Ultrastructural localization of acid phosphatase in thyrocytes of rats with thyroid hyperfunction

Acid phosphatase was detected electron-cytochemically in lysosomes which appeared in large numbers in the follicular cells of the hyperplastic rat thyroid gland. The remaining types of granules (mature secretory granules, lipid granules), the number of which also increased appreciably during functional stress on the thyrocytes, did not contain the reaction product. By the character of distribution of acid phosphatase the lysosomes could be subdivided into three main groups: those with a dense homogeneous residue and those with residue in the form of densely or infrequently distributed circular grains. The heterogeneity of the lysosomes may be attributed to differences in their functional state. Besides in the lysosomes, the reaction product was found in pale cells as a sprinkling of widely scattered dark grains. These drops evidently appeared as the result of fusion of droplets of colloid with lysosomes. The acid phosphatase of the lysosomes in this case participates in hydrolysis of the secretion product of the cells with the formation of the active substances.Electron Microscopy Room, N. K. Kol'tsov Institute of Developmental Biology, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 5, pp. 622–625, May, 1977.     

Ultrastructural localization of Tartrate-resistant acid phosphatase (purple acid phosphatase) activity in chicken cartilage and bone

Tartrate-resistant acid adenosine triphosphatase activity at pH 6.5, using a lead-salt method, was localized at light and electron microscopic levels in cartilage and bone matrices, osteocalsts, and chondrocalsts. Cartilage matrix staining occurred after vascular invasion of the growth plate. In osteoclasts, activity was present in lysosomes, extracellular ruffled border channels, and the underlying cartilage and bone matrices. Staining artifacts occurred at lower pH levels (pH 5.4, 5.0). Adenosine diphosphate, p-nitrophenylphosphate, thiamine pyrophosphate, and α-naphthylphosphate also acted as substrates; but no activity was observed when adenosine monophosphate, adenylate-(β,γ-methylene) diphosphate, and β-glycerophosphate were used. The activity was inhibited by NaF, dithionite, and a high concentration of p-chloromercuribenzoic acid, and activated by simultaneous addition of FeCl₂ and ascorbic acid, as has been shown in biochemical studies. These histochemical results support the view that the adenosine triphosphate hydrolyzing activity at pH 6.5 is due to tartrate-resistant acid phosphatase (TRAP). There were some differences in ultrastructural localization between TRAP and tartrate-sensitive acid phosphatase (TSAP) activities in osteoclasts: TSAP activity was more intense in lysosomes and Golgi complexes and TRAP was stronger in the cartilage and bone matrices. It is suggested, therefore, that most of TRAP is in an inactive form in cells and is activated when secreted.     

Annulate lamellae of the jejunal absorptive cells in 21-day-starved rats

The ultrastructure of annulate lamellae of the jejunal absorptive cells in control and 21 d starved rats was investigated. Annulate lamellae were only rarely encountered in the jejunal absorptive cells of control rats, and then frequently in small stacks continuous with the rough-surfaced endoplasmic reticulum. In contrast, there was a relatively frequent incidence of annulate lamellae in the jejunal absorptive cells of 21 d starved rats, and larger stacks of annulate lamellae were also observed in spite of marked ultrastructural changes of these cells. The annulate lamellae were also continuous with the rough-surfaced endoplasmic reticulum, which was degenerating. The degenerative process of the absorptive cells following starvation might be related to the origin and function of the annulate lamellae.     

Ultrastructural localization of thiamine pyrophosphatase activity in the intestinal epithelial cells of adult rats

Ultrastructural localization of thiamine pyrophosphatase (TPPase) activity was studied in the small and large intestinal epithelial cells of adult rats. In the duodenal and jejunal absorptive cells, TPPase activity was localized on the microvilli, on the lateral cell membrane, and on the lysosome-like bodies. In the ileal epithelial cells, TPPase activity was detected on the lysosome-like bodies, and weak phosphatase activity was observed on the microvilli and on the cisternae of the rough-surfaced endoplasmic reticulum (rER). In the principal cells of the caecum, the ascending colon, and the descending colon, the lysosome-like bodies and the cisternae of the rER showed TPPase activity. Almost all Golgi lamellae, vesicles and GERL elements (NOVIKOFF 1964) in the duodenal absorptive cells were strongly TPPase positive. The 1st to 3rd of the inner Golgi lamellae and the vesicles of the jejunal, ileal, and large intestinal epithelial cells usually showed positive TPPase activity, while the GERL elements were totally negative.     

Ultrastructural localization of acid phosphatase in denervated and diabetic striated muscles.

Catabolism in denervated and diabetic rat skeletal muscle undergoing accelerated protein degradation has been investigated with methods for demonstrating acid phosphatase ultrastructurally. Control muscles displayed strong acid phosphatase activity in lateral sacs and in sparse secondary hysosomes distributed mainly near nuclear poles. Muscles from diabetic rats and, to a lesser extent, 2-day denervated rats, revealed increased secondary lysosomes apparently derived from fusion of mitochondria with acid-phosphatase-reactive vesicles and cisternae. The latter were interpreted as possibly originating from T tubules. Reaction product was also noted in the junctional folds of the motor end plate of a denervated muscle. At the longer post denervation intervals studied, deposits indicative of acid phosphatase were dispersed throughout the sarcoplasm with greater concentration in the I band and appeared more abundant in denervated than in contralateral control muscles. The enzymatic basis for the sarcoplasmic deposits and other deposits was confirmed by their absence from cytochemical controls, which included incubation in substrate-free medium, heat or NaF inactivation of enzyme, and exposure sequentially to PbNO3 and NaH2PO or PbNO3 and beta-glycerophosphate.     

Ultrastructural localization of alkaline phosphatase activity in the developing thyroid gland of the rat

The localization of alkaline phosphatase activity during morphogenesis of the thyroid gland was studied at the fine structural level in Holtzman rats from the fifteenth day of foetal life till the first day after birth. The present work deals with the formation of thyroid follicles in the median thyroid primordium only. Since the rat thyroid does not develop synchronously, the three stages described may overlap during a given day of development. In the precolloid stage that extends roughly from the fifteenth to the nineteenth day of development, alkaline phosphatase activity is localized in Golgi saccules and vesicles and also in smooth membrane tubules and vesicles found in the cytoplasm near the lateral plasma membranes. At the end of this stage the lateral plasma membranes become strongly reactive and a cluster of positive vesicles and tubules appears immediately under the junctional zone. The second phase, the early colloid stage (17–19 days), is characterized by the formation of the colloid cavity in center of the disc-like junctional zone: at this moment the newly formed apical plasma membrane bearing the microvilli shows the reaction product. During the third stage (18 days onwards), which starts with a gradual increase in the diameter of follicular lumina, a drastic fall in alkaline phosphatase activity is observed. In one day old rats, follicular cells are completely negative. These findings are briefly discussed in connection with thyroid cell differentiation.     

Ultrastructural localization of the NADPH-diaphorase activity in the Leydig cells of aging mice

Recently, it has been shown that nitric oxide may inhibit the Leydig cell steroidogenesis. The present paper describes, by means of NADPH-diaphorase histochemistry, the ultrastructural localization of the enzyme nitric oxide synthase in the Leydig cells of young adult and aging mice. In the young adult mice, the enzymatic reaction was mainly located in the mitochondria and in some clustered cistern? of the smooth endoplasmic reticulum. The nuclear envelope was faintly labeled. In the aging mice, most Leydig cells showed an enhanced enzymatic reaction. Labeled mitochondria were increased in number, and labeled areas of the smooth endoplasmic reticulum were more numerous and extended. In addition, a strong enzymatic reaction was recognized in the nuclear envelope. We conjecture that the impaired steroidogenesis observed in the testis of aging mammals might, at least in part, depend on the increased nitric oxide production in the Leydig cells. Accepted: 15 January 2001     

Origin of the apical transcytic membrane system in jejunal absorptive cells of neonates

We investigated the origin of the apical transcytic membrane system in jejunal absorptive cells of neonatal rats using light, electron, and immunofluorescence microscopy. In rats just after birth, intraluminally injected horseradish peroxidase (HRP), used as a macromolecular tracer, was observed only in the apical endocytic membrane system including the lysosomes, of jejunal absorptive cells in vivo. No tracer, however, was found in the intercellular space between the jejunal absorptive cells and the submucosa. Immunoreactive neonatal Fc receptor (FcRn) was localized in the perinuclear region of these absorptive cells whereas immunoglobulin G (IgG) was not found in these absorptive cells. In contrast, in rats 2 h after breast-feeding, intraluminally injected HRP was observed in the apical endocytic membrane system and in the apical transcytic membrane system of the absorptive cells. Moreover, HRP was found in the intercellular space between the jejunal absorptive cells and the submucosa. Furthermore, FcRn and IgG were widely distributed throughout the absorptive cells, and IgG was detected in both the intercellular space and the submucosa. These data suggest that initiation of breast-feeding induces the transportation of membrane-incorporated FcRn from its perinuclear localization to the apical plasma membrane domain. This transportation is achieved through the membrane system, which mediates apical receptor-mediated transcytosis via the trans-Golgi network. Subsequently, the apical plasma membrane containing the FcRn binds to maternal IgG, is endocytosed into the absorptive cells, and is transported to the basolateral membrane domain.     

Ultrastructural localization of xanthine oxidoreductase activity in isolated rat liver cells

Xanthine oxidoreductase (XOR) can exist in a dehydrogenase form (XD) and an oxidase form (XO). The D-form uses NAD as cofactor and the O-form uses oxygen as second substrate and produces oxygen radicals. Both enzymes have a high affinity for hypoxanthine and xanthine as substrate and produce uric acid, a potent antioxidant. In the present study, XOR activity was demonstrated with the ferricyanide method in permeabilized isolated rat liver cells at the electron microscopical level. Moreover, ultrastructural localization of XO activity in these cells was studied with the cerium salt method. Activity of both XOR and XO was found in matrix and core of peroxisomes of rat liver parenchymal cells. Only XOR activity was present as well in the cytoplasm of rat liver parenchymal cells. In Kupffer cells and sinusoidal endothelial cells, XOR activity was demonstrated in vesicles and occasionally on granular endoplasmic reticulum. XO activity was not found in Kupffer cells and sinusoidal endothelial cells. The presence of uric acid oxidase activity in matrix and core of peroxisomes as was found previously suggests further breakdown of purines to allantoin in peroxisomes. It is suggested that the major function of XOR activity in the cytoplasm of rat liver parenchymal cells and in sinusoidal cells is not the production of oxygen radicals, but rather the production of uric acid which can act as a potent antioxidant.     

Ultrastructural localization of acid phosphatase in rosetted T and B lymphocytes of normal human blood.

Using electron microscopy and cytochemical techniques, the authors determined the distribution of acid phosphatase (AcPase) within the organelles of lymphocytes from blood rosetted with either neuraminidase-treated sheep erythrocytes (En) or sheep erythrocytes coated with antibody and complement (EACs). Subsequently, the various reactive organelles of the rosetted lymphocytes were counted, affording a comparison of T and B cells. It was found that AcPase was present in approximately 80% of T cells and 45% of B cells and was most frequently observed in secondary lysosomes of varying size and content. Although more T cells than B cells were reactive for AcPase, the extent of reaction in some B cells clearly precludes the use of AcPase for differentiating the two cell lines. It should be recognized that while the En rosetting procedure detects T cells in a nonselective manner, the EAC rosette is a marker of a major subpopulation of B lymphocytes, ie, those bearing complement receptors. We believe that the distribution of lysosomal enzymes in B and T lymphocytes probably reflects the functional state of individual cells rather than being a reliable indicator of cell lineage. A surprising finding (which could be established only by a fine-structural study) was the fact that 20% of circulating "resting" T cells contained reaction product for AcPase within endoplasmic reticulum and the perinuclear cisterna indicating that these cells are actively synthesizing AcPase, probably due to a foregoing inductive event. Such stimulus could be the result of recent endocytosis of surface receptors in combination with antigen, antibody, or immune complexes and/or recent mitosis, or possibly some unrelated autophagic incident.     

Alkaline phosphatase and tartrate resistant acid phosphatase activity in cells of prolymphocytic leukemia

Summary In a typical case of prolymphocytic leukemia, blood smears and lymph node imprints have been investigated cytologically and cytochemically. It could be shown that many leukemic cells in both blood smears and lymph node imprints contained tartrate resistant acid phosphatase activity. Furthermore, the lymph node imprints disclosed many cells with a positive alkaline phosphatase reaction. Such a reaction hitherto has not been described in malignant cells of lymphoproliferative diseases. The cytochemical results underline that prolymphocytic leukemia indeed is a separate entity which can be differentiated from hairy cell leukemia and chronic lymphatic leukemia not only morphologically but also cytochemically. In addition, the case shows that leukemic blood cells are not inevitably identical with those occurring in organ infiltrates.     

Ultrastructural localization of acetylcholinesterase activity in Hirschsprung's disease

Localization of acetylcholinesterase activity in congenital megacolon was studied by light and electron microscopy. Acetylcholinesterase activity was strongly positive at the light microscopic level in the Auerbach's plexus of the normal segment and in proliferated nerve fibers of the aganglionic segment. The reaction product observed by electron microscopy was deposited in and between the plasma membranes of the ganglion cells, nerve fibers, and their terminals. The product was also observed in the rough endoplasmic reticulum, nuclear envelope, and Golgi apparatus of the ganglion cells. Acetylcholinesterase activity in the aganglionic segment was observed in and between the plasma membranes of nerve fibers and nerve terminals, which terminated in proximity to smooth muscle cells. Reaction deposits were also observed in the interspace between nerve terminals and smooth muscle cells, suggesting direct innervation of smooth muscles by extrinsic nerve fibers.     

Nuclear localization of non-specific acid phosphatase(s) activity in rat vaginal epithelium

The nuclear localization of non-specific acid phosphatase(s) in rat vaginal epithelial cells (VEC) by histochemical and biochemical techniques has been reported in the present work. By different histochemical methods, the enzyme activity was predominantly localized in certain nuclei but not in all of them, however, within the nucleus nucleoli are free from the enzyme activity. This enzyme is diffused in the nucleoplasm and perhaps tightly associated with the nuclear proteins. Very little activity was seen in the cytoplasm in the selective population of VEC. After 12 h treatment of estradiol 17beta in vivo the VEC lose the enzyme activity in the basal and intermediate layers, and the activity remains within the nuclei of luminal cells. Enzyme assay in low and high salt extracts of the isolated nuclei of the VEC with or without the enzyme inhibitors indicates that this enzyme may be present in isoforms in the VEC. No activity was observed in these cells at alkaline pH.