Cytochemical localisation of acid phosphatase in the phagocytising conjunctival epithelium of the guinea pig

The ultrastructural localisation of acid phosphatase activity was investigated on the guinea pig conjunctival epithelium incubated in vivo with a suspension of latex spheres. Deposits of acid phosphatase reaction product were concentrated on the elements of GERL, the phagocytic vacuoles, and the cell membrane. Acid phosphatase activity in GERL was intense in basal and suprabasal cells and decreased towards the superficial cells. Phagosomes containing latex spheres and reaction product of acid phosphatase were observed mainly in the centrospheral region of the superficial and intermediate epithelial cells. Acid phosphatase activity in phagocytising cells was not increased as compared to that in non-phagocytising cells. The observations indicate that existing acid phosphatase in unstimulated conjunctival epithelial cells is released into heterophagosomes brought within the lysosomal compartment. The number of secondary phagosomes seems to be increased by intercellular transport of latex spheres to the acid phosphatase rich cells in the deep layers of the epithelium.     

The relationships between the Golgi apparatus, GERL, and lysosomes of fetal rat liver Kupffer cells examined by ultrastructural phosphatase cytochemistry

Kupffer cells are the sinusoidal macrophages of the liver. Using ultrastructural phosphatase cytochemical methods, we examined the relationship between the Golgi apparatus, GERL, and lysosomes of Kupffer cells in fetal rat livers identified, in part, by their ability to phagocytize intravenously injected latex spheres. Thiamine pyrophosphatase (TPPase) activity was localized to the inner Golgi saccules and some vesicles in the Golgi region but not to GERL. A TPPase-like activity, demonstrable in lysosomes, was abolished by sodium fluoride but not suppressed by the alkaline phosphatase inhibitors L-cysteine and L-p-bromotetramisole. Acid phosphatase (AcPase) was localized by GERL, some coated vesicles, and in lysosomes, but not to the Golgi stacks. Continuities between GERL and lysosomes were observed. Phagosomes containing internalized latex spheres received TPPase and AcPase sequentially. TPPase was localized in phagosomes immediately after latex administration. AcPase activity was not found here until at least 10 minutes following the injection of the particulates. Our findings indicate that Kupffer cell lysosomes are derived from GERL, but also suggest that phagosomes may receive material packaged by the Golgi apparatus as well as GERL.     

Ultrastructural localization of acid phosphatase activity in the small intestinal absorptive cells of postnatal rats

Summary The ultrastructural localization of acid phosphatase activity was investigated in ultrathin (0.05 m) and semithin (0.5 m) sections of the small intestinal epithelial cells of postnatal rats. Until around the 15th day of neonatal life acid phosphatase activity in the duodenal and jejunal epithelial cells was observed on the microvillous membrane, the membrane of the tubulo-vacuolar system, the lateral cell membrane, the lysosomes, the Golgi apparatus and the GERL of Novikoff (1963). After about the 15th neonatal day, the tubulo-vacuolar system enzyme disappeared from both cells. Acid phosphatase activity then became localized on the microvillous membrane, the lateral cell membrane, the lysosomes, the Golgi apparatus, and the GERL, as in adult rats. During the suckling period, acid phosphatase in the ileal cells could be seen on the microvillous membrane, the lateral cell membrane, the Golgi apparatus, the GERL, the membrane of tubulo-vacuolar system and the supranuclear vacuole. At weaning, however, the tubulovacuolar system and the supranuclear vacuole enzyme disappeared, and only the lysosomes and the GERL of these cells showed acid phosphatase activity, as in the adult rat. It was concluded that the acid-phosphatase-containing tubulo-vacuolar system and the supranuclear vacuole in the epithelial cells of the distal intestine of suckling rats may possess a strong phagolysosomal function as well as having an absorptive capacity.     

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.     

Cytochemical localization of acid phosphatase and trimetaphosphatase activities in exocrine acinar cells

Acid phosphatase activity, a lysosomal marker, is commonly demonstrated using the Gomori technique with cytidine 5'-monophosphate or beta-glycerophosphate as substrate. Using this lead capture method on mouse and rat exorbital lacrimal, parotid, and pancreatic acinar cells, reaction product was localized in GERL, forming secretory granules, and secondary lysosomes. However, a different cytochemical localization was observed for inorganic trimetaphosphatase, another lysosomal enzyme. When the technique for trimetaphosphatase activity, a metal chelation method, was applied to exocrine acinar cells, reaction produce was conspicuously absent from GERL and forming secretory granules, but was present in secondary lysosomes, occasionally in Golgi saccules, and in previously unreported basal elongated lysosomes. The differences in the localization of the two enzymatic activities emphasizes the importance of employing more than one substrate where possible, and raises questions concerning the mechanism of delivery of acid hydrolases to secondary lysosomes.     

Submicroscopic localization of acid phosphatase in the dental cyst epithelium

Summary Distribution of acid phosphatase was studied in the dental cyst epithelium. Enzyme activity was found to be localized in primary and secondary lysosomes and vacuoles of the Golgi complex. Longer incubation revealed positive reaction in the Golgi complex vesicles and cisternae, in the endoplasmic reticulum, and perinuclear' space. Acid phosphatase reaction was bound also to membranous structures occurring in the intercellular spaces. These structures originated probably from disintegrated cells. Localization of acid phosphatase reaction in the dental cyst epithelium was found to be essentially the same as in the oral cavity epithelium. Smaller differences between both types of epithelium may be conditioned by different type and arrangement of the organelles in the corresponding epithelial layers.     

Studies on the nature and localization of acid phosphatase in normale and lens-regenerating urodele eyes. I. Histochemical localization

Histochemical procedures for acid phosphatase in normal and lens-regenerating eyes of the urodele Diemictylus viridescens demonstrate activity in a variety of structures. In the normal urodele eye, acid phosphatase is present in conjunctival and corneal epithelial cells and associated glands, in blood vessel endothelium and posterior epithelial cells of the iris, in the anterior lens epithelium, and in the cytoplasm of the optic nerve. Acid phosphatase in the lens-regenerating eye is localized in the same structures as in the normal eye as well as in increased amounts in the corneal epithelial cells and stromal macrophages at the lentectomy wound site and in the posterior portion of the developing lens during completion of differentiation of primary into mature lens fibers characterized by loss of many intracellular organelles. On the basis of these histochemical findings, it is proposed that hydrolytic lysosomal enzymes play an important role in the processes of cellular and intracellular destruction and synthesis which occur during Wolffian lens regeneration in the urodele.     

LYSOSOMAL PACKAGING IN DIFFERENTIATING AND DEGENERATING ANURAN LATERAL MOTOR COLUMN NEURONS

The role of the Golgi apparatus and the Golgi-endoplasmic reticulum-lysosome complex (GERL) in the genesis of lysosomes was examined in differentiating and degenerating motor neurons of anuran larvae. Acid phosphatase, aryl sulfatase, and thiolacetic acid esterase were utilized as marker enzymes for the lysosomal system, while nucleoside diphosphatase and thiamine pyrophosphatase labeled the inner saccule(s) of the Golgi apparatus. Reduced osmium tetroxide was routinely deposited in the outer Golgi saccule regardless of the state of neuronal maturation. In all young neurons, the disposition of acid hydrolase reaction product paralleled the formation of GERL, with no lytic activity in the Golgi apparatus per se. Hypertrophy of the Golgi apparatus and GERL was observed in the early phases of degeneration, and both organelles apparently exhibit extensive hydrolytic activity. Dense bodies, autophagic vacuoles, and primary lysosomes were found arising from GERL, while the Golgi apparatus may produce primary lysosomal granules during regression. On the other hand, in differentiating neurons, hydrolytic activity was restricted to GERL and an occasional dense body and autophagic vacuole. These studies illustrate a parallelism between the development of GERL and genesis of primary and secondary lysosomes during neuronal cytodifferentiation, and implicate GERL and possibly the Golgi apparatus in lysosomal packaging in degenerating neurons.     

Relationship between the golgi apparatus, gerl, and secretory granules in acinar cells of the rat exorbital lacrimal gland

The method of secretory granuleformation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemical techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A. B. Novikoff, N. Quintana, and J.-J. Hauw. 1971. J. Cell Bio. 50:859-886). In the lacrimal gland. GERL was located adjacent to the inner Golgi saccule, or separated from it by a variable distance. Portions of GERL were often closely paralleled by modified cisternae of rough endoplasmic reticulum (RER), which lacked ribosomes on the surface adjacent to GERL. Diaminobenzidine reaction product of the secretory enzyme peroxidase was localized in the cisternae of the nuclear envelope, RER, peripheral Golgi vesicles, Golgi saccules, and immature and mature secretory granules. GERL was usually free of peroxidase reaction product or contained only a small amount. Thiamine pyrophosphatase reaction product was present in two to four inner Golgi saccules; occasionally, the innermost saccule was dilated and fenestrated, and contained less reaction product than the next adjacent saccule. Acid phosphatase (AcPase) reaction product was present in GERL, immature granules, and, rarely, in the innermost saccule, but not in the rest of the Golgi saccules. Thick sections of AcPase preparations viewed at 100 kV revealed that GERL consisted of cisternal, and fenestrated or tublular portions. The immature granules were attached to GERL by multiple connections to the tublular portions. These results suggest that, in the rat exorbital lacrimal gland, the Golgi saccules participate in the transport of secretory proteins, and that GERL is involved in the formation of secretory granules.     

PROGRAMMED CELL DEATH IN THE HONEY-BEE (APIS MELLIFERA L.) LARVAE MIDGUT

The histochemical and cytochemical localization of acid phosphatase has been used in an attempt to map the sites of cellular lysis and death. Reaction product was found both in the brush border of the midgut epithelium and in the basal membrane. Vacuolar acid phosphatase activity was found in the regenerative epithelial cells. Extra-cisternal reaction product was associated with the endoplasmic reticulum which was dilated in lysed areas of the cytoplasm. Free acid and alkaline phosphatase activity was found in the basal area of the midgut epithelial cells and the former also occurred in the haemocoel. In the tracheoblastic cells only vacuolar acid phosphatase activity was seen. Chromatin aggregates were distributed throughout the nucleus and the nuclear envelope showed some infolding. Certain mature epithelial cells proved positive for anti-histone associated DNA fragmentation indicative of programmed cell death.     

Effects of secretory stimulation on the Golgi apparatus and GERL of rat parotid acinar cells

The structure and cytochemistry of the Golgi apparatus and GERL of rat parotid acinar cells was studied after in vivo secretory stimulation with isoproterenol. Discharge of mature secretory granules was complete within 1 hr after isoproterenol injection, but immature granules in the Golgi region or near the lumen were not released. At early times (1-5 hr) after isoproterenol, acid phosphatase (AcPase) activity was markedly increased in GERL and immature secretory granules compared to uninjected controls. GERL appeared increased in extent and numerous continuities with immature granules were observed. Reaccumulation of mature secretory granules was first evident at 5 hr, and was almost complete by 16 hr after isoproterenol. Thiamine pyrophosphatase (TPPase) activity, normally restricted to the trans Golgi saccules, was frequently present in immature granules during this time. Narrow cisternae resembling GERL, occasionally in continuity with immature granules, also contained TPPase reaction product. By 16-24 hr after stimulation, the activity and distribution of AcPase and TPPase were similar to control cells. These results demonstrate the dynamic nature of the Golgi apparatus and GERL in parotid acinar cells, and emphasize the close structural and functional relationship between these two structures.     

The light and electron microscopic distribution of acid phosphatase activity in human normal oesophageal epithelium

Acid phosphatase activity in human normal oesophageal epithelium was studied with light and electron microscopic techniques. The maximum activity was found to be in the prickle and lower functional layers. Electron microscopic examination revealed activity to be localized in GERL, lysosomes and membrane coating granules. These last structures probably secreted their content into the intercellular space in the central part of the functional layer. Thick sections (0.5 micron) with tilting showed GERL to consist of anastomosing tubules.     

Studies on the secretory process in exocrine pancreas cells. II. C57 black and beige mice

Published electron microscopic and cytochemical studies (thiamine pyrophosphatase and acid phosphatase) on exocrine pancreas cells of guinea pig, hamster, rat and rabbit have demonstrated that the nascent secretory granules, or condensing vacuoles, are part of GERL. The studies reported here show this to be true of the mouse pancreatic exocrine cells as well, thus permitting comparison of this cell type in the C57 black mouse and its "beige" mutant. This is of considerable interest because GERL is very much enlarged in these cells of the beige mouse. Most of GERL consists of wide dilated portions filled with electron-opaque materials that appear to be packaged into huge residual body-type lysosomes ("anomalous granules"). Acid phosphatase activity is demonstrable not only in these portions of GERL, but also in the condensing vacuoles as in pancreatic acinar cells in the black mouse where these dilated lysosome-producing regions are not present.     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum an nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Summary Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum and nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.     

Cytochemical studies of GERL and its role in secretory granule formation in exocrine cells

Synopsis the structure and cytochemistry of GERL was studied in several different exocrine secretory cells, including the exorbital lacrimal gland, parotid, lingual serous (von Ebner's), submandibular, and sublingual salivary glands, and exocrine pancreas of the rat; the lacrimal, parotid and pancreas of the guinea-pig; and the lacrimal gland of the monkey. GERL was morphologically and cytochemically similar in all cell types studied. It was located in the inner Golgi region and consisted of cisternal and tubular portions. Immature secretory granules were in continuity with GERL through multiple tubular connections. Modified cisternae of endoplasmic reticulum, with ribosomes only on one surface, closely paralleled parts of GERL. GERL and immature granules were intensely reactive for acid phosphatase activity, while the inner Golgi saccules were reactive for thiamine pyrophosphatase and nucleoside diphosphatase activities. In the rat exorbital lacrimal and parotid glands, reaction product for endogenous peroxidase, a secretory enzyme, was present in the endoplasmic reticulum, Golgi saccules, immature and mature secretory granules. GERL was usually free of reaction product or contained only a small amount. The widespread occurrence of GERL in secretory cells, and its intimate involvement with the formation of granules, suggest that it is an integral component of the secretory process.     

Fine structural and histochemical studies on salivary glands of Peripatoides novae-zealandiae (Onychophora) with special reference to acid phosphatase distribution

The Onychophora feed on small arthropods and produce saliva when ingesting prey. Although saliva undoubtedly helps to liquefy the food its constituents have not yet been fully described. The salivary glands, two long tubes of glandular epithelium, are known to secrete a powerful protease, however, besides other enzymes and mucus. In Peripatoides novae-zealandiae there are protein-secreting cells of three types, referred to here as columnar, cuboidal and modified cells, and mucus cells. The anterior two-thirds of the gland show most cell diversity, while the posterior region consists mainly of columnar cells. These are the most numerous elements overall and they probably secrete salivary protease. In thick resin sections the granules of all protein-secreting cells stain strongly with methylene blue. Those of columnar cells are markedly uneven in size and accumulate distally, eventually filling the cytoplasm. More proximal Golgi regions may be discernible. Mucus cells are all of one type and their secretion droplets are stained lightly by methylene blue. The electron microscope shows that distal microvilli, desmosomes and septate junctions are common to all gland cells. In columnar cells, secretory material is contributed by Golgi complexes and by rough endoplasmic reticulum. Early secretory vacuoles containing dense material are seen in the concavity of Golgi regions. They are precursors to larger condensing vacuoles whose contents have a more flocculent appearance, and which may attain 3–4 μm in diameter. These evolve into secretory granules, usually of uneven texture, which are up to 2–5 μm in diameter. Histochemical tests for acid phosphatase show moderate amounts of enzyme throughout the gland. In whole mounts and sections the strongest reaction is in a band of cuboidal cells along the anterior median border. Columnar cells show a diffuse cytoplasmic reaction towards the base and sometimes distal to the nucleus, and mucus cells may also react strongly round the nucleus. Cytoplasm near the lumen shows little reaction. The secretory granules do not appear to contain active enzyme. Under the electron microscope a positive reaction for acid phosphatase is seen in lysosomal derivatives near the base and lateral periphery of gland cells. These bodies are probably autophagic vacuoles and they may contain membranous whorls and possibly old secretion granules. Acid phosphatase is involved also in the elaboration of new secretory granules in both columnar and mucus cells. Dense reaction product is found in a system of interconnected tubules and cisternae near the innermost face of the Golgi complex, which is interpreted as GERL. Acid phosphatase is present in the peripheral zone of adjacent early secretory vacuoles, and interconnections occur between GERL and secretory vacuoles. It is suggested that GERL tubules containing the enzyme may fuse with early secretory vacuoles and release acid phosphatase at their periphery. The acid phosphatase reaction is negative in large condensing vacuoles and most secretory granules. These findings are consistent with what is known from mammalian cells, including those of salivary glands.     

Demonstration of acid phosphatase-containing vacuoles in hyphal tip cells of Sclerotium rolfsii.

A lysosomal system was demonstrated in hyphal tip cells of Sclerotium rolfsii by light and electron microscopy observations of the sites of acid phosphatase activity visualized by a modified Gomori lead nitrate method. The cytochemical reaction product was found to be present in numerous vacuoles, each aout 0.5 mum in diameter, which were seen as chains of spheres when viewed with the light microscope. They usually did not occur in the first 30 to 40 mum of the hyphal tip cell, but were concentrated in a zone extending from 30 to 200 mum from the hyphal apex. As shown by the electron microscope, the vacuoles were sometimes interconnected by narrow channels. Acid phosphatase reaction product was also occasionally localized in vacuoles of the older hyphal cells, but never in apical vesicles, lipid bodies, or microbodies. It is proposed that this vacuolar system may orginate from the endoplasmic reticulum.     

Microanalysis of epithelial and mesenchymal acid hydrolase activities in the developing palate

Microdissection of lyophilized sections of fetal heads permits the analysis of relatively pure samples of epithelium and mesenchyme. These techniques were applied to a study of acid phosphatase and beta-glucuronidase activities in the developing palate in A/Jax mice. Acid phosphatase was found to be more concentrated in the palatal epithelium than the underlying mesenchyme. Conversely, beta-glucuronidase was more concentrated in the mesenchyme than the epithelium. A disparate developmental pattern of acid phosphatase and beta-glucuronidase activity was observed in the oral epithelium: acid phosphatase activity increased from intra-uterine Day 17 into neonatehood and beta-glucuronidase activity decreased towards term. Analysis of cortisone-induced palatal shelves showed increased activity of both acid phosphatase and beta-glucuronidase in the presumptive fusing epithelium.     

Ultrastructural localization of acid phosphatase activity in the small intestinal absorptive cells of adult rats.

Ultrastructural localization of acid phosphatase activity was investigated in ultrathin (0.05 micron) and semithin (0.5 and 0.75 micron) sections of the small intestinal epithelial cells of adult rats. The results showed that the enzyme activity was localized on the membrane of microvilli, lateral cell membranes, lysosomes, the Golgi complex, and the GERL of Novikoff (a part of the smooth-surfaced endoplasmic reticulum located in close proximity to the inner Golgi saccules) of duodenal absorptive cells. The lysosomes contained within the duodenal and jejunal absorptive cells appeared to be mainly heterolysosomes rather than autolysosomes. The enzyme activity of absorptive cells was lower in the jejunum than in the duodenum, and was barely detectable except in the GERL and lysosomes of the ileum. The average numbers of lysosomes having a diameter of 0.2 approximately 1.0 microns, per cell profile in sections of 214 duodenal, 226 jejunal and 318 ileal epithelial cells were 8.9 +/- 0.189, 6.4 +/- 0.155 and 3.5 +/- 0.027 (mean +/- SE), respectively. From these results, it was assumed that both the Golgi apparatus and GERL produce some lysosomes in the duodenal and jejunal absorptive cells, but only GERL does so in the ileum. It was considered also that because of an unexpectedly high number of lysosomes containes within the epithelial absorptive cells of the proximal intestine of adult rats, these cells may possess the strong heterophagic, as well as absorptive capacity.