Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function

Abstract

Transient receptor potential melastatin 4 (TRPM4) channels are Ca(2+)-activated nonselective cation channels that have been recently identified as regulators of detrusor smooth muscle (DSM) function in rodents. However, their expression and function in human DSM remain unexplored. We provide insights into the functional role of TRPM4 channels in human DSM under physiological conditions. We used a multidisciplinary experimental approach, including RT-PCR, Western blotting, immunohistochemistry and immunocytochemistry, patch-clamp electrophysiology, and functional studies of DSM contractility. DSM samples were obtained from patients without preoperative overactive bladder symptoms. RT-PCR detected mRNA transcripts for TRPM4 channels in human DSM whole tissue and freshly isolated single cells. Western blotting and immunohistochemistry with confocal microscopy revealed TRPM4 protein expression in human DSM. Immunocytochemistry further detected TRPM4 protein expression in DSM single cells. Patch-clamp experiments showed that 9-phenanthrol, a selective TRPM4 channel inhibitor, significantly decreased the transient inward cation currents and voltage step-induced whole cell currents in freshly isolated human DSM cells. In current-clamp mode, 9-phenanthrol hyperpolarized the human DSM cell membrane potential. Furthermore, 9-phenanthrol attenuated the spontaneous phasic, carbachol-induced and nerve-evoked contractions in human DSM isolated strips. Significant species-related differences in TRPM4 channel activity between human, rat, and guinea pig DSM were revealed, suggesting a more prominent physiological role for the TRPM4 channel in the regulation of DSM function in humans than in rodents. In conclusion, TRPM4 channels regulate human DSM excitability and contractility and are critical determinants of human urinary bladder function. Thus, TRPM4 channels could represent promising novel targets for the pharmacological or genetic control of overactive bladder.

Keywords: 9-phenanthrol; patch-clamp electrophysiology; smooth muscle; transient receptor potential channels; urinary bladder.

Fig. 1.

Transient receptor potential melastatin 4 (TRPM4) channel mRNA expression in human detrusor smooth muscle (DSM) whole tissue and native freshly isolated human DSM single cells. Gel electrophoresis imaging illustrates RT-PCR detection of TRPM4 channel mRNA transcripts (196 bp) in DSM whole tissue and DSM single cells (n = 4, N = 4). Human brain and prostate samples were used as positive controls (+). No products were observed in the negative control (−RT), in which reverse transcriptase (RT) was not added to the reaction.

Fig. 2.

Western blot, immunohistochemical, and immunocytochemical detections of TRPM4 channel protein in human DSM tissue and DSM single cells. A: Western blot showing TRPM4 channel protein expression in human DSM tissue. Arrow indicates ∼134-kDa band, consistent with expected molecular mass of TRPM4 channel protein. Lack of an immunoreactive band in the presence of competing peptide (CP) confirmed specificity of the primary antibody. HEK-293 cell lysate was used as a positive control. B: confocal images showing immunohistochemical detection of TRPM4 channel protein expression in human DSM tissue. Red staining (bottom left) indicates TRPM4 channels; blue staining indicates cell nuclei (top left); green staining indicates α-smooth muscle actin (α-SMA, top right); merged image (bottom right) illustrates overlap of all 3 images. C: confocal images illustrating immunocytochemical detection of TRPM4 channel protein expression in isolated human DSM cells. Red staining (bottom left) indicates TRPM4 channels; blue staining indicates cell nucleus (top left); green staining indicates α-SMA (top right); merged image (bottom right) illustrates overlap of all 3 images. Results were verified in 4 separate experiments using DSM whole tissue or multiple DSM cells isolated from 4 patients. D and E: immunohistochemistry and immunocytochemistry experiments, respectively. In control experiments, no staining was visible after absorption of the primary antibody with a competing peptide (+CP control). Merged image (bottom right) illustrates overlap of all 3 images.

Fig. 3.

Inhibition of transient inward cationic currents (TICCs) by the TRPM4 channel-selective inhibitor 9-phenanthrol in freshly isolated human DSM cells. A: original recording illustrating the inhibitory effect of 30 μM 9-phenanthrol on TICC activity in a human DSM single cell recorded at −70 mV. B: summary data illustrating inhibitory effects of 30 μM 9-phenanthrol on TICCs, analyzed as total open channel probability (NP_o) before and after application of 30 μM 9-phenanthrol (n = 15, N = 9). *P < 0.05.

Fig. 4.

Inhibition of TRPM4 channels with 9-phenanthrol attenuates voltage step-induced whole cell currents in human DSM cells. A: representative recordings illustrate the inhibitory effect of 30 μM 9-phenanthrol on the amplitude of the voltage step-induced whole cell current in human DSM cell. Inhibitory effect of 9-phenanthrol was reversed after washout of the human DSM cell with fresh 9-phenanthrol-free bath solution. B: summary data of current-voltage relationships in the absence (control), in the presence, and after washout of 30 μM 9-phenanthrol (n = 7, N = 7). *P < 0.05.

Fig. 5.

Inhibition of TRPM4 channels with 9-phenanthrol hyperpolarizes the resting membrane potential (RMP) in human DSM cells. A: representative current-clamp recording illustrating hyperpolarizing effects of 30 μM 9-phenanthrol on RMP in a human DSM cell. B: summary data illustrating hyperpolarizing effects of 30 μM 9-phenanthrol on the RMP in human DSM cells (n = 4, N = 3). *P < 0.05.

Fig. 6.

Inhibition of TRPM4 channels with 9-phenanthrol attenuates spontaneous phasic and tonic contractions of human DSM isolated strips. A: representative myograph recording demonstrating inhibition of spontaneous phasic contractions and tone of a human DSM isolated strip by 30 μM 9-phenanthrol. B: summary data illustrating decrease in spontaneous phasic contraction amplitude, muscle force integral, frequency, duration, and tone of human DSM strips by 30 μM 9-phenanthrol (n = 11, N = 5). ***P < 0.005.

Fig. 7.

Inhibition of TRPM4 channels with 9-phenanthrol significantly reduces carbachol-induced phasic and tonic contractions of human DSM isolated strips. A: representative myograph recording obtained from a human DSM strip depicting the inhibitory effect of 30 μM 9-phenanthrol on 0.1 μM carbachol-induced phasic contractions. B: summary data illustrating inhibitory effects of 30 μM 9-phenanthrol on amplitude, muscle force integral, frequency, duration, and tone of carbachol-induced contractions of human DSM isolated strips (n = 17, N = 5). ***P < 0.005.

Fig. 8.

Cumulative addition of 9-phenanthrol inhibits continuous 10-Hz electrical field stimulation (EFS)-induced contractions of human DSM isolated strips. A: representative myograph recording from a human DSM strip illustrating effects of 0.1–30 μM 9-phenanthrol on 10-Hz EFS-induced contractions. B: cumulative concentration-response curves for inhibitory effects of 9-phenanthrol on 10-Hz EFS-induced contraction amplitude and muscle force integral of human DSM isolated strips (n = 16, N = 11).

Fig. 9.

Inhibition of TRPM4 channels with 9-phenanthrol decreases the amplitude of 3.5- to 50-Hz EFS-induced contractions of human DSM isolated strips. A: representative recording of EFS-induced contractions (stimulation frequency = 3.5–50 Hz) in the absence of 9-phenanthrol (control) and 10 min after application of 30 μM 9-phenanthrol. B: frequency-response curves in the presence or absence of 30 μM 9-phenanthrol illustrating a decrease in amplitude of EFS-induced contractions of human DSM isolated strips (n = 22, N = 16). **P < 0.01, ***P < 0.005.

Fig. 10.

Species-related differences in inhibitory effects of 9-phenanthrol on human and rodent DSM excitability and contractility. A: effects of 30 μM 9-phenanthrol on voltage step-induced whole cell currents recorded in human and guinea pig DSM cells, represented as percentage of whole cell current (n = 7, N = 7 for human; n = 10, N = 5 for guinea pig). *P < 0.05; **P < 0.01. B: effects of 9-phenanthrol (30 μM) on spontaneous phasic DSM contractions in humans (n = 11, N = 5), guinea pigs (n = 7, N = 7), and rats (n = 8, N = 7). **P < 0.01; ***P < 0.005. Rat and guinea pig data are from Smith et al. (44, 45).