Fewtrell C, Sherman E
Department of Pharmacology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853.
Biochemistry. 1987 Nov 3;26(22):6995-7003. doi: 10.1021/bi00396a021.
The intracellular calcium indicator and buffer quin2 has been used to generate a large calcium buffering capacity in the cytoplasm of rat basophilic leukemia cells. Above 3 mM intracellular quin2, there is no further increase in the initial rate of antigen-induced 45Ca uptake, suggesting that 45Ca buffering by quin2 is now sufficient to prevent the immediate efflux of 45Ca from the cells. Thus, the initial rate of 45Ca uptake should reflect the true unidirectional influx of calcium that occurs when immunoglobulin E (IgE) receptors are aggregated by antigen. The antigen-induced calcium permeability pathway appears to be saturable, with a Km of about 0.7 mM and a Vmax of 0.9 nmol of calcium (10(6) cells)-1 min-1. Although net 45Ca uptake reaches a plateau a few minutes after antigen stimulation, the increase in plasma membrane permeability is maintained for at least an hour, provided that receptors for IgE remain aggregated. The initial rate of 45Ca influx correlates well with the subsequent secretion of [3H]serotonin in response to different concentrations of antigen. Both 45Ca uptake and [3H]serotonin secretion are maximal when only 10% of the receptors are occupied with antigen-specific IgE. Thus, 45Ca influx correlates more closely with secretion than with the number of IgE receptors aggregated by antigen.
细胞内钙指示剂兼缓冲剂喹啉-2已被用于在大鼠嗜碱性白血病细胞的细胞质中产生较大的钙缓冲能力。当细胞内喹啉-2浓度高于3 mM时，抗原诱导的45Ca摄取初始速率不再进一步增加，这表明喹啉-2对45Ca的缓冲作用已足以防止45Ca立即从细胞中流出。因此，45Ca摄取的初始速率应反映当免疫球蛋白E（IgE）受体被抗原聚集时发生的真正的钙单向流入。抗原诱导的钙通透性途径似乎是可饱和的，其米氏常数（Km）约为0.7 mM，最大反应速度（Vmax）为0.9 nmol钙（10^6个细胞）-1分钟-1。尽管抗原刺激后几分钟净45Ca摄取达到平台期，但只要IgE受体保持聚集状态，质膜通透性的增加就会持续至少一小时。45Ca流入的初始速率与随后不同浓度抗原刺激下[3H]5-羟色胺的分泌密切相关。当只有10%的受体被抗原特异性IgE占据时，45Ca摄取和[3H]5-羟色胺分泌均达到最大值。因此，45Ca流入与分泌的相关性比与被抗原聚集的IgE受体数量的相关性更紧密。
