CALCIUM-DEPENDENCE OF CATECHOLAMINE RELEASE FROM BOVINE ADRENAL-MEDULLARY CELLS AFTER EXPOSURE TO INTENSE ELECTRIC-FIELDS
CALCIUM-DEPENDENCE OF CATECHOLAMINE RELEASE FROM BOVINE ADRENAL-MEDULLARY CELLS AFTER EXPOSURE TO INTENSE ELECTRIC-FIELDS
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DOI：
10.1007/bf01872259
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发表时间：
1982-01-01
影响因子：
2.4
通讯作者：
BAKER, PF
中科院分区：
文献类型：
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作者：
KNIGHT, DE;BAKER, PF
通过使分离的肾上腺髓质细胞经受短时间的强电场，有可能在不损害细胞进行胞吐作用的能力的情况下进入细胞内部。在2kV/cm，τ=200 μsec的单次暴露后，肾上腺髓质细胞的行为就好像它们的质膜含有两个有效半径为2nm的孔。在37°C下，这些“等效孔”保持通畅长达1小时。这些“孔”的形成和稳定性不受浴液中Ca含量的影响。细胞在含有5 mmMg-ATP和EGTA的谷氨酸钾培养基中出现“渗漏”，使游离钙接近10− 8 m，释放的细胞总儿茶酚胺量不到1%。当暴露于10 - 5 mCa时，这些细胞可以释放高达30%的儿茶酚胺。这种钙依赖性释放不受钙通道阻滞剂如D 600的影响。响应于钙挑战的儿茶酚胺释放似乎仅发生在前几分钟期间，而Ca浓度正在变化，并且释放的程度取决于所达到的最终Ca浓度。半最大释放发生在约1 μmCa，并且该值与用于缓冲离子化Ca的EGTA浓度无关。Ca ~（2+）与儿茶酚胺释放的关系最符合需要2个Ca ~（2+），Ca ~（2+）诱发的儿茶酚胺释放与多巴胺-β-羟化酶（DβH）的释放有关，而与乳酸脱氢酶的释放无关。释放的DβH/儿茶酚胺比值与刺激的完整细胞和灌流腺体中的DβH/儿茶酚胺比值相同。DβH和儿茶酚胺在体外介质中出现的时间过程是相同的。暴露于10− 8 mCa的“渗漏”细胞的透射电子显微镜显示与未受刺激的完整细胞没有显著差异。暴露于10− 5 mCa的“渗漏”细胞的细胞质含有大的膜界空泡。当辣根过氧化物酶存在时，分泌发生时，在液泡中发现这种标记物。在不存在Mg-ATP的情况下用Ca平衡的细胞可以通过添加Mg-ATP触发以经历胞吐作用。在缺乏Mg的情况下，单独的ATP是无效的。在测试的各种其他核苷酸中，没有一种像ATP那样有效。Mg-ATP影响胞吐作用的程度，而不影响其对Ca的表观亲和力。氯离子取代谷氨酸作为主要阴离子，导致儿茶酚胺和DβH的Ca依赖性释放显著减少。氯化物引起的钙非依赖性释放的儿茶酚胺，胞吐的程度大大减少，并在表观亲和力的胞吐钙的减少一个小的增加。在所研究的各种阴离子中，它们支持钙依赖性胞吐作用的有效性顺序为谷氨酸->乙酸->Cl->Br->SCN-，用Na或蔗糖代替K或在pH 6.6 ~ 7.8范围内改变pH值对胞吐作用没有明显影响。提高游离Mg浓度降低了Ca依赖性胞吐的程度，也降低了其对钙的表观亲和力。“渗漏”细胞中的钙依赖性胞吐在很大程度上不受以下因素的影响：（i）烟碱受体的各种激动剂和拮抗剂;（ii）破坏微管和微丝的试剂;（iii）鬼笔环肽;（iv）钒酸盐;（v）阴离子渗透性抑制剂;（vi）蛋白酶抑制剂;和（vii）消散囊泡pH梯度和电位的试剂。它被部分抑制（i）某些抗精神病药物;（ii）渗透压升高。
By subjecting isolated adrenal medullary cells to intense electric fields of brief duration it is possible to gain access to the cell interior without impairing the ability of the cell to undergo exocytosis. After a single exposure to a field of 2 kV/cm, τ=200 μsec, adrenal medullary cells behave as if their plasma membrane contains two pores of effective radius 2 nm. At 37°C these ‘equivalent pores’ remain patent for up to 1 hr. The formation and stability of these ‘pores’ is not affected by the Ca content of the bathing solution. The ‘pores’ permit externally applied catecholamine and Ca-EGTA to equilibrate rapidly with the cell water.Cells rendered ‘leaky’ in K glutamate medium containing 5mmMg-ATP and EGTA to give an ionized Ca close to 10−8mrelease less than 1% of their total catecholamine. These same cells can release up to 30% of their catecholamine when exposed to 10−5mCa. This Ca-dependent release is unaffected by Ca-channel blockers such as D600. Catecholamine release in response to a calcium challenge only seems to occur during the first few minutes whilst the Ca concentration is changing, and the extent of release depends on the final Ca concentration achieved. Half-maximal release occurs at about 1 μmCa, and this value is independent of the EGTA concentration used to buffer the ionized Ca. The relation between ionized Ca and catecholamine release is best fitted by a requirement for 2 Ca ions.Calcium-evoked release of catecholamine is associated with the release of dopamine-β-hydroxylase (DβH) but not lactate dehydrogenase. The ratio DβH/catecholamine released is the same as that in stimulated intact cells and perfused glands. The time course of appearance in the external medium of DβH and catecholamine is identical. Transmission electron microscopy of ‘leaky’ cells exposed to 10−8mCa reveals no marked differences from unstimulated intact cells. The cytoplasm of ‘leaky’ cells exposed to 10−5mCa contains large membrane-bounded vacuoles. When secretion is caused to take place in the presence of horseradish peroxidase, this marker is found within the vacuoles.Ca-dependent release of both catecholamine and DβH requires Mg-ATP. Cells equilibrated with Ca in the absence of Mg-ATP can be triggered to undergo exocytosis by the addition of Mg-ATP. In the absence of Mg, ATP alone is ineffective. Of a variety of other nucleotides tested, none is as effective as ATP. Mg-ATP affects the extent of exocytosis and not its apparent affinity for Ca.Replacement of glutamate as the major anion by chloride results in a marked reduction in Ca-dependent release of both catecholamine and DβH. Chloride causes a small increase in Ca-independent release of catecholamine, a large reduction in the extent of exocytosis, and a decrease in the apparent affinity of exocytosis for Ca. Of a variety of anions examined, their order of effectiveness at supporting Ca-dependent exocytosis is glutamate−>acetate−>Cl−>Br−>SCN−.Exocytosis is not obviously affected by replacing K by Na or sucrose or by altering the pH over the range pH 6.6 to 7.8. Raising the free Mg concentration reduces the extent of Ca-dependent exocytosis and also its apparent affinity for calcium. Calcium-dependent exocytosis in ‘leaky’ cells is largely unaffected by (i) a variety of agonists and antagonists of the nicotinic receptor; (ii) agents that disrupt microtubules and microfilaments; (iii) phalloidin; (iv) vanadate; (v) inhibitors of anion permeability; (vi) protease inhibitors; and (vii) agents that dissipate the vesicle pH gradient and potential. It is partially inhibited by (i) certain antipsychotic drugs; (ii) a rise in osmotic …