Satoki Ueno

Ultracytochemical localization of Ca++-ATPase activity in the paraphyseal epithelial cells of the frog, Rana esculenta.

SummaryCa++-ATPase activity was studied ultracytochemically (cf. Ando et al. 1981) in the paraphysis cerebri of the frog. An intense reaction was demonstrated on the plasmalemma of the microvilli at the apical pole of paraphyseal cells; in contrast, the basolateral plasmalemma showed only a slight staining. In addition, mitochondria, gap junctions, cilia, and cytoplasmic elements (e.g., microfilaments) displayed Ca++-ATPase activity. Variation of the Ca++-concentration in the incubation medium from 0.1 mM to 100 mM altered the Ca++-ATPase activity of the cell organelles. The substitution of Ca-by Mg-ions resulted in a conspicuous decrease in the enzyme activity, especially on the apical plasmalemma. Ca++-ATPase activity is claimed to be involved in a number of extra-and intracellular functions. In comparison to the epithelium of the adjacent choroid plexus the paraphyseal epithelial cell is thought to be a principal Ca-ion regulator of the cerebrospinal fluid in frogs.

TISSUE CULTURE OF THE RETINAL PIGMENT EPITHELIAL CELLS

Pure culture of the retinal pigment epithelial (RPE) cells of the chick embryo was carried out and the morphological characteristics of each developmental stage were observed under light microscope and transmission and scanning electron microscopes.

Ultracytochemical study of Ca++-ATPase and K+-NPPase activities in retinal photoreceptors of the guinea pig.

SummaryCa++-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.

Histochemical and cytochemical demonstration of Ca++-ATPase activity in the stellate cells of the adenohypophysis of the guinea pig

SummaryThe 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.

Ultracytochemical localization of Ca++-ATPase activity in pituicytes of the neurohypophysis of the guinea pig.

SummaryCa++-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.

A new histo-and cytochemical method for demonstration of cyclic 3′, 5′-nucleotide phosphodiesterase activity in retinal rod photoreceptor cells of the rat

SummaryCyclic 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.

Silicone Oil Particles Trapped in the Subretinal Space: Complications after Substitution of the Vitreous

Silicone oil particles trapped in the subretinal space, following total replacement of the vitreous by oil, were found in the aphakic eye of a 51-year-old man. The eye had been operated on two times for retinal detachment in other institutes which resulted in a grade D-1 proliferative vitreoretinopathy. The silicone particles resembled beads and were slightly mobile among subretinal strands by changing the eye position. The particles freely moved into the vitreous cavity through a retinotomy site and were removed with the silicone from the vitreous cavity because of ocular pain. Subconjunctival granulation was removed and histological examination showed silicone oil particles in the vitreous cavity, and cellular infiltration.

Ultracytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in the lacrimal gland of the rat.

SummaryThe electron-microscopic localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na-K-ATPase complex was studied in the exorbital lacrimal gland of the untreated rat with the use of a newly developed one-step lead-citrate method (Mayahara and Ogawa 1980; Mayahara et al. 1980). In the rat lacrimal gland fixed for 15 min in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, an electron-dense reaction product was observed on the plasma membrane of the basal infoldings and the lateral interdigitations of the ductal cells. The most intense reaction product — and thus the major site of the Na-K-ATPase activity — was evident on the basolateral membranes of the cells of the large interlobular ducts; a weak reaction was seen on the basolateral, extensively folded plasma membranes of the small intercalated ducts; no reaction product was observed on the plasma membranes of the acinar cells. Addition of 1) 10 mM ouabain, 2) p-chloromercuri-phenyl-sulfonic acid (PCMB-S), 3) elimination of K-ions from the incubation medium, or 4) preheating abolished completely the K-NPPase reaction. The activity was also substrate-dependent.Mg-ATPase-activity was observed not only in the basolateral membranes of all ductal cells but also in the basal part of the acinar cells and on the walls of blood vessels. This reaction was neither inhibited by ouabain nor activated by K-ions. The precipitate of the Mg-ATPase-activity was localized at the extracellular side of the plasma membrane, whereas the K-NPPase-reaction product was restricted to the cytoplasmic side of the plasmalemma.In contrast, non-specific alkaline-phosphatase (ALPase) activity was missing in cells of the large interlobular ducts, but obvious on the apical plasmalemma of cells lining the small intercalated ducts.With respect to its localization and reactivity pattern the activity of the K-NPPase (member of the Na-K-ATase complex) differs markedly from the Mg-ATPase- and ALPase-activity.

Localization of ATPases in Retinal Receptor Cells

Ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K- NPPase ) activity of the Na-K-ATPase complex and the Ca-ATPase activity were studied ultracytochemically in the inner and outer segments of guinea pig photoreceptor cells by means of newly developed methods. The K- NPPase activity was demonstrated on the plasmalemma of the inner segments, whereas the Ca-ATPase activity was restricted to the matrix side of the inner membrane of the mitochondria which were accumulated in the inner segments. In contrast to the enzyme activities in the inner segments no such activity could be detected on the plasma and disk membranes of the outer segments. The functional meaning of the enzyme activities is discussed.

Localization and function of cyclic guanosine monophosphate-phosphodiesterase activity in the retinal rods of the rat by means of a newly developed cytochemical method

SummaryCyclic guanosine monophosphate-phosphodiesterase (cGMP-PDEase) activity was studied histo- and cytochemically in the retinal rods of the rat with the use of a newly developed technique. Intense activity of cGMP-PDEase was evenly distributed over the outer segments of the rods. Reaction product was observed on the plasmalemma and on the disk membranes of the outer segments. A weak reaction product occurred also on the plasmalemma of the inner segments; however, no precipitate was found in the perinuclear and synaptic portions of the rod cells. The enzyme activity was strongly inhibited by 2 mM theophilline and by 2 mM 3-isobutyl-1-methylxanthine (IBMX). To confirm the specificity of this new cGMP-PDEase method, the localization of 5′nucleotidase (5′GMPase) was also studied. In contrast to the activity of cGMP-PDEase, the activity of 5′GMPase was distributed on the plasma membrane of the photoreceptor cells extending over a wide range from the synaptic endings in the outer plexiform layer to the tip of the outer segments.