Distinct Age‐dependent Effects of Glibenclamide on Guinea Pig Urinary Bladder Smooth Muscle Non‐selective Cation Currents and Spontaneous Phasic Contractions

Abstract

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 InformationNIH R01‐DK106964 and P20‐DK123971 to Georgi V. Petkov.