TRPM4 channel: a new player in urinary bladder smooth muscle function in rats

Tissue preparation.

Fifty-three male Sprague-Dawley rats, weighing on average 353.0 ± 43.8 g, were used in this study. All animals were kept under standard laboratory conditions (12:12-h light-dark). Rats were euthanized with CO₂ followed by a thoracotomy according to the protocol reviewed and approved by the Institutional Animal Care and Use Committee of the University of South Carolina (Animal Use Protocol no. 1747). The urinary bladders were removed and placed in ice-cold Ca²⁺-free dissection solution (DS). Each bladder was sliced open and rinsed free of urine. The urothelial layer and lamina propria were removed by careful dissection, and only the DSM tissue was used in this study.

DSM single-cell isolation.

Rat DSM single cells were isolated based on the protocol described previously (3, 4, 18). Briefly, small DSM strips (5–10 mm long; 2–4 mm wide) were cut from the rat DSM specimens. Next, two to three DSM strips were incubated in 2 ml of DS supplemented with 1 mg/ml BSA, 1 mg/ml papain (Worthington, Lakewood, NJ), and 1 mg/ml dithiothreitol for 25 min at 37°C. Then, the DSM strips were transferred to prewarmed 2 ml of DS containing 1 mg/ml BSA, 0.5 mg/ml collagenase type II (Sigma-Aldrich, St. Louis, MO), 0.5 mg/ml trypsin inhibitor (Sigma-Aldrich), and 100 μM CaCl₂ for 7–14 min at 37°C. Enzyme-treated DSM strips were then washed two to three times with fresh DS containing BSA. Individual cells were released from the DSM tissue by passing the strips through the tip of a Pasteur pipette. Several drops of the isolated DSM cell suspension were placed onto a recording chamber and allowed to adhere to the glass bottom for ∼20 min. Freshly isolated elongated, distinct, bright, and shiny DSM cells, when viewed illuminated under an Axiovert 40CFL microscope (Carl Zeiss, Thornwood, NY), were selected for patch-clamp recordings, live-cell intracellular Ca²⁺ imaging, single-cell RT-PCR, and immunocytochemistry.

RNA extraction, RT-PCR, and sequencing.

For RT-PCR experiments, mucosa-free whole DSM tissue or freshly isolated, pooled single DSM cells were used as previously described (3, 4). Briefly, freshly isolated DSM cells were left to settle at the bottom of a chamber for ∼5 min and washed several times before individual single cell selection by suction into a glass pipette. Approximately 50–100 DSM single cells were collected and expelled into a 1.5-ml centrifuge tube with RNAlater (Qiagen, Hilden, Germany) and then pelleted at 7,000 g for 5 min. Total RNA was isolated from whole DSM tissue kept in RNAlater, as well as from freshly isolated, pooled single DSM cells using the RNeasy Mini Kit (Qiagen). Extracted RNA was reverse-transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (Promega) and oligo (dT) primers. The TRPM4 channel specific primer pair sequence was selected based on a previous publication (7) and synthesized by Integrated DNA Technologies (IDT, Coralville, IA). The sense and antisense primers were 5′-GTCATCGTGAGCAAGATGATGAA-3′ and 3′-GTCCACCTTCTGGGACGTGC-5′, respectively. The cDNA produced was PCR amplified using GoTaq Green Master Mix (Promega) and specific primers for the TRPM4 channel. The PCR annealing temperature for the primer pair was optimized using a mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany). Rat prostate tissue was used as a positive control based on a previous report (33). Negative control experiments were performed in the absence of reverse transcriptase (−RT). PCR products were purified using the GenElute PCR Clean-Up Kit (Sigma-Aldrich) and sequenced at the University of South Carolina Environmental Genomics facility for sequence conformation.

Western blotting.

Mucosa-free DSM isolated tissue strips (30–50 mg) were placed in Tris-buffered saline (TBS) and sonicated using a model 100 Dismembrator (Thermo Fisher Scientific, Pittsburgh, PA). The mixture was centrifuged at 10,000 g for 5 min at 4°C. The mixture was then placed in a 37°C water bath for 10 min to enhance phase separation. The supernatant was collected, and the protein concentration was determined using Nano Drop 2000 (Thermo Fisher Scientific). Protein was mixed with 2× Laemmli buffer (1:1) and put on a rotator for 5–10 min to homogenize. The mix was then denatured in a water bath at 100°C for 5 min. Subsequently, equal amounts of protein (∼50 μg) were loaded into adjacent lanes subjected to 4–20% precast SDS-PAGE for 50 min at 150 V (Bio-Rad Mini-PROTEAN Tetra Cell) and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA) at 100 mA for 50 min using a semidry blot. The membrane was blocked with 5% dry milk/Tris-buffered saline-Tween 20 buffer for 1 h at room temperature. The membrane was incubated with the primary antibody, anti-TRPM4 (1:300 dilution; ab63080, Abcam), overnight at 4°C. The membrane was washed with Tris-buffered saline-Tween-20 three to four times and incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:3,000) in blocking buffer for 1 h at room temperature. Bound antibodies were detected by a Pierce Fast echochemiluminescence substrate kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The antibody specificity was verified by preincubation of the antibody with the respective competing peptide.

Immunohistochemistry and immunocytochemistry.

For immunocytochemical studies, freshly isolated DSM cells were dropped on a poly-l-lysine-coated slide and allowed to settle for 30 min at room temperature. The DSM cells were fixed with 4% paraformaldehyde for 10 min. DSM cells were then washed three times for 15 min each with PBS/0.01 M glycine/0.1% Triton X-100 solution and blocked in 1% BSA/PBS containing 5% normal donkey serum for 10 min. DSM cells were then incubated with the rabbit polyclonal primary antibody, anti-TRPM4 (1:500 dilution; ab63080, Abcam), diluted in 1% BSA/PBS at 37°C for 1 h. After incubation with the primary antibody, cells were rinsed two times for 15 min with 1% BSA in PBS and were again blocked in 1% BSA/PBS containing 5% normal donkey serum for 10 min. DSM cells were then labeled with the secondary antibody [Cy3-conjugated anti-rabbit IgG, at 1:500, 1% BSA/PBS (Jackson ImmunoResearch, West Grove, PA)] overnight in the dark at 4°C. After labeling, cells were washed three times for 15 min with 1% BSA/PBS and then washed with PBS for 15 min. After washing, the cells were incubated with Alexa Fluor 488 phalloidin (diluted in PBS, 1:50) for 2 h in the dark. After incubation with Alexa Fluor 488 phalloidin, DSM cells were then washed two more times with PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI; diluted in PBS 1:5,000) for 15 min and washed again with PBS. 1,4-Diazabicyclo[2.2.2]octane (DABCO) was placed onto the slides, and the slides were coverslipped. Images at ×63 oil objective were acquired with a LSM 510 META confocal microscope (Carl Zeiss, Göttingen, Germany). The cells for each group were imaged with the same laser power, gain settings, and pinhole for the controls and antibody treatment.

Immunohistochemical experiments were performed using freshly isolated mucosa-free native rat DSM tissue. Rat DSM tissue was sliced into 120-μm-thick sections using a vibratome model G tissue slicer (Oxford Laboratories, Foster City, CA). Sections were then stained with primary antibodies directed toward TRPM4 (1:100; ab63080, Abcam) and α-smooth muscle actin (1:100, ab21027). Nuclear staining was achieved with DAPI. Secondary antibodies were tagged with Cy3-conjugated anti-rabbit IgG at 1:100 (used to stain for TRPM4) and Alexa Fluor 488 donkey anti-goat IgG (Life Technologies, A11055) at 1:100 (used to stain for α-smooth muscle actin).

Electrophysiological (patch-clamp) recordings.

We employed the amphotericin B perforated whole cell configuration of the patch-clamp technique, which preserves the native environment and the signal transduction pathways of the cells (16, 18, 26). Spontaneous inward whole cell currents were recorded using an Axopatch 200B amplifier, Digidata 1440A, and pCLAMP version 10.2 software (Molecular Devices, Union City, CA). An eight-pole Bessel filter 900CT/9L8L (Frequency Devices, Ottawa, IL) was used to filter the recorded currents. The patch-clamp pipettes were prepared from borosilicate glass (Sutter Instruments, Novato, CA) and pulled using a Narishige PP-830 vertical puller (Narishige Group, Tokyo, Japan). The patch-clamp pipettes were coated with dental wax to reduce capacitance and polished with a Micro Forge MF-830 fire polisher to give a final tip resistance of ∼4–6 MΩ. DSM cells were held at −70 mV, and spontaneous inward currents were recorded. At this potential, the activity of BK channels is negligible, and thus we did not detect any transient BK currents (TBKCs) (16, 18). After obtaining a stable perforated patch-clamp recording, measurements were made for at least 5–10 min before and after the addition of 9-phenanthrol. All patch-clamp experiments were conducted at room temperature (22–23°C).

Live-cell Ca²⁺ imaging.

Measurement of DSM cell intracellular Ca²⁺ levels was performed as previously described (19). Briefly, suspension of freshly isolated single DSM cells (500 μl) was added into a poly-l-lysine-coated dish and allowed to settle for at least 30 min to adhere to the glass bottom. Fura 2-AM solution was added to the cell suspension at a final concentration of 2 μM and incubated for 30 min in the dark at room temperature. DSM cells were washed three times with a fresh bath solution. Ca²⁺ imaging was acquired with a ×40/1.35 oil Iris objective and an OLYMPUS IX81 motorized inverted research microscope. The light was generated by a TILL LB LS/30 300 Walt Xenon Arc Lamp and then filtered into monochromatic light (340 and 380 nm) with an emission wavelength of 510 nm. A region without any cells was selected as a background, which was subtracted from the measured values. The elicited light was detected with a digital CCD camera (Hamamatsu Photonics C10600–10B-H), with an exposure time of 200 ms. Image acquisition and hardware control were performed with MetaFluor 7.7.2.0 software (Molecular Devices, Sunnyvale, CA).

Isometric DSM tension recordings.

Isometric DSM tension recordings were conducted as previously described (3, 18, 30). Briefly, small mucosa-free DSM strips (∼2–4 mm wide × 5–10 mm long) were cut from the bladder and transferred to a small petri dish containing Ca²⁺-free DS. Each strip was attached to an isometric force-displacement transducer and suspended in a 10-ml temperature-controlled (37°C) water bath containing physiological saline solution (PSS) and aerated with 95% O₂-5% CO₂ (pH 7.4). The DSM strips were initially stretched to 1 g of tension and washed with fresh PSS solution every 15 min during an equilibration period of 45–60 min. After the equilibration period, DSM strips were separated into four experimental groups. The first group consisted of DSM isolated strips that exhibited substantial spontaneous phasic contractions after the initial stretch of tension following the equilibration period. In the second, third, and fourth group, DSM contractions were induced by carbachol (0.1 μM), a cholinergic agonist; by KCl (20 or 60 mM), a depolarizing agent; or by electrical field stimulation (EFS), respectively. To minimize the effects of neurotransmitter release, the DSM strips exhibiting spontaneous phasic contractions, carbachol-induced, 20 or 60 mM KCl-induced contractions were performed in the presence of 1 μM tetrodotoxin (TTX), a selective blocker of the neuronal Na⁺ channels. The spontaneous, carbachol-induced, and KCl-induced phasic and tonic contractions were allowed to stabilize for ∼30 min. Then increasing concentrations of 9-phenanthrol (0.1–30 μM) were applied at 10-min intervals directly into the bath solution. In the fourth experimental group, nerve-evoked contractions were induced by EFS using a pair of platinum electrodes mounted in the tissue bath in parallel to the DSM strip. The EFS pulses were generated using a PHM-1521 stimulator (MED Associates, Georgia, VT), and the EFS pulse parameters were 0.75-ms pulse width, 20-V pulse amplitude, 3-s stimulus duration, and polarity was reversed for alternating pulses. For EFS studies, after the equilibration period, DSM strips were subjected to either continuous repetitive stimulation with a frequency of 10 Hz at 1-min intervals or increasing frequencies from 0.5 to 50 Hz at 3-min intervals. The contractions were recorded using the Myomed myograph system (MED Associates).

Solutions and drugs.

The Ca²⁺-free DS had the following composition (in mM): 80 monosodium glutamate, 55 NaCl, 6 KCl, 2 MgCl_2, 10 HEPES, and 10 glucose, adjusted to pH 7.3 with NaOH. For DSM contraction studies, the PSS was prepared daily and had the following composition (mM): 119 NaCl, 4.7 KCl, 24 NaHCO₃, 1.2 KH₂PO₄, 2.5 CaCl_2, 1.2 MgSO_4, and 11 glucose, aerated with 95% O₂-5% CO₂ (pH 7.4). The extracellular (bath) solution used in the patch-clamp and Ca²⁺ imaging experiments contained (in mM) 134 NaCl, 6 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, and 10 HEPES, pH adjusted to 7.4 with NaOH. The patch-clamp pipette solution contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl₂, 10 HEPES, and 0.05 EGTA, pH adjusted to 7.2 with NaOH and supplemented with freshly dissolved (every 1–2 h) 200 μg/ml amphotericin B in DMSO. BSA and amphotericin B were obtained from Thermo Fisher Scientific. All other drugs were obtained from Sigma-Aldrich. 9-Phenanthrol was dissolved daily in DMSO. Carbachol and atropine were dissolved in double distilled water, and TTX was dissolved in citrate buffer. The final concentration of DMSO in the bath solutions did not exceed 0.05%.

Statistical analysis.

MiniAnalysis software (Synaptosoft, Decatur, GA) was used to analyze the DSM contraction parameters. Specifically, the DSM contractile activity was quantified by measuring the average phasic contraction amplitude (the difference between the force-time baseline curve and the maximum peak of the contractions), the frequency (contractions/min), muscle force integral (calculated by integrating the area under the force-time baseline curve), the phasic contraction duration (defined as width of the individual phasic contraction at 50% of the amplitude), and the DSM tone (the difference between the zero line and the force-time baseline curve). Statistical analyses were performed with GraphPad Prism 4.03 software (GraphPad Software, La Jolla, CA) and CorelDraw Graphic Suite × 3 software (Corel) was used to illustrate the data. IC₅₀ values were obtained using a sigmoidal fitting function in GraphPad Prism 4.03 software and are reported as means (95% confidence interval). To compare the phasic contraction parameters for spontaneous, 0.1 μM carbachol-induced, or 20 mM KCl-induced contractions, data were normalized to the last 5 min of the contractions before the addition of the first concentration of 9-phenanthrol (taken to be 100%) and expressed as percentages. For the EFS-induced contractions, the contraction amplitude at EFS frequency of 50 Hz under control conditions was taken to be 100%, and the data were normalized. To evaluate the effect of the cumulative concentrations of 9-phenanthrol (0.1–30 μM), the last 5 min before addition of each concentration to the bath was analyzed. In the electrophysiology experiments, the channel open probability (NP_o) values were calculated in the absence and presence of 9-phenanthrol as described previously (9, 10). The spontaneous inward whole cell current activity at −70 mV was calculated as the sum of the NP_o values of multiple open states using the build-in single-channel analysis feature of the Clampfit software. The baseline/channel closed state was obtained in the presence of 9-phenanthrol. The single-channel TRPM4 current of 1.75 pA was chosen in the data analysis based on the reported unitary conductance of the TRPM4 channel (25 pS) (21). Data are summarized as means ± SE for n (the number of DSM strips or cells) isolated from N (the number of rats). In Ca²⁺ imaging experiments, the effects of 9-phenanthrol were analyzed before and at least 10 min after application allowing the levels to reach steady state. Data were compared using a two-way ANOVA followed by a Bonferroni posttest or two-tailed paired Student's t-test. A P value <0.05 was considered statistically significant.

Rat DSM expresses mRNA for TRPM4 channels.

We detected expression of mRNA message for TRPM4 channels in whole DSM tissue (Fig. 1). In whole DSM tissue, detection of mRNA message could come directly from DSM cells or non-DSM cells such as fibroblasts, neuronal cells, and vascular smooth muscle. To investigate the expression of TRPM4 channels in DSM cells directly, RT-PCR experiments were performed in isolated, pooled single DSM cells. The single-cell RT-PCR results confirmed that mRNA message for TRPM4 channels was detected in the DSM cells (Fig. 1). A lack of genomic DNA contamination was confirmed using the negative control reactions lacking RT (Fig. 1).

Rat DSM expresses TRPM4 channel protein.

To confirm the presence of TRPM4 channel protein in mucosa-free DSM tissue, we used the Western blot technique and a TRPM4 channel-specific antibody (Fig. 2A). The expected molecular mass of TRPM4 channel protein was ∼134 kDa, consistent with the experimentally determined molecular mass. In control experiments, preabsorption of the primary antibody in the presence of its antigenic competing peptide resulted in the loss of the band, indicating the specificity of the antibody for its intended epitope (Fig. 2A). Thus the Western blot experiments confirmed the presence of TRPM4 channel protein in rat DSM whole tissue. Furthermore, immunohistochemical analysis also confirmed the expression and localization of TRPM4 channel protein in rat DSM whole tissue (Fig. 2B).

To determine whether isolated single DSM cells express TRPM4 channel protein, immunocytochemistry was carried out with a TRPM4 channel-specific antibody in isolated single DSM cells. As illustrated in Fig. 2D, immunolabeling confirmed the expression of TRPM4 channel protein in freshly isolated DSM cells and demonstrated that TRPM4 channel protein is localized to the plasma membrane. Control treatments were further conducted by preabsorption of the primary antibody with its antigenic competing peptide to verify the specificity of the antibody for its intended epitope and demonstrated no specific staining (Fig. 2, C and E).

Attenuation of spontaneous inward current activity by the selective TRPM4 channel inhibitor 9-phenanthrol in freshly isolated rat DSM cells.

To examine the role of the TRPM4 channel in DSM cell excitability, we conducted electrophysiological studies using the perforated patch-clamp technique and the TRPM4 channel-selective inhibitor 9-phenanthrol. At a holding potential of −70 mV, we observed stable spontaneous basal activity along with numerous TICCs as illustrated in Fig. 3A. This activity is reminiscent of the TRPM4-mediated TICCs recently reported in rat cerebral arterial myocytes (10). We then examined the effect of 9-phenanthrol on the spontaneous inward current in DSM cells. We applied 9-phenanthrol at 30 μM, since this is a threefold higher concentration than its IC₅₀ value identified in recombinant cells (11) or cerebral artery myocytes (10). The addition of 30 μM 9-phenanthrol attenuated the spontaneous inward current activity by ∼50% from an NP_o of 4.41 ± 0.7 in the absence of the compound to 2.2 ± 0.5 in its presence (n = 10, N = 6; P < 0.01; Fig. 3B). In the majority of the cells examined including the representative one shown in Fig. 3A, 9-phenanthrol (30 μM) reduced the apparent inward current. This suggests that TRPM4 channels are tonically active under the experimental conditions of this study and are important regulators of rat DSM cell excitability.

Selective inhibition of TRPM4 channels with 9-phenanthrol reduces intracellular Ca²⁺ levels in freshly isolated rat DSM cells.

To study the effects of TRPM4 channel inhibition with 9-phenanthrol on intracellular Ca²⁺ levels, real-time live-cell Ca²⁺ imaging was carried out using the ratiometric dye fura 2. The control resting Ca²⁺ level was measured for at least 10 min, and then 30 μM 9-phenanthrol was added into the bath solution and Ca²⁺ levels were recorded for at least 30 additional min until reaching a steady-state level. Under control conditions, the fluorescent ratio (340/380 nm) was 0.996 ± 0.049, and in the presence of 9-phenanthrol the fluorescent ratio (340/380 nm) decreased to 0.607 ± 0.004 (n = 19, N = 6; P < 0.005). The responses of the TRPM4 channel-selective inhibitor 9-phenanthrol on the intracellular Ca²⁺ levels are likely associated with diminished Ca²⁺ influx via VDCCs due to decreased DSM membrane excitability.

Inhibition of spontaneous phasic contractions by the TRPM4 channel-selective inhibitor 9-phenanthrol in rat DSM isolated strips.

In this experimental series, functional studies were carried out to elucidate the inhibitory effect of the TRPM4 channel blocker 9-phenanthrol on the spontaneous contractile activity of rat DSM isolated strips. After initial spontaneous phasic contractions stabilized (30–60 min), cumulative addition of 9-phenanthrol (0.1–30 μM) to rat DSM isolated strips was carried out as exemplified in Fig. 4A. As illustrated, 9-phenanthrol dramatically decreased the DSM spontaneous contractile activity. The concentration-response curves for the effect of cumulative addition of 9-phenanthrol (0.1–30 μM) on the contractile parameters in rat DSM isolated strips are illustrated in Fig. 4B. 9-Phenanthrol caused a concentration-dependent inhibition of the spontaneous contraction amplitude, muscle force integral, and frequency with IC₅₀ values ranging from 1.4 to 4.6 μM (n = 8, N = 7; Table 1). Control experiments showed that the vehicle (DMSO) had no effect on DSM spontaneous phasic contraction amplitude, muscle force integral, duration, frequency, and tone (Table 2).

Selective TRPM4 channel inhibition with 9-phenanthrol decreases pharmacologically induced contractions of rat DSM isolated strips.

In the next experimental series, we sought to test the effect of cumulative addition of 9-phenanthrol (0.1–30 μM) under conditions of either muscarinic receptor stimulation or sustained membrane depolarization. Carbachol (0.1 μM) rapidly and robustly increased the basal tension and spontaneous contractions of DSM strips (Fig. 5A). Cumulative addition of 9-phenanthrol (0.1–30 μM) significantly decreased the amplitude, muscle force integral, and frequency of the carbachol-induced contractions of DSM isolated strips with IC₅₀ values ranging from 1.7 to 3.3 μM (n = 11, N = 7; Fig. 5B; Table 1). In comparison, carbachol-induced DSM tone was reduced to a lesser extent with a maximum inhibition value of 10.5 ± 18.4% (Table 1). Control experiments showed that the vehicle (DMSO) had no effect on carbachol-induced phasic contraction amplitude, muscle force integral, duration, frequency, and tone (see Table 2).

Additionally, we used 20 mM KCl to mildly depolarize rat DSM strips and induce phasic contractions. As illustrated in Fig. 6A, exposure of DSM strips to cumulative addition of 9-phenanthrol (0.1–30 μM) caused a significant inhibition of the 20 mM KCl-induced contractions. Cumulative addition of 9-phenanthrol significantly decreased the amplitude, muscle force integral, and frequency of the 20 mM KCl-induced DSM contractions with IC₅₀ values ranging from 1.9 to 5.5 μM (n = 8, N = 7; Fig. 6B; Table 1). Control experiments showed that the DMSO vehicle at 0.05% had no effect on 20 mM KCl-induced contraction parameters (Table 2).

We also tested the effect of 9-phenanthrol on depolarization-mediated DSM contraction elicited by a higher K⁺ concentration (60 mM KCl). The 60 mM KCl-induced tonic contractions were manifested by an initial peak followed by recovery and stabilization at a steady-state level. The effect of 9-phenanthrol during the steady-state phase of the 60 mM KCl-induced tonic contraction is illustrated in Fig. 7A. In the 20 mM KCl solution, cumulative addition of 9-phenanthrol (0.1–30 μM) significantly inhibited DSM tone to 45.8 ± 7.5% at 30 μM (Fig. 7B; Table 1). However, cumulative addition of 9-phenanthrol (0.1–30 μM) had a substantially reduced inhibitory effect on DSM tone in the presence of the 60 mM KCl solution (n = 12, N = 8; Fig. 7B).

Attenuation of nerve-evoked contractions by TRPM4 channel-selective inhibitor 9-phenanthrol in rat DSM isolated strips.

Activation of parasympathetic nerves causes DSM contractions during bladder voiding. In this experimental series, we sought to explore the ability of cumulative addition of 9-phenanthrol (0.1–30 μM) to reduce DSM neurogenic contractions evoked by repetitive EFS of 10-Hz frequency. These experiments were conducted in the absence of TTX. A representative myographic recording in Fig. 8A shows that cumulative addition of 9-phenanthrol (0.1–30 μM) reduced the EFS-induced contractions at a stimulation frequency of 10 Hz in DSM isolated strips. Figure 8C summarizes the mean-concentration responses and demonstrates significant inhibitory effects on nerve-evoked contraction amplitude and muscle force integral at a stimulation frequency of 10 Hz with IC₅₀ values of 2.3 and 2.8 μM, respectively (n = 8, N = 6, Table 1). Control experiments showed that the DMSO vehicle had no effect on 10-Hz EFS-induced contraction amplitude, muscle force integral, duration, and tone (Table 2).

In addition, we sought to test the effect of cumulative addition of 9-phenanthrol (0.1–100 μM) on purinergic-mediated DSM contractions. DSM purinergic contractions were evoked by repetitive EFS of 10-Hz frequency in the presence of atropine (1 μM) to block the muscarinic receptors. As illustrated in Fig. 8B, in the presence of atropine, cumulative addition of 9-phenanthrol (0.1–100 μM) had less of an inhibitory effect on purinergic-mediated, nerve-evoked contraction amplitude and muscle force integral at a stimulation frequency of 10 Hz with IC₅₀ values of 20.5 and 20.8 μM, respectively (n = 23, N = 5; Fig. 8C; Table 1). Therefore, 9-phenanthrol is more effective at inhibiting cholinergic than purinergic DSM contractions.

In another group of experiments, DSM isolated strips were stimulated by increasing EFS frequencies (0.5–50 Hz). Following a frequency-response curve under control conditions, DSM strips were incubated for 10 min with 9-phenanthrol (3 μM) and a frequency-response curve in the presence of 9-phenanthrol was generated again. As illustrated in Fig. 9A, 9-phenanthrol significantly decreased the maximum amplitude generated in response to EFS frequencies of 7.5, 10, 12.5, 15, 20, 30, 40, and 50 Hz (n = 11, N = 7; P < 0.05; Fig. 9B). At the highest frequency tested of 50 Hz, addition of 3 μM 9-phenanthrol to the bath solution inhibited the EFS-induced contraction amplitude in rat DSM isolated strips to 45.2 ± 14.7% compared with the control responses (Fig. 9B). This experimental series further suggests that selective inhibition of the TRPM4 channel can reduce nerve-evoked contractions at various stimulation frequencies in rat DSM isolated strips.