Distribution and properties of an adenosine triphosphatase in the tanycyte ependyma of the IIIrd ventricle of the rat.

The distribution, histochemical properties and ultrastructural localization of adenosine triphosphate (ATP) hydrolyzing enzymes in the tanycyte ependyma of the third ventricle have been studied in female Wistar rats. Using a calcium-cobalt procedure and a lead capture technique, splitting of ATP could be demonstrated in perikarya and processes of tanycytes in the region of the ventromedial nucleus. The reaction showed no dependence on magnesium or sodium ions, did not occur with other monodi-, and tri-phosphates as substrates, and was inhibited by p-chlormercuribenzoate (PCMB) and sodium fluoride, but not by ouabain. With the calcium-cobalt method the highest intensity of reaction was found at pH 9.4, whereas the lead method gave optimal results at pH 6--8. At the ultrastructural level, the reaction product was found at the outer surface of the plasma membranes of tanycytes and reached its highest concentrations in the region of the region of the apical microvilli; From the findings it is concluded that splitting of ATP in tanycytes is due to a true ATPase. The enzyme might be involved in an active transport of substances by tanycytes.     

Electron-microscopic localization of acetyl coenzyme A carboxylase in cells of Claviceps purpurea

Summary The ultrastructural localization of acetyl-CoA carboxylase activity was studied in two strains of the ascomycetous fungus Claviceps purpurea differing in the ergot alkaloid synthesis. Mycelia were harvested by centrifugation of saprophytic submerged cultures, fixed in cold 3% glutaraldehyde in 0.05 M cacodylate buffer pH 7.2 and washed repeatedly in the same buffer. The incubation medium of Yates et al. (1969) had to be modified in the molarity of ATP. The best results were obtained with a medium of the following composition: 50 mM cacodylate buffer pH 7.2, 4 mM ATP, 3.5 mM lead nitrate, 13.5 mM sodium citrate, 3.75 mM sodium bicarbonate, 1.25 mM manganese chloride, 0.4 mM acetyl-CoA and 2 mM biotin. The fixation is a prerequisite for a distinct localization. The enzyme activity was detected only in cells producing high amount of clavine alkaloids. It was confined to the membranes of endoplasmic reticulum and their derivatives: tonoplast of vacuoles, tiny vesicles and amorphous material inside vacuoles. The reaction product was very fine and localized in both leaflets of the membranes. The specificity of the reaction was confirmed by negative results in control preparations: boiled cells incubated in the complete medium, cells incubated in the medium supplemented with avidin or in the media from which either ATP, or acetyl CoA, or sodium bicarbonate, or biotin were omitted. It is suggested that the activity of acetyl-CoA carboxylase is linked to the synthesis of clavine alkaloid precursors which occurs in the endoplasmic reticulum and its derivatives.With technical assistance of J. Martínková     

Regulation of secretion from the adrenal medulla. Evidence for adenylate cyclase activity in secretory vesicle membranes.

Adenylate cyclase activity has been found in purified secretory vesicle membranes from the adrenal medulla. Activity was detected both by formation of radioactive cAMP from [alpha-32P]ATP and by the competitive protein binding assay for cAMP. Activity was highest at pH 8.0 to 8.5, and was stimulated by sodium fluoride and GppNHp, a GTP analogue known to stimulate adenylate cyclase activity in plasma membrane preparations. The reaction rate was strongly dependent on the molar ratio of Mg2+:ATP in the system. This is the first demonstration of adenylate cyclase in a secretory vesicle membrane.     

Ecto-ATPase activity in the rat cardiac muscle: biochemical characteristics and histocytochemical localization

We used a combined biochemical and histocytochemical approach to study ecto-ATPase in the rat cardiac muscle. The reaction medium employed for histocytochemical detection was optimized in biochemical assays to achieve the highest enzyme activity and lowest inhibition by the capture agent used for visualization of the reaction product. Approximately 70% of the enzyme activity was retained in samples after the fixation procedure. Divalent cations stimulated ecto-ATPase. High activity was detectable within a wide pH range. Histocytochemical reaction was observed at sites at which extracellular ATP can potentially exert its actions on the cardiac muscle: nerve endings, plasma membranes of cardiac myocytes and capillary endothelial cells, and T-tubules. Product of the reaction was found exclusively at the outer surface of the cells. In controls, enzyme activity was abolished by diethyl pyrocarbonate and slightly stimulated by digitonin and concanavalin A, whereas sodium orthovanadate, N-ethylmaleimide, and sodium azide yielded no effect. Our results support the view that cardiac ecto-ATPase is involved in important physiological functions and suggest that its activity may be regulated by the release of ATP from nerve endings.     

Effects of ATP and ADP on hydrogen-deuterium exchange in heavy meromyosin. An examination of difference spectra.

The exchange reaction of peptide hydrogens with deuterium has been followed by measuring the decrease of the amide II band for heavy meromyosin (HMM). The difference spectra between HMM and HMM + ATP, between HMM and HMM + ADP, and between HMM + ATP and HMM + ADP have been examined as functions of time in order to detect small differences in the kinetic behavior of these different states of HMM. It has been found that, at 14 degrees C and 26 degrees C (pH 8.0), the exchange reaction is slightly slower for HMM + ATP than for HMM, and slightly slower for HMM + ADP than for HMM + ATP. This indicates that the secondary structure of HMM changes its flexibility during the ATP splitting cycle.     

The dependence on internal pH of Ca2+-fluxes across sarcoplasmic reticulum vesicular membranes

The interdependence of the competition between Ca2+ and hydrogen ions for the internally located low-affinity Ca2+ binding sites of sarcoplasmic reticulum vesicles and the pH-dependent splitting rate of phosphoenzyme was investigated. Sarcoplasmic reticulum vesicles were preincubated at a selected pH and passive Ca2+ loading, active Ca2+ uptake at the same pH as well as active Ca2+ uptake at a distinct pH (pH-jump method) were observed. In addition, Cai-Cao exchange in the absence and presence of ADP and ATP-ADP exchange were measured. The overall ATP splitting rate was assayed with leaky vesicles in the presence of varied Ca2+ concentration and four different pH. All experiments were carried out at Ca2+ concentrations sufficient to saturate the externally located activating high-affinity binding sites at all pH and in the absence of affecting concentrations of monovalent cations. Active Ca2+ transport (particularly evident applying the pH-jump method) is facilitated at low intravesicular pH, reflecting the favoured Ca2+ release to the intravesicular space, in contrast to the reverse pH-dependence of passive Ca2+ accumulation and the initial rate of Cai-Cao exchange, both favoured by elevated internal Ca2+ binding capacity. The rates of ATP splitting, the continuing slow rate of Cai-Cao exchange, and the ATP-ADP exchange are optimal at an intermediate proton concentration, reflecting the influence of protons on partial reaction steps occurring later in the reaction cycle and the accelerated exchange of Ca2+ at the internal low-affinity sites as well as the establishment of a new pseudo equilibrium between the possible reaction intermediates. The pool of rapidly exchangeable Ca2+ is enlarged whereas the rate of slow exchange is unaltered or diminished (pH 7.8) by ADP.     

Cytochemical localization of phosphatase in differentiating secondary vascular cells

Summary Phosphatase activity was studied in the cambium and differentiating vascular cells of beech by using a modified Washstein and Meisel method. After fixation in glutaraldehyde or crotonaldehyde and incubation in a medium containing ATP and lead nitrate at pH 7.2, a deposit of electron-opaque granules was found in the nucleoli, nucleoplasm, nuclear envelope, endoplasmic reticulum, plastids, mitochondria, and at the plasmalemma. Although located at these different sites, the distribution varied both inter- and intra-cellularly. This is thought to be a true reflection of the variation in activity between closely adjacent cells in this part of the stem.Some reaction was obtained when ADP replaced ATP in the reaction mixture, but there was no reaction at all when both ATP and ADP were omitted. Fixation in hydroxyadipaldehyde, or incubation at pH 6.4 both produced very little reaction.     

Prenatal and early postnatal development of the glial cells in the median eminence of the rat

Summary The development of the glial cells of the rat median eminence (ME), including the supraependymal cells, was investigated from embryonic day (ED) 14 through postnatal day (PD) 7, and pituicyte development from ED 12 through ED 17. The anlage of the ME and neurohypophysis shows a neuroepithelial-like structure at ED 12. From ED 13 to 15, the cells of both regions start to differentiate. At the ultrastructural level, only one cell type appears. At the beginning of ED 16, glioblasts of the oligodendrocyte and astrocyte series migrate laterally (from the region of the arcuate nucleus) into the ME. Also at this time the first distinctive structural features appear in the neurohypophysial anlage, the cells of which later develop into pituicytes. Starting at ED 18, tanycytes and astrocytic tanycytes arise in the ME from local glial cells, and somewhat later oligodendroblasts and astroblasts are formed from immigrant glioblasts. Due to their common features, the pituicytes, tanycytes and astrocytic tanycytes apparently represent different forms of the same parent cell type. Microglial and supraependymal cells are first seen at ED 12. Initially, they resemble the prenatal phagocytic connective tissue cells and mature in the fetus into typical electron-dense microglia and macrophage-like supraependymal cells. Both cell types are apparently of mesodermal origin. The microglial elements of the ME probably migrate from the mesenchyma through the basement into the nervous tissue. The intraventricular macrophages of the infundibular region may originate from microglia, epiplexal cells and subarachnoid macrophages.Dedicated to Prof. I. Törö, Budapest, on the occasion of his 80th birthday     

大鼠第三脑室室管膜NADPH-d阳性伸展细胞的形态与分布

应用 Ｎ Ａ Ｄ Ｐ Ｈｄ 组织化学方法研究了大鼠第三脑室视前区室管膜的伸展细胞⒚结果表明：１）第三脑室视前区室管膜存在 Ｎ Ａ Ｄ Ｐ Ｈｄ 阳性的伸展细胞,其基突伸向视前区并与神经元或毛细血管相接触;２） Ｎ Ａ Ｄ Ｐ Ｈｄ 阳性的伸展细胞在第三脑室侧壁常见、室底少见、室顶未发现其存在;３） Ｎ Ａ Ｄ Ｐ Ｈｄ 阳性的伸展细胞的形态与分布,在雌、雄大鼠间不存在明显的性别差异⒚尽管 Ｎ Ａ Ｄ Ｐ Ｈｄ 阳性的伸展细胞的生理功能不十分清楚,但本研究为伸展细胞作为脑脑脊液环路的一部分,介导下丘脑对脑脊液中化学变化的感受提供了形态学依据,并提示一氧化氮可能参与了这一过程⒚     

Immunogold electron microscopic localization of glial fibrillary acidic protein (GFAP) in neurohypophyseal pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

Summary The post-embedding immuno gold staining (IGS) technique was used for the ultrastructural localization of glial fibrillary acidic protein (GFAP) in pituicytes and tanycytes of the neurohypophysis. IGS was applied to LR White embedded neurohypophyseal tissue of the Mongolian gerbil (Meriones unguiculatus), a species which contains abundant GFAP-positive pituicytes and tanycytes. GFAP-immunoreactivity could be demonstrated on pituicytic intermediate filaments (IF's) in situ. Thus, it was shown that pituicytes contain GFAP in its filamentous form, what had been a matter of speculation. At the ultrastructural level, gerbil tanycytes and tanycyte-like cells in the external zone of the median eminence were characterized by a great amount of densely packed IF's, which were labeled by both GFAP- or vimentin-antibodies. Sequential immunostaining of serial semithin sections with GFAP- and vimentin-antibodies revealed an invariable coexpression of the two IF proteins in somata and processes of these median eminence cells.     

Localization of ATPase Activity on the Plasmalemma of Scutellar Epithelial Cells of Germinating Barley (Hordeum vulgare L.)

Chaffey, N. J. and Harris, N. 1985. Localization of ATPase activityon the plasmalemma of scutellar epithelial cells of germinatingbarley (Hordeum vulgare L.).—J. exp. Bot 36: 1612–1619. ATPase activity has been localized at an ultrastructural levelin the absorptive region of the scutella of germinating barley(Hordeum vulgare L.). The enzyme is localized on the plasmalemmaof the epithelial cells. Using the Gomori reaction the depositionof reaction product on the plasmalemma, which is dependent uponthe presence of supplied ATP, was precluded or reduced by theinhibitors orthovanadate, mercuric chloride and DCCD, whilstß-glycerophosphate would not act as an alternativesubstrate. The mitochondria demonstrated phosphatase activitywith both ATP and ß-glycerophosphate as substrate.The results are discussed in relation to the active uptake ofmetabolites by the scutellum during germination and the structuralmodification of the plasmalemma of the epithelial cells to formplasmatubules. Key words: ATPase, Hordeum vulgare L., localization (ultrastructural)     

32P]ATP synthesis in steady state from [32P]Pi and ADP by Na+/K(+)-ATPase from ox brain and pig kidney. Activation by K+

The ouabain-sensitive synthesis of [32P]ATP from [32P]Pi and ADP (vsyn) was measured in parallel with the ouabain-sensitive hydrolysis of [32P]ATP (vhy) at steady state, at varying concentrations of sodium, potassium, magnesium, inorganic phosphate, ADP, ATP and oligomycin, and at varying pH. Na+ was necessary for ATP synthesis, but vsyn was decreased by high sodium concentrations. Oligomycin, depending on the Na+ concentration, either decreased or did not affect vsyn. Potassium, at low concentrations (1-5 mM) increased vsyn at all magnesium and sodium concentrations tested, lower potassium concentrations being needed to activate vsyn at lower sodium concentrations. vsyn was optimal below pH 6.7, decreasing abruptly at higher values of pH. At pH 6.7, vsyn was a hyperbolic function of the concentration of inorganic phosphate. In the presence of potassium, half-maximal rate was obtained at [Pi] congruent to 40 mM, whereas a higher concentration was needed to obtain half-maximal rate in the absence of K+. In contrast, increasing the concentration of ADP caused a nonhyperbolic activation of vsyn, the pattern obtained in the presence of potassium being different from that obtained in its absence. Increasing the ATP concentration above 0.5 mM decreased vsyn. The data are used to elucidate (1) which reaction steps are involved in the ATP-synthesis catalysed by the Na+/K(+)-ATPase at steady state in the absence of ionic gradients and (2) the mechanism by which K+ ions stimulate the reaction.     

Some properties of the catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase from pigeon breast muscle]

A method for the preparation of a homogenous catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase from pigeon breast muscle was developed. The molecular weight of the enzyme as determined by electrophoresis in the presence of sodium dodecyl sulfate was found to be 42000. The pH optimum of the catalytic subunit was around 8.0. The active site of the catalytic subunit was studied using some derivatives of ATP, containing different reactive groups in the triphosphate chain of the molecule. It may be assumed that the pH optimum of the enzyme inactivation by adenosine 5'-chloromethylpyrophosphonate and the protective effect of ATP suggest covalent binding of the imidazole ring in the enzyme active site. The kinetic mechanism of the protein kinase reaction was studied using the initial rate experiments and reaction product inhibition. The results obtained were consistent with a random Bi-Bi kinetic mechanism.     

Effect of long-term suppression and long-term stimulation of ACTH secretion on the ultrastructure of tanycytes[]

The ultrastructure of the tanycyte ependyma in males of Wistar albino rats (160--180 g of weight) was studied under experimental long-term suppression of ACTH secretion and its long-term stimulation. The former effect was accomplished by daily (for 8 days) intraperitoneal administration of dexamethasone phsophate; the stimulation of ACTH secretion was achieved by bilateral adrenalectomy. The long-term suppression of the hypophyseal adrenocorticotrophic function was followed by the ultrastructural markers of an increased transport and secretory activity in the tanycytes (mainly in beta-tanycytes, i. e. the ependyma lining floor of the third ventricle): the increased amount of microvesicles in the Golgi area and in the vicinity of the particular membranous cavity system (MCS, according to the authors' definition), the appearance of some figures of exocytosis, the elevated amount of lipid inclusions. On the contrary, the long-term stimulation of ACTH secretion was followed by the ultrastructural changes of the opposite character. The data obtained are in agreement with the previously established fact that there is a negative correlation between the tanycyte activity and the hypophyseal adrenocorticotrophic activity. It is supposed that this correlation is regulated by a feedback mechanism and attest to the involvement of tanycytes in the inhibitory control of ACTH secretion. A possible substrate inhibiting ACTH secretion by the tanycytes is discussed.     

Immunogold electron microscopic localization of glial fibrillary acidic protein (GFAP) in neurohypophyseal pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

The post-embedding immuno gold staining (IGS) technique was used for the ultrastructural localization of glial fibrillary acidic protein (GFAP) in pituicytes and tanycytes of the neurohypophysis. IGS was applied to LR White embedded neurohypophyseal tissue of the Mongolian gerbil (Meriones unguiculatus), a species which contains abundant GFAP-positive pituicytes and tanycytes. GFAP-immunoreactivity could be demonstrated on pituicytic intermediate filaments (IF's) in situ. Thus, it was shown that pituicytes contain GFAP in its filamentous form, what had been a matter of speculation. At the ultrastructural level, gerbil tanycytes and tanycyte-like cells in the external zone of the median eminence were characterized by a great amount of densely packed IF's, which were labeled by both GFAP- or vimentin-antibodies. Sequential immunostaining of serial semithin sections with GFAP- and vimentin-antibodies revealed an invariable coexpression of the two IF proteins in somata and processes of these median eminence cells.     

Sodium ions as substitutes for protons in the gastric H,K-ATPase

In view of the striking homology among various ion-translocating ATPases including Na,K-ATPase, Ca-ATPase, and H,K-ATPase, and the recent evidence that protons can replace cytoplasmic sodium as well as potassium in the reaction mechanism of the Na,K-ATPase (Polvani, C., and Blostein, R. (1988) J. Biol. Chem. 263, 16757-16763), we studied the role of sodium as a substitute for protons in the H,K-ATPase reaction. Using hog gastric H,K-ATPase-rich inside-out membrane vesicles we observed 22Na+ influx which was stimulated by intravesicular potassium ions (K+i) at pH 8.5 but not at pH 7.1. This sodium influx was observed in medium containing ATP and was inhibited by vanadate and SCH28080, a selective inhibitor of the gastric H,K-ATPase. At least 2-fold accumulation of sodium was observed at pH 8.5. Experiments aimed to determine the sidedness of the alkaline pH requirement for K+i-dependent sodium influx showed that K+i-activated sodium influx depends on pHout and is unaffected by changes in pHin. These results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na,K-ATPase and the gastric H,K-ATPase enzymes.     

Functional identification of electrogenic Na+-translocating ATPase in the plasma membrane of the halotolerant microalga Dunaliella maritima

The hypothesis that the primary Na+-pump, Na+-ATPase, functions in the plasma membrane (PM) of halotolerant microalga Dunaliella maritima was tested using membrane preparations from this organism enriched with the PM vesicles. The pH profile of ATP hydrolysis catalyzed by the PM fractions exhibited a broad optimum between pH 6 and 9. Hydrolysis in the alkaline range was specifically stimulated by Na+ ions. Maximal sodium dependent ATP hydrolysis was observed at pH 7.5-8.0. On the assumption that the ATP-hydrolysis at alkaline pH values is related to a Na+-ATPase activity, we investigated two ATP-dependent processes, sodium uptake by the PM vesicles and generation of electric potential difference (Deltapsi) across the vesicle membrane. PM vesicles from D. maritima were found to be able to accumulate 22Na+ upon ATP addition, with an optimum at pH 7.5-8.0. The ATP-dependent Na+ accumulation was stimulated by the permeant NO3- anion and the protonophore CCCP, and inhibited by orthovanadate. The sodium accumulation was accompanied by pronounced Deltapsi generation across the vesicle membrane. The data obtained indicate that a primary Na+ pump, an electrogenic Na+-ATPase of the P-type, functions in the PM of marine microalga D. maritima.     

Ultrastructural localization of adenosine triphosphatase activity in lymphocytes activated in vitro by phytohaemagglutinin

Summary The ultrastructural localization of Ca²⁺, Mg²⁺-activated ATPase was studied in phytohaemagglutinin activated lymphocytes and in normal unstimulated lymphocytes. Cells, fixed in paraformaldehyde-glutaraldehyde, were incubated in a medium containing 3mm ATP, 5mm CaCl₂ and 2.4mm Pb(NO₃)₂ in 0.1m tris buffer at pH 8.5, the optimum pH for histochemical demonstration of this enzyme. Reaction product was localized i the endoplasmic reticulum, nuclear membrane, Golgi apparatus and mitochondria and on the membrane surrounding large electron-dense bodies. Cytoplasmic vesicles and the plasma membrane were negative. Activity in unstimulated lymphocytes showed a similar localization but the amount of endoplasmic reticulum was much less than in activated lymphocytes.The pH of the medium was critical for the localization of the enzyme. At pH 7.5, the cytoplasmic reaction was almost completely inhibited but a dense precipitate was present on the outer surface of the plasma membrane. The reaction was stimulated by either Ca²⁺ or Mg²⁺ and was greatly decreased in the absence of these cations or in the presence ofp-chloromercuribenzoate orN-ethylmaleimide. Oligomycin inhibited selectively the reaction in mitochondria but not the reaction at other sites. While the reaction in mitochondria showed complete substrate specificity, a mild reaction was obtained at the other sites with uridine diphosphate or sodium -glycophosphate as substrate. ATP was, however, the preferential substate.     

Hypothalamic ependymal cells express the glucose transporter GLUT2, a protein involved in glucose sensing

Kinetic analysis of vitamin C uptake has demonstrated that specialized cells take up ascorbic acid (AA), the reduced form of vitamin C, through sodium-AA cotransporters. Recently, two different isoforms of sodium-vitamin C cotransporters (SVCT 1, 2) that mediate high affinity Na⁺-dependent l -ascorbic acid have been cloned. SVCT2 was detected mainly in choroid plexus cells and neurons, however, there are no evidences of SVCT2 expression in glial cells. High concentrations of vitamin C has been demonstrated in brain hypothalamic area. The hypothalamic glial cells, known as alpha and beta tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. Our hypothesis postulates that tanycytes take up reduced vitamin C from the portal blood and cerebrospinal fluid generating an high concentration of this vitamin in brain hypothalamic area. In situ immunohistochemical analyses demonstrated that SVCT2 transporter is selectively expressed in apical region of tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of SVCT2 isoform in these cells. Reduced vitamin C uptake was temperature and sodium dependent. Kinetic analysis showed an apparent Km of 20 μ m and a Vmax of 45 pmol/min per million cells for the transport of ascorbic acid. The expression of SVCT2 was confirmed by immunoblots and RT–PCR. Tanycytes may perform a neuroprotective role concentrating the vitamin C in the hypothalamic area.
Acknowledgements:   Supported by Grands FONDECYT 1010843 and DIUC-GIA 201.034.006-1.4 from Concepción University.