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  • 1
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Cyclic 3′, 5′-mononucleotide phosphodiesterase (cyclic nucleotide PDEase) activity was studied histo-and cytochemically in the retinal rod photoreceptor cells of the rat by means of a newly developed technique utilizing the intrinsic 5′ nucleotidase activity instead of an exogenous 5′ nucleotidase source (snake venom). Cyclic GMP and cyclic AMP were used as substrates. When cyclic GMP was used as a substrate, the intense activity of phosphodiesterase (PDEase) was distributed over the entire rod outer segments; reaction product was observed on the plasmalemma and on the disk membranes of the outer segments. A slight reaction was also observed on the plasmalemma of the inner segments. However, no precipitate was found in the perinuclear and synaptic regions of the rod photoreceptors. In contrast, when cyclic AMP was utilized as a substrate, a moderate reaction was seen in the synaptic region of the plexiform layer. The intensity of the reaction in the outer segments was much reduced in comparison to the results with cyclic GMP. The enzyme activity was almost completely inhibited by 2 mM 3-isobutyl-1-methylxanthine (IBMX) or 2 mM theophylline, which were potent inhibitors of PDEase. To confirm the propriety of our new cytochemical method, the localization of 5′ nucleotidase was also studied utilizing 5′ AMP or 5′ GMP as substrates. In contrast to the activity of cyclic nucleotide PDEase, the activity of 5′ nucleotidase was distributed on all membranes of the photoreceptors from the synaptic outer plexiform layer to the tip of outer segments. After inhibition of the intrinsic 5′ nucleotidase activity with the use of 1 mM Ni-ions or 10 mM NaF no demonstration of cyclic nucleotide PDEase activity was possible; the existence of intrinsic 5′ nucleotidase activity is necessary for the release of free phosphateions from 5′ AMP (5′ GMP), which are a prerequisite for the histochemical reaction. For comparison, some sections were incubated with the conventional cyclic nucleotide PDEase incubation medium containing snake venom from Ophiophagus hannah. With this conventional method, morphological preservation was extremely poor, and moreover, the reaction itself was weaker than that with the presently described method.
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  • 2
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The histo- and cytochemical localization of Ca++-ATPase activity in the adenohypophysis of the guinea pig was studied utilizing a newly developed method (Ando et al. 1981). An intense reaction was observed in the wall of the blood vessels and between non-secretory cells (stellate cells) and endocrine cells of the pars distalis. Under the electron microscope the Ca++-ATPase reaction product was located extracellularly in relation to the plasmalemma of the stellate cells. This reaction was dependent on Ca++ and the substrate, ATP, and reduced by the addition of 0,1 mM quercetin to the standard incubation medium. Preheating of the sections before incubation completely inhibited the enzyme activity. When Mg++ in different concentrations were substituted for Ca++ in the incubation medium the reaction was always reduced. Both Ca++ and Mg++ in the incubation medium also reduced the reaction. The plasmalemma of the endocrine cells contains no demonstrable amount of Ca++-ATPase activity. The function of the Ca++-ATPase activity is discussed in relation to the regulation of the extracellular Ca++ concentration which seems to be important with respect not only to the secretory process of the endocrine cells but also to the metabolism of the adenohypophysis.
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  • 3
    ISSN: 1432-0878
    Keywords: Ca++-ATPase ; K+-NPPase ; Na+-K+ATPase ; Ultracytochemistry ; Photoreceptor cells, retinal ; Guinea pig
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Ca++-ATPase activity was demonstrated histochemically at light- and electron-microscopic levels in inner and outer segments of retinal photoreceptor cells of the guinea pig with the use of a newly developed one-step lead-citrate method (Ando et al. 1981). The localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity, which represents the second dephosphorylative step of the Na+-K+-ATPase system, was studied by use of the one-step method newly adapted for ultracytochemistry (Mayahara et al. 1980). In retinal photoreceptor cells fixed for 15 min in 2% paraformaldehyde the electron-dense Ca++-ATPase reaction product accumulated significantly on the inner membranes of the mitochondria but not on the plasmalemma or other cytoplasmic elements of the inner segments. The membranes of the outer segments remained unstained except the membrane arrays in close apposition to the retinal pigment epithelium. The cytochemical reaction was Ca++- and substrate-dependent and showed sensitivity to oligomycin. When Mg++-ions were used instead of Ca++-ions, a distinct reaction was also found on mitochondrial inner membranes. In contrast to the localization of the Ca++ -ATPase activity, the K+-NPPase activity was demonstrated only on the plasmalemma of the inner segments, but not on the mitochondria, other cytoplasmic elements or the outer segment membranes. This reaction was almost completely abolished by ouabain or by elimination of K+ from the incubation medium.
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  • 4
    ISSN: 1432-0878
    Keywords: CA++-ATPase ; Pituitary gland, pars nervosa ; Pituicytes ; Neurosecretion ; Guinea pig
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Ca++-ATPase activity (cf. Ando et al. 1981) was examined both light- and electron-microscopically in the neurohypophysis of the guinea pig. Apart from a strong activity within the walls of the blood vessels, in the parenchyma of the neurohypophysis the reaction product of the Ca++-ATPase activity was restricted to the plasmalemma of the pituicytes. This reaction was completely dependent upon Ca++ and the substrate, ATP; the reaction was inhibited by 0.1 mM quercetin, an inhibitor of Ca++-ATPase. A reduction of the enzyme activity occurred by 1) adding Mg++ to the standard incubation medium, and 2) substituting Ca++ with Mg++ at varing concentrations. In all experiments the neurosecretory fibers were devoid of Ca++-ATPase activity. The function of the Ca++-ATPase activity in the plasmalemma of the pituicytes is discussed in connection with the regulation of the extracellular Ca++ concentration, which seems to be important with respect to the discharge of secretory material from the neurosecretory fibers.
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