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.

During development, maturation, or aging, the expression and function of urinary bladder smooth muscle (UBSM) ion channels can change, thus affecting micturition. Increasing evidence supports a novel role of transient receptor potential melastatin‐4 (TRPM4) channels in UBSM physiology. However, it remains unknown whether the functional expression of these key regulatory channels fluctuates in UBSM over different life stages. Here, we examined TRPM4 channel protein expression (Western blot) and the effects of TRPM4 channel inhibitors, 9‐phenanthrol and glibenclamide, on phasic contractions of UBSM isolated strips obtained from juvenile (UBSM‐J, 5–9 weeks old) and adult (UBSM‐A, 6–18 months old) male guinea pigs. Compared to UBSM‐J, UBSM‐A displayed a 50–70% reduction in total TRPM4 protein expression, while the surface‐to‐intracellular expression ratio (channel trafficking) remained the same in both age groups. Consistent with the reduced total TRPM4 protein expression in UBSM‐A, 9‐phenanthrol showed lower potencies and/or maximum efficacies in UBSM‐A than UBSM‐J for inhibiting amplitude and muscle force of spontaneous and 20 mM KCl‐induced phasic contractions. Compared to 9‐phenanthrol, glibenclamide also attenuated both spontaneous and KCl‐induced contractions, but with less pronounced differential effects in UBSM‐A and UBSM‐J. In both age groups, regardless of the overall reduced total TRPM4 protein expression in UBSM‐A, cell surface TRPM4 protein expression (~80%) predominated over its intracellular fraction (~20%), revealing preserved channel trafficking mechanisms toward the cell membrane. Collectively, this study reports novel findings illuminating a fundamental physiological role for TRPM4 channels in UBSM function that fluctuates with age.

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.

Introduction Voltage‐gated K+ (KV) channels, specifically type 7 (KV7), are emerging key regulators of urinary bladder smooth muscle (UBSM) excitability and contractility. The KV7 channel family consists of 5 pore‐forming α‐subunits (KV7.1–KV7.5) capable of assembling as homotetrameric (e.g. KV7.4) or heterotetrameric (e.g. KV7.4/KV7.5) channels; thus, further increasing species/tissue expression diversity and regulatory potential. However, much remains unknown about KV7 channel properties in UBSM and the translational value of the common experimental animal model ‐ the rat. Hypothesis KV7 channels, especially homomeric or heteromeric KV7.4 and KV7.5 subtypes, regulate rat UBSM excitability and contractility. Methods UBSM tissue without mucosa was obtained from adult male Sprague Dawley rats. UBSM homogenates were used for Western blots to detect KV7 channel subtypes with or without surface biotinylation. UBSM isometric tension recordings were carried out in the presence of tetrodotoxin for spontaneous, 20 mM K+‐induced and 1 µM carbachol‐induced contractions or in its absence for the electrical field stimulated (EFS) contractions. For electrophysiological amphotericin‐B perforated patch‐clamp studies, single UBSM cells were prepared using enzymatic dissociation. UBSM cell membrane potential was recorded in current‐clamp (I=0) mode at room temperature. The following KV7 channel modulators were tested: XE991 (KV7.1‐KV7.5 subtype blocker), retigabine (KV7.2‐KV7.5 activator), and ICA‐069673 (selective activator for KV7.2/KV7.3, which at higher concentrations preferentially enhances KV7.4 and KV7.4/KV7.5 but not KV7.5 channels). Results Western blot experiments detected KV7.4 and KV7.5 channels in UBSM under both non‐ and surface‐biotinylation assay conditions. The latter condition revealed that for both KV7.4 and KV7.5 subtypes surface localization predominated (>75 %). In isometric tension experiments, retigabine attenuated contractility in a concentration‐dependent manner for all four contraction protocols (EC50 values: 2.6‐58.7 µM for the amplitude and force, with complete inhibition of contractility at 100 µM, n=3‐10). XE991 (10 µM) depolarized the membrane potential of isolated UBSM cells (Δ10.5±3.6 mV, n=5, p<0.05). In contrast, retigabine (10 µM, Δ‐6.5±1.8 mV, n=4, p<0.04) and ICA‐069673 (10 µM, Δ‐11.5±2.4 mV, n=4, p<0.02), when applied separately, induced UBSM cell hyperpolarization. In the presence of XE991, ICA‐069673 (10 µM) did not significantly change the membrane potential of isolated UBSM cells (Δ0.4±1.2 mV, n=4, p>0.05). Conclusions Our data show that both KV7.4 and KV7.5 channel subtypes are expressed in rat UBSM and display preferential cell membrane localization. Pharmacological modulation of KV7 channels can control UBSM cell excitability and tissue contractility. The hyperpolarizing effect of ICA‐069673, which is blocked by XE991, revealed a critical role of KV7.4 homomeric and/or KV7.4/KV7.5 heteromeric channels in UBSM function.

Glibenclamide blocks ATP‐sensitive K+ channels (Kir6.x‐SUR) via sulphonylurea receptor (SUR) subunit engagement. Glibenclamide can also inhibit TRPM4‐SUR channel complexes via SUR interaction or TRPM4 channels directly. Recently, our group has found that glibenclamide inhibits whole‐cell non‐selective cation (NSC) currents and spontaneous phasic contractions of guinea pig urinary bladder smooth muscle (UBSM) cells and isolated tissue strips, respectively, although less effectively than 9‐phenanthrol, a TRPM4 channel inhibitor. Here, the age‐dependent effects of glibenclamide and 9‐phenanthrol were determined on guinea pig UBSM whole‐cell NSC currents and spontaneous phasic contractions. Urinary bladders were obtained from young (5–8 wk old, 350–650 g) and retired breeder (RB, 0.5–1.5 yr old, >800 g) male Hartley guinea pigs (Charles River). Mucosa was removed and UBSM strips prepared for isometric tension recordings (spontaneous phasic contractions) or for subsequent enzymatic treatment yielding freshly isolated UBSM cells. UBSM cells were then used in amphotericin‐perforated patch‐clamp recordings either (1) whole‐cell NSC currents optimized by blocking K+ and Ca2+ selective currents or (2) cell membrane potential (I=0) with K+ and Ca2+ channel currents intact. Glibenclamide (100 μM) and 9‐phenanthrol (100 μM) inhibited the voltage‐step‐induced NSC currents in RB‐group UBSM cells; at +106 mV, the respective decreases were 36.6±4.8% (n=6, p<0.0001) and 42.9±3.9% (n=9, p<0.001). In UBSM cells of young guinea pigs, 9‐phenanthrol (100 μM, at +106 mV, 50.4±2.9%, n=7, p<0.001) — but not glibenclamide (100 μM, at +106 mV, 4.6±8.3%, n=6) — caused attenuation in the whole‐cell NSC currents. Glibenclamide (100 μM) did not change the membrane potential in UBSM cells of either young (control: −27.8±4.0 mV; change: +4.2±2.4 mV, n=9, p=0.11) or RB (control: −11.6±4.0 mV; change: −0.42±0.8 mV, n=4, p=0.65) guinea pigs. Of note, a majority of UBSM cells of young (5/9 cells), but not RB‐group (0/4 cells), responded to glibenclamide with depolarization (>3 mV). Spontaneous phasic contractions were concentration‐dependently inhibited by glibenclamide and 9‐phenanthrol in UBSM strips of both age groups (n=5–10). Only glibenclamide displayed age‐dependent differential effects on the muscle force (ANOVA, p=0.04), and the phasic contraction amplitude just failed to reach the statistical significance (p=0.07). IC50 values of glibenclamide for the two parameters were 2–3‐fold more potent in young‐ (~14, 15 μM) than RB‐group UBSM strips (31, 45 μM) with high maximum inhibitions (72–93%, n=7–10). This supports the concept that in RB guinea pigs glibenclamide‐sensitive NSC currents oppose UBSM relaxation. In summary, glibenclamide but not 9‐phenanthrol exhibits age‐dependent effects on whole‐cell NSC currents and spontaneous phasic contractions. In old but not young guinea pigs, glibenclamide‐sensitive NSC channels may contribute to the regulation of UBSM excitability and contractility. Support or Funding Information NIH R01‐DK106964 and P...