mM) preincubated for 30 minutes before the addition of agonist.
Contractile Responses in the Presence of Second Messenger Inhibitors. NKA and LMN-NKA were fixed at the submaximal concentration of 0.3 mM and were added at 60-minute intervals. Muscle strips were washed twice between each application of agonist. Female muscle strip desensitization was not observed under control conditions, whereas slight desensitization occurred in male strips. For both control and treated muscle strips, results were expressed as a percentage of the average of two initial contractile responses under control conditions (before inhibitors were applied). In treated muscle strips, preincubation with second messenger inhibitors occurred for 30 minutes using increasing concentrations (3, 30, 100, 300 mM) of jpet.aspetjournals.org either U73122 hydrate (PLC inhibitor), 2-aminoethoxydiphenyl borate (2-APB, IP 3 receptor inhibitor), GF109203X [protein kinase C (PKC) inhibitor], or Y-27632 dihydrochloride (Rho kinase inhibitor). The inactive analog of U73122 hydrate, U-73343 (100 mM), did not produce an effect on the agonist-induced contractions.
Neuronal Tachykinin Release by Electrical Field Stimulation. Muscle strips were suspended between two platinum rings at 1 g tension, and preparations were allowed to equilibrate for 30 minutes. Electrical field stimulation (EFS) was conducted using cumulative frequencies (0.5, 5, 10, 50 Hz) of 1-millisecond duration, in 80-V trains lasting 10 seconds (Liu et al., 2009). Each frequency of EFS was given 10 minutes apart. Immediately after each stimulation, 300ml bath fluid was collected and stored at 280°C, and an equal volume of Krebs-Henseleit solution was added back to each bath. The amount of released NKA and SP was later analyzed using radioimmunoassay kits (RK-046-15 for NKA and RK-061-05 for SP; Phoenix Pharmaceuticals, Burlingame, CA).
Protein Expression of NK 2 Receptors by Western Blot. Total protein was extracted from frozen human sigmoid colonic smooth muscle using lysis buffer containing 1 mM EGTA, 10 mM Tris-HCl, and protease inhibitor cocktail tablets (Sigma-Aldrich, St Louis, MO). Protein samples (20 mg) were denatured at 95°C for 8 minutes and loaded into 50-ml wells of 10% Mini-PROTEAN precast gels (Bio-Rad Laboratories, Hercules, CA). Electrophoresis was conducted at 100 V for 1.5 hours. Protein transfer onto polyvinylidene difluoride membranes was carried out using the Trans-Blot Turbo Transfer Pack and System (Bio-Rad Laboratories) at 25 V for 7 minutes. Immunodetection was achieved by incubating with the anti-NK 2 receptor antibody (ATR-002; Alomone Laboratories, Jerusalem, Israel) at a dilution of 1: 1000 for 1 hour at room temperature. The specificity of the NK 2 antibody was verified using corresponding control peptides, the same as previously confirmed (Delvalle et al., 2018), as well as by conducting positive and negative control Western blots. The anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (G9545, 1:50,000; Sigma-Aldrich) was used as the internal control. After washing, the membranes were incubated with anti-rabbit IgG (whole molecule) peroxidase secondary antibody (A4914, 1:5000; Sigma-Aldrich) for 1 hour at room temperature. Visualization of protein bands was performed using SuperSignal West Pico PLUS Chemiluminescent Substrate (34577; Thermo Scientific, Waltham, MA), and densitometry analysis was carried out using Image Studio Lite 5.2 (LI-COR Biosciences, Lincoln, NE).
Gene Expression of Second Messenger Molecules by Real-Time Polymerase Chain Reaction. Human ascending and sigmoid colonic smooth muscle samples were frozen in RNAlater (R0901; Sigma-Aldrich). Total RNA was extracted by using the Trizol method (Invitrogen) and was purified by DNase treatment (3 U at 37°C, 20 minutes). Single-strand cDNA (sscDNA) was synthesized using the SuperScript III First-Strand Synthesis Supermix (Invitrogen) in a reaction mixture containing 2 mg RNA and 50 ng ml 21 random hexamers, at one cycle of 25°C for 10 minutes, 50°C for 50 minutes, and 85°C for 5 minutes. RNase H treatment occurred (2 U ml 21 ) at 37°C for 20 minutes. sscDNA was diluted to a working concentration of 500 ng ml 21 .
Gene expression was determined by real-time PCR using the SYBR Select Master Mix (Applied Biosystems, Austin, TX). The primers were designed with Primer3, and primer sequences and PCR amplification efficiencies are listed in Supplemental Table 1. Each PCR reaction was performed in a volume of 10 ml containing 5 ml Master Mix, 10 mM forward and reverse primers, and 80 ng ml 21 sscDNA. The solutions were dispensed into 384-well plates by the Eppendorf epMotion 5075 Automated Pipetting System. GAPDH was used as a housekeeping gene. Inter-run comparisons were achieved using a designated calibrator (one colonic smooth muscle sample) in each real-time PCR assay. PCR amplification occurred for one cycle at 95°C (2 minutes), 40 cycles at 95°C (15 seconds), 60°C (15 seconds), and 68°C (20 seconds), followed by 10 minutes of gradual temperature elevation from 60 to 95°C for the melting curve analysis.
Data Analysis. Data were analyzed using GraphPad Prism 7 (GraphPad Software, San Diego, CA), and statistical significance was achieved when P , 0.05. The results obtained from ascending and sigmoid colonic muscle strips did not differ, so data from both regions were combined in the analyses.
For the functional studies, data were expressed as tension (grams). Similar results were seen when the data were expressed as tension (grams) per strip weight (grams). However, ACh could not be used as a reliable internal standard because contractile responses varied between the experimental groups. Results are presented as means 6 S.E.M. unless otherwise stated. Concentration-response curves were generated using nonlinear regression analysis to determine maximal contractile responses (E max ) and half maximal EC 50 values. The data were analyzed by two-way ANOVA followed by Bonferroni multiple comparisons test. For the antagonist ibodutant, Schild plots were created, and pA 2 values were taken as the x-intercept of the Schild regression analysis. The apparent pK B values of each individual antagonist concentration were also calculated using the equation: is the antagonist concentration (Arunlakshana and Schild, 1959;Kenakin, 2009). Data were analyzed using one-way ANOVA with Bonferroni's multiple comparisons test.
For the NKA and SP release study, basal levels of NKA and SP were expressed as nanograms per strip weight (nanograms per gram), and comparisons of median values were made using Mann-Whitney analysis. EFS-induced release was expressed as a percentage of the basal level and presented as means 6 S.E.M. Gender differences were assessed by two-way ANOVA with Bonferroni's multiple comparisons test, whereas comparisons between basal and EFS-induced tachykinin levels were made at each EFS frequency using one-sample t test analysis.
For the expression studies, NK 2 receptor protein levels were expressed as a percentage of GAPDH. The mRNA level of genes encoding second messengers was expressed as fold change normalized to GAPDH and relative to the calibrator, using the following equation: (Pfaffl, 2001). Median values were compared using Mann-Whitney analysis or the Kruskal-Wallis test Drugs. NKA was purchased from Abcam (ab120185), and LMN-NKA was custom synthesized by China Peptides (Pudong, Shanghai, China). Both were reconstituted in 0.01 M acetic acid with b-mercaptoethanol. ACh was purchased from Sigma-Aldrich (A6625) and reconstituted in water. Ibodutant was a gift from our collaborators at The Menarini Group (Florence, Italy) and was prepared in DMSO. The second messenger inhibitors 2-APB (1224), GF109203X (0741), and Y-27632 dihydrochloride (1254) were purchased from Tocris Bioscience (Noble Park, Victoria, Australia), and the inhibitor U73122 hydrate (U6756) was obtained from Sigma-Aldrich. The second messenger inhibitors were dissolved in DMSO. Aliquots of all reagents were stored at 220°C.

Results
Contractile Responses to NK 2 Receptor Agonists. The endogenous NK 2 receptor ligand NKA and the selective NK 2 receptor agonist LMN-NKA evoked prominent contractile responses in human colonic smooth muscle strips (Fig. 1). There were no gender differences in contractions produced by NKA (n = 11 females, 10 males) (Fig. 1A). On the other hand, LMN-NKA elicited contractile responses that were significantly greater in females (n = 11) compared with males (n = 11, **P = 0.0023, two-way ANOVA) (Fig. 1B). In line with this, the E max of NKA did not vary between the genders (1.8 6 0.27), whereas the E max of LMN-NKA was significantly greater in females (2.0 6 0.18) compared with males (1.2 6 0.17, **P = 0.0029, unpaired t test). When comparing the E max of NKA and LMN-NKA in males, values were close to statistical significance (P = 0.0742), suggesting that suppressed responses to LMN-NKA in male strips underlie the observed gender difference. There was no correlation between age and E max for either agonist, in either gender. There were no gender differences in EC 50 values. For NKA, the EC 50 values and 95% confidence intervals were 65.63 nM (25-167) in females and 75.31 nM (28-200) in males. For LMN-NKA, the EC 50 values and 95% confidence intervals were 87.06 nM (45-166) in females and 28.76 nM (10-78) in males.
Potency of NK 2 Receptor Antagonist Ibodutant. Ibodutant antagonized the contractile responses elicited by NKA and LMN-NKA in the human colonic smooth muscle strips of females (n = 11) and males (n = 9-11) and produced a rightward shift of the agonist concentration-response curves (Fig. 2). Schild plots were generated to calculate pA 2 values for ibodutant against both agonists, and its competitive antagonism property was confirmed, since ibodutant did not change the maximum contractilities of the agonists, and the slopes of Schild plots are not different from unity (Figs. 2 and 3; Table 1). The apparent pK B values of each individual antagonist concentration were also calculated and presented as mean values in Table 1.
When against NKA, ibodutant displayed a significantly higher degree of antagonism in females (pA 2 = 8.51 6 0.38) compared with males (pA 2 = 7.23 6 0.21, *P , 0.05, one-way ANOVA) (Table 1). On the other hand, no gender differences were observed in the potency of ibodutant when against LMN-NKA (pA 2 = 8.30 6 0.36 for females and 8.26 6 0.22 for males) (Table 1). Surprisingly, ibodutant appeared agonistdependent in the male colon, being less potent against NKA compared with LMN-NKA (*P , 0.05, one-way ANOVA). These results were similar to the apparent pK B values calculated from the individual concentration of ibodutant (Table 1). There was no significant difference in the potency of ibodutant against the different agonists in female colonic muscle strips. The pA 2 values of ibodutant obtained from our study were lower than that previously reported in the human colon (pK B = 9.1), and gender-related differences were not described (Santicioli et al., 2013). It is noted that in their study, the concentration-response curves to the NK 2 receptor agonist [bAla 8 ]NKA(4-10) were cumulatively constructed (Santicioli et al., 2013). Nevertheless, a 60-minute interval between each addition of the agonists was applied in our study to avoid desensitization. The different protocols may contribute to the discrepancies between the two studies.
Basal and EFS-Induced Release of NKA and SP. NKA and SP released from human colonic smooth muscle strips were measured to uncover any gender differences that could be contributing to the observed distinctions in contractility (Fig. 4). The basal level of NKA did not vary between females (4.7 6 2.0, n = 14) and males (2.8 6 0.8, n = 15) (Fig. 4A). On the other hand, a higher amount of SP was spontaneously released under resting conditions from muscle strips of females (5.5 6 1.0, n = 10) compared with those of males (2.6 6 0.4, n = 9, *P = 0.0279, Mann-Whitney test) (Fig. 4B).
The neuronal release of NKA and SP elicited by cumulative frequencies of EFS was then recorded and expressed as a percentage of the basal level. EFS-induced release of NKA (Fig. 4C) and SP (Fig. 4D) was significantly greater than basal amounts at 5 and 10 Hz for both genders (one-sample t test). At 50 Hz, there was a significant increase in the amount of NKA released from male smooth muscle only (*P = 0.0467, onesample t test) (Fig. 4C). Interestingly, SP release in response to 0.5 Hz was significantly lower than basal levels in male Downloaded from strips (****P , 0.0001, one-sample t test), and a trend of decrease was also observed in female strips (P = 0.0934) (Fig. 4D). Overall, there were no significant differences in EFS-induced tachykinin release between the genders. NK 2 Receptor Protein Expression. Western blot and densitometry analysis of human colonic sigmoid smooth muscle were performed to uncover any gender differences in the protein expression of NK 2 receptors (Fig. 5). The NK 2 receptor protein band was consistent with an ∼45-kDa protein size, as indicated by the manufacturer (Fig. 5A). Data are expressed as a percentage of GAPDH levels (∼36-kDa protein size). There was no difference in NK 2 receptor protein expression between females (n = 25) and males (n = 15) (Fig. 5B). There was a noticeable trend increase in the expression of the NK 2 receptor in specimens older than 50 years of age compared with younger ones (Fig. 5C). Correlation analysis revealed an increased NK 2 receptor expression with age in females (r = 0.5023, *P = 0.0105,
NK 2 Receptor Localization Throughout the Smooth Muscle Layers. Strong NK 2 receptor immunoreactivity was detected throughout the circular and longitudinal smooth muscle layers of the human sigmoid colon (n = 6) (Fig. 6). Extensive colocalization with a-SMA (n = 2) indicates that NK 2 receptors are present on smooth muscle cells (Fig. 6, A-C). On the other hand, NK 2 receptors did not appear to be expressed on interstitial cells of Cajal (n = 2), as evidenced by double labeling with c-kit (Fig. 6, D-I). NK 2 receptor staining was colocalized with the neuronal marker b-tubulin on some neuronal processes throughout the smooth muscle (Fig. 6, J-L). No obvious gender differences were observed in the distribution of NK 2 receptors throughout the smooth muscle layers.
NK 2 Receptor Localization within the Myenteric Ganglia. Dense NK 2 receptor immunoreactivity was expressed within the myenteric ganglia of the human sigmoid colon (n = 6) (Fig. 7). Although weak colocalization with the neuronal marker HuC/D was observed, strong NK 2 receptor staining was detected on the structures surrounding the HuC/D positive cells (n = 2) (Fig. 7, A-C). Similarly, double labeling with b-tubulin (n = 6) suggests that NK 2 receptors are present on a limited number of enteric neurons (Fig. 7, D-F). On the other hand, NK 2 receptor staining was extensively colocalized with the glial cell markers S100 (Fig. 7, G-I, n = 6) and GFAP (Fig. 7, J-L, n = 2) within the myenteric ganglia, as well as on glial processes throughout the muscle layers. There were no obvious gender differences in the localization of NK 2 receptor expression within the myenteric ganglia.
NK 2 Receptor-Mediated Signaling Pathways. Contractile responses of human colonic smooth muscle strips to NKA and LMN-NKA were examined in the presence of various second messenger inhibitors to determine whether the gender differences observed in contractility could be attributed to TABLE 1 Summary of Schild plot results for ibodutant against NKA and the selective NK 2 receptor agonist LMN-NKA in human colonic circular smooth muscle pA 2 values and slopes were generated from Schild plot and expressed as means 6 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 pK B of individual ibodutant concentrations calculated using the equation pK B = log (dose ratio 2 1) 2 log [B], where [B] is the antagonist concentration (Arunlakshana and Schild, 1959;Kenakin, 2009  jpet.aspetjournals.org receptor signaling (Fig. 8). Following the blockade of PLC by U73122, contractile responses produced by NKA in male strips (n = 11) were significantly attenuated (***P = 0.0005, two-way ANOVA) (Fig. 8A). NKA-induced contractions in female strips (n = 9) were also reduced, but to a lesser extent (**P = 0.0058), and a gender difference was observed at the highest concentration (300 mM) of inhibitor (*P = 0.0138, twoway ANOVA) (Fig. 8A). Similar observations were made using  LMN-NKA as the agonist (Fig. 8B). Contractions elicited by LMN-NKA in both male (n = 10) and female (n = 9) strips were reduced by PLC inhibition (****P , 0.0001 and **P = 0.0078, respectively, two-way ANOVA), and overall, this inhibitory effect was more pronounced in males compared with females (***P , 0.0008, two-way ANOVA) (Fig. 8B).
Since activation of PLC triggers separate transduction pathways involving IP 3 receptors and PKC, the effect of inhibiting these signaling proteins was also examined. Blockade of IP 3 receptors by 2-APB did not affect contractions elicited by NK 2 receptor activation in either gender (n = 6 females, five males) (Fig. 8, C and D). On the other hand, PKC inhibition by GF109203X produced concentration-dependent reductions of NK 2 receptor-mediated contractile responses in both female (n = 6) and male (n = 6) strips (****P , 0.0001 for both genders and agonists, two-way ANOVA) (Fig. 8, E and F). During smooth muscle contraction, the Ga q/11 -PLC cascade may occur alongside Ga 12/13 -Rho kinase signaling, so the involvement of this latter pathway was also examined. In both genders (n = 9 for each), contractile responses evoked by NKA were significantly diminished by Rho kinase blockade with Y-27632 in a concentration-dependent manner (****P , 0.0001, two-way ANOVA) (Fig. 8G). Similarly, inhibition of Rho kinase caused a concentration-dependent attenuation of LMN-NKA-induced contractions in both female (n = 9) and male (n = 10) strips (****P , 0.0001; ***P , 0.0004, respectively, two-way ANOVA) (Fig. 8H). No gender-related distinctions were observed for PKC and Rho kinase inhibition (Fig. 8, E-H).
Since gender differences were observed for PLC inhibition, real-time PCR was conducted to uncover any gender variations in the expression of genes encoding the PLCb family of isoenzymes, as well as the Ga q family of G proteins (Fig. 9). There were no observed differences in the mRNA expression of  = 2), the neuronal marker b-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 mm. Representative images were taken from a female specimen aged 62 years old (A-F) and a male specimen aged 49 years old (G-L).  . 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). G proteins Ga q , Ga 11 , and Ga 14 (Fig. 9, A-C) or PLCb 1-4 isoenzymes (Fig. 9, D-G) between male (n = 25-27) and female (n = 26 to 27) human colonic smooth muscle samples.

Discussion
The present study has uncovered several gender differences in NK 2 receptor activity and expression in the human colon. NK 2 receptor-mediated contractility is more pronounced in females compared with males, and this is accompanied by a greater potency of the NK 2 receptor antagonist ibodutant, a higher basal release of SP, and an increased NK 2 receptor protein expression with age. In addition, the involvement of PLC-mediated signaling appears to be less prominent in females compared with males. The present findings also appreciate the importance of NK 2 receptor-mediated Ca 2+ sensitization pathways via PKC and, for the first time, Rho kinase in the human colon.
Male colonic smooth muscle exhibited reduced responsiveness to the selective NK 2 receptor agonist LMN-NKA. This finding is in contrast to our previous report, in which contractile responses induced by LMN-NKA in human colonic muscle strips were similar in both genders (Burcher et al., 2008). However, in that earlier study, the data were expressed as a percentage of the maximum response to ACh, so this may underlie the discrepancy. On the other hand, there were no gender differences in contractility produced by the natural NK 2 receptor ligand NKA in the current study, and this is consistent with our previous findings (Burcher et al., 2008). The differences observed for NKA and LMN-NKA could be due to the activation of NK 1 receptors by NKA, since natural tachykinins preferentially, but not exclusively, bind to all three tachykinin receptor subtypes (Nakamura et al., 2011).
The NK 2 receptor antagonist ibodutant displays a significantly higher degree of antagonism in females compared with males when against NKA. This is in line with the results obtained from human clinical trials and may underlie the gender discrepancies reported for the efficacy of ibodutant in patients with diarrhea-predominant IBS (Tack et al., 2017). Conversely, the potency of ibodutant against LMN-NKA did not vary between genders, and this is consistent with previous findings using [b-Ala 8 ]NKA(4-10) as the selective NK 2 receptor agonist (Santicioli et al., 2013). Ibodutant has been shown to display a high affinity for human NK 2 receptors in [ 125 I]NKA binding studies using transfected cells (pK i = 10.1) (Cialdai et al., 2006) and colonic smooth muscle (pK i = 9.9) (Santicioli et al., 2013). Its affinity values at NK 1 (pK i = 6.1) and NK 3 (pK i = 6.2) receptors were shown to be four orders of magnitude lower, confirming the selectivity of ibodutant for NK 2 receptors (Cialdai et al., 2006). Although the mechanisms underlying agonist-dependent differences in the genderspecific antagonistic activity of ibodutant remain unclear, they may be occurring to different extents in males and females. Nevertheless, the potency of ibodutant against NKA, which we have observed to be greater in females, would more closely reflect the physiologic setting.
Interestingly, the antagonistic activity of ibodutant at the NK 2 receptor appears agonist-dependent in males with a higher potency against LMN-NKA compared with NKA. NK 2 receptors can couple to both Ga q/11 and Ga s proteins, and different ligands can stabilize distinct NK 2 receptor states, leading to the activation of different downstream pathways (Palanche et al., 2001;Maillet et al., 2007;Valant et al., 2009). Thus, NKA-and LMN-NKA-dependent conformations of the NK 2 receptor may be responsible for the agonist-specific effect of ibodutant in the male colon.
In terms of tachykinin peptide release, basal levels of NKA were similar in both genders, whereas a higher amount of SP was released from female colonic smooth muscle. There were no gender-related variations in EFS-induced tachykinin release. In a previous study, KCl-induced NKA and SP levels were higher in colonic smooth muscle from female jpet.aspetjournals.org guinea pigs subjected to colitis compared with controls, although this difference was not seen in males (Bellucci et al., 2016). The overall amount of SP released was lower compared with NKA in guinea pig colonic smooth muscle (Bellucci et al., 2016), and this is consistent with the current findings. It appears that alternative splicing of the tachykinin precursor 1 gene (encoding SP and NKA) favors NKA production in colonic enteric neurons. The reduced level of SP at lower frequency of EFS (0.5 Hz) may be due to an autoregulatory mechanism, whereby SP exerts a feedback inhibition of its own release by activating NK 1 receptors on enteric nerve terminals (Lomax et al., 1998).
NK 2 receptor protein expression increases with age in females, supporting the evidence that changes in sex hormone levels influence tachykinin peptide and receptor expression (Villablanca and Hanley, 1997;Pinto et al., 2009). Sex hormones have also been shown to influence intestinal smooth muscle contractions. In human colonic circular muscle strips, carbachol-evoked contractions were reduced in the presence of estrogen, and this effect was prevented by estrogen receptor antagonism (Hogan et al., 2009). Similarly, progesterone inhibited spontaneous and EFS-induced contractions in mice colonic circular muscle strips and decreased fecal output in two diarrheal mouse models (Li et al., 2012). Since sex hormones affect tachykinin expression and colonic contractile responses, they may also be contributing to the age-related changes of NK 2 receptor protein expression in females. In other words, NK 2 receptor expression and activity may be regulated by sex hormones.
NK 2 receptor immunoreactivity was densely localized on colonic circular and longitudinal smooth muscle but was absent on interstitial cells of Cajal, reinforcing previous immunofluorescence findings in the human colon (Renzi et al., 2000;Warner et al., 2000;Jaafari et al., 2007;Nakamura et al., 2011). This suggests that NK 2 receptors mediate colonic contractions by direct activation of smooth muscle cells, without regulating intestinal pacemaker cell activity.
Strong NK 2 receptor staining was also detected throughout the myenteric ganglia, suggesting that apart from mediating direct myogenic effects, NK 2 receptors also play a neuroregulatory role in colonic motor pathways. Although weak NK 2 receptor colocalization with neuronal markers was observed, intense NK 2 receptor labeling was found on myenteric glial cells. Glial cells were traditionally considered to provide passive support to neurons, but an abundance of evidence now exists linking impaired glial function to altered intestinal motility (Aube et al., 2006;Nasser et al., 2006;McClain et al., 2014). Interestingly, tachykinin receptor agonists can induce glial cell activation in the mouse colon (Broadhead et al., 2012). Furthermore, NKA has been shown to activate neuronal and glial networks during neuroinflammation and neurodegeneration (Delvalle et al., 2018). However, this effect is via NK 2 receptors on surrounding sensory neurons and nerve varicosities, since NK 2 receptors were undetectable on mouse enteric glial cells (Delvalle et al., 2018). This earlier study used the same NK 2 receptor antibody as in the current study, indicating a species difference in the expression of NK 2 receptors on enteric glial cells.
U73122 showed a more prominent effect in males compared with females. This does not appear to be associated with the gene expression of PLC, since the mRNA expression levels of all PLCb isoenzymes were similar in both genders. The mechanism underlying the gender distinction is unclear but may involve differences in the protein levels of phosphorylated PLCb isoenzymes, which have not been determined in the current study. In addition to PLC inhibition, U73122 has been shown to inhibit the Ca 2+ pump on the sarcoplasmic reticulum to deplete Ca 2+ (MacMillan and McCarron 2010).
Interestingly, NK 2 receptor-mediated contractile responses in the human colon do not appear to be related to IP 3 receptor activation. In accordance, inositol phospholipid hydrolysis stimulated by LMN-NKA has been previously shown to be much weaker in human colonic smooth muscle compared with rat bladder positive controls (Warner et al., 2000). In addition, NKA-evoked contractions of human colonic smooth muscle were unaffected by intracellular Ca 2+ depletion (O'Riordan et al., 2001). Thus, NK 2 receptor-induced intracellular Ca 2+ release by IP 3 receptor activation is a minor pathway in mediating human colon contractility.
NKA and LMN-NKA evoked contractile responses that were significantly diminished by PKC and Rho kinase inhibitors, suggesting that Ca 2+ sensitization contributes to NK 2 receptor-mediated contractility of the human colon. Ca 2+ sensitization is the phenomenon by which smooth muscle contractions can be sustained independently of Ca 2+ , by PKCand Rho kinase-mediated inhibition of myosin light-chain phosphatase, which prevents the dephosphorylation of myosin light chain (Puetz et al., 2009;Perrino, 2016). Previously, the contribution of PKC and Rho kinase to Ca 2+ sensitization evoked by muscarinic receptor activation has been demonstrated in membrane-permeabilized rat colonic muscle strips (Takeuchi et al., 2004(Takeuchi et al., , 2007. This is the first report demonstrating the role of PKC and Rho kinase signaling in NK 2 receptor-activated smooth muscle contractions of the human colon.
In conclusion, the present study has revealed an increased NK 2 receptor activity in females, which may underlie the gender differences in the treatment of diarrhea-predominant IBS. These gender-related differences may be due to the influence of ovarian hormones, since the expression of NK 2 receptors increases with age in females. These findings highlight that gender differences should be considered in the therapeutic development of NK 2 receptor agents. The pronounced localization of NK 2 receptors on enteric glial cells in the human colon suggests that aside from direct myogenic activation, NK 2 receptors may also have an important neuroregulatory role. Furthermore, the current study indicates the importance of both Ga q/11 -PKC and Ga 12/13 -Rho kinase signaling pathways in NK 2 receptor-mediated contractile responses in the human colon.