Wood L C, Neufeld E F
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024.
J Biol Chem. 1990 Aug 5;265(22):12796-800.
As a step toward understanding the metabolic consequences of the cystic fibrosis (CF) mutation, we have examined the kinetics of 36Cl efflux in cells cultured from sweat glands, a tissue that is affected in the disease. Epithelial cells, derived from the secretory coil of sweat glands of CF and control individuals, were cultured in serum-free medium, and primary cultures used for efflux experiments. Cell layers were equilibrated with Na36Cl in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered balanced salt solution for 45 min at 37 degrees C, washed in 0.25 M sucrose, and incubated in nonradioactive buffer for measurement of 36Cl efflux. Efflux from CF and control cells followed biphasic kinetics and was described by the equation Y = Ae-kat + Be-kbt. All efflux was inhibited at 6 degrees C. The fast component of efflux, Ae-kat, of both control and CF cells was inhibited by the anion channel blockers 4,4'-diisothiocyanato-2,2'-stilbene disulfonic acid, 9-anthracene carboxylate, and diphenylamine 2-carboxylate, implicating release through chloride channels. At 23 degrees C, the kinetics of 36Cl efflux from CF and control cells were indistinguishable, but efflux from control cells could be accelerated by cAMP analogs and isoproterenol. At 37 degrees C, 36Cl efflux was more rapid from control cells than from CF cells, but could not be stimulated further by beta-adrenergic agents. In both cases, the increased rate of efflux was due to a severalfold increase in the A parameter of the fast component. These differential responses constitute a "CF phenotype" of secretory sweat gland cells in culture that may be useful for further investigation of the metabolic defect in cystic fibrosis.
作为了解囊性纤维化（CF）突变代谢后果的一个步骤，我们研究了从汗腺培养的细胞中³⁶Cl外流的动力学，汗腺是该疾病中受影响的组织。从CF患者和对照个体的汗腺分泌盘衍生的上皮细胞在无血清培养基中培养，并用于外流实验的原代培养物。细胞层在37℃下用4-（2-羟乙基）-1-哌嗪乙磺酸缓冲的平衡盐溶液中的Na³⁶Cl平衡45分钟，在0.25M蔗糖中洗涤，并在非放射性缓冲液中孵育以测量³⁶Cl外流。CF细胞和对照细胞的外流遵循双相动力学，并由方程Y = Ae⁻ᵏᵃᵗ + Be⁻ᵏᵇᵗ描述。所有外流在6℃时均受到抑制。对照细胞和CF细胞外流的快速成分Ae⁻ᵏᵃᵗ受到阴离子通道阻滞剂4,4'-二异硫氰酸根合-2,2'-二苯乙烯二磺酸、9-蒽甲酸酯和二苯胺-2-羧酸酯的抑制，这表明通过氯离子通道释放。在23℃时，CF细胞和对照细胞的³⁶Cl外流动力学无法区分，但对照细胞的外流可被cAMP类似物和异丙肾上腺素加速。在37℃时，对照细胞的³⁶Cl外流比CF细胞更快，但不能被β-肾上腺素能药物进一步刺激。在这两种情况下，外流速率的增加是由于快速成分的A参数增加了几倍。这些差异反应构成了培养的分泌汗腺细胞的“CF表型”，这可能有助于进一步研究囊性纤维化的代谢缺陷。
