Daniels A J, Dean G, Viveros O H, Diliberto E J
Mol Pharmacol. 1983 Mar;23(2):437-44.
Chromaffin cells in primary culture take up [14C]ascorbic acid from the incubation medium. Cells, stimulated immediately after a short labeling period with [14C]ascorbate, secrete ascorbic acid concomitantly with catecholamines (CA) through a nicotinic receptor-mediated Ca2+-dependent process. A proportional release of CA and [14C]ascorbic acid was observed through a large range of secretion rates obtained by varying the concentration of nicotine or by changing the concentrations of Ca2+ and Na+ in the external medium. However, under the same conditions of stimulation, different cell preparations secrete 2-10 times more CA than [14C]ascorbate (as percentage of cell content). Furthermore, a different time course of secretion was observed for CA and [14C]ascorbate for each of several secretagogues. In addition, Ba2+ is a much more potent stimulus for CA secretion than for secretion of [14C]ascorbate, and Ca2+ channel blockers are more potent in inhibiting CA secretion than [14C]ascorbate secretion. These data suggested the possibility that newly taken up ascorbate was being secreted from a compartment altogether distinct from the chromaffin vesicle. This hypothesis was confirmed by subcellular distribution studies, where only a minor fraction of newly taken up [14C]ascorbate was found in the vesicular fraction (P2) from homogenates of chromaffin cells prepared after a short incubation with [14C]ascorbate. However, the subcellular distribution of [14C]ascorbate follows that of endogenous ascorbate when a short pulse with the label is chased by a prolonged equilibration period in the absence of ascorbate, indicating that a transfer has occurred from the extravesicular compartment(s) to the CA storage organelle. Endogenous ascorbate, which is found both inside and outside the chromaffin vesicle, was also found to be secreted from chromaffin cells, indicating that ascorbic acid could be released simultaneously from two different subcellular compartments.
原代培养的嗜铬细胞从孵育培养基中摄取[14C]抗坏血酸。在用[14C]抗坏血酸盐进行短时间标记后立即受到刺激的细胞，通过烟碱受体介导的Ca2+依赖性过程，与儿茶酚胺（CA）同时分泌抗坏血酸。通过改变尼古丁浓度或改变细胞外培养基中Ca2+和Na+的浓度获得一系列分泌率，观察到CA和[14C]抗坏血酸呈比例释放。然而，在相同的刺激条件下，不同的细胞制剂分泌的CA比[14C]抗坏血酸盐多2-10倍（占细胞含量的百分比）。此外，对于几种促分泌素中的每一种，CA和[14C]抗坏血酸盐的分泌时间进程不同。此外，Ba2+对CA分泌的刺激作用比对[14C]抗坏血酸盐分泌的刺激作用更强，Ca2+通道阻滞剂对CA分泌的抑制作用比对[14C]抗坏血酸盐分泌的抑制作用更强。这些数据表明，新摄取的抗坏血酸盐可能从一个与嗜铬囊泡完全不同的区室分泌出来。亚细胞分布研究证实了这一假设，在与[14C]抗坏血酸盐短时间孵育后制备的嗜铬细胞匀浆的囊泡部分（P2）中，仅发现一小部分新摄取的[14C]抗坏血酸盐。然而，当在无抗坏血酸盐的情况下进行长时间平衡期追踪短时间标记的[14C]抗坏血酸盐时，其亚细胞分布与内源性抗坏血酸的分布一致，表明已发生从囊泡外区室向CA储存细胞器的转移。在内源性抗坏血酸中，嗜铬囊泡内外均有发现，也发现其从嗜铬细胞中分泌出来，表明抗坏血酸可同时从两个不同的亚细胞区室释放。
