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

Abstract

Members of the transient receptor potential (TRP) channel superfamily, including the Ca(2+)-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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.

The Novel KV7.2/KV7.3 Channel Opener ICA-069673 Reveals Subtype-Specific Functional Roles in Guinea Pig Detrusor Smooth Muscle Excitability and Contractility

“…Isometric DSM tension recordings were performed as previously described using DSM isolated strips without urothelium (Chen et al, 2010;Hristov et al, 2012a;Afeli et al, 2013;Smith et al, 2013). For spontaneous phasic, carbachol-induced, and KCl-induced contractions, the neuronal voltage-gated Na 1 channel blocker tetrodotoxin (TTX; 1 mM) was included prior to the application of increasing concentrations of ICA-069673 (100 nM-30 mM).…”

Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

“…The animals were euthanized with CO 2 and then subjected to a thoracotomy according to Animal Use Protocol 1747, reviewed and approved by the Institutional Animal Care and Use Committee of the University of South Carolina. DSM tissue dissection and preparation of DSM strips were performed as previously described (15,39,45,47).…”

“…Guinea pig DSM single cells were enzymatically isolated using papain and collagenase, as previously described (15,39,45,47). Single DSM cells were used for patch-clamp and live-cell Ca 2ϩ -imaging studies within 12 h after isolation.…”

“…Isometric DSM tension recordings were conducted using mucosa-free DSM strips (ϳ2-3 mm wide ϫ 5-6 mm long), as previously described (15,39,45,47). In the first set of experimental series, the DSM strips exhibiting spontaneous phasic contractions were allowed to equilibrate for Ն30 min.…”

New life in overactive bladder. Focus on “Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function”

“…The Petkov lab has previously reported a role of TRPM4 in the function of the DSM of the guinea pig bladder (9). However, in the paper highlighted here, Petkov and coworkers (5) have gone to the next research echelon by examining the role of TRPM4 in human DSM tissues (from patients without OAB symptoms) and isolated DSM cells.…”