Paradiso A M, Brown H A, Ye H, Harden T K, Boucher R C
Department of Pharmacology, University of North Carolina at Chapel Hill School of Medicine 27599-7020, USA.
Exp Lung Res. 1999 Jun;25(4):277-90. doi: 10.1080/019021499270196.
The Ca(2+)-mobilizing actions of adenosine 5'-triphosphate (ATP), bradykinin, and histamine were compared in phenotypically distinct human nasal epithelial (HNE) cell types and as a function of time in cell culture. Single-cell measurements of intracellular free Ca2+ (Ca2+i, Fura-2 fluorescence) were recorded in ciliated cells 1-2 days in primary culture, and in nonciliated cells 1-2 days (keratin 14-positive) or 4-5 days (keratin 18-positive) after seeding. No difference in basal Ca2+i was noted between ciliated and nonciliated cell preparations. For ciliated and nonciliated cells studied 1-2 days in culture, ATP, bradykinin, and histamine elicited a cytosolic Ca2+ response in 100% of the cells examined. For nonciliated HNE cells maintained 4-5 days in culture, ATP (10(-4) M) increased cytosolic Ca2+ in all cells tested, but only 85% of the cells responded to bradykinin (10(-5) M) addition, and 65% to histamine (10(-4) M) stimulation. In terms of the absolute change of Ca2+i (delta Ca2+i, peak-basal value), the efficacy was ATP > bradykinin > histamine for the 3 HNE cell preparations. However, the delta Ca2+i in response to agonists was smaller in nonciliated HNE cells studied 1-2 days or 4-5 days in culture as compared to the ciliated cell preparation. Thapsigargin (300 nM), an agent that mobilizes Ca2+i, was equally effective in raising cytosolic Ca2+ in nonciliated (1-2 days and 4-5 days in culture) and ciliated HNE cells. These data show that ciliated cells consistently respond to all agonists, whereas the cytosolic Ca2+ response to ATP, bradykinin, and histamine in nonciliated cells was quantitatively reduced at a comparable time period (1-2 days) and became smaller and less frequent in nonciliated cell preparations maintained 4-5 days in culture. These results demonstrate time-dependent differences in the magnitude and frequency of cytosolic Ca2+ responses to certain agonists, strongly indicating that measurements of Ca2+i in HNE cells must account for the heterogeneity of the cell types and the time cells are maintained in primary culture.
比较了三磷酸腺苷（ATP）、缓激肽和组胺在表型不同的人鼻上皮（HNE）细胞类型中的钙离子动员作用，以及在细胞培养过程中随时间的变化。在原代培养1 - 2天的纤毛细胞中，以及接种后1 - 2天（角蛋白14阳性）或4 - 5天（角蛋白18阳性）的非纤毛细胞中，记录细胞内游离钙离子（Ca2+i，Fura - 2荧光）的单细胞测量值。在纤毛细胞制剂和非纤毛细胞制剂之间，未观察到基础Ca2+i有差异。对于培养1 - 2天的纤毛细胞和非纤毛细胞，ATP、缓激肽和组胺在所有检测的细胞中均引发了胞质Ca2+反应。对于在培养中维持4 - 5天的非纤毛HNE细胞，ATP（10(-4)M）使所有测试细胞中的胞质Ca2+增加，但只有85%的细胞对添加缓激肽（10(-5)M）有反应，65%的细胞对组胺（10(-4)M）刺激有反应。就Ca2+i的绝对变化（δCa2+i，峰值 - 基础值）而言，对于这3种HNE细胞制剂，效力为ATP > 缓激肽 > 组胺。然而，与纤毛细胞制剂相比，在培养1 - 2天或4 - 5天的非纤毛HNE细胞中，对激动剂的δCa2+i较小。毒胡萝卜素（300 nM），一种动员Ca2+i的试剂，在非纤毛（培养1 - 2天和4 - 5天）和纤毛HNE细胞中升高胞质Ca2+的效果相同。这些数据表明，纤毛细胞始终对所有激动剂有反应，而在相当的时间段（1 - 2天）内，非纤毛细胞中对ATP、缓激肽和组胺的胞质Ca2+反应在数量上有所减少，并且在培养4 - 5天的非纤毛细胞制剂中变得更小且频率更低。这些结果证明了对某些激动剂的胞质Ca2+反应在幅度和频率上存在时间依赖性差异，强烈表明在HNE细胞中测量Ca2+i时必须考虑细胞类型的异质性以及细胞在原代培养中维持的时间。
