Hamilton Kirk L, Kiessling Matt
Dept. of Physiology, School of Medical Sciences, Univ. of Otago, PO Box 913, Dunedin, New Zealand.
Am J Physiol Cell Physiol. 2006 Jan;290(1):C152-64. doi: 10.1152/ajpcell.00187.2005. Epub 2005 Aug 31.
We investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one(DCEBIO) on the Cl- secretory response of the mouse jejunum using the Ussing short-circuit current (Isc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in Isc (EC50 41 +/- 1 microM). Pretreating tissues with 0.25 microM forskolin reduced the concentration-dependent increase in Isc by DCEBIO and increased the EC50 (53 +/- 5 microM). Bumetanide blocked (82 +/- 5%) the DCEBIO-stimulated Isc consistent with Cl- secretion. DCEBIO was a more potent stimulator of Cl- secretion than its parent molecule, 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated Isc by >80% indicating the participation of CFTR in the DCEBIO-stimulated Isc response. Clotrimazole reduced DCEBIO-stimulated Isc by 67 +/- 15%, suggesting the participation of the intermediate conductance Ca2+-activated K+ channel (IKCa) in the DCEBIO-activated Isc response. In the presence of maximum forskolin (10 microM), the DCEBIO response was reduced and biphasic, reaching a peak response of the change in Isc of 43 +/- 5 microA/cm2 and then falling to a steady-state response of 17 +/- 10 microA/cm2 compared with DCEBIO control tissues (61 +/- 6 microA/cm2). The forskolin-stimulated Isc in the presence of DCEBIO was reduced compared with forskolin control tissues. Similar results were observed with DCEBIO and 8-BrcAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated Isc, providing evidence that DCEBIO increased Cl- secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl- secretion of the mouse jejunum and that DCEBIO targets components of the Cl- secretory mechanism.
我们使用尤斯灌流短路电流（Isc）技术研究了5,6-二氯-1-乙基-1,3-二氢-2H-苯并咪唑-2-酮（DCEBIO）对小鼠空肠Cl⁻分泌反应的影响。DCEBIO刺激Isc呈浓度依赖性持续增加（半数有效浓度[EC50]为41±1微摩尔/升）。用0.25微摩尔/升福斯高林预处理组织可降低DCEBIO引起的Isc浓度依赖性增加，并提高EC50（53±5微摩尔/升）。布美他尼可阻断（82±5%）DCEBIO刺激的Isc，这与Cl⁻分泌一致。DCEBIO是比其母体分子1-乙基-2-苯并咪唑啉酮更强效的Cl⁻分泌刺激剂。格列本脲或5-硝基-2-（3-苯丙胺基）苯甲酸（NPPB）可使DCEBIO刺激的Isc降低>80%，表明囊性纤维化跨膜传导调节因子（CFTR）参与了DCEBIO刺激的Isc反应。克霉唑可使DCEBIO刺激的Isc降低67±15%，提示中间电导钙激活钾通道（IKCa）参与了DCEBIO激活的Isc反应。在存在最大浓度福斯高林（10微摩尔/升）时，DCEBIO反应降低且呈双相性，与DCEBIO对照组织（61±6微安/平方厘米）相比，Isc变化的峰值反应为43±5微安/平方厘米，然后降至17±10微安/平方厘米的稳态反应。与福斯高林对照组织相比，在存在DCEBIO时福斯高林刺激的Isc降低。在绕过腺苷酸环化酶的情况下，用DCEBIO和8-溴腺苷-3',5'-环一磷酸（8-BrcAMP）观察到了类似结果。蛋白激酶A（PKA）抑制剂H89可降低DCEBIO激活的Isc，这证明DCEBIO通过环磷酸腺苷/蛋白激酶A依赖性方式增加Cl⁻分泌。这些数据表明DCEBIO刺激小鼠空肠的Cl⁻分泌，且DCEBIO作用于Cl⁻分泌机制的组成部分。
