应急大鼠肾上腺髓质嗜铬细胞颗粒数目与其钙含量的关系

采用电镜细胞立体形态计量法及电镜Ｘ射线显微定量分析术,对制动应急大鼠的肾上腺髓质细胞内嗜铬颗粒数密度和颗粒内Ｃａ浓度变化进行测量。结果显示,两者在制动过程中均呈进行性下降,但颗粒内钙浓度的下降快于颗粒数目的减少（Ｐ〈０．０１）。该结果支持颗粒内钙释放入胞质,参与胞质内游离钙浓度的升高,进而激发颗粒胞吐的假设,为主宰嗜铬颗粒是一种细胞内钙库并参与细胞分泌提供了实验依据。     

新生小牛肾上腺髓质嗜铬细胞的原代培养及其儿茶酚胺的分泌

目的：原代培养新生小牛肾上腺髓质嗜铬细胞,观察离体条件下肾上腺髓质细胞的生长和儿茶酚胺的分泌特性。方法：密度梯度离心和差速贴壁法分离和纯化肾上腺髓质细胞,ELISA检测培养上清液中肾上腺素和去甲肾上腺素的浓度变化。结果：肾上腺髓质嗜铬细胞生长良好。4～6周起细胞出现终极分化。培养液中儿茶酚胺浓度在第2～8 d内稳定,第10 d开始明显下降。结论：应用单细胞培养法可以成功建立原代肾上腺髓质嗜铬细胞,并可在原代培养前8d内进行细胞行为学的研究。     

大鼠肾上腺髓质儿茶酚胺分泌的在体伏安法测定

应用碳纤微电极直接测定大鼠肾上腺髓质中儿茶酚胺浓度的在体伏安法。首次报道了大鼠肾上腺髓质中儿茶酚胺的基础浓度为０．１－０．５μｍｏｌ／Ｌ,缺血时髓质中儿茶酚胺的浓度显著增加,严重缺血时可达到５－３０μｍｏｌ／Ｌ。肾上腺髓质能自发分泌儿茶酚胺,缺血时自发分泌儿茶酚胺的幅度和频率都大大增加,提示缺血对大鼠是一种强烈的应激,测定结果还表明,乙酰胆碱能剂量依赖性地刺激肾上腺髓质嗜铬细胞分泌儿茶酚胺。     

FK506结合蛋白12.6调节小鼠嗜铬细胞分泌

FK506结合蛋白12.6(FKBP12.6)能够结合并调控钙离子释放通道兰尼碱受体2型(RyR2)的开放,可能是儿茶酚胺分泌的重要调控器.利用FKBP12.6敲除小鼠模型,我们研究了FKBP12.6在肾上腺嗜铬细胞胞吐中的作用.结果表明,FKBP12.6在小鼠肾上腺嗜铬细胞中表达,而敲除FKBP12.6小鼠的嗜铬细胞中有正常的去极化引起的钙电流和胞吐作用.然而,FKBP12.6敲除会导致嗜铬细胞中出现增强的咖啡因引起的细胞整体钙瞬变和咖啡因引起的胞吐作用.结果提示,FKBP12.6调控肾上腺嗜铬细胞儿茶酚胺的分泌,这种调控作用是通过调节钙离子的释放而实现的.FKBP12.6是嗜铬细胞分泌的重要蛋白.     

人肾上腺髓质素原N端20肽受体——MrgX2的分离鉴定

肾上腺髓质素原N端20肽[proadrenomedullin N-terminal 20 peptide,PAMP（1～20）/PAMP-20]及其类似物PAMPl2（二者均来源于肾上腺髓质素前体肽prepro-adrenomedullin,prepro-ADM）,是具有舒张血管、降低血压效应的内源性多肽,其降压效应通过抑制交感神经末梢和肾上腺嗜铬细胞分泌儿茶酚胺而实现。     

尿甲氧基肾上腺素及甲氧基去甲肾上腺素在嗜铬细胞瘤诊断中的作用

目的:测定尿液中甲氧基肾上腺素和甲氧基去甲肾上腺素,并评价其在嗜铬细胞瘤诊断中的作用。方法:选择酶联免疫分析法,对2014年1月至2015年1月广州军区广州总医院有嗜铬细胞瘤筛查指征的患者的24 h尿甲氧基肾上腺素和甲氧基去甲肾上腺素进行测定。结果:嗜铬细胞瘤患者24 h尿甲氧基肾上腺素和甲氧基去甲肾上腺素含量明显高于肾上腺皮质腺瘤、肾上腺增生、原发性醛固酮增多症等其他肾上腺占位性疾病患者,同时也显著高于原发性高血压患者及健康人参考区间上限值;以甲氧基去甲肾上腺素806.5μg或甲氧基肾上腺素385.5μg为切点,诊断嗜铬细胞瘤的敏感度为83.3%、特异性为90%;甲氧基去甲肾上腺素和甲氧基肾上腺素的受试者工作特征曲线下面积分别为0.912±0.057和0.886±0.076。结论:酶联免疫法检测24 h尿甲氧基去甲肾上腺素和甲氧基肾上腺素的方法准确度和特异性均较高,可以作为嗜铬细胞瘤的初步筛查、排除、临床诊断的可靠的实验室检查方法。     

尿去甲基肾上腺素/甲氧基肾上腺素测定对嗜铬细胞瘤的方法学比较

目的:通过对尿儿茶酚胺代谢产物去甲肾上腺素、甲氧基肾上腺素的测定,综合分析其对嗜铬细胞瘤的早期临床诊断价值。方法:利用酶联免疫分析法和传统柱层析法,对正常人和临床诊断为嗜铬细胞瘤及肾上腺占位病变并伴有阵发性高血压患者的24h尿去甲基肾上腺素/甲氧基肾上腺素和3-甲氧基-4-羟基苦杏仁酸(VMA)进行测定。结果:与VMA相比,24h尿去甲基肾上腺素/甲氧基肾上腺素在嗜铬细胞瘤患者中的测定值要显著高于其他肾上腺占位性病变伴高血压患者和正常人群,二者方法学有显著性差异。结论:酶联免疫分析法检测24h尿去甲基肾上腺素/甲氧基肾上腺素具有灵敏度高、特异性好的特点,为临床从肾上腺占位性病变并伴有阵发性高血压患者中筛查嗜铬细胞瘤提供了一种有价值的参考方法。     

糖皮质激素对大鼠肾上腺髓质嗜铬细胞瘤细胞ACh诱发电流的快速抑制作用

本研究采用全细胞膜片箝 技术,以大鼠肾上腺髓质嗜铬细胞瘤细胞为标本,观察了糖皮质激素对乙胆碱诱发电流的快速作用,并初步探讨了其可能机制。     

5-羟色胺对大鼠腎上腺皮质功能的影响

5-羟色胺(5-HT)是一个具有广泛生理活性的重要体液因子,它与肾上腺的关系亦为许多学者所注意。Reid首先发现5-HT可以直接刺激肾上腺髓质释放肾上腺素。后来Halberg又发现它可以使小鼠循环血液中嗜伊红细胞的数目减少。1957年Moussatche等认为5-HT具有间接刺激肾上腺皮质,使其功能升高的作用。     

心房钠尿肽(ANP)对大鼠卵巢细胞生成雌激素与孕激素的影响

应用大鼠卵巢黄体细胞、颗粒细胞培养以及放射免疫分析法,观察了α型心房钠尿肽(α-ANP)对性甾体激素孕酮(P)和雌二醇(E_2)分泌的影响,结果发现,0.1—10ng/ml 浓度的α-ANP 促进离体培养的大鼠黄体细胞分泌孕酮,并呈量效关系。α-ANP 也促进大鼠卵泡颗粒细胞分泌孕酮,但对分泌雌二醇没有影响。说明α-ANP 也影响卵巢分泌功能。     

An osmotic mechanism for exocytosis from dissociated chromaffin cells

Dissociated chromaffin cells from bovine adrenal medulla were stimulated to secrete epinephrine and dopamine beta-hydroxylase with a variety of secretagogues in a study designed to test the hypothesis that the chemiosmotic lysis reaction of isolated chromaffin granules might in some way be related to the mechanism of release during exocytosis. Increasing the osmotic strength of the incubation medium with either NaCl or sucrose led to suppression of secretion of epinephrine from the cells regardless of whether secretion was induced with veratridine or acetylcholine. Suppression of secretion was approximately exponential with respect to osmotic strength. Epinephrine secretion occurred only if the medium contained a permeant anion such as chloride, and secretion induced by veratridine was suppressed when Na isethionate replaced NaCl in the medium. In an extensive study with different monovalent anions veratridine supported epinephrine secretion according to the following activity series: Br-, I-, NO3- greater than methylsulfate, SCN- greater than Cl greater than acetate much greater than isethionate. A similar series, except for the potency of NO3-, was observed with A23187 as agonist. In general, the anion series for granule lysis was analogous. However, there was a poor quantitative correlation between the anion dependence of chemiosmotic granule lysis and the anion dependence of cell secretion. Anion transport inhibitors such as probenecid and pyridoxal phosphate also inhibited secretion while the stilbene disulfonates were inactive. The ineffectiveness of the stilbene disulfonates further distinguished chemiosmotic granule lysis from cell secretion. Secretion of catecholamines, induced by veratridine or nicotine, a cholinergic agonist, was suppressed when NaCl in the medium was replaced by isosmotic sucrose and unexpectedly low levels of dopamine beta-hydroxylase were observed in some cases. In sum, these properties of secreting chromaffin cells resembled some properties of isolated chromaffin granules incubated in ATP and Cl-, but were different in a number of instances. We, therefore, have interpreted our data to indicate that while some mechanistic relationships may indeed exist between the release event in exocytosis from chromaffin cells and the chemiosmotic lysis reaction characteristic of isolated chromaffin granules, an understanding of the energetics of exocytosis awaits the discovery of reasons for the quantitative differences between the two systems.     

Uptake of calcium in chromaffin granules of bovine adrenal medulla stimulated in vitro

The calcium content of bovine adrenal medulla perfused in vitro has been shown to increase about 30% in response to extensive acetylcholine stimulation. The calcium accumulated during secretion was mainly associated with the mitochondria and chromaffin granule fractions and to a lesser extent in the microsome fraction. While the calcium taken up by the mitochondria and microsomes was partly or totally removed by treatment with EDTA, the chelating agent had no effect on the granule content of calcium. The uptake of calcium in the mitochondria and microsomes during secretion is consistent with a function of these organelles in regulating the cellular calcium concentration. It is suggested that also the chromaffin granules may act as a “Ca-pump” in the chromaffin cell of the adrenal medulla.     

Restoration of Catecholamine Content of Previously Depleted Adrenal Medulla In Vitro: Importance of Synthesis in Maintaining the Catecholamine Stores

The functional integrity of adrenal chromaffin storage vesicles was studied in the perfused rat adrenal gland subjected to intense exocytosis. Continuous perfusion with 55 mM K+-Krebs solution produced a large and uninterrupted secretion of catecholamines. Total amounts secreted within 45 min were 4.66 micrograms and represented almost 30% of the total tissue catecholamine content. If perfusion with excess K+ was extended to 90 min, the secretion increased further to 5.76 micrograms. Despite such a large secretory response, the catecholamine content of the K+-stimulated adrenal medulla was comparable to that of unstimulated control, suggesting an enhanced resynthesis to maintain the normal levels. Pretreatment of rats with alpha-methyl-p-tyrosine, and including this agent in the perfusion medium during stimulation with K+, caused a marked reduction in catecholamine content. The degree of depletion depended on the extent of stimulation with K+ (45% in 45 min and 60% in 90 min). Although depleted catecholamine stores did not show spontaneous recovery in 2 h, inclusion of tyrosine, L-3,4-dihydroxyphenylalanine or dopamine (but not epinephrine or norepinephrine) completely restored the catecholamine content of previously depleted adrenal medulla. Repletion achieved by tyrosine was time dependent (evident in 30 min and maximum in 2 h) and blocked by alpha-methyl-p-tyrosine but not by calcium deprivation. The ratio of epinephrine to norepinephrine remained constant during various stages of the experiment, suggesting both types of vesicles were equally affected by different treatments. The secretory response (10 Hz for 30 s) was unaffected even though tissue catecholamine stores were significantly depleted (50%). In summary, we have demonstrated that catecholamine content of the isolated perfused adrenal gland can be reduced by stimulation of exocytotic secretion in the presence of tyrosine hydroxylase inhibitor. Since the depleted stores can be fully refilled by synthesis of catecholamines from its precursors, it is suggested that chromaffin vesicles may be reutilized for the purpose of synthesis, storage, and secretion of adrenal medullary hormones.     

Effects of changes in osmolality on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis

Recent evidence indicates that osmotic forces may play a role in exocytosis. To examine this possibility and to investigate the osmotic properties of storage granules within cells, we investigated the effects of changes of osmolality on stability and function of cultured bovine chromaffin cells. Cell volume measurements indicated that the cells behaved as osmometers and that the intracellular osmolality rapidly equilibrated with the osmolality of the extracellular medium. Hyperosmotic solutions strongly inhibited nicotinic agonist-stimulated secretion but did not alter nicotinic agonist-stimulated Ca(2+) uptake. Hyperosmotic solutions also strongly inhibited elevated potassium- stimulated secretion but only weakly inhibited elevated K(+)-stimulated Ca(2+) uptake. Thus, hyperosmotic solutions inhibited secretion at a step after calcium entry. Cells exposed to 165 mOs(1) solutions did not lyse and retained their capacity to store and secrete catecholamine upon stimulation. Significant intracellular lysis of chromaffin granules occurred within cells exposed to lower osmolalities. In contrast, 75 percent of the catecholamine was released from granules from cultured cells or from fresh adrenal medulla incubated in vitro at 210 mOs. The data provide evidence for a role for osmotic forces in exocytosis and suggest that if osmotic stress of the granule occurs during exocytosis, then water influx into chromaffin granules increases granule volume by at least 70 percent. The results also indicate that the osmotic properties of the granules are altered upon homogenization and subcellular fractionation of the cells.     

In Adrenal Medulla Synaptophysin (Protein p38) Is Present in Chromaffin Granules and in a Special Vesicle Population

We have analyzed the properties and subcellular localization of synaptophysin (protein p38) in bovine adrenal medulla. In one-dimensional immunoblotting the adrenal antigen appears identical to synaptophysin of rat synaptic vesicles. In two-dimensional immunoblotting it migrates as a heterogeneous band varying in pI from 4.5 to 5.8. Subcellular fractionation by various sucrose gradients revealed that synaptophysin was present in two different cell particles. More than half of the antigens present in adrenal medulla were confined to special membranes that sedimented both with the "large granules" and with microsomal elements. These membranes could be removed from the large granule sediment by washing. In gradients it equilibrated in regions of low sucrose density. These membranes did not contain any markers for chromaffin granules. Less than half of the amount of synaptophysin present in adrenal medulla copurified with chromaffin granules. Despite several variations in the fractionation scheme synaptophysin could not be removed from chromaffin granules. After washing of granule membranes with alkaline solution synaptophysin still cosedimented in gradients with typical granule markers. The concentration of synaptophysin in membranes of chromaffin granules is low (less than 10%) when compared with synaptic vesicles. It is concluded that in adrenal medulla synaptophysin is present in special membranes, probably in high concentration, and in membranes of chromaffin granules, either in a low concentration in all or in a higher concentration in some of them.     

Electron probe microanalysis of the subcellular compartments of bovine adrenal chromaffin cells. Comparison of chromaffin granules in situ and in vitro

The elemental and water content of cultured bovine adrenal chromaffin cells and their secretory chromaffin granules have been measured and compared with isolated chromaffin granules using quick freezing, ultracryomicrotomy, and electron microprobe analysis methods. In units of millimole/kilogram dry weight (+/- S.E.) granules in situ contained: P, 523 +/- 32; K+, 124 +/- 9; S, 82 +/- 3; Cl-, 74 +/- 9; Ca2+, 13 +/- 2; Mg2+, 6 +/- 2; and Na+, -2 +/- 2. Following routine isolation in isotonic sucrose buffer, granule K and Cl- had decreased while granule Na+ increased. Cl- exhibited a consistent decrease to 35-40 mmol/kg dry weight. Granule Na+ and K+ concentrations ranged from 43 to 12 mmol/kg and 28 to 60 mmol/kg dry weight, respectively, depending on the Na+ and K+ content of the buffer. Despite the redistribution of monovalent ions, granule Ca2+, granule P, being in the form of ATP, and granule S, being in the form of protein, were not significantly changed. The stability of these elements is consistent with the existence of a stable storage complex for Ca2+, ATP, and protein. Using the granule as an internal standard with a water content of 66%, the water contents of external space, nucleus, cytoplasm, and mitochondria were estimated to be 89, 88, 82, and 70%, respectively. Wet weight concentrations for each element were calculated for granules and cytoplasm from which the transgranular concentration gradients for K+, Cl-, and Na+ were determined. Cl-, a permeant anion, was 2-fold higher in the granule than in the cytoplasm while K+, a slightly permeant cation, had an opposite distribution ratio slightly less than two. Together, the K+ and Cl- data suggest the presence of an inside-positive granule membrane potential of approximately 10-16 mV. The surprising lack of Na+ from the granule matrix suggests a hugh inward gradient for Na+ even though the Na+ content of chromaffin cell cytoplasm is low at 5 mmol/kg water. The lack of an outward Na+ gradient is important in that it indicates that the previously described electroneutral Na+-Ca2+ exchange system, by which isolated granules accumulate Ca2+, does not operate in mature granules in situ. Consequently, if chromaffin granules regulate internal calcium during stimulus secretion coupling, a mechanism other that Na+-Ca2+ exchange is necessary.     

Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling

The exocytotic exposure and retrieval of an antigen of chromaffin granule membranes were studied with chromaffin cells isolated from bovine adrenal medulla. Cells were incubated with an antiserum against glycoprotein III followed by fluorescein- or gold-labeled anti-IgG. Immunofluorescence on the cell surface was present in a patchy distribution irrespective of whether bivalent antibodies or Fab fragments were used. During subsequent incubation these fluorescent membrane patches were internalized within 45 min. At the ultrastructural level immunogold-labeled patches were present on the surface of stimulated cells. During incubation (5 min to 6 h) these immunolabeled membrane patches became coated, giving rise to coated vesicles and finally to smooth vesicles. These latter vesicles were found spread throughout the cytoplasm including the Golgi region, but Golgi stacks did not become labeled. Part of the immunolabel was transferred to multivesicular bodies, which probably represent a lysosomal pathway. 30 min after incubation immunolabel was also found in electron-dense vesicles apparently representing newly formed chromaffin granules. After 6 h of incubation immunolabel was found in vesicles indistinguishable from mature chromaffin granules. These results provide direct evidence that after exocytosis membranes of chromaffin granules are selectively retrieved from the plasma membrane and are partly recycled to newly formed chromaffin granules, providing a shuttle service from the Golgi region to the plasma membrane.     

Cholinergic stimulation of chromaffin cells induces rapid coating of the plasma membrane

Stimulation of primary bovine adrenal chromaffin cells by carbachol produced a 6-fold increase in cell surface coated pits within 30 s. This coat appeared not to be recruited from a preformed pool at the plasma membrane, but from some pool transparent to electron microscopy. The number of coated pits appeared to decrease rapidly after 1 to 2 min stimulation, but processing for electron microscopy using tannic acid to enhance contrast indicated that both coated pits and closed coated vesicles were increased relative to unstimulated cells for up to 30 min. Analyses of purified adrenal medulla coated vesicles showed a lipid composition close to that expected for cell surface membrane, but there were only trace levels of plasma membrane marker enzymes. Coated vesicles contained significant amounts of both membrane and content proteins characteristic of the chromaffin granule, suggesting that medulla coated vesicles preferentially carry secretion granule proteins. The kinetics of stimulus-dependent formation of coated membrane in the cortical zone of chromaffin cells is closely similar to that observed for secretion granule membrane retrieval.     

Retrieving vesicles in secretion-induced rat chromaffin cells contain fodrin

We examined the distribution of fodrin and cytochrome b561 in secretion-induced rat chromaffin cells (epinephrine cells) by immunofluorescence and immunoelectron microscopy. Fasted rats injected with a large dose of insulin were perfusion-fixed and frozen sections of the adrenal medulla were immunolabeled. Fodrin, a peripheral membrane protein, was distributed only in the cell periphery in control cells, but was observed in the cell interior after the insulin treatment; many of the markers were found around small vesicles, 50-200 nm in diameter, and large vacuoles, more than 500 nm in diameter. On the other hand, cytochrome b561, an integral membrane protein, was seen only in the chromaffin granules in control cells, and appeared in small vesicles in the stimulated cells but not in large vacuoles. By double immunolabeling it was shown that cytochrome b561 coexisted with fodrin in the small vesicles. The coexistence of the two proteins was confirmed by the labeling of subcellular particles immunoadsorbed from the insulin-treated adrenal medulla homogenate; vesicles immunoisolated with anti-fodrin antibody on polyacrylamide beads were positively immunolabeled with anti-cytochrome b561 antibody. The present results show that during massive secretion fodrin is taken into the cell interior by vesicles, which may be a mechanism that retrieves the secretory granule membrane from the cell surface.