Preparation of Canine LGA
After an overnight fast, 96 mongrel adult healthy dogs of either sex, weighing 15–30 kg, obtained from the Section of Surgical Teaching, Jilin University, were sacrificed by rapid exsanguination from the common carotid artery under deep anaesthesia with sodium pentobarbital (30 mg kg−1, i.v.). The connective tissues and fat were carefully dissected under a dissecting microscope (SZ61, Olympus, Japan) avoiding over-pulling and clamping.
Immunostaining of MLNR
Immunohistochemical staining of MLNR
The LGAs were procured from six dogs. The paraffin-embedded tissues were cut into 4-μm sections, deparaffinised, rehydrated, boiled in retrieval solution and washed in PBS. After blocking in normal horse serum for 30 min, the sections were incubated with rabbit anti-dog MLNR antibody (1:100; RaQualia Pharma Inc., Taketoyo, Japan) overnight at 4°C . The next day, the sections were incubated with biotinylated goat anti-rabbit antibody (Sigma, St. Louis, MO, USA) and horseradish peroxidase-conjugated avidin (Sigma, Shanghai, China) for 30 min at room temperature. The stained sections were then visualised using 3,3′-diaminobenzidine (Sigma, St. Louis, MO, USA). For negative controls, the primary antibody was replaced with affinity-purified pre-immune IgG.
Immunofluorescence staining of MLNR
The LGAs were procured from three dogs. The 6-μm paraffin-embedded sections were deparaffinised, incubated in 0.1% Triton X-100 at 4°C for 1 h and washed three times with PBS. After 5-min digestion with proteinase K, the sections were washed with PBS and blocked in 5% normal goat serum for 30 min. Subsequently, the sections were incubated with rabbit anti-dog MLNR antibody (1:100, RaQualia Pharma Inc., Taketoyo, Japan) and mouse anti-dog cluster of differentiation 34 (CD34) antibody (1:100; Affymetrix, Santa Clara, CA, USA) overnight at 4°C. After three 10-min washes with PBS, the sections were incubated with fluorescein isothiocyanate-labelled (green) goat anti-rabbit immunoglobulin (Ig)G (1:50; Abbkine, California, USA) and Cy3-labelled (red) goat anti-mouse IgG (1:100; Abbkine, California, USA) for 1 h at room temperature. The sections were washed again three times with PBS for 10 min, incubated with Hoechst 33342 (Sigma, Shanghai, China) for nuclear staining for 5 min, washed twice with PBS for 5 min each and finally mounted. The slides were visualised and photographed under a confocal microscope (FV1000; Olympus, Tokyo, Japan). For negative controls, the primary antibody was replaced with affinity-purified pre-immune IgG. All images were processed using FV10-ASW 1.7 software (Olympus, Tokyo, Japan).
Porcine motilin (Peptide Institute Inc., Osaka, Japan), acetylcholine chloride (ACh) (Sigma, Shanghai, China), GM-109 (Peptide Institute Inc.), atropine sulfate (Atropin; Neptunus, Shenzhen, China), N-nitro-L-arginine methyl ester (L-NAME) (Sigma), tetraethylammonium chloride (TEA) (Yuanye, Shanghai, China) and ethylene glycol tetra-acetic acid (EGTA) (Sigma) were dissolved in distilled water. N-ethylmaleimide (NEM) (Sigma) was dissolved in distilled water but shielded from light, and the pH was controlled between 7.0 and 7.35. U73122 (MCE, Shanghai, China), 2-aminoethyl diphenylborinate (2-APB) (Sigma), chelerythrine chloride (MCE), (N-2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamidehydrochloride (H89) (MCE), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (Sigma), indomethacin (MCE), 18α-glycyrrhetinic acid (18α-GA) (Sigma), glibenclamide (MCE) and verapamil (MCE) were dissolved in dimethylsulfoxide (DMSO) (Solarbio, Beijing, China). U46619 (Sigma) was dissolved in 96% ethanol to 0.4 mM stock and further diluted with distilled water before use. Concentrations refer to the final concentration of the drugs in the bath and are expressed as mol L−1 (M). The DMSO and ethanol in the bath were set to <0.4% and 0.1% (v/v), respectively, which produced no changes in the tonus of the vascular preparations.
Record of Isometric Vascular Tone
Each LGA was approximately 18–30 mm long and was cut into six to ten 3-mm length rings which were immediately mounted between two L-shaped stainless-steel hooks (300 μm in diameter) in the organ bath of the multi-wire myograph system (DMT620, Demark). The organ bath contained a 5-mL modified Krebs–Henseleit bicarbonate buffer (Krebs solution) composed of 118.0 mM NaCl, 4.7 mM KCI, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3 and 10 mM glucose (pH 7.4)  and continuously supplied with 95% O2 and 5% CO2. The temperature and pH of the buffer were maintained at 37°C and 7.40. Tension was recorded using LabChart Data Acquisition Software (LabChart 8.0; ADInstruments, Australia). The rings were passively stretched to a tension of approximately 15 mN, maintained for approximately 60 min and washed every 15 min. After stabilisation of the tension, the viability of the rings was assessed by 60 mM KCl (60 mM KCl Krebs solution made by replacing NaCl with equimolar KCl) at least twice.
Subsequently, U46619 (a thromboxane A2 analogue) (5 × 10−8 M) was used. Once a sustained contract tension was reached, one concentration of motilin was examined to avoid tachyphylaxis . Endothelium-denuded rings were made by gently scraping the endothelial cells with a pair of pointed metal forceps before mounting. In the inhibitor groups, the rings were first incubated with different inhibitors for 15–40 min before adding U46619; rings from the same LGA incubated with saline were considered as the control group. In the Ca2+-free Krebs solution, Ca2+ was replaced with 1 mM EGTA. The endothelium integrity or functional removal was verified by acetylcholine (10−5 M) at the end of each test, with the relaxation rate (RR) >80% or <10%, respectively.
The RR was expressed as a percentage decrease in the tension induced by U46619 and/or high-potassium solution according to the following formula:
In the calculation of the inhibition rate (IR), the tension was normalised to the corresponding values of the control group:
where T is a sustained tension, L is the lowest tension, i represents the inhibitor group and c represents the control group.
Concentration–response curves were analysed by nonlinear regression analysis with variable slopes in GraphPad Prism 6 (GraphPad Software, San Diego California, USA), from which the ECx (x% maximal effective concentration) and HillSlope were obtained. The pA2 value was calculated according to the Van Rossum equation:
where, A is the molar concentration of the antagonist and CR is the ratio of the EC50 value (EC50 with an antagonist/EC50 without an antagonist) .
Measurement of NO and cGMP Levels in LGA
Tissue collection and homogenisation were conducted based on the methods provided by Schachter et al . LGAs from three dogs were pooled together to provide sufficient tissue for analysis at each time. When NO was detected, seven groups were created. Three groups were first incubated with 5 × 10−8 M U46619 (5 × 10−8 M) followed by saline, motilin (9 × 10−8 M) or acetylcholine (10−5 M) respectively. Four groups were incubated with GM109 (10−5 M), L-NAME (10−4 M), H89 (5 × 10−6 M) or chelerythrine (2 × 10−6 M) before U46619 (5 × 10−8 M) and motilin (9 × 10−8 M) were added. When cGMP was detected, the groups were the same as those in which NO was detected, except that the ODQ (10−5 M) group was included. U46619 (5 × 10−8 M) was the negative group (blank column), motilin (9 × 10−8 M) added after U46619 (5 × 10−8 M) was the positive control group (motilin column), inhibitors added before U46619 and motilin were the experimental groups and acetylcholine (10−5 M) was used to confirm the activity of endothelial cells (acetylcholine group).
Tissues were homogenised in the following solution: 10.0 mM Tris-HCL, 0.1 mM EDTA-2Na, 10 mM sucrose and 136.7 mM NaCl (pH 7.4) in 1:9 ratio of weight (g) to volume (mL). The homogenate mixture was then centrifuged at 4°C, 2.4 g (5000 rpm) for 10 min, and the supernatant was used for detection. We strictly adhered to the protocol for the detection process. The total protein concentration was assayed using a bicinchoninic acid (BCA) total protein assay kit (A045-3; Jiancheng, Nanjing, China) and bovine serum albumin was used as the standard and expressed in μmol mL−1. The cGMP concentration was assayed using a canine cGMP ELISA kit (Cat No. ela05471Ca, SANCHEZ, Colorado, USA) and expressed in picomoles of cGMP per milligram of protein (pmol mg−1). The NO concentration was assayed using a nitrate reductase system kit (S0023; Beyotime, Haimen, China) and expressed in micromoles of NO per gram of protein (μmol g−1).
All numerical data are presented as means ± standard error of the mean (SEM) with n equal to the number of dogs in the multi-wire myograph system and N equal to the number of repetitions in other experiments. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software). Data analyses were performed using a paired or unpaired t -test for paired or unpaired data, and one-way analysis of variance followed by Dunnett’s post-hoc test for multiple comparisons. P < 0.05 was considered statistically significant.