Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, South Carolina (W.X., N.L., Q.C., G.V.P.); and Department of Urology, Fourth Hospital of China Medical University, Shenyang, China (N.L.).
J Pharmacol Exp Ther. 2014 Apr;349(1):56-65. doi: 10.1124/jpet.113.210708. Epub 2014 Jan 23.
Elevation of intracellular cAMP and activation of protein kinase A (PKA) lead to activation of large conductance voltage- and Ca(2+)-activated K(+) (BK) channels, thus attenuation of detrusor smooth muscle (DSM) contractility. In this study, we investigated the mechanism by which pharmacological inhibition of cAMP-specific phosphodiesterase 4 (PDE4) with rolipram or Ro-20-1724 (C(15)H(22)N(2)O(3)) suppresses guinea pig DSM excitability and contractility. We used high-speed line-scanning confocal microscopy, ratiometric fluorescence Ca(2+) imaging, and perforated whole-cell patch-clamp techniques on freshly isolated DSM cells, along with isometric tension recordings of DSM isolated strips. Rolipram caused an increase in the frequency of Ca(2+) sparks and the spontaneous transient BK currents (TBKCs), hyperpolarized the cell membrane potential (MP), and decreased the intracellular Ca(2+) levels. Blocking BK channels with paxilline reversed the hyperpolarizing effect of rolipram and depolarized the MP back to the control levels. In the presence of H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride], a PKA inhibitor, rolipram did not cause MP hyperpolarization. Rolipram or Ro-20-1724 reduced DSM spontaneous and carbachol-induced phasic contraction amplitude, muscle force, duration, and frequency, and electrical field stimulation-induced contraction amplitude, muscle force, and tone. Paxilline recovered DSM contractility, which was suppressed by pretreatment with PDE4 inhibitors. Rolipram had reduced inhibitory effects on DSM contractility in DSM strips pretreated with paxilline. This study revealed a novel cellular mechanism whereby pharmacological inhibition of PDE4 leads to suppression of guinea pig DSM contractility by increasing the frequency of Ca(2+) sparks and the functionally coupled TBKCs, consequently hyperpolarizing DSM cell MP. Collectively, this decreases the global intracellular Ca(2+) levels and DSM contractility in a BK channel-dependent manner.
细胞内环磷酸腺苷（cAMP）水平的升高和蛋白激酶 A（PKA）的激活会导致大电导电压和 Ca2+激活的钾（BK）通道的激活，从而减弱逼尿肌平滑肌（DSM）的收缩性。在这项研究中，我们研究了通过罗利普兰或 Ro-20-1724（C15H22N2O3）抑制 cAMP 特异性磷酸二酯酶 4（PDE4）来抑制豚鼠 DSM 兴奋性和收缩性的机制。我们使用高速线扫描共聚焦显微镜、比率荧光 Ca2+成像以及新鲜分离的 DSM 细胞的穿孔全细胞膜片钳技术，以及 DSM 分离条带的等长张力记录。罗利普兰引起 Ca2+火花和自发瞬态 BK 电流（TBKCs）的频率增加，细胞膜电位（MP）超极化，并降低细胞内 Ca2+水平。用巴氯芬阻断 BK 通道会逆转罗利普兰的超极化作用，并将 MP 去极化回对照水平。在 PKA 抑制剂 H-89[N-[2-[[3-(4-溴苯基)-2-丙烯基]氨基]乙基]-5-异喹啉磺酰胺二盐酸盐]存在的情况下，罗利普兰不会引起 MP 超极化。罗利普兰或 Ro-20-1724 降低了 DSM 自发性和 carbachol 诱导的相收缩幅度、肌肉力、持续时间和频率，以及电场刺激诱导的收缩幅度、肌肉力和张力。巴氯芬恢复了被 PDE4 抑制剂预处理抑制的 DSM 收缩性。罗利普兰对预先用巴氯芬处理的 DSM 条带的 DSM 收缩性的抑制作用降低。这项研究揭示了一种新的细胞机制，即通过增加 Ca2+火花和功能偶联的 TBKCs 的频率，从而超极化 DSM 细胞 MP，从而抑制 PDE4 可导致豚鼠 DSM 收缩性的抑制。总的来说，这以 BK 通道依赖性的方式降低了整体细胞内 Ca2+水平和 DSM 收缩性。
