Transient Receptor Potential (TRP) channels are the most recently discovered superfamily of non-selective cation channels that are involved in multiple cell types and regulate important physiological functions. However, very little is known about the pharmaco-physiological properties of the TRP channels in human detrusor smooth muscle (DSM). They are thought to play a role in increasing DSM cellular excitability and contractility. Recently, expression of TRP melastatin type 4 (TRPM4) channels has been established in human and animal (e.g. rat and guinea pig) DSM cells [1,2,3]. The TRPM4 channel is a voltage-dependent calcium-activated nonselective cation channel displaying preferential plasma membrane expression and sensitivity to inhibition by 9-phenanthrol. When tested in human, guinea pig, and rat DSM cells and strips (mucosa-free), 9-phenanthrol decreased excitability and contractility supporting the hypothesis that TRPM4 channels regulate DSM excitation-contraction coupling [1,2]. It is worth noting that 9-phenanthrol exhibited stronger effects on DSM whole cell cation currents in human than in guinea pig suggesting species differences [3]. Since 9-phenanthrol displays weak selectivity, medicinal chemistry efforts are underway to design improved TRPM4 channel modulators. The recently developed compound 4-chloro-2-[2-(2-chloro-phenoxy)-acetylamino]-benzoic acid (CAB) has been characterized as a highly selective TRPM4 inhibitor. This novel ion channel blocker exhibits improved selectivity and ~10-fold higher potency at TRPM4 channels than 9-phenanthrol. Here, we examined the effects of CAB on DSM excitation-contraction coupling in both human and guinea pig and compared these results to those of 9-phenanthrol reported in these species previously [1,2].  The aims for this study were two-fold: (a) to provide additional evidence for a role of TRPM4 channels in the regulation of DSM excitability and contractility and (b) to reveal whether CAB displays species-dependent pharmacological effects on human and guinea pig DSM.

Human DSM tissue harvesting: Full thickness urinary bladder specimens were obtained from twelve adult patient-donors (average age: 68.1 years old, range: 54 – 84; race: nine Caucasian, two African-American, and one unknown; gender: two female and ten male) undergoing open bladder surgeries. The specimens were placed in ice-cold physiological solution and transported to the laboratory. Mucosa, adjacent connective and adipose tissues, and blood vessels were removed from specimens by sharp dissection isolating DSM whole tissues. DSM strips were then prepared for subsequent investigations.

Guinea pig DSM tissue harvesting: Eighteen adult male Hartley-Albino guinea pigs (651.5 g, 577.3 – 879.5 g; median, 25th and 75th quartiles) were euthanized with either CO2 or isofluorane, followed by thoracotomy. The whole bladders were then excised and transferred to ice-cold physiological solution. The bladders were opened and the mucosa was removed. DSM strips (3 - 10 mm long and 2 - 4 mm wide) were then prepared for isometric tension recording and single DSM cell isolation.

Fresh single-DSM cell isolation and perforated whole-cell patch-clamp electrophysiological recordings: Enzymatic dissociation of mucosa-free DSM was utilized to obtain freshly-isolated DSM cells (both human and guinea pig) to be used in amphotericin-B perforated patch-clamp electrophysiological experiments. This was performed by a two-step process using papain and collagenase type II as previously described [1,2,3]. The conditions for recording voltage-step induced cation whole-cell currents (K+-free) and membrane potential (at physiological K+) followed the standard experimental procedures [1,2,3]. 

Isometric tension recordings: Human and guinea pig mucosa-free DSM strips were mounted for isometric tension recordings as previously described [1,2]. DSM strips were placed in thermostatically regulated (37⁰C) tissue baths filled with calcium-containing physiological salt solution. The effects of CAB on DSM spontaneous phasic and 20 mM KCl-induced contractions were determined by cumulative addition of the compound.

CAB (10-100 uM) inhibited both human DSM spontaneous phasic and 20 mM KCl-induced phasic contractions (n=3-6) in a concentration-dependent manner. The results reveal that CAB more effectively attenuated spontaneous phasic contractions (amplitude, force, duration, and frequency IC50’s  <10 uM, complete inhibitions at 30 uM, n=3) than 20 mM KCl-induced contractions (amplitude, force, and duration IC50’s: 58.1–146.8 uM, maximum inhibitions: 46-74% with either no or very weak effect on contraction frequency, n=6). In contrast, CAB (100 uM and lower contractions) did not effectively inhibit guinea pig DSM spontaneous phasic and 20 mM KCl-induced phasic contractions (n=3-21). For all guinea pig DSM phasic contraction parameters (amplitude, force, and duration and frequency), the maximum inhibitions observed at 100 uM CAB were 10-35% (and IC50’s >100 uM), except for the 20 mM KCl-induced phasic contraction frequency, which did not decrease. 

In human DSM isolated cells, CAB (30-50 uM) reduced voltage step-induced cation currents (voltage-clamp) with relatively weak maximum efficacy (at +96 mV, the normalized current decreased from 1.00 to 0.90±0.04, n=7, p<0.0001) and under the recording conditions of physiological K+ hyperpolarized the membrane potential (current-clamp, I=0) from 2.4 to 21.9 mV (n=3). In contrast, CAB did not change the voltage-step induced cation currents (30-100 uM, n=8) and cell membrane potentials (30 uM, n=5) in guinea pig DSM cells.

CAB attenuated both human DSM strip contractility and DSM cell excitability. Compared to 9-phenanthrol examined previously on human DSM [2], CAB displayed quantitatively comparable or weaker effects than 9-phenanthrol. For example, on voltage step-induced cation currents at +96 mV, CAB and 9-phenanthrol inhibited the currents by ~10% (this study) and ~40% [2], respectively. On the cell membrane potential (current-clamp) and on spontaneous and KCl-induced DSM phasic contractions, CAB exhibited similar effects to 9-phenanthrol. In guinea pig, CAB at concentrations active on TRPM4 channels (30-100 uM) effectively changed neither DSM contractility nor cell excitability under the same experimental conditions where 9-phenanthrol showed robust attenuations [1]. CAB, hence, displayed differential species-dependent activity on DSM function.

The novel selective TRPM4 channel blocker CAB attenuated cell excitability and contractility in human DSM providing strong support for TRPM4 channels in regulating urinary bladder function. The effects of CAB on human DSM function were either comparable or less robust than those of the poorly selective TRPM4 channel blocker 9-phenanthrol. In guinea pig, CAB was either ineffective or caused very weak inhibitions on DSM functional parameters revealing species-dependent pharmacological effects for this novel selective TRPM4 channel inhibitor. This study further emphasizes the critical importance of conducting translational studies directly on human bladder DSM specimens rather using experimental animals due to target-specific species differences such as demonstrated here for the TRPM4 channel.

Malysz, J., Maxwell, S.E., Yarotskyy, V., and Petkov, G.V. (2020). TRPM4 channel inhibitors 9-phenanthrol and glibenclamide differentially decrease guinea pig detrusor smooth muscle whole-cell cation currents and phasic contractions. Am J Physiol Cell Physiol 318, C406-C421.
Hristov, K.L., Smith, A.C., Parajuli, S.P., Malysz, J., Rovner, E.S., and Petkov, G.V. (2016). Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function. Am J Physiol Cell Physiol 310, C600-611.
Parajuli, S.P., Hristov, K.L., Sullivan, M.N., Xin, W., Smith, A.C., Earley, S., Malysz, J., and Petkov, G.V. (2013). Control of urinary bladder smooth muscle excitability by the TRPM4 channel modulator 9-phenanthrol. Channels (Austin) 7, 537-540.