Muderrisoglu A E, Ciotkowska A, Rutz B, Hu S, Qian S, Tamalunas A, Stief C G, Hennenberg M
Department of Urology, LMU University Hospital, LMU Munich, Munich, Germany.
Department of Medical Pharmacology, Istanbul Medipol University, Istanbul, Türkiye.
Front Pharmacol. 2024 Jul 24;15:1446831. doi: 10.3389/fphar.2024.1446831. eCollection 2024.
Mirabegron is available for treatment of overactive bladder (OAB). However, mechanisms underlying symptom improvements and long-term effects on bladder smooth muscle cells are uncertain. Contractility and growth of bladder smooth muscle contribute to OAB, and depend on smooth muscle phenotypes, and on muscarinic receptor expression. Here, we examined prolonged exposure to mirabegron (20-48 h) on phenotype markers, muscarinic receptor expression, and phenotype-dependent functions in human bladder smooth muscle cells (hBSMC).
Expression of markers for contractile (calponin, MYH11) and proliferative (MYH10, vimentin) phenotypes, proliferation (Ki-67), and of muscarinic receptors were assessed by RT-PCR. Proliferation, viability, actin organization and contractions in cultured hBSMC were examined by EdU, CCK-8, phalloidin staining and matrix contraction assays.
Calponin-1 mRNA decreased with 100 nM and 150 nM mirabegron applied for 20 h (0.56-0.6 fold of controls). Decreases were resistant to the β-AR antagonist L-748,337 (0.34-0.55 fold, 100-150 nM, 20 h). After 40 h, decreases occured in the presence of L-748,337, but not without L-748,337. MYH11 mRNA increased with 150 nM mirabegron (40 h, 1.9 fold). This was partly preserved with L-748,337, but not observed after 20 h mirabegron exposure. Vimentin mRNA reduced with 150 nM mirabegron after 20 h, but not after 40 h, with and without L-748,337 (0.71-0.63 fold). MYH10 mRNA expression remained unaffected by mirabegron. Exposure to 150 nM mirabegron increased Ki-67 mRNA after 20 h in the presence of, but not without L-748,337, and after 40 h without, but not with L-748,337. Proliferation rates and actin organization were stable with 50-150 nM mirabegron (24 h, 48 h). Viability increased significantly after mirabegron exposure for 20 h, and by trend after 40 h, which was fully sensitive to L-748,337. M2 mRNA was reduced by 20 h mirabegron, which was resistant to L-748,337. Carbachol (3 µM) enhanced time-dependent contractions of hBSMC, which was inhibited by mirabegron (150 nM) in late phases (24 h), but not in early phases of contractions. Mirabegron induces dynamic phenotype alterations and M2 downregulation in hBSMC, which is paralleled by time-shifted anticontractile effects. Phenotype transitions may be involved in improvements of storage symptoms in OAB by mirabegron.
米拉贝隆可用于治疗膀胱过度活动症（OAB）。然而，症状改善的潜在机制以及对膀胱平滑肌细胞的长期影响尚不确定。膀胱平滑肌的收缩性和生长与OAB有关，并且取决于平滑肌表型以及毒蕈碱受体表达。在此，我们研究了人膀胱平滑肌细胞（hBSMC）长时间暴露于米拉贝隆（20 - 48小时）对表型标志物、毒蕈碱受体表达以及表型依赖性功能的影响。
通过逆转录聚合酶链反应（RT-PCR）评估收缩性（钙调蛋白、肌球蛋白重链11，MYH11）和增殖性（肌球蛋白重链10，波形蛋白）表型的标志物、增殖（Ki-67）以及毒蕈碱受体的表达。通过EdU、CCK-8、鬼笔环肽染色和基质收缩试验检测培养的hBSMC中的增殖、活力、肌动蛋白组织和收缩情况。
在应用100 nM和150 nM米拉贝隆20小时后，钙调蛋白-1 mRNA下降（为对照的0.56 - 0.6倍）。这种下降对β-肾上腺素能受体拮抗剂L-748,337有抗性（0.34 - 0.55倍，100 - 150 nM，20小时）。40小时后，在存在L-748,337的情况下出现下降，但在没有L-748,337时未出现下降。150 nM米拉贝隆作用40小时后，MYH11 mRNA增加（1.9倍）。在存在L-748,337时部分得以保留，但在米拉贝隆暴露20小时后未观察到这种情况。150 nM米拉贝隆作用20小时后波形蛋白mRNA下降，但在40小时后，无论有无L-748,337均未下降（0.71 - 0.63倍）。MYH10 mRNA表达不受米拉贝隆影响。在存在L-748,337但无L-748,337时，暴露于150 nM米拉贝隆20小时后Ki-67 mRNA增加，40小时后在无L-748,337但有L-748,337时未增加。50 - 150 nM米拉贝隆（24小时、48小时）作用下增殖率和肌动蛋白组织稳定。米拉贝隆暴露20小时后活力显著增加，40小时后有增加趋势，这对L-748,337完全敏感。米拉贝隆作用20小时使M2 mRNA减少，这对L-748,337有抗性。卡巴胆碱（3 μM）增强hBSMC的时间依赖性收缩，在收缩后期（24小时）米拉贝隆（150 nM）可抑制这种收缩，但在收缩早期无此作用。米拉贝隆诱导hBSMC中动态表型改变和M2下调，这与时间延迟的抗收缩作用平行。表型转变可能参与米拉贝隆改善OAB的储尿期症状。
