Age‐dependent decrease in TRPM4 channel expression but not trafficking alters urinary bladder smooth muscle contractility

Abstract

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.

Differential effects of TRPM4 channel inhibitors on Guinea pig urinary bladder smooth muscle excitability and contractility: Novel 4‐chloro‐2‐[2‐(2‐chloro‐phenoxy)‐acetylamino]‐benzoic acid (CBA) versus classical 9‐phenanthrol

“…The modest CBA‐induced inhibition observed at the highest concentrations likely reflects non‐selectivity 36 . In contrast, 9‐phenanthrol strongly inhibited guinea pig UBSM contractility 3,13,15 . In our experiments, the maximum concentration of DMSO to dissolve CBA was 0.3% (v/v).…”

“…The isometric UBSM tension recording experiments were conducted as previously described. 3,13,15 Briefly, UBSM strips were clipped between a stationary mount and a force-displacement transducer and then placed in tissue baths filled with Ca 2+containing physiological salt solution (PSS) thermostatically controlled at 37°C. UBSM strips were stretched to an initial level of ~10 mN (1 g) and washed with fresh PSS every 15 min for 45-60 min equilibration period.…”

“…Two major lines of evidence have been provided for the role of TRPM4 channels in UBSM function. First, UBSM tissues and cells showed robust expression of TRPM4 mRNA and protein 3–5,7,12,13 . Second, 9‐phenanthrol, a widely used TRPM4 channel inhibitor, 14 caused attenuation of UBSM cell excitability and inhibition of contractility in UBSM strips 3–7,12,13,15 .…”

“…First, UBSM tissues and cells showed robust expression of TRPM4 mRNA and protein 3–5,7,12,13 . Second, 9‐phenanthrol, a widely used TRPM4 channel inhibitor, 14 caused attenuation of UBSM cell excitability and inhibition of contractility in UBSM strips 3–7,12,13,15 . The presence of TRPM4 channels and pharmacological studies using TRPM4 channel modulators, especially 9‐phenanthrol, have also revealed a role of TRPM4 channels in insulin secretion (pancreatic β‐cells), cardiac excitability (ventricular cells), immune response (lymphocytes and monocytes), and vascular smooth muscle excitability and contractility 10,11,16–18 .…”

Pharmacological Modulation and (Patho)Physiological Roles of TRPM4 Channel—Part 2: TRPM4 in Health and Disease

“…Provence et al suggested a spatial and functional coupling between TRPM4 channels and IP3 receptors in detrusor smooth muscle cells obtained from healthy humans [171]. Moreover, in guinea pigs, an overall reduction of the TRPM4 protein was reduced during aging and in line with that, the action of 9-phenanthrol (inhibition of the amplitude and muscle force of spontaneous and KCl-induced phasic contractions) was less pronounced in adult animals compared with juvenile ones [176].…”