Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, ...

University of Tennessee Health Science Center

We recently reported key physiologic roles for Ca-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca, inositol 1,4,5-trisphosphate receptor (IPR)-mediated Ca release from the sarcoplasmic reticulum represents a potential Ca source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IPRs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IPRs and are activated by IPR-mediated Ca release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IPRs in human DSM cells. As the TRPM4 channels and IPRs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca-mediated TRPM4-IPR regulatory mechanism. To investigate IPR regulation of TRPM4 channel activity, we sought to determine the consequences of IPR pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IPRs with the selective IPR inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IPRs have a key role in mediating the Ca-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IPRs are spatially and functionally coupled in human DSM.

Regulation of transient receptor potential melastatin 4 channel by sarcoplasmic reticulum inositol trisphosphate receptors: Role in human detrusor smooth muscle function.

Estrogens have an important role in regulating detrusor smooth muscle (DSM) function. However, the underlying molecular and cellular mechanisms by which estrogens control human DSM excitability and contractility are not well known. Here, we used human DSM specimens from open bladder surgeries on 27 patients to elucidate the mechanism by which 17-estradiol regulates large conductance voltage- and Ca-activated K (BK) channels, the most prominent K channels in human DSM We employed single BK channel recordings on inside-out excised membrane patches, perforated whole-cell patch-clamp on freshly isolated DSM cells, and isometric tension recordings on DSM-isolated strips to investigate the mechanism by which 17-estradiol activates BK channels. 17-Estradiol (100 nmol/L) rapidly increased depolarization-induced whole-cell K currents in DSM cells. The 17-estradiol stimulatory effects on whole-cell BK currents were completely abolished by the selective BK channel inhibitor paxilline (1 mol/L), clearly indicating that 17-estradiol specifically activates BK channels. 17-Estradiol also increased the frequency of ryanodine receptor-mediated transient BK currents. Single BK channel recordings showed that 17-estradiol (100 nmol/L) significantly increased the BK channel open probability of inside-out excised membrane patches, revealing that 17-estradiol activates BK channels directly. 17-Estradiol reduced spontaneous phasic contractions of human DSM-isolated strips in a concentration-dependent manner (100 nmol/L-1 mol/L), and this effect was blocked by paxilline (1 mol/L). 17-Estradiol (100 nmol/L) also reduced nerve-evoked contractions of human DSM-isolated strips. Collectively, our results reveal that 17-estradiol plays a critical role in regulating human DSM function through a direct nongenomic activation of BK channels.

Nongenomic modulation of the large conductance voltage- and Ca-activated K channels by estrogen: A novel regulatory mechanism in human detrusor smooth muscle.

Voltage-gated K7 channels (K7.1 to K7.5) are important regulators of the cell membrane potential in detrusor smooth muscle (DSM) of the urinary bladder. This study sought to further the current knowledge of K7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound -(2,4,6-trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of K7.2, K7.4, and K7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 M) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the K7 channel inhibitor XE991 (10 M). ML213 (0.1-30 M) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 M) decreased global intracellular Ca concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 M) caused an increase in the amplitude of whole-cell K7 currents. Further, in current-clamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 M), consistent with ML213 activation of K7 channel currents. Preapplication of XE991 (10 M) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for K7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric K7.4/K7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive K7.4- and K7.5-containing channels are essential regulators of DSM excitability and contractility.

K7 Channel Pharmacological Activation by the Novel Activator ML213: Role for Heteromeric K7.4/K7.5 Channels in Guinea Pig Detrusor Smooth Muscle Function.

Multiple types of Cl channels regulate smooth muscle excitability and contractility in vascular, gastrointestinal, and airway smooth muscle cells. However, little is known about Cl channels in detrusor smooth muscle (DSM) cells. Here, we used inside-out single channel and whole cell patch-clamp recordings for detailed biophysical and pharmacological characterizations of Cl channels in freshly isolated guinea pig DSM cells. The recorded single Cl channels displayed unique gating with multiple subconductive states, a fully opened single-channel conductance of 164 pS, and a reversal potential of -41.5 mV, which is close to the  of -65 mV, confirming preferential permeability to Cl. The Cl channel demonstrated strong voltage dependence of activation (half-maximum of mean open probability, , ~-20 mV) and robust prolonged openings at depolarizing voltages. The channel displayed similar gating when exposed intracellularly to solutions containing Ca-free or 1 mM Ca. In whole cell patch-clamp recordings, macroscopic current demonstrated outward rectification, inhibitions by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid, and insensitivity to chlorotoxin. The outward current was reversibly reduced by 94% replacement of extracellular Cl with I, Br, or methanesulfonate (MsO), resulting in anionic permeability sequence: Cl>Br>I>MsO. While intracellular Ca levels (0, 300 nM, and 1 mM) did not affect the amplitude of Cl current and outward rectification, high Ca slowed voltage-step current activation at depolarizing voltages. In conclusion, our data reveal for the first time the presence of a Ca-independent DIDS and niflumic acid-sensitive, voltage-dependent Cl channel in the plasma membrane of DSM cells. This channel may be a key regulator of DSM excitability.

Properties of single-channel and whole cell Cl currents in guinea pig detrusor smooth muscle cells.

Cl channels serve as key regulators of excitability and contractility in vascular, intestinal, and airway smooth muscle cells. We recently reported a Cl conductance in detrusor smooth muscle (DSM) cells. Here, we used the whole cell patch-clamp technique to further characterize biophysical properties and physiological regulators of the Cl current in freshly isolated guinea pig DSM cells. The Cl current demonstrated outward rectification arising from voltage-dependent gating of Cl channels rather than the Cl transmembrane gradient. An exposure of DSM cells to hypotonic extracellular solution (Δ 165 mOsm challenge) did not increase the Cl current providing strong evidence that volume-regulated anion channels do not contribute to the Cl current in DSM cells. The Cl current was monotonically dependent on extracellular pH, larger and lower in magnitude at acidic (5.0) and basic pH (8.5) values, respectively. Additionally, intracellularly applied phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] analog [PI(4,5)P-diC8] increased the average Cl current density by approximately threefold in a voltage-independent manner. The magnitude of the DSM whole cell Cl current did not depend on the cell surface area (cell capacitance) regardless of the presence or absence of PI(4,5)P-diC8, an intriguing finding that underscores the complex nature of Cl channel expression and function in DSM cells. Removal of both extracellular Ca and Mg did not affect the DSM whole cell Cl current, whereas Gd (1 mM) potentiated the current. Collectively, our recent and present findings strongly suggest that Cl channels are critical regulators of DSM excitability and are regulated by extracellular pH, Gd, and PI(4,5)P.

Extracellular pH and intracellular phosphatidylinositol 4,5-bisphosphate control Cl currents in guinea pig detrusor smooth muscle cells.

Non-selective 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.

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

Georgi V. Petkov

Department of Pharmaceutical Sciences,
College of Pharmacy,
Department of Urology,
College of Medicine,
Department of Pharmacology,
Department of Pharmaceutical Sciences, College of Pharmacy,
Department of Urology, College of Medicine,
Department of Pharmacology, College of Medicine

University of Tennessee Health Science Center

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents