2023 Volume 46 Issue 7 Pages 997-1003
Platelet-activating factor (PAF) not only acts as a mediator of platelet aggregation, inflammation, and allergy responses but also as a constrictor of various smooth muscle (SM) tissues, including gastrointestinal, tracheal/bronchial, and pregnancy uterine SMs. Previously, we reported that PAF induces basal tension increase (BTI) and oscillatory contraction (OC) in mouse urinary bladder SM (UBSM). In this study, we examined the Ca2+ influx pathways involved in PAF-induced BTI and OC in the mouse UBSM. PAF (10−6 M) induced BTI and OC in mouse UBSM. However, the PAF-induced BTI and OC were completely suppressed by extracellular Ca2+ removal. PAF-induced BTI and OC frequencies were markedly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors (verapamil (10−5 M), diltiazem (10−5 M), and nifedipine (10−7 M)). However, these VDCC inhibitors had a minor effect on the PAF-induced OC amplitude. The PAF-induced OC amplitude in the presence of verapamil (10−5 M) was strongly suppressed by SKF-96365 (3 × 10−5 M), an inhibitor of receptor-operated Ca2+ channel (ROCC) and store-operated Ca2+ channel (SOCC), but not by LOE-908 (3 × 10−5 M) (an inhibitor of ROCC). Overall, PAF-induced BTI and OC in mouse UBSM depend on Ca2+ influx and the main Ca2+ influx pathways in PAF-induced BTI and OC may be VDCC and SOCC. Of note, VDCC may be involved in PAF-induced BTI and OC frequency, and SOCC might be involved in PAF-induced OC amplitude.
Platelet-activating factor (PAF) is a phospholipid mediator that causes platelet aggregation.1) PAF is known to be involved in inflammation, allergic reactions, and shock,1) and has been demonstrated to act as a constrictor of various smooth muscle (SM) tissues, including gastrointestinal,2–6) tracheal/bronchial,7–9) and pregnant uterine SMs.10,11) In addition to these SMs, PAF was previously found to induce basal tension increase (BTI) and oscillatory contraction (OC) in mouse urinary bladder (UB) SM (UBSM).12) Although PAF-induced contractile responses in gastrointestinal and pregnant uterine SMs were demonstrated to depend on Ca2+ influx via voltage-dependent Ca2+ channel (VDCC),3–5,10) the dependence of Ca2+ influx on PAF-induced BTI/OC in mouse UBSM and these Ca2+ influx pathways remain unclear. We examined the Ca2+ influx pathway involved in PAF-induced BTI/OC in mouse UBSM.
Male mice (age, 8–12 weeks old; weight, 33–52 g; Japan SLC, Hamamatsu, Japan) were housed under controlled conditions (temperature, 21–22 °C; relative air humidity, 50 ± 5%) at a fixed 12 h light-dark cycle (08 : 00–20:00) with access to food and water ad libitum. This study was approved by the Toho University Animal Care and User Committee (approval numbers: 21-52-444/22-53-444) and was conducted in accordance with the user guidelines of the Laboratory Animal Center of the Faculty of Pharmaceutical Sciences, Toho University.
Measurement of PAF-Induced BTI and OC in Mouse UBSMPAF-induced BTI/OC were isometrically measured according to a previous report.12) Briefly, UBSM strips were suspended under resting tension (0.5 g) in a 20 mL organ bath filled with Locke–Ringer solution containing (mM) NaCl, 154; KCl, 5.63; CaCl2, 2.16; MgCl2, 2.1; NaHCO3, 5.95; and glucose, 2.78. The solution was stored at 32 ± 1.0 °C and bubbled with a mixture of 95% O2/5% CO2. The UBSM strips were equilibrated for 20 min and then contracted using acetylcholine (ACh, 10−4 M) at least three times at 10-min intervals. After stabilization of the UBSM basal tension, verapamil (10−5 M), diltiazem (10−5 M), nifedipine (10−7 M) (VDCC inhibitors), ethanol (EtOH) (nifedipine vehicle), verapamil plus LOE-908 (a receptor-operated Ca2+ channel (ROCC) inhibitor, 3 × 10−5 M), verapamil plus LOE-908 vehicle (dimethyl sulfoxide (DMSO)), or verapamil plus SKF-96365 (a store-operated Ca2+ channel (SOCC)/ROCC inhibitor, 3 × 10−5 M) was administered. The inhibitor cocktail (phentolamine (an α-adrenoceptor antagonist, 10−6 M)/propranolol (a β-adrenoceptor antagonist, 10−6 M)/atropine (a muscarinic receptor antagonist, 10−6 M)/suramin (a purine P2 receptor antagonist, 10−4 M)/tetrodotoxin (a Na+ channel inhibitor, 3 × 10−7 M)), anti-foam (0.005%), and bovine serum albumin (BSA, 0.25%) were also administered, and the strips were incubated for 60 min. The inhibitor cocktail was used to eliminate the potential effects of peripheral nerve-derived neurotransmitters. After a 60-min incubation, PAF (10−6 M) was administered, and the strips were incubated for 60 min. Thereafter, papaverine (PPV, 10−4 M) was administered to relax the UBSM strips. To determine the effects of Ca2+-free solution, the inhibitor cocktail was added and incubated for 20 min. The bath solution was replaced with Ca2+-free Locke–Ringer solution containing 0.2 mM ethylene glycol tetraacetic acid (EGTA), the inhibitor cocktail, anti-foam, and BSA at 10 min before PAF administration. All experiments were performed in the presence of indomethacin (a cyclooxygenase inhibitor, 3 × 10−6 M).
Measurement of Intracellular Ca2+ Concentrations ([Ca2+]i) in 293T CellsMeasurement of [Ca2+]i in 293T cells was performed as previously described.13–15) Briefly, seeded 293T cells in a 96-well plate were incubated with recording medium [(mM): 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 20; NaCl, 115; KCl, 5.4; MgCl2, 0.8; CaCl2, 1.8; glucose, 13.8; pH 7.4] containing Fura-2 AM (5 × 10−6 M) for 30–60 min at 37 °C/5% CO2. The cells were then rinsed with Fura-2 AM-free/Ca2+-free medium, and their fluorescence intensities were measured using a microplate reader (Nivo, PerkinElmer, Inc., MA, U.S.A.) at 25 °C in Ca2+-free medium. Following treatment for 10 min in the presence of LOE-908/SKF-96365 (3 × 10−5 M each), cyclopiazonic acid (CPA, 10−5 M) was applied. After 5-min incubation, Ca2+ (1.8 mM) was applied and the fluorescence intensity was measured for 5 min. Changes in the ratio of fluorescence intensities at 510 nm emission generated by excitation at 340 nm/380 nm (F340/380) were considered the relative changes in [Ca2+]i. After the experiment, ionomycin (5 µM) and Mn2+ (50 mM) were applied to determine background fluorescence; this background fluorescence was subtracted from the fluorescence intensities of all measurements.
DrugsThe following drugs were used in this study: PAF C-16/CPA (Cayman Chemical, Ann Arbor, MI, U.S.A.); SKF-96365 (Cayman Chemical/Tokyo Chemical Industry Co., Ltd., Tokyo, Japan); (±)-verapamil hydrochloride/diltiazem hydrochloride/DL-propranolol hydrochloride/atropine sulfate salt monohydrate/indomethacin (Sigma-Aldrich Co., St. Louis, MO, U.S.A.), nifedipine (Tokyo Chemical Industry Co., Ltd.); LOE-908 (Nippon Boehringer Ingelheim Co., Ltd., Hyogo, Japan/Bio-Techne, Minneapolis, MN, U.S.A.); ACh chloride (Daiichi Sankyo Co., Ltd., Tokyo, Japan); tetrodotoxin/anti-foam (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan.); suramin sodium (FUJIFILM Wako Pure Chemical Corporation/Merck KGaA, Darmstadt, Germany); Ionomycin calcium (Merck KGaA); phentolamine mesylate (Novartis Pharma KK, Tokyo, Japan), and BSA (fatty acid free, pH 7.0; Nacalai Tesque Inc., Kyoto, Japan). PAF was dissolved in ethanol (EtOH) to prepare a stock solution of 2 × 10−3 M and stored at −80 °C. When PAF was used, the EtOH solvent was evaporated, and PAF was re-dissolved/diluted with 0.25% BSA to prepare a 2 × 10−4 M solution. Indomethacin/nifedipine/ionomycin were dissolved in EtOH to prepare stock solutions of 10−2 M/2 × 10−4 M/10−2 M, respectively. LOE-908/CPA were dissolved in DMSO to prepare a stock solution of 6 × 10−2 M/1.5 × 10−2 M, respectively. Anti-foam was diluted 10-fold with EtOH. All other drugs were dissolved/diluted with distilled water.
Data AnalysisPAF-induced BTI and the amplitude/frequency of PAF-induced OC were analyzed as previously reported.12,16) Briefly, BTI/OC were calculated over 3 min using the LabChart™ (Version 7) software (ADInstruments Pty. Ltd., Bella Vista, NSW, Australia): immediately before PAF administration (control) and 7–10, 17–20, 27–30, 37–40, 47–50, 57–60 min after PAF administration. The cut-off value was set to 2% of the third 10−4 M ACh-induced contractions for these analyses. BTI displayed basal tension changes before and after PAF administration. BTI and OC amplitude are shown as relative values, with the third 10−4 M ACh-induced contraction set to 100%. Data are expressed as mean ± standard error of the mean (S.E.M.), where n refers to the number of experiments. Statistical analyses were performed using two-way ANOVA followed by Šidák’s test or Student’s/Welch’s t-test on GraphPad Prism™ (Version 6) (GraphPad Software, Inc., San Diego, CA, U.S.A.). Statistical significance was set at p < 0.05.
PAF (10−6 M) induced BTI (Fig. 1C) and OC (amplitude, Fig. 1D; frequency, Fig. 1E) in mouse UBSM (Fig. 1A) over 60 min in Ca2+-containing solution. However, the PAF-induced BTI/OC were completely suppressed in 0.2 mM EGTA-containing/Ca2+-free solution (Figs. 1B–E).
The Ca2+-free solution contained 0.2 mM ethylene glycol tetraacetic acid (EGTA). Bovine serum albumin (BSA, 0.25%) and anti-foam (0.005%) were added to the bath medium after the bath solution was changed. Data are expressed as the mean ± S.E.M. (each n = 5). * p < 0.05, ** p < 0.01 vs. the corresponding 2.16 mM Ca2+ value (two-way ANOVA followed by Šidák’s test). Horizontal axes: basal tension and oscillatory contraction (OC) were analyzed over 3 min during the following periods: immediately before administration of PAF (Ctrl, control); 7–10 min (10), 17–20 min (20), 27–30 min (30), 37–40 min (40), 47–50 min (50), and 57–60 min (60) after PAF administration. Vertical axis: basal tension increases (c, % of 10−4 M ACh) show over 3 min basal tension (minimum average amplitude of OC or average basal tension (if OC was not detected)) changes before and after PAF administration; amplitude (d, % of 10−4 M ACh) shows average amplitude of OC over 3 min; frequency (e, counts/min) shows the values dividing the total number of OC over 3 min by 3 min. ACh: acetylcholine, Inhibitors: atropine (10−6 M), suramin (10−4 M), phentolamine (10−6 M), propranolol (10−6 M), and tetrodotoxin (3 × 10−7 M), PPV: papaverine (10−4 M), w: wash out.
Verapamil (10−5 M, Fig. 2A), diltiazem (10−5 M, Fig. 2B), and nifedipine (10−7 M, Fig. 2C) significantly suppressed PAF-induced BTI (Figs. 2Ac, Bc, Cc) and OC frequency (Figs. 2Ae, Be, Ce) compared to those in the absence of these inhibitors. These inhibitors did not significantly inhibit PAF-induced OC amplitude (Figs. 2Ad, Bd, Cd) compared to that in the absence of the inhibitors, although these inhibitors slightly suppressed OC amplitude over 7–10 min after PAF administration.
Data are expressed as the mean ± S.E.M. (each n = 10). ** p < 0.05, ** p < 0.01 vs. the corresponding value in the absence of any inhibitor (two-way ANOVA followed by Šidák’s test). Horizontal axes: basal tension and oscillatory contraction (OC) were analyzed over 3 min during the following periods: immediately before administration of PAF (Ctrl, control); 7–10 min (10), 17–20 min (20), 27–30 min (30), 37–40 min (40), 47–50 min (50), and 57–60 min (60) after administration of PAF. Vertical axis: basal tension increases (c, % of 10−4 M ACh) show over 3 min basal tension (minimum average amplitude of OC or average basal tension (if OC was not detected)) changes before and after administrations of PAF; amplitude (d, % of 10−4 M ACh) shows average amplitude of OC over 3 min; frequency (e, counts/min) shows the values dividing the total number of OC over 3 min by 3 min. ACh: acetylcholine (10−4 M), Inhibitors: atropine (10−6 M), suramin (10−4 M), phentolamine (10−6 M), propranolol (10−6 M), tetrodotoxin (3 × 10−7 M), bovine serum albumin (BSA, 0.25%), and anti-foam (0.005%), EtOH: ethanol (0.05%), PPV: papaverine (10−4 M), w: wash out.
LOE-908 (3 × 10−5 M) plus verapamil (Fig. 3Ab) only slightly suppressed PAF-induced BTI (Fig. 3Ac) and OC amplitude (Fig. 3Ad) and frequency (Fig. 3Ae) compared to those in the presence of verapamil alone (Fig. 3Aa), although the decrease in BTI over 17–20 min and OC amplitude over 57–60 min after PAF administration was significant.
Data are expressed as the mean ± S.E.M. (each n = 20). * p < 0.05, ** p < 0.01 vs. the corresponding verapamil plus DMSO/verapamil value (two-way ANOVA followed by Šidák’s test). Horizontal axes: basal tension and oscillatory contraction (OC) were analyzed over 3 min during the following periods: immediately before administration of PAF (Ctrl, control); 7–10 min (10), 17–20 min (20), 27–30 min (30), 37–40 min (40), 47–50 min (50), and 57–60 min (60) after administration of PAF. Vertical axis: basal tension increases (c, % of 10−4 M ACh) show over 3 min basal tension (minimum average amplitude of OC or average basal tension (if OC was not detected)) changes before and after administrations of PAF; amplitude (d, % of 10−4 M ACh) shows average amplitude of OC over 3 min; frequency (e, counts/min) shows the values dividing the total number of OC over 3 min by 3 min. ACh: acetylcholine (10−4 M), Inhibitors: atropine (10−6 M), suramin (10−4 M), phentolamine (10−6 M), propranolol (10−6 M), tetrodotoxin (3 × 10−7 M), bovine serum albumin (BSA, 0.25%), and anti-foam (0.005%), PPV: papaverine (10−4 M), w: wash out.
SKF-96365 (3 × 10−5 M) plus verapamil almost completely suppressed PAF-induced OC amplitude (Figs. 3Bb, Bd) compared to that in the presence of verapamil alone (Fig. 3Ba). In addition, SKF-96365 strongly suppressed PAF-induced BTI (Fig. 3Bc) and OC frequency (Fig. 3Be), which was observed even in the presence of verapamil.
Effect of LOE-908 and SKF-96365 on [Ca2+]i Increases in the Presence of CPA in 293T CellsIn Ca2+-free medium, CPA (10−5 M) transiently increased [Ca2+]i in 293T cells (Figs. 4Aa, Ba). This increase could be elicited via Ca2+ release from sarcoplasmic reticulum (SR) by CPA-induced SR Ca2+-ATPase inhibition.17) This increase was enhanced by LOE 908 (3 × 10−5 M, Fig. 4Aa) and suppressed by SKF-96365 (3 × 10−5 M, Fig. 4Ba). CPA could activate SOCC by causing SR depletion.18) When Ca2+ (1.8 mM) was added to the medium in the presence of CPA, a rapid increase in [Ca2+]i, presumed to be mediated by SOCC, was observed (Figs. 4Aa, Ba). LOE-908 (3 × 10−5 M, Figs. 4Aa, Ab) did not suppress this increase; however, it was strongly suppressed by SKF-96365 (3 × 10−5 M, Figs. 4Ba, Bb). Thus, SKF-96365 is speculated to inhibit SOCC-mediated Ca2+ influx.
A: mean Fura-2 fluorescence intensity ratio (F340/380) changes induced by CPA (10−5 M) and Ca2+ (1.8 mM) in Ca2+-free medium in the presence and absence of LOE-908 (3 × 10−5 M, Aa) and SKF-96365 (3 × 10−5 M, Ab). Arrows indicate the administration of CPA (10−5 M) and Ca2+ (1.8 mM). B: summarized data of the peak ratio (F340/380) within 5 min after Ca2+ (1.8 mM) administration in the presence and absence of LOE-908 (3 × 10−5 M, Ba) and SKF-96365 (3 × 10−5 M, Bb). Data are expressed as the mean ± S.E.M. (n = 8 each). ** p < 0.01 vs. control (Welch’s t-test). DMSO: 0.05% dimethyl sulfoxide.
PAF-induced BTI/OC in mouse UBSM were demonstrated to depend on Ca2+ influx, and VDCC/SOCC were implied as the main Ca2+ influx pathways in PAF-induced BTI/OC.
We demonstrated the extracellular Ca2+ dependence of PAF-induced BTI/OC in mouse UBSM, as PAF-induced BTI/OC were completely suppressed by extracellular Ca2+ removal, indicating their dependence on Ca2+ influx.
To determine the possible involvement of VDCC-mediated Ca2+ influx in PAF-induced BTI/OC, we tested the effects of VDCC inhibitors on PAF-induced BTI/OC. PAF-induced BTI was strongly suppressed in the presence of all tested VDCC inhibitors. Therefore, Ca2+ influx via VDCC is considered to play an important role in PAF-induced BTI. These results are consistent with those of previous reports in which Ca2+ influx via VDCC was demonstrated to play an important role in PAF-induced contractile responses in guinea pig (GP) ileum SM/gallbladder SM and rat fundus SM/pregnant uterus SM.3–5,10) PAF-induced OC frequency was strongly suppressed by all tested VDCC inhibitors, whereas PAF-induced OC amplitude was not significantly suppressed by these inhibitors. Therefore, PAF-induced OC is considered to involve VDCC-dependent/independent Ca2+ influx. These results are consistent with those of a previous study in which carbachol-induced contraction of mouse UBSM was revealed to be caused by VDCC-dependent major Ca2+ influx and VDCC-independent minor Ca2+ influx.19)
We proceeded to determine the possible involvement of ROCC-mediated Ca2+ influx in PAF-induced BTI/OC. As LOE-908, which inhibits ROCC, has been reported to inhibit VDCC,20) we determined the effects of LOE-908 on residual PAF-induced BTI/OC in the presence of verapamil to eliminate this effect. LOE-908 had a minor inhibitory effect on residual PAF-induced BTI/OC in the presence of verapamil, suggesting that ROCC may not contribute to VDCC-independent Ca2+ influx. However, ROCC activation may contribute to VDCC activation. Notably, TRP channels are considered ROCC candidates.21) TRPC4β channel activation may activate VDCC by depolarizing the cell membrane in mouse UBSM.22)
The possible involvement of SOCC-mediated Ca2+ influx in PAF-induced BTI/OC was investigated. To date, many pharmacological studies have used SKF-96365 as a selective inhibitor for SOCC to identify this Ca2+ influx route in chemically stimulated responses.16,23–27) We also showed that a CPA-induced increase in [Ca2+]i within 293T cells, which was insensitive to LOE 908, was strongly inhibited by SKF-96365, suggesting that this inhibitor is a useful pharmacological tool for SOCC. However, SKF-96365 also inhibits VDCC/ROCC in addition to SOCC.28) As LOE 908 had a minor inhibitory effect on the PAF-induced BTI/OC, we determined the effects of SKF-96365 on residual PAF-induced BTI/OC in the presence of verapamil to eliminate the VDCC inhibitory effect. SKF-96365 almost completely suppressed the residual PAF-induced OC amplitude in the presence of verapamil. Accordingly, SOCC might serve as the VDCC-independent Ca2+ influx responsible for PAF-induced OC amplitude. SOCC candidates include Orai channels, whose activity is modulated by stromal interaction molecules (STIMs), SR Ca2+ sensors.29) SOCC, including Orai–STIM pathways, has been reported to play an important role in human UBSM contractile response.29) Therefore, the PAF-induced OC amplitude in mouse UBSM might also be caused by Ca2+ influx via SOCC, including Orai–STIM pathways. However, to clarify the Orai–STIM pathway as a key Ca2+ entry route to induce PAF-produced OC in UBSM, further studies employing STIM1/2- and Orai1/2/3-targeted knockdown experiments with small interfering RNA or knockout mice of these SOCC-related molecules are necessary.
Next, we discussed why PAF-induced OC is caused by both VDCC/SOCC. UBSM OC is suggested to be related to mitochondrial Ca2+ handling, Ca2+ release from SR ryanodine receptors, and SOCC-mediated Ca2+ influx due to SR depletion (Orai activation by STIM).30,31) Therefore, PAF might cause OC by activating these pathways, including SOCC. The role of VDCC in UB Ca2+ mobilization has been reported as follows: 1) the frequency of Ca2+ sparklet production in mouse UBSM was reduced by diltiazem, but the amplitude of this production was not affected32); and 2) nicardipine (a VDCC inhibitor) disrupted the synchrony of Ca2+ transients within/between cell populations in mouse UB pericytes.33) Thus, in PAF-induced OC, VDCC may synchronize Ca2+ mobilization within/between cell populations, resulting in an increase in the frequency of OC elicited through the SOCC pathways.
Finally, we discussed the significance of this study. Recently, 1) the accumulation of PAF caused by smoking in UB tissues34,35) and 2) the association between smoking and overactive bladder (OAB)36,37) were reported. These reports suggest PAF can induce OAB in smokers. Thus, our findings indicate VDCC/SOCC inhibitors may be effective in patients with OAB that smoke.
This work was supported in part by the JSPS KAKENHI Grants-in-Aid for Scientific Research (C) (20K11519 to Y.T., K.O., K.Y., and 21K11686 to K.O., Y.T., K.Y.) and a Grant-in-Aid for Early-Career Scientists (21K17666 to K.Y.).
The authors declare no conflict of interest.
