Grubb B, Lazarowski E, Knowles M, Boucher R
Department of Medicine, University of North Carolina, Chapel Hill 27599-7020.
Am J Respir Cell Mol Biol. 1993 Apr;8(4):454-60. doi: 10.1165/ajrcmb/8.4.454.
It has been proposed that a combination of an activated adenylyl cyclase and a high concentration of a phosphodiesterase inhibitor (isobutylmethylxanthine [IBMX], 5 mM) stimulates Cl- secretion mediated by the heterologously expressed cystic fibrosis transmembrane regulator protein carrying the most common cystic fibrosis (CF) mutation (delta F508). We tested whether Cl- secretion could be stimulated by this protocol in vitro and in vivo in CF airway epithelia expressing endogenous delta F508 CFTR protein. In cultured airway preparations, forskolin (a direct adenylyl cyclase activator) stimulated Cl- secretion in amiloride-pretreated normal (delta Isc = 7.1 +/- 1.7 microA.cm-2) but not CF tissues (delta Isc = -02 +/- 0.1 microA.cm-2). Unexpectedly, IBMX partially inhibited the forskolin-induced Cl- secretion in normal tissues; IBMX addition had no effect on CF tissues. Direct measurements of cell cAMP concentrations revealed that 0.1 mM IBMX and forskolin produced the maximum levels of cell cAMP levels attainable with this drug combination, and 5 mM IBMX was without further effect. The combination of forskolin (10(-5) M) and isoproterenol, an adenylyl cyclase activator (10(-5) M), produced approximately 3 times higher levels of cAMP than forskolin/IBMX but also did not induce Cl- secretion in CF tissues. Studies of Cl- secretion in vivo, assessed by the transepithelial electric potential difference (PD), showed that isoproterenol (10(-5) M) stimulated Cl- secretion (delta PD = -16.3 +/- 4.3 mV; n = 4) in nasal epithelia of normal subjects but not in CF patients homozygous for the delta F508 mutation (delta PD = -2.6 +/- 1.9 mV; n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)
有人提出，激活的腺苷酸环化酶与高浓度的磷酸二酯酶抑制剂（异丁基甲基黄嘌呤[IBMX]，5 mM）联合使用，可刺激由携带最常见囊性纤维化（CF）突变（ΔF508）的异源表达囊性纤维化跨膜调节蛋白介导的Cl⁻分泌。我们测试了该方案在体外和体内表达内源性ΔF508 CFTR蛋白的CF气道上皮细胞中是否能刺激Cl⁻分泌。在培养的气道制剂中，福斯可林（一种直接的腺苷酸环化酶激活剂）刺激了用阿米洛利预处理的正常组织（ΔIsc = 7.1±1.7 μA/cm²）中的Cl⁻分泌，但未刺激CF组织（ΔIsc = -0.2±0.1 μA/cm²）。出乎意料的是，IBMX部分抑制了正常组织中福斯可林诱导的Cl⁻分泌；添加IBMX对CF组织没有影响。细胞cAMP浓度的直接测量显示，0.1 mM IBMX和福斯可林产生了该药物组合可达到的最大细胞cAMP水平，而5 mM IBMX没有进一步作用。福斯可林（10⁻⁵ M）和异丙肾上腺素（一种腺苷酸环化酶激活剂，10⁻⁵ M）的组合产生的cAMP水平比福斯可林/IBMX高约3倍，但也未在CF组织中诱导Cl⁻分泌。通过跨上皮电位差（PD）评估的体内Cl⁻分泌研究表明，异丙肾上腺素（10⁻⁵ M）刺激了正常受试者鼻上皮中的Cl⁻分泌（ΔPD = -16.3±4.3 mV；n = 4），但未刺激ΔF508突变纯合的CF患者（ΔPD = -2.6±1.9 mV；n = 5）。（摘要截断于250字）
