Compound‐dependent Effects of TRPM4 Channel Modulators on Guinea Pig Detrusor Smooth Muscle Excitability and Contractility

Abstract

Guinea pig detrusor smooth muscle (DSM) cells express transient receptor potential melastatin 4 (TRPM4) channels, proposed to regulate DSM excitability and contractility1. Supporting evidence with the TRPM4 channel inhibitor 9‐phenanthrol showed reduced guinea pig DSM phasic contractions, and induced DSM cell hyperpolarization and decreased voltage‐step evoked cation currents1. Recently, a concern has been raised about selectivity of 9‐phenanthrol2–4. Here, we used whole‐cell amphotericin‐B perforated and inside‐out single‐channel patch‐clamp techniques, and isometric DSM tension recordings to address the hypothesis that TRPM4 channels regulate DSM excitation‐contraction coupling by examining the novel selective TRPM4 channel inhibitor 4‐chloro‐2‐[2‐(2‐chloro‐phenoxy)‐acetylamino]‐benzoic acid (CBA, compound 5 in reference5), along with non‐selective TRPM4 channel modulators: flufenamic acid (FFA)5 and BTP2 (or YM‐58483)6, a blocker and an activator, respectively. DSM cell voltage‐step induced cation currents were not increased when held at +26 or +6 mV in‐between voltage steps in comparison to the standard −74 mV (n=7), inconsistent with presence of TRPM4 currents6. Separate applications of CBA (30 μM) or BTP2 (10 μM) in DSM cells did not change the voltage‐step evoked currents (n=5–6). In contrast, FFA (100 μM) caused an increase at positive voltages, 1.4‐fold at +106 mV (p<0.05, n=7). In the same DSM cells, 9‐phenanthrol (100 μM) added following washout of FFA or in the presence of either CBA or BTP2 reduced the currents, 42–57% at +106 mV (p<0.05, n=4–7). 9‐Phenanthrol but not CBA, FFA, or BTP2 altered cell capacitance, 1.1‐fold increase (p<0.05). Excised single‐channel recordings revealed a non‐TRPM4 channel cation conductance of ~15 pS that was blocked by 9‐phenanthrol (30 μM). CBA, added cumulatively up to 100 μM, showed either no or very weak effects (maximum inhibitions: 8–38%) on spontaneous and 20 mM KCl‐induced phasic contraction parameters (n=10–13). For 300 μM CBA, inhibitions were higher (up to 68%) on all contraction parameters except for the duration of KCl‐induced contractions, which increased 2‐fold (n=7–9). 9‐Phenanthrol examined in parallel showed robust reductions in DSM phasic contraction parameters (IC50 values: 1.1–21 μM and maximum inhibitions: 35–86%, n=11–20). In summary, 9‐phenanthrol ‐ in contrast to other tested TRPM4 channel modulators: CBA, FFA, or BTP2 ‐effectively reduced DSM cell voltage‐step induced cation currents and DSM strip contractions. Our results reveal differential effects of TRPM4 channel modulators on guinea pig DSM excitability and contractility. Future studies are needed to determine the underlying mechanisms and to elucidate the function of TRPM4 in the urinary bladder. Interpretation of the results with 9‐phenanthrol requires caution especially related to its perceived TRPM4 channel selectivity.Support or Funding InformationNIH DK106964 to Georgi V. PetkovThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.