scholarly journals TRPM4 channel inhibitors 9-phenanthrol and glibenclamide differentially decrease guinea pig detrusor smooth muscle whole-cell cation currents and phasic contractions

2020 ◽  
Vol 318 (2) ◽  
pp. C406-C421 ◽  
Author(s):  
John Malysz ◽  
Sarah E. Maxwell ◽  
Viktor Yarotskyy ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Complete Removal ◽  
Isometric Tension ◽  
Detrusor Smooth Muscle ◽  
Voltage Step ◽  
Transient Receptor Potential Melastatin ◽  
Trpm4 Channel

Nonselective cation channels, consistent with transient receptor potential melastatin-4 (TRPM4), regulate detrusor smooth muscle (DSM) function. TRPM4 channels can exist as homomers or assemble with sulfonylurea receptors (SURs) as complexes. We evaluated contributions of TRPM4/SUR-TRPM4 channels to DSM excitability and contractility by examining the effects of TRPM4/SUR-TRPM4 channel modulators 9-phenanthrol, glibenclamide, and diazoxide on freshly-isolated guinea pig DSM cells (amphotericin-B perforated patch-clamp electrophysiology) and mucosa-free DSM strips (isometric tension recordings). In DSM cells, complete removal of extracellular Na+ decreased voltage-step-induced cation (non-K+ selective) currents. At high positive membrane potentials, 9-phenanthrol at 100 μM attenuated voltage step-induced currents more effectively than at 30 μM, revealing concentration-dependent, voltage-sensitive inhibition. In comparison to 9-phenanthrol, glibenclamide (100 μM) displayed lower inhibition of cation currents. In the presence of glibenclamide (100 μM), 9-phenanthrol (100 μM) further decreased the currents. The SUR-TRPM4 complex activator diazoxide (100–300 μM) weakly inhibited the currents. 9-Phenanthrol, but not glibenclamide or diazoxide, increased cell capacitance (a cell surface area indicator). In contractility studies, glibenclamide displayed lower potencies than 9-phenanthrol attenuating spontaneous and 20 mM KCl-induced DSM phasic contractions. While both compounds showed similar maximum inhibitions on DSM spontaneous phasic contractions, glibenclamide was generally less efficacious on 20 mM KCl-induced phasic contractions. In summary, the observed differential effects of 9-phenanthrol and glibenclamide on DSM excitability and contractility support unique mechanisms for the two compounds. The data suggest that SUR-TRPM4 complexes do not contribute to DSM function. This study advances our understanding of pharmacological effects of glibenclamide and 9-phenanthrol on DSM cell cation currents.

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

2016 ◽  
Vol 310 (7) ◽  
pp. C600-C611 ◽  
Author(s):  
Kiril L. Hristov ◽  
Amy C. Smith ◽  
Shankar P. Parajuli ◽  
John Malysz ◽  
Eric S. Rovner ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Transient Receptor Potential ◽  
Single Cells ◽  
Receptor Potential ◽  
Detrusor Smooth Muscle ◽  
Rt Pcr ◽  
Transient Receptor Potential Melastatin ◽  
Transient Receptor ◽  
Trpm4 Channel

Transient receptor potential melastatin 4 (TRPM4) channels are Ca2+-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.

20-HETE inotropic effects involve the activation of a nonselective cationic current in airway smooth muscle

2003 ◽  
Vol 285 (3) ◽  
pp. L560-L568 ◽  
Author(s):  
Martin Cloutier ◽  
Shirley Campbell ◽  
Nuria Basora ◽  
Sonia Proteau ◽  
Marcel D. Payet ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Airway Smooth Muscle ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Isometric Tension ◽  
Transient Receptor Potential Channels ◽  
Inotropic Effects ◽  
Cationic Current ◽  
Transient Receptor

20-Hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such as vasoactivity, mitogenicity, and ion transport in various tissues. Our goal was to quantify the effects of 20-HETE on the electrophysiological properties of airway smooth muscle (ASM). Isometric tension measurements, performed on guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect with an EC50 value of 1.5 μM. This inotropic response was insensitive to GF-109203X, a PKC inhibitor. The sustained contraction, requiring Ca2+ entry, was partially blocked by either 100 μM Gd3+ or 1 μM nifedipine, revealing the involvement of noncapacitative Ca2+ entry and L-type Ca2+ channels, respectively. Microelectrode measurements showed that 3 μM 20-HETE depolarized the membrane potential in guinea pig ASM by 13 ± 2mV( n = 7), as did 30 μM 1-oleoyl-2-acetyl- sn-glycerol. Depolarizing effects were also observed in the absence of epithelium. Patch-clamp recordings demonstrated that 1 μM 20-HETE activated a nonselective cationic inward current that may be supported by the activation of transient receptor potential channels. The presence of canonical transient receptor potential mRNA was confirmed by RT-PCR in guinea pig ASM cells.

scholarly journals Novel role for the transient potential receptor melastatin 4 channel in guinea pig detrusor smooth muscle physiology

2013 ◽  
Vol 304 (5) ◽  
pp. C467-C477 ◽  
Author(s):  
Amy C. Smith ◽  
Kiril L. Hristov ◽  
Qiuping Cheng ◽  
Wenkuan Xin ◽  
Shankar P. Parajuli ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Transient Receptor Potential ◽  
Critical Role ◽  
Protein Detection ◽  
Muscle Physiology ◽  
Detrusor Smooth Muscle ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Trpm4 Channel

Members of the transient receptor potential (TRP) channel superfamily, including the Ca2+-activated monovalent cation-selective TRP melastatin 4 (TRPM4) channel, have been recently identified in the urinary bladder. However, their expression and function at the level of detrusor smooth muscle (DSM) remain largely unexplored. In this study, for the first time we investigated the role of TRPM4 channels in guinea pig DSM excitation-contraction coupling using a multidisciplinary approach encompassing protein detection, electrophysiology, live-cell Ca2+ imaging, DSM contractility, and 9-phenanthrol, a recently characterized selective inhibitor of the TRPM4 channel. Western blot and immunocytochemistry experiments demonstrated the expression of the TRPM4 channel in whole DSM tissue and freshly isolated DSM cells with specific localization on the plasma membrane. Perforated whole cell patch-clamp recordings and real-time Ca2+ imaging experiments with fura 2-AM, both using freshly isolated DSM cells, revealed that 9-phenanthrol (30 μM) significantly reduced the cation current and decreased intracellular Ca2+ levels. 9-Phenanthrol (0.1–30 μM) significantly inhibited spontaneous, 0.1 μM carbachol-induced, 20 mM KCl-induced, and nerve-evoked contractions in guinea pig DSM-isolated strips with IC50 values of 1–7 μM and 70–80% maximum inhibition. 9-Phenanthrol also reduced nerve-evoked contraction amplitude induced by continuous repetitive electrical field stimulation of 10-Hz frequency and shifted the frequency-response curve (0.5–50 Hz) relative to the control. Collectively, our data demonstrate the novel finding that TRPM4 channels are expressed in guinea pig DSM and reveal their critical role in the regulation of guinea pig DSM excitation-contraction coupling.

scholarly journals Specific detection of the endogenous transient receptor potential (TRP)-1 protein in liver and airway smooth muscle cells using immunoprecipitation and Western-blot analysis

2002 ◽  
Vol 364 (3) ◽  
pp. 641-648 ◽  
Author(s):  
Hwei Ling ONG ◽  
Jinglong CHEN ◽  
Tim CHATAWAY ◽  
Helen BRERETON ◽  
Lei ZHANG ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Western Blot ◽  
Airway Smooth Muscle ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Airway Smooth Muscle Cells ◽  
Transient Receptor

Although there are numerous reports of the presence of mRNA encoding the transient receptor potential (TRP)-1 protein in animal cells and of the detection of the heterologously expressed TRP-1 protein by Western-blot analysis, it has proved difficult to unequivocally detect endogenous TRP-1 proteins. A combination of immunoprecipitation and Western-blot techniques, employing a polyclonal antibody and a monoclonal antibody respectively, was developed. Using this technique, a band of approx. 80kDa was detected in extracts of H4-IIE rat liver hepatoma cell line and guinea-pig airway smooth muscle (ASM) cells transfected with human TRPC-1 cDNA. In extracts of untransfected H4-IIE cells, ASM cells, rat brain and guinea-pig brain, a band of approx. 92kDa was detected. Reverse transcriptase PCR experiments detected cDNA encoding both the α- and β-isoforms of TRP-1 in H4-IIE cells. Treatment of protein extracts with peptide N-glycosidase F indicated that the 92kDa band represents an N-glycosylated protein. Western blots conducted with a commercial polyclonal anti-(TRP-1) antibody (Alm) detected a band of 120kDa in extracts of H4-IIE cells and guinea-pig ASM cells. A combination of immunoprecipitation and Western-blotting techniques with the Alm antibody did not detect any bands at 92kDa or 120kDa in extracts of H4-IIE and ASM cells. It is concluded that (a) the 92-kDa band detected in untransfected H4-IIE and ASM cells corresponds to the N-glycosylated β-isoform of endogenous TRP-1, (b) the combined immunoprecipitation and Western-blot approach, employing two different antibodies, provides a reliable and specific procedure for detecting endogenous TRP-1 proteins, and (c) that caution is required in developing and utilizing anti-(TRP-1) antibodies.

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