Animals
Five-week-old male C57BL/6JJmsSlc mice (N =130; Japan SLC Inc., Shizuoka, Japan) were purchased and housed in plastic cages (170 W × 240 D × 125 H mm, Clea, Tokyo, Japan) under a 12-h light (200 lx of fluorescent light) / dark cycle (12L12D, 0800 light ON, 2000 light OFF), maintained at a constant temperature (23 ± 1 °C). Food (CE-2; CLEA) and water were provided ad libitum. All animal experiments were approved by the Committee of Animal Care and Use of the Aichi Medical University. All experimental procedures were conducted in accordance with the institutional guidelines for the use of experimental animals.
IOP measurement
IOP measurements were performed using a tonometer (Icare TonoLab, TV02; Icare Finland Oy, Espmoo, Finland), as previously reported 8,19. All mice were kept under 12L12D conditions for more than 2 weeks before IOP measurements. Unanesthetized mice were gently held using a sponge. IOPs were measured during the light phase under light (200 lx) conditions and during the dark phase under dim red-light conditions. To analyze nocturnal IOP increase, IOP was obtained by measuring the IOP at zeitgeber time (ZT) 10 and ZT15. ZT0 (0800) was defined as the time of light ON. For diurnal changes in IOP, IOP was measured at ZT6 and ZT15 2 weeks after bead injection. For the analysis of dobutamine-mediated IOP induction, IOP was measured before drug instillation at ZT4 and measured at ZT9.
Intraocular injection and detection of fluorescence particles
To investigate the effect of AH inflow on nocturnal IOP increase, mice were anesthetized by isoflurane inhalation (2%; WAKO, Saitama, Japan), supplemented with topical proparacaine HCl (0.5%; P2156, Tokyo, Tokyo Chemical Industry [TCI]), and were treated with an intraocular injection of an Na+/K+ATPase inhibitor ouabain (100 µM / 0.1% DMSO phosphate-buffered saline [PBS], 2 µL) into the right eye with a 34-gauge needle (0.18 × 8 mm, Pasny; Unisis) connected to a Hamilton syringe at ZT10. To precisely control the small volume (3 μL) of anterior chamber injection, 3 μL PBS (0.1% DMSO) was injected into the left eye with a 34-gauge needle connected to a Hamilton syringe.
For microbead injection to prevent AH outflow, mice were anesthetized by isoflurane inhalation (2%; WAKO, Japan), supplemented with topical proparacaine HCl. IOP elevation was induced unilaterally in adult C57BL/6J mice by injection into the anterior chamber of 3 µL of 1/10 diluted fluosphere polystyrene microspheres (15 µm, yellow-green fluorescent, F8844, Invitrogen, Carlsbad, CA) of the right eye with a 34-gauge needle connected to a Hamilton syringe. Microbeads were then resuspended in PBS at 5.0 × 106 beads/mL. To precisely control the small volume (3 μL) of anterior chamber injection, 3 μL PBS was injected into the left anterior chamber with a 34-gauge needle connected to a Hamilton syringe.
To visualize the AH outflow in the SC, mice were anesthetized by isoflurane inhalation (2%; WAKO, Japan), supplemented with topical proparacaine HCl, and were treated with an intraocular injection of 3 µL of carboxylate-modified microspheres (0.5 µm, yellow-green fluorescent, 2% solids, F8813, Invitrogen) at ZT0 and ZT12. After 6 h, the injected mice were anesthetized, and their anterior eyes were extracted and fixed in 4% paraformaldehyde (26126-25, Nakarai) / PBS for 5 min. After being washed with PBS, anterior eye cups were placed in Hanks‘ Balanced Salt Solution (HBSS; 082-08961 WAKO) in a 96-well microplate to observe the fluorescence from the bottom by digital fluorescent microscope Dino-Lite Edge M Fluorescence TGFBW (Opto Science Inc.), and the fluorescence intensity (525 nm) was measured using a microplate reader SpectraMax M5 (Molecular Devices).
iHTMC culture
The immortalized human TM-SV40 cell line (iHTMC) derived from primary human SC and TM region was purchased from Applied Biological Materials Inc. (T-371-C, ABM Inc., Richmond, BC, Canada) and cultured in TM cell medium (6591, Sciencell, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (0010, Sciencell), 1% growth supplement (TMCGS, 6592, Sciencell) and 1% penicillin/streptomycin (0503, Sciencell) in type I collagen-coated 100-mm dish (3020-100, IWAKI, Japan). Experiments were performed in type I collagen-coated plates. Upon reaching confluence, iHTMCs were split 1:4 using 0.05% trypsin/PBS. Cell viability was determined using trypan blue (0.4%) exclusion.
Phagocytosis assay and drug treatment
For phagocytosis assay, iHTMCs (T0371, abm) were plated in collagen I-coated 96-well microplates (4860-010; IWAKI) at a density of 5.0 × 103 cells/well in trabecular meshwork cell medium (TMCM) supplemented with 1% penicillin/streptomycin and growth factors (6591; Sciencell). To measure phagocytosis, pHrodo Green Zymosan Bioparticles (P35365; ThermoFisher) were resuspended in PBS and vortexed to disperse. After 90% confluence, the medium was removed by aspiration, and 100 μL of serum-free TMCM was immediately added. After 24 h, the medium was replaced with serum-free TMCM containing pHrodo Zymosan (2.5 µg / well) in the presence of several kinds of drugs, and the plate was placed in the Incucyte ZOOM instrument (Essen Bioscience, Ann Arbor, MI, USA), installed in a 5% CO2 incubator at 37 °C. Each well was imaged at 3 points, every 0.5 h or 1 h for more than 72 h using the phase and green fluorescence channels and the 10× objective. No pHrodo Zymosan was used for fluorescent control using background fluorescent intensity because of autofluorescence in TMCM, and vehicle control included 0.1% DMSO. At the end of the experiment, the fluorescence intensity at each time point in each well was measured using the IncuCyte ZOOM 2015A software (Essen Bioscience). To perform the detailed analysis of the pulse stimulation, we calculated the difference from the control. We calculated the average daily fluorescence intensity normalized to that of the control DMSO treatment group to show statistical changes. The phagocytosis assay was independently repeated three times using four biological replicates. Data were combined and averaged, and the standard error was calculated.
Drug treatment
For the phagocytosis assay, iHTMCs were simultaneously treated with (−)-NE (0.1, 1, and 10 µM; S9507, Selleck) and/or dexamethasone (Dex; 0.1, 1, and 10 µM; 11107-51, Nakarai Tesk) and pHrodo Zymosan. To confirm the phagocytic activity in iHTMC, we treated to iHTMCs with the phagocytosis inhibitor Cytochalasin D (0.01 nM; 11330, Cayman). For detailed analysis of pulse stimulation, serum-free TMCM including NE (1, 10, and 100 µM) or Dex (0.1, 1, and 10 µM) were added to iHTMC for 30 min. After being washed with TMCM, pHrodo zymosan-containing TMCM was added to iHTMC. For analysis of effect of agonists on phagocytosis, we treated several kinds of agonists for AR agonist L-Adrenaline (A0173, TCI), β1-AR agonists (L-Noradrenaline Bitartrate Monohydrate [L-NE, A0906, TCI] and dobutamine hydrochloride [15582, Cayman], β1β2-AR agonist [Isoproterenol Hydrochloride (I0260, TCI), selective α1-AR agonist [L-Phenylephrine (P0395, TCI)], direct-acting α2-AR agonist [Clonidine HCl (038-14291, WAKO)], β2-AR agonist [Formoterol fumarate hydrate (F0881, TCI)], short-acting β2-AR agonist (Salbutamol Hemisulfate [S0531, TCI]), PGE2 (0.1, 1, and 10 µM; P1884, TCI), cAMP inducers (Forskolin [10 µM FSK; F0855, TCI] and 3-Isobutyl-1-methylxanthine [10 µM IBMX; 095-03413, WAKO]), cAMP analogs as PKA activator Sp-cAMP (10 µM; 14983, Cayman) and as Epac/PKA activator 8-CPT-cAMP (10 µM; 12011, Cayman) to iHTMC. For analysis of antagonists for ARs, NE (5 µM) were simultaneously added with antagonists; β1β2-AR antagonist Timolol Maleate (1, 10, and 100 µM; T2905, TCI), β1-AR antagonist Betaxolol hydrochloride (1, 10, and 100 nM; B4474, TCI), β1-AR antagonist ICI-118.551 hydrochloride (0.01, 0.1, and 1 µM; HY-13951, MCE), α2-AR antagonist phentolamine mesylate (100 µM; P1985, TCI), PKA inhibitor KT5720 (0.001, 0.01, 0.1, and 1 µM; 10011011, Cayman), EAPC1/2 inhibitor ESI09 (0.1, 1, 10, and 100 µM; 19130, Cayman), PTEN inhibitor bpV (pic) (0.001, 0.01, 0.1, and 1 µM; SML0885, Sigma-Aldrich), and 3α-aminocholestane (0.01, 0.1, 1, and 10 µM; 3AC; HY-19776, MCE). To regulate PIP3 content, we treated iHTMC with the PIP3 antagonist PITenin-7 (0.01, 0.1, 1, and 10 µM; 524618, Calbiochem), PI3Kαβδ inhibitor LY294002 (70920, Cayman), and selective PI3Kγ inhibitor CAY10505 (HY-13530, MCE).
Western blot analysis
Western blot analysis was performed as previously our report 71. Protein extraction was performed using cell lysis buffer (9803, Cell Signaling Technology, Tokyo, Japan) containing protease inhibitor cocktail (P8340, Sigma) and phosphatase inhibitor cocktail 1 (P2850, Sigma) according to the manufacturer’s instructions. Total protein transferred to the PVDF membrane was detected using EzStainAQua MEM (WSE-7160, ATTO) and used for normalization. After destaining, membranes were incubated with the following primary antibodies: rabbit monoclonal antibodies against phospho-CREB (Ser133) (1:1000; 9198, Cell Signaling Technology), CREB (1:1000; 9192, Cell Signaling Technology), Akt (pan) (C67E7) (1:1000; 4691, Cell Signaling Technology), and rabbit polyclonal antibody against phospho-PKCα/βII (Thr638/641) (1:1000; 9375, Cell Signaling Technology), phospho-Akt (Thr308) (1:1000; 9275, Cell Signaling Technology), phosphor-CaMKII alpha (Thr286) (1:1000; ab5683,abcam), ERK1 / ERK2 (1:1000; A16686, ABclonal), phospho-ERK1(T202/Y204) /ERK2(T185/Y187) (1:1000; AP0472, ABclonal), INPP5D (SHIP1) (1:1000; A0122, ABclonal), phosphor-INPP5D (Tyr1021) (1:1000; PA903060, CSB), and mouse monoclonal antibody against CaMKIIα/β/γ/δ (G-1) (1:500; sc-5306, Santa Cruz Biotechnology). Membranes were washed and then incubated with HRP-conjugated goat polyclonal antibody against mouse and rabbit IgG (1:10,000; 7074, Cell Signaling Technology). Chemiluminescent images were detected using an Amersham Imager 600 (Cytiva Lifescience).
cAMP measurement
cAMP measurements were performed with a homogeneous TR-FRET immunoassay using the LANCE cAMP Detection Kit (AD0262, PerkinElmer, USA), according to the manufacturer’s instructions (PerkinElmer). After confluence, iHTMCs in collagen I-coated 96-well microplates (4860-010; IWAKI) were washed with PBS (0.2 g/L EDTA), and then washed with stimulation buffer (HBSS, 5 mM HEPES, 0.5 mM IBMX, and 0.01% BSA at pH 7.4). After aspiration, iHTMC was added to 10 µL of tested compounds with FSK (0.001, 0.01, 0.1, and 1 µM), L-NE (0.01, 0.1, 1, and 10 µM), and dobutamine (0.001, 0.01, 0.1, 1, and 10 µM), and 10 µL of Alexa Fluor 647 anti-cAMP antibody diluted with stimulation buffer. The cells were then stimulated for 60 min at room temperature. The antagonist response analysis was performed using L-NE or dobutamine as the reference agonist. To analyze the antagonist effect on β1-AR, the β1-AR agonist L-NE or dobutamine was used at submaximal concentrations (10 µM and 1 µM, respectively) to stimulate cAMP accumulation. These agonists and the β1-AR antagonist betaxolol (0.05, 0.5, 5, and 50 µM) were simultaneously added. After incubation, the reaction was stopped, and cells were lysed by the addition of 20 µL working solution (10 µL Eu-cAMP and 10 µL ULight-anti-cAMP), and incubated for 1 h at room temperature. The TR-FRET signal was read using a microplate reader SpectraMax M5 (Molecular Devices). cAMP concentrations were determined using GraphPad Prism software (version 6.0; GraphPad Software Inc., San Diego, CA).
Small interfering RNA knockdown
iHTMCs were seeded in quadruplicate into collagen I-coated 96-well microplates (4860-010; IWAKI) at a density of 5.0 × 103 cells/well with serum-free TMCM (1% penicillin/streptomycin; #6591; Sciencell). Twenty-four hours later, cells were transfected with 0.04 µM Accell SMARTpool siRNA against ADRB1, ADRB2, RAPGEF3, RAPGEF4, and PRKACA (Table EV1, Dharmacon), Accell GAPDH pool-human siRNA (D-001930) as a positive control, and Accell Non-targeting pool siRNA (D-001910) as a negative control using 100 µL Accell Delivery Media (B-005000-100; Dharmacon) according to the manufacturer’s instructions. Twenty-four hours later, NE (1 µM; 20 µL/well) or 0.1% DMSO with PBS containing pHrodo Green Zymosan Bioparticles (2.5 µg / well; P35365) were added. After 24 h of exposure to siRNA, total RNA was extracted and purified as described above for knockdown confirmation by qPCR.
Drug instillation
Drug instillation was performed as described in our previous report 19. Unanesthetized 8-week old male mice were used in this study. For nocturnal IOP increase analysis, mice were instilled with a single drop (30 μL) of betaxolol (100 µM / 0.1% DMSO PBS), KT5720 (100 µM), ESI09 (100 µM), 3AC (1 mM), SHIP1 inhibitor K118 (1 mM; B0344, Echelon Biosciences), and bpV (pic) (100 µM) at ZT10 using a micropipette into bilateral eyes, and IOP was measured at ZT15. To analyze the inhibitory efficacy of dobutamine-induced IOP increase, the above antagonists were preinstillated at ZT4, and then, after 10 min, a single drop of dobutamine (100 µM / 0.1% DMSO PBS) was added. During instillation, the mice were gently restrained with the necks held back.
PIP3 extraction and quantification
PIP3 measurements were performed using ELISA kits (Echelon Biosciences, K-2500s) according to the manufacturer’s instructions. iHTMCs were seeded at a density of 1.2 × 106 cells/collagen I coated 6 well dish (IWAKI). After 14 h of treatment with dobutamine (1 μM) with or without agonists, KT5720 (10 μM), ESI09 (1 μM), bpV (pic) (1 μM), and 3AC (10 μM), the media were removed by aspiration and 1 mL of ice-cold 0.5 M tricarboxylic acid (TCA) was immediately added. Cells were scraped, transferred into a 1.5-mL tube on ice, and centrifuged at 3,000 rpm for 7 min at 4°C. The pellet was resuspended in 5% TCA/1 mM EDTA (0.5 mL), vortexed for 30 s, and centrifuged at 3,000 rpm for 5 min at room temperature. After discarding the supernatant, this washing step was repeated once more. Next, neutral lipids were extracted by adding 0.5 mL of MeOH:CHCl3 (2:1) and continuously vortexing for 10 min at room temperature. After centrifugation at 3,000 rpm for 5 min, the supernatant was discarded, and the extraction step was repeated one more time. The acidic lipids were extracted by adding 0.5 mL MeOH:CHCl3:12 M HCl (80:40:1) with continuous vortexing for 25 min at room temperature. Extracts were centrifuged at 3,000 rpm for 5 min, and the supernatant was transferred to a new 1.5 mL tube; 0.15 mL of CHCl3 and 0.27 mL of 0.1 M HCl were added to the supernatant, vortexed, and centrifuged at 3,000 rpm for 5 min to separate organic and aqueous phases. The organic (lower) phase 0.3 mL was transferred into a new vial for PIP3 measurement. All the samples were dried for 4 h at 4°C. PIP3 samples were resuspended in 125 μL of PBS-Tween+3% Protein Stabilizer (provided by the Echelon kit). Samples were sonicated in an ice-water bath for 5 min, vortexed, and spun down before being added to the ELISA. All experiments were performed three times, each performed in triplicate. The lipid amount (60 μL) was used and was always run twice for each sample. After color reaction for 30 min in the dark, the 96-well plate was read by measuring the absorption at 450 nm with the SpectraMax M5 (Molecular Device). PIP3 concentrations were determined using GraphPad Prism software (version 6.0; GraphPad Software Inc., San Diego, CA).
Statistical analysis
All data are shown as the mean ± standard error of the mean (SEM). Statistical comparisons were made using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA) or Excel-Toukei 2012 software (Social Survey Research Information Co. Ltd., Osaka, Japan). Paired or Student’s t-tests were used to compare two groups, and one-way ANOVA with Tukey’s multiple comparison test or Dennett’s multiple comparison test for more than two groups. Differences were considered statistically significant at p < 0.05.