Himpens B, De Smedt H, Casteels R
Physiological Laboratory, K. U. Leuven, Gasthuisberg, Louvain, Belgium.
Am J Physiol. 1993 Oct;265(4 Pt 1):C966-75. doi: 10.1152/ajpcell.1993.265.4.C966.
The free calcium concentrations in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of cultured renal LLC-PK1 epithelial cells were estimated by confocal laser microscopy. No difference between the resting mean [Ca2+]n and [Ca2+]c was found. During stimulation with maximal effective concentrations of arginine vasopressin (AVP) or the purinergic agonist ATP, the transient Ca2+ rise was followed mostly by a decline to basal levels. A differential rise could be observed when the increase in [Ca2+]n attained higher values than [Ca2+]c. In 50-60% of the cells, epidermal growth factor (EGF) also induced a transient Ca2+ rise, and a differential increase ([Ca2+]n > [Ca2+]c) was found. The G protein-linked stimuli AVP and ATP were however quantitatively much more efficacious at stimulating the [Ca2+]n and [Ca2+]c increases than was EGF. To investigate whether AVP, ATP, and EGF released Ca2+ from distinct or overlapping stores, the agonists were sequentially added. AVP and ATP applied after EGF in Ca(2+)-free medium elicited an increase in [Ca2+]n and [Ca2+]c that was not significantly lower than the release of Ca2+ in control cells without EGF prestimulation. Similarly, the amplitude of the Ca2+ responses attained by EGF in cells prestimulated by ATP or AVP was comparable to the response in naive cells. Neither EGF, ATP, nor AVP evoked a Ca2+ signal after thapsigargin treatment, indicating that the intracellular Ca2+ pools stimulated by all these agonists are part of the thapsigargin-sensitive Ca2+ pools. In contrast, when ATP was applied after AVP in Ca(2+)-containing as well as in Ca(2+)-free solutions, the Ca2+ transients were lower as compared with the response without preincubation. No differential rise could be found in Ca(2+)-free conditions. An explanation could be the use of different phospholipase C isozymes by the different receptor types, which possibly gives rise to the mobilization of different Ca2+ pools.
通过共聚焦激光显微镜估算培养的肾LLC-PK1上皮细胞核内（[Ca2+]n）和细胞质内（[Ca2+]c）的游离钙浓度。静息状态下的平均[Ca2+]n和[Ca2+]c之间未发现差异。在用最大有效浓度的精氨酸加压素（AVP）或嘌呤能激动剂ATP刺激期间，Ca2+的瞬时升高之后大多是降至基础水平。当[Ca2+]n的升高值高于[Ca2+]c时，可以观察到差异升高。在50%-60%的细胞中，表皮生长因子（EGF）也诱导了Ca2+的瞬时升高，并且发现了差异增加（[Ca2+]n>[Ca2+]c）。然而，与EGF相比，G蛋白偶联刺激物AVP和ATP在刺激[Ca2+]n和[Ca2+]c增加方面在数量上更有效。为了研究AVP、ATP和EGF是否从不同或重叠的储存库中释放Ca2+，依次添加了激动剂。在无钙培养基中，EGF之后施加AVP和ATP引起[Ca2+]n和[Ca2+]c的增加，这并不显著低于未进行EGF预刺激的对照细胞中的Ca2+释放。同样，在由ATP或AVP预刺激的细胞中，EGF引起的Ca2+反应幅度与未处理细胞中的反应相当。在用毒胡萝卜素处理后，EGF、ATP和AVP均未引发Ca2+信号，表明所有这些激动剂刺激的细胞内Ca2+池是毒胡萝卜素敏感Ca2+池的一部分。相比之下，在含Ca2+以及无Ca2+溶液中，AVP之后施加ATP时，与未预孵育的反应相比，Ca2+瞬变较低。在无Ca2+条件下未发现差异升高。一种解释可能是不同受体类型使用了不同的磷脂酶C同工酶，这可能导致动员不同的Ca2+池。
