Ultracytochemical localization of acetylcholine-like cations in excited motor end-plates by means of ionic fixation

Using rapid ionic fixation with molybdic or tungstic heteropolyanions (strong precipitating agents of quaternary ammonium cations such as choline and acetylcholine), acetylcholine-like cations were localized as point-like precipitates in the synaptic vesicles of resting (electrically nonstimulated) motor nerve terminals. When performed at low temperature, the same procedure revealed spot-like precipitates (presumed to be exocytotically released acetylcholine-like cations) in the synaptic cleft in the vicinity of the active zone. These precipitates were often seen in paired forms. Unlike resting motor-nerve terminals, excited terminals (electrical stimulation with occasional 4-aminopyridine pretreatment) after ionic fixation exhibited, at first, laminar precipitates both in the vicinity of the active zone inside the nerve terminals and in the synaptic space. In the vicinity of the active zone, the laminar precipitates were directed towards the synaptic membrane, while those in the synaptic space showed no orientation. Ionic fixation also revealed diffused precipitates both around the synaptic vesicles and on the axoplasmic side of the presynaptic membrane. Finally, the same fixation procedure demonstrated the presence of empty synaptic vesicles (without point-like precipitates) in close contact with the presynaptic membrane. The laminar and diffused precipitates are presumed to be two different forms of the same salts of acetylcholine-like cations that are insolubilized by ionic fixation in both the nerve terminals and the synaptic space of excited motor end-plates.     

Electron-microscope cytochemistry of acetylcholine-like cation by means of low-temperature "ionic fixation"

A fresh preparation of frog neuromuscle was fixed at low temperatures (0 degree-4 degrees C) by means of an "ionic-fixation" procedure which is based on the precipitation of quaternary ammonium cations, such as choline and acetylcholine, with molybdic or tungstic heteropolyanions. A low temperature was used to slow down drastically the biological processus of vesicular exocytosis so that ionic fixation, the speed of which is only slightly influenced by temperature variation, could be performed efficiently. In addition to the conventional point-like precipitate in the synaptic vesicle which is considered to be vesicular acetylcholine, numerous spot-like precipitates were observed in the synaptic cleft. Most of these were contiguous to the active zone, and some were in a paired form and corresponded to the double rows of the synaptic vesicles in contact with active zones. It is concluded that these spot-like precipitates were acetylcholine-like cations of the synaptic vesicles which had been discharged into the synaptic cleft by exocytosis and captured by the ionic fixation procedure. The results are discussed in relation to the vesicular and non-vesicular hypothesis of acetylcholine release.     

The ultrastructural ionic fixation technique localizing acetylcholinesterase activity,reveals simultaneously acetylcholine-like cation in the synaptic vesicles of the motor nerve terminals

Summary A new cytochemical technique is proposed for side by side localization of acetylcholine and of acetylcholinesterase activity of motor end-plate at ultrastructural level. The technique is based on the simultaneous ionic fixation of vesicular acetylcholine and of histochemical copper thiocholine precipitate with phosphomolybdic acid: the molybdic heteropolyanion forms insoluble salts with these two quaternary ammonium cations, providing in situ acetylcholine phosphomolybdate and copper thiocholine phosphomolybdate. Both of them are osmium resistant; the electron dense precipitates allow for a fine localization of acetylcholine and acetylcholinesterase activity at electron microscopic level.     

The ultrastructural ionic fixation technique localizing acetylcholinesterase activity, reveals simultaneously acetylcholine-like cation in the synaptic vesicles of the motor nerve terminals

A new cytochemical technique is proposed for side by side localization of acetylcholine and of acetylcholinesterase activity of motor end-plate at ultrastructural level. The technique is based on the simultaneous "ionic fixation" of vesicular acetylcholine and of histochemical copper thiocholine precipitate with phosphomolybdic acid: the molybdic heteropolyanion forms insoluble salts with these two quaternary ammonium cations, providing in situ "acetylcholine phosphomolybdate" and "copper thiocholine phosphomolybdate". Both of them are osmium resistant; the electron dense precipitates allow for a fine localization of acetylcholine and acetylcholinesterase activity at electron microscopic level.     

Ultracytochemical localization of estrogen-stimulated guanylate cyclase in rat uterus

Estrogens are known to increase cyclic guanosine monophosphate (cGMP) levels in the uterus of rats by enhancing guanylate cyclase (GC) activity. In the present study, the cytochemical localization of GC activity was studied in the uteri of immature and ovariectomized rats after treatment with diethylstilbestrol (DES), progesterone, estrogen antagonist (CI628), and a combination of DES and CI628. Twenty-four hours after the first dose of DES, moderate to strong guanylate cyclase activity was indicated by lead phosphate precipitate on the luminal microvillar and basolateral surfaces of epithelial cells, whereas strong activity was found on the plasma membranes of fibroblasts, endothelial cells, and myometrial cells. The enzyme activity in the epithelial cells declined slightly 24 hr after the second daily dose of DES. Uterine tissues from DES-treated rats that were preheated at 60 degrees C for 30 min or preincubated with a GC inhibitor showed no reaction product. Guanylate cyclase activity was not observed cytochemically in the uterine tissues of the vehicle control (immature or ovariectomized) or progesterone-and CI628-treated animals. Weak guanylate cyclase activity was observed on the plasma membranes of epithelial cells and endothelial cells after doses of DES and CI628 were given simultaneously. The biochemical assays of the total homogenate in vitro indicated that uterine GC showed about a twofold increase after one dose of DES and a 1.3-fold increase following two doses (one dose per day) of DES when compared with their respective nontreated controls, or with progesterone-treated uteri. GC was found in particulate (09%) and cytosol (10%) fractions.These data demonstrated that DES stimulated uterine guanylate cyclase activity, while progesterone and CI628 were ineffective at the doses used. Estrogen antagonist CI628 doses not completely suppress the effect of DES.     

Ultracytochemical localization of adenylate cyclase activity in rat thymocytes

Summary Ultrastructural localization of adenylate cyclase (AC) activity was investigated in suspensions of unfixed isolated rat thymocytes using a medium containing 0.6 mM 5-adenylylimidodiphosphate (AMP-PNP) as a substrate, 10 mM MgSO₄ as an activator, 5 mM theophylline as an inhibitor of 3,5-AMP-phosphodiesterase and 2 mM lead nitrate as a capturing agent. AC activity was demonstrated in plasma membrane, perinuclear space, endoplasmic reticulum, Golgi complex, centriole microtubules and mitochondria. AC was activated with 10^–4 M adrenalin in the presence of 5-guanylylimido-diphosphate (GMP-PNP) as well as with 10^–2 M NaF. In the cells incubated in a medium devoid of theophylline and containing 5-AMP instead of AMP-PNP, 5-nucleotidase activity was observed in the same cell structures as AC activity. Hydrolysis of 5-AMP in the nucleus was much stronger than that of AMP-PNP. 10 mM NaF markedly inhibited hydrolysis of 5-AMP in all cell structures. No staining was observed with 2 mM -glycerophosphate as a substrate. Incubation of unfixed thymocytes in media containing AMP-PNP, 5-AMP or p-nitrophenyl phosphate, but not -glycerophosphate, induced both in the nucleus and in the cytoplasm in some cells an appearance of a transitory reticular formation consisting of about 30 nm thick strands which could penetrate the nuclear envelope and plasma membrane and form connections with adjacent cells. The transitory reticular formation seems to belong to the cytoskeleton and to be involved in cell aggregation.     

Ultracytochemical localization of guanylate cyclase in early human placenta

Previous biochemical and cytochemical studies have indicated that in human term placenta the enzyme guanylate cyclase (GC) is associated mostly with the cytosolic fraction of homogenates and localized on the syncytiotrophoblast microvillous border. In the present study we have shown cytochemically the GC particulate form in early human placenta using guanylyl-imidodiphosphate [Gpp(NH)p] as substrate and NaN3 as activator. In samples of placental villi taken from the 6th to 12th week of pregnancy, the GC reaction product was always found on the apposing Langhans cytotrophoblast and syncytiotrophoblast plasma membranes. Furthermore, GC was present on cells in mitosis of the Langhans cytotrophoblast. From the 11th week GC was also visible on basal plasma membranes of Langhans cytotrophoblast and on endothelial cells of fetal capillaries. In samples of human term placenta GC was detectable on the syncytiotrophoblast microvillous border. This suggests a shift of enzyme localization during pregnancy.     

Variations in the distribution of motor end-plates in the avian pectoralis

Most avian muscles consist of serially arranged, overlapping fibers that do not extend the length of the muscle. This condition appears to be plesiomorphic with respect to diapsid reptiles. The presence of this serialfibered architecture is evidenced by bands of stained motor end-plates (meps) perpendicular to the columns of fibers and dividing each column into a series of “segments.” The avian pectoralis was chosen for a study of variation in the distribution of meps within a single muscle. We report the interspecific variation for 158 specimens in 63 species. We also use additional specimens to examine intraspecific variation. Setting aside hummingbirds, which have an unique and clearly derived condition, the number of mep bands along a column of fibers near the shoulder falls within a remarkably small range. The number of segments is not obviously related to phylogenetic relatedness or to any characteristic of flight or ecology and is only slightly related to size. The largest specimens do average more segments per column, but there are no trends among small to medium-sized species, suggesting that there is an upper limit to fiber length. However, the shape of the sternum and pattern of connective tissue in the pectoralis alleviate the need for additional fibers in many large birds. These findings suggest that the architecture of the avian pectoralis is subject to some as yet unexplained selection that stabilizes the number of myofibers and/or motor neurons. The findings provide few clues as to whether the significant factors are phylogenetic, functional, ontogenetic, or some combination of these. © 1993 Wiley-Liss, Inc.     

Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles ofSaccharomyces cerevisiae

The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures ofSaccharomyces cerevisiae. Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures. The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0°C for 20 min, approximately one-half of the enzyme activity was preserved. The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0. The osmicated formazan deposits featured envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes. The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles. In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria. Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.     

Ultracytochemical study of adenylate cyclase localization in the adenohypophysis of rats

Using an electron cytochemical method and adenylylimidodiphosphate (AMP--PNP) as substrate, the localization of adenylate cyclase activity was determined in the rat's adenohypophysis. This activity was discovered in the perinuclear space, in the canaliculi of the endoplasmic reticulum and Golgi complex, in mitochondria, on the external surface of the plasma membrane. In sinusoidal capillaries, the reaction product was localized on the plasma membrane, in perinuclear space, endoplasmic reticulum and mitochondria. The addition of isoproterenol and sodium fluoride to the incubation medium led to a rise in adenylate cyclase activity.     

Ultracytochemical localization of acid phosphatase in Humicola lutea conidia and mycelia

Electron microscopic cytochemical procedures were used to determine the cellular location of acid phosphatase in the fungus Humicola lutea grown in casein-containing medium lacking in mineral orthophosphates. In our investigations acid phosphatase in nongerminating conidia was localized on the outer side of the cell wall, in the cell wall, and on the exterior surface of the plasma membrane. The reaction product of acid phosphatase in germinating conidia was seen in the outer wall layer while in young mycelium on the cell surface and in the exocellular space. The relationship between phosphatase activities localized in the cell wall and their role in the enzymatic degradation of the phosphoprotein casein providing available phosphates for cell growth is discussed.     

Ultracytochemical localization of H＋—adenosine triphosphatase activity in autophagic vacuoles induced by vinblastine in rat liver

H^-adenosine triphosphatase (H^ -ATPase) activity was demonstrated cytochemically in autophagic vacuoles(AVs) of rat hepatocytes using a modification of the method for the demonstration of neutral p-nitrophenyl phosphatase(p-NPPase) activity[1].When an inhibitor of H^ -ATPase,N-ethylmaleimide (NEM) or 4,4‘-diisothiocyanostilbene-2,2‘disulfonic acid,disodium salt (DIDS) was included in the incubation medium the enyzme activity was abolished indicating that p-NPPase demonstrated in this study represents H^ -ATPase.Autophagy was induced by a single intraperitoneal injection of vinblastine sulfate(VBL).The number of AVs increased remarkably in hepatocytes from 40 min after VBL treatment.H^ -ATPase activity was observed mainly on the membranes of lysosomes and AVs.However,early forms of AVs containing only incompletely digested material showed no H^ -ATPase activity.Most AVs revealing a positive reaction seemed to be in advanced stages of development.Acid phosphatase acticity was demonstrable in mature but not in early forms of AVs.The present investigation showed that membranes of advanced stage AVs possess an H^ -ATPase which may be derived from lysosomal membranes.     

Ultracytochemical localization and microprobe quantitation of calcium stores in the insect oocyte

Detection of calcium in the follicles of Galleria mellonella (Lepidoptera) was performed using two cytochemical methods. Calcium precipitation was obtained either with ammonium oxalate (AO) or with N,N-naphtaloylhydroxylamine (NHA). In both cases the X-ray "on line" analysis monitored the presence of calcium in the oocytes, which was correlated with the accumulation of yolk spheres. Concentration of calcium in oocytes filled with yolk and treated with AO amounted to 9 mmoles per 1,000 g tissue wet weight. This value is similar to that calculated previously for follicles untreated with any reagent and prepared for the analysis by the freeze-drying technique (Prze?ecka et al. 1980). Examination of the ultrastructure of oocytes treated with NHA revealed calcium precipitate at the follicular epithelium/oocyte interface, in endocytotic canaliculi and vesicles formed by the oocyte plasma membrane, in ooplasm, and in yolk spheres. In oocytes treated with AO, the calcium-precipitate intermingled with the precipitate produced by the osmium alone. The presumed cause of this phenomenon is discussed.     

毛竹茎秆纤维细胞发育过程中ATP酶的超微细胞化学定位研究

采用磷酸铅沉淀技术,对毛竹茎秆纤维细胞发育过程中的ATP酶进行了超微细胞化学定位研究.在初生壁形成时期,大量的ATP酶的活性产物沉积在质膜、质膜内陷、运输小泡、胞间连丝等膜体系以及细胞核和各种细胞器上;在次生壁形成的初期,ATP酶在多泡小体和裂解的液泡膜上出现,凝聚并边缘化的染色质上仍然具有ATP酶活性;随着次生壁的逐渐加厚,在前四年中持续存在具有ATP酶活性的质膜内陷结构,以后消失;而在六年生纤维细胞的质膜、运输小泡、纹孔、胞间连丝和凝聚化的染色质上仍然发现有明显的ATP酶分布,并发现在染色质上ATP酶活性会随着凝聚程度的加深而增强.结果表明,ATP酶在毛竹茎秆纤维细胞壁的整个形成过程中发挥重要作用,而纤维细胞的次生壁形成过程是一个由核基因控制的主动的PCD过程;并证实毛竹茎秆纤维细胞的发育有别于其它木本植物纤维细胞的发育过程,这种纤维细胞是一种典型的长寿细胞.