乙酰胆碱和A23187对离体大鼠肾上腺髓质细胞肾上腺素分泌的作用

通过胆碱能激动剂乙酰胆碱及离子诱导剂A23187（以下简称激动剂）作用于分离的肾上腺髓质细胞,以引起离体细胞的刺激－分泌耦联过程,运用细胞立体形态计量法计算分泌过程中的嗜铬颗粒数目的变化,运用电镜X射线显微分析法测量分泌过程中嗜铬颗粒内钙含量的变化,并运用高 液相色谱分析法测定离体细胞在激动剂作用后的肾上腺素分泌情况,结果发现,分离的肾上腺髓质细胞嗜铬颗粒内钙含量在激动剂作用10min时有明显下降,颗粒数目在激动剂作用过程中呈缓慢下降趋势,而细胞悬液中的肾上腺素含量在激动剂作用20min以后有明显的升高,激动剂作用引起的离体肾上腺髓质的细胞分泌时颗粒内钙含量的下降早于颗粒数目减少或肾上腺素升高,提示颗粒释放的Ca^2 可能是引起细胞分泌的原因之一。     

Calcium binding by subcellular fractions of bovine adrenal medulla

Significantly more calcium per gram protein was found in a relatively pure granule fraction isolated from fresh bovine adrenal medulla than in predominantly mitochondrial fractions isolated from the same tissue. Sixty-four and 55% of the calcium associated with chromaffin granule and mitochondrial fractions, respectively, was released into the supernatant upon lowering the tonicity of the medium. The per cent calcium released by this procedure was significantly greater for granules than for mitochondria (p < 0.05). The amount of calcium per gram protein released into the supernatant also was greater in granule fractions than in mitochondrial fractions (p < 0.05). These data, coupled with a previous report that 10^?3 M EDTA does not markedly decrease the calcium content of whole granules, indicate that the excess calcium of the granule fractions relative to the mitochondrial fractions is maintained within the particles of that fraction. The functional significance of the relatively large amount of calcium in chromaffin granules is not clear. The presence of 150 mM sodium chloride or potassium chloride decreases calcium binding by granule or mitochondrial fragments incubated in 2.2 mM calcium chloride in 0.2 M Tris, pH 7, by about 50%. EDTA, 10^?3 M, removes all but a small residual of the calcium associated with the granule or mitochondrial fragments whereas lowering the concentration of Tris increases calcium binding to about the same extent in both these subcellular fractions. The calcium-binding properties of granule and mitochondrial fragments therefore appear to be quantitatively and qualitatively similar. Inhibition of catecholamine release by relatively high concentrations of sodium may be explained by competitive inhibition of calcium binding. Calcium binding by granule fragments decreases with an increase in hydrogen ion concentration.     

The adenosine-triphosphatase activity of adrenal chromaffin granules

1. The preparation of a fraction containing highly purified chromaffin granules from the bovine adrenal medulla is described. 2. The fraction contains an adenosine-triphosphatase activity that is stimulated by Mg²⁺ and that cannot be explained by contamination with mitochondria or microsomes. 3. It is suggested that the adenosine-triphosphatase activity is related to the uptake of cate-cholamines by the chromaffin granules.     

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。     

Presence of the Novel Pituitary Protein „7B2” in Bovine Chromaffin Granules: Possible Co-Release of 7B2 and Catecholamine as Induced by Nicotine

We observed the presence of the novel pituitary protein "7B2" and its release in the bovine adrenal medulla. The 7B2 concentration (mean +/- SEM) in extracts of the bovine adrenal medulla was 952 +/- 155 pg/mg tissue (n = 6). 7B2 was distributed in the chromaffin granule fraction prepared from the bovine adrenal medulla and was released by high K+ and/or nicotine from cultured cells of the bovine adrenal medulla. Co-release of 7B2 with catecholamine induced by nicotine from the cultured bovine chromaffin cells was also observed. In an analysis of the bovine adrenal medulla chromaffin granule fraction on gel permeation chromatography, there was a major peak with an apparent molecular weight of 45,000, whereas a major peak with an apparent molecular weight of 20,000 was found in that on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On reverse-phase HPLC, a major peak with a retention time of 35 min was observed in the bovine chromaffin granule fraction and in the bovine anterior pituitary extract. These findings indicate that 7B2 is a secretory protein in the bovine adrenal medulla. The possibility that 7B2 might be released with catecholamine, possibly in response to stress, warrants investigation.     

Identification and subcellular localization of catalase activity in bovine adrenal medulla and cortex

Catalase activity was detected in homogenates of bovine adrenal cortex and medulla. Analysis by equilibrium density centrifugation in isoosmotic metrizamide-sucrose gradients revealed that 70% of the medullary catalase activity was soluble while most of the remainder was found in a particulate form with a density of 1.175 g/ml. This was distinct from the densities of lysosomes, mitochondria, and chromaffin granules. Catalase activity in adrenal cortex was primarily (90%) soluble with only 6% being particulate, with a density of 1.185 g/ml. d-Amino acid, uric acid, and α-hydroxyacid oxidase activities, often associated with peroxisomes in other tissues, were absent from homogenates and catalase-containing gradient fractions from either cortex or medulla. There was an indication that some catalase activity was associated with chromaffin granules on the basis of density gradient analysis of both medullary homogenates and crude granule preparations. When granule fractions were subjected to osmotic shock, catalase activity distributed between soluble and sedimentable fractions differently from epinephrine and dopamine β-hydroxylase activity. The sedimentable catalase activity remained associated with chromaffin granule membranes upon isopycnic centrifugation. We concluded that catalase activity in both adrenal cortex and medulla was largely cytoplasmic, but that both tissues contained at least some catalase in dense organelles. Catalase activity which may be associated with chromaffin granules represents a small fraction of the total activity in the medulla.     

Lipids of the adrenal medulla: Lysolecithin, a characteristic constituent of chromaffin granules

1. The lipid composition of microsomes, mitochondria and chromaffin granules, obtained from homogenates of bovine adrenal medulla, has been investigated. 2. The three types of particle showed characteristic differences of phospholipid and cholesterol content. The lipid composition of microsomes and mitochondria resembled that of corresponding particles from other tissues. The chromaffin granules contained 19% of the cholesterol and 14% of the phospholipids of the low-speed supernatant. 3. Thin-layer chromatography indicated the presence of these phospholipids in extracts from each particle: lecithin, lysolecithin, phosphatidylethanolamine (partly plasmalogen), phosphatidylserine, phosphatidylinositol and sphingomyelin. 4. On quantitative analysis of the phospholipids, chromaffin granules were found to contain a high concentration of lysolecithin (17% of the lipid phosphorus). Mitochondria and microsomes, on the other hand, contained very little lysolecithin (less than 2% of the lipid phosphorus).     

Actin associated with membranes of monoamine storage organelles.

A histo-immunofluorescence technique using anti-actin antibodies has been applied to various subcellular fractions of bovine adrenal medulla and rabbit blood platelets. In the adrenal medulla only the membranes of the chromaffin granules, but not the fractions containing other subcellular particles (microsomes, mitochondria) showed marked immunofluorescence was present in the membranes of the 5-hydroxy-tryptamine organelles as well as in other subcellular particles. It is concluded that in the adrenal medulla, actin is specifically associated with the membranes of the amine storage organelles, whereas in platelets the protein shows a rather general subcellular distribution.     

An intracellular opiate receptor

The adrenal medulla contains an intracellular opiate receptor associated with the chromaffin granule. This receptor may participate in regulation of the catecholamine content of the granule.     

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.     

Calelectrin, a Calcium-Dependent Membrane-Binding Protein Associated with Secretory Granules in Torpedo Cholinergic Electromotor Nerve Endings and Rat Adrenal Medulla

Calelectrin, a calcium-dependent membrane-binding protein of subunit molecular weight 32,000 has been isolated from the electric organ of Torpedo, and shown to occur in cholinergic neurones and in bovine adrenal medulla. In this study a monospecific antiserum against the Torpedo protein has been used to study the localization of calelectrin in the rat adrenal gland. The cortex was not stained, whereas in the medulla the cytoplasm of the chromaffin cells was stained in a particulate manner. An identical staining pattern was obtained with an antiserum against the chromaffin granule enzyme dopamine beta-hydroxylase, although the two antisera did not cross-react with the same antigen. The purified protein aggregates bovine chromaffin granule membranes and cholinergic synaptic vesicles and also self aggregates in a calcium-dependent manner. Negative staining results demonstrate that calcium induces a transformation of the purified protein from circular structures 30-80 nm in diameter into a highly aggregated structure. Calelectrin may have a structural or regulatory role in the intracellular organization of secretory cells.     

Phosphorylation of the membrane components of chromaffin granules: synthesis of diphosphatidylinositol and presence of phosphatidylinositol kinase in granule membranes.

Adenosine triphosphate (ATP) induces the release of catecholamines, endogenous ATP, and soluble protein from chromaffin granules isolated from the adrenal medulla. When ATP exerts this action, it is hydrolyzed by enzymes present in the granule membrane, and part of the Pi liberated from ATP is transferred to the protein and lipid of the granule membrane. The phosphorylated lipid component, which was identified by thin-layer and ion-exchange chromatography as diphosphatidylinositol, was formed from ATP and monophosphatidylinositol. This latter phospholipid was the substrate for the enzyme phosphatidylinositol kinase. Both substrate and enzyme are components of the granule membranes, because they have a similar subcellular distribution as dopamine beta-hydroxylase (a granule membrane marker). The formation of diphosphatidylinositol was Mg(2 plus)-dependent, it was further stimulated by Mn(2 plus), it was inhibited by N-ethylmaleimide and the reaction had an optimal pH of 5. The synthesis of diphosphatidylinositol was also shown to occur in chromaffin granules "in situ". during the stimulation of the adrenal medulla by acetylcholine.     

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.     

Pituitary adenylate cyclase-activating peptide (PACAP) recruits low voltage-activated T-type calcium influx under acute sympathetic stimulation in mouse adrenal chromaffin cells

Low voltage-activated T-type Ca_v3.2 calcium channels are expressed in neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that naïve adrenal chromaffin cells express a nominal Ca_v3.2-dependent conductance. However, Ca_v3.2 conductance is up-regulated following chronic hypoxia or long term exposure to cAMP analogs. Thus, although a link between chronic stressors and up-regulation of Ca_v3.2 exists, there are no reports testing the specific role of Ca_v3.2 channels in the acute sympathoadrenal stress response. In this study, we examined the effects of acute sympathetic stress on T-type Ca_v3.2 calcium influx in mouse chromaffin cells in situ. Pituitary adenylate cyclase-activating peptide (PACAP) is an excitatory neuroactive peptide transmitter released by the splanchnic nerve under elevated sympathetic activity to stimulate the adrenal medulla. PACAP stimulation did not evoke action potential firing in chromaffin cells but did cause a persistent subthreshold membrane depolarization that resulted in an immediate and robust Ca²⁺-dependent catecholamine secretion. Moreover, PACAP-evoked secretion was sensitive to block by nickel chloride and was acutely inhibited by protein kinase C blockers. We utilized perforated patch electrophysiological recordings conducted in adrenal tissue slices to investigate the mechanism of PACAP-evoked calcium entry. We provide evidence that stimulation with exogenous PACAP and native neuronal stress stimulation both lead to a protein kinase C-mediated phosphodependent recruitment of a T-type Ca_v3.2 Ca²⁺ influx. This in turn evokes catecholamine release during the acute sympathetic stress response.     

Synexin protein is non-selective in its ability to increase Ca2+-dependent aggregation of biological and artificial membranes

Synexin, a soluble protein which increases the specificity of Ca²⁺ to aggregate isolated bovine chromaffin granules was prepared from bovine adrenal medullary tissue by the method of Creutz, Pazoles and Pollard (J. Biol. Chem. $\text{253}$, 2858–2866, 1978). We also find that synexin increases both the initial rate and final amplitude of Ca²⁺-promoted aggregation of granule membranes. This effect is Ca²⁺-specific. However in contrast to Creutz $\text{et}\text{al}$, we find that synexin also potentiates aggregation of adrenal medulla and liver mitochondria and microsomes as well as phosphatidylserine vesicles. This lack of membrane specificity argues against the suggestion of Creutz $\text{et}\text{al}$ that synexin specifically binds the granule to the plasma membrane prior to exocytosis $\text{in}\text{vivo}$.     

Transsynaptic Regulation of Galanin, Neurotensin, and Substance P in the Adrenal Medulla: Combinatorial Control by Second-Messenger Signaling Pathways

The adrenomedullary content of neurotensin and substance P was examined 1, 6, and 12 days after hypoglycemic shock. The neurotensin content was increased 60-fold within 24 h and remained elevated for up to 12 days, whereas the substance P content was increased approximately sevenfold within 24 h of insulin treatment and returned to control levels by 12 days poststimulation. Because protein kinase A, protein kinase C, and calcium influx in the rat adrenal medulla are all stimulated following splanchnic nerve stimulation, the differential regulation of neurotensin and substance P biosynthesis following stimulation of these three pathways was examined in bovine chromaffin cells in vitro. Neurotensin levels were up-regulated by elevated potassium, forskolin, and phorbol ester in bovine chromaffin cells. Substance P levels were up-regulated by elevated potassium and forskolin but not by phorbol ester treatment. When chromaffin cells were treated with phorbol ester in combination with forskolin, neurotensin levels were increased in a synergistic fashion, whereas phorbol ester antagonized the forskolin-induced elevation of substance P levels. Earlier, it was reported that galanin biosynthesis, like neurotensin biosynthesis, is upregulated by depolarization, phorbol ester stimulation, and forskolin treatment in chromaffin cells in vitro. Here we report that galanin is also, like neurotensin, increased greater than 60-fold after stimulation of the rat adrenal medulla in vivo. Neuropeptide-specific combinatorial effects of stimulating the calcium, protein kinase A, and protein kinase C signaling pathways may underlie the quantitative differences between galanin and neurotensin compared with substance P up-regulation in rat adrenal medulla after splanchnic nerve stimulation in vivo.     

An immunohistochemical study of the adrenal medulla of the ox

Summary The adrenal medulla of ox was studied by an indirect immunofluorescent technique using anti-ox chromaffin granule serum. The serum had a weak cross reaction with ox brain stem and splenic nerve. There was a species cross reaction with sheep, pig and horse. Immunoelectrophoresis showed five components in the serum against ox adrenal lysates. The whole adrenal medulla of ox was found to fluoresce by the immunofluorescent technique but not the cortex. The adrenals of sheep, pig and horse behaved similarly using the anti-ox serum. A serum prepared against ox chromogranin-A, the most abundant soluble protein of the chromaffin granules, was also used for immunofluorescence. Again both the adrenaline and noradrenaline storing cells fluoresced, but not the cortex.     

pH modulation of large conductance potassium channel from adrenal chromaffin granules

We report here that large conductance K(+) selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of (86)Rb(+) into chromaffin granules prepared from bovine adrenal gland medulla. The (86)Rb(+) influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432+/-9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca(2+)-activated K(+) channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.     

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.     

Primary cultures of bovine chromaffin cells synthesize and secrete vasoactive intestinal polypeptide (VIP)

Dispersed cells of the bovine adrenal medulla express immunoreactive vasoactive intestinal polypeptide (VIP) after 24 hours in culture, although VIP could not be detected in extracts of bovine adrenal medulla or cortex. Immunoreactive VIP eluted from a reversed-phrase chromatography column with the same retention time as authentic porcine VIP_1–28. VIP in chromaffin cells in culture appears to be contained in a secretory granule pool, since it, like methionine-enkephalin (met-enk) was released into the medium after exposure of cells to nicotine, carbachol, veratridine and elevated potassium in a dose-dependent manner. Doseresponse curves for VIP and enkephalin release by the above secretagogues were similar but not identical. Enkephalins and VIP may either be contained in separate subpopulations of chromaffin cells or co-stored in the same cells.