The presence of carbonic anhydrase in orbital glands. An enzyme-histochemical study in cynomolgus monkeys and rabbits

The distribution of carbonic anhydrase (CA) was studied in the lacrimal gland of the cynomolgus monkey as well as in the lacrimal, infra-orbital and harderian glands of the rabbit. In the lacrimal gland of the cynomolgus monkey, a number of acini with positive staining were found; however, another group of acini did not stain. In the positively stained acinar cells, large amounts of reaction product were located in the cytoplasm, but only weak staining was observed in the membranes. In the endothelial cells of capillaries a strong staining reaction was only seen in those vessels which were adjacent to the acinar cells containing CA. In the lacrimal and infra-orbital glands of the rabbit, there was intense staining of the cell membranes in all acinar cells and weak staining of the cytoplasm in a few acinar cells. Stained capillaries were also found here, but these were not as numerous as in the lacrimal gland of the cynomolgus monkey. In the harderian gland of the rabbit, there was no staining in the white lobe. In the red lobe the acinar cells displayed distinct staining exclusively in the basolateral membranes. There was no staining of capillaries in the harderian gland. In none of the glands studied was there staining of the epithelial cells of the excretory ducts. The functional significance of these findings is discussed.     

The presence of carbonic anhydrase in orbital glands

Summary The distribution of carbone anhydrase (CA) was studied in the lacrimal gland of the cynomolgus monkey as well as in the lacrimal, infra-orbital and harderian glands of the rabbil. In the lacrimal gland of the cynomolgus monkey a number of acini with positive staining were found; however, another group of acini did not stain. In the positively stained acinar cells, large amounts of reaction product were located in the cytoplasm, but only weak staining was observed in the membranes. In the endothelial cells of capillaries a strong staining reaction was only seen in those vessels which were adjacent to the acinar cells containing CA. In the lacrimal and infra-orbital glands of the rabbit, there was intense staining of the cell membranes in all acinar cells and weak staining of the cytoplasm in a few acinar cells. Stained capillaries were also found here, but these were not as numerous as in the lacrimal gland of the cynomolgus monkey. In the harderian gland of the rabbit, there was no staining in the white lobe. In the red lobe the acinar cells displayed distinct staining exclusively in the basolateral membranes. There was no staining of capillaries in the harderian gland. In none of the glands studied was there staining of the epithelial cells of the excretory ducts. The functional significance of these findings is discussed.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Ultrastructural investigation of ACTH immunoreactivity in arcuate and supraoptic nuclei of the rat

Summary Fine structural localization of an ACTH-like substance was obtained in neurons of the rat arcuate nucleus using immuno-electron microscopy, whereas it could not be confirmed that ACTH-containing cell bodies are present in the supraoptic nucleus. The immunoreactive cells of the arcuate nucleus appeared to be more numerous than the unreactive neurons. Immunostaining was carried out before embedding in resin. Empty vesicles of irregular shape were found in dendrites of immunoreactive arcuate neurons, but their significance and nature remain enigmatic. The reaction product was distributed uniformly throughout the cytoplasm of the ACTH-positive cells, except that the mitochondria, rough endoplasmic reticulum and Golgi vesicles and cisternae were devoid of PAP molecules. This distribution differed from the localization reported in ACTH-secreting cells of the rat anterior pituitary, where the reaction product was found in the rough endoplasmic reticulum and Golgi complex as well as in secretory granules.     

Diffusion artefacts and tissue fixation in thyroperoxidase cytochemistry

The distribution of endogenous peroxidase activity in rat, mouse and human thyroid follicle cells was studied with electron microscopic cytochemistry after incubation in 3-3'-diaminobenzidine (DAB). In all three species enzyme activity was found at the apical plasma membrane (facing the follicle lumen) as well as in intracellular compartments. The enzyme activity in the apical plasma membrane was more sensitive to changes in fixation conditions than the activity in intracellular compartments. Under optimal conditions more than 90% of the follicle cells in normal rat thyroids displayed a cytochemical reaction at the apical plasma membrane. In all three species the reaction product at the apical plasma membrane formed a gradient which extended into the colloid which otherwise was unreactive. Evidence obtained indicated that this gradient was not due to the presence of soluble peroxidase in the lumen but most likely signified the diffusion of the reaction product from the membrane-bound enzyme.     

Histochemical, ultrastructural and X-ray microprobe analytical studies of localization of calcium in the mucous lining of the rat duodenum

Localization of calcium in a rapid frozen and freeze substituted duodenum of normal, starved or calcium-repleted rat was examined using either of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, a sensitive histochemical calcium stain or electron microscopy. In normally-fed rats, a majority of absorptive cells of the duodenum showed numerous discrete red granular GBHA reactions, approximately 1 micron or less in diameter, located primarily along their lateral plasma membranes and within intercellular spaces. Electron microscopy also revealed electron-dense granules, 30-100 nm in diameter, showing a similar distribution as the GBHA granules in the respective absorptive cells, and confirmed their absence in mitochondria and other intracellular compartments. Some of the absorptive cells located exclusively at the tip of each villus contained highly GBHA-reactive tubulo-vesicular structures extending throughout the cytoplasm. However, they displayed virtually no granular GBHA reaction. In these cells, electron microscopy revealed numerous electron-dense granules in the nucleus, mitochondria and in other unidentified organelles. X-ray microprobe analyses of ultrathin sections confirmed the presence of calcium within electron-dense granules associated with both types of absorptive cells. The number and intensity of all GBHA reactions fluctuated according to luminal calcium concentration. In calcium-repleted rats, strong GBHA reactions appeared in a narrow zone of lamina propria at the tip of the villus, overlaid, predominantly, with absorptive cells showing tubulo-vesicular GBHA reactions. These results suggest the existence of distinct types of absorptive epithelial cells in the rat duodenum, with respect to patterns of calcium localization which they display.     

Transneuronal uptake of horseradish peroxidase in the central nervous system of dipterous insects

Summary Horseradish peroxidase (HRP) applied to lesioned neurons in the retina and thoracic ganglia of the flies Musca, Calliphora and Drosophila labeled axon terminals, dendrites and perikarya of the severed neurons after anterograde or retrograde passage. In addition, HRP reaction product secondarily labeled intact neurons that are contiguous with injured nerve cells. In many cases labeling of optic lobe neurons remote from primarily filled ones was also seen (here called tertiary labeling). HRP labeling was extensive and both primarily and transneuronally filled neurons could be resolved in almost as much detail as Golgi-impregnated or cobalt-silver-labeled cells. Electron microscopy showed that in both primarily and secondarily filled neurons, reaction product was distributed diffusely in the cytoplasm.Transneuronal uptake of HRP was specific to certain types of neurons in the brain and thus displayed certain pathways. The pathways resolved by transneuronal labeling with HRP extend from the optic lobes to the thoracic ganglia and include visual neurons previously identified electrophysiologically and anatomically.Transneuronal HRP uptake, although believed to occur in vivo, could not be shown to be dependent on synaptic activity. Three other heme peptides tested were taken up by injured neurons, but showed no transneuronal labeling: lactoperoxidase, cytochrome c, and microperoxidase.     

Electron microscopic immunocytochemical investigation on the postnatal development of the vasopressin system in the rat

Summary The present ultrastructural results indicate that, in the rat, the vasopressin-synthesizing perikarya of the supraoptic nucleus (NSO) attain a certain degree of maturity earlier than those of the paraventricular nucleus (NPV). In the neonate rat, the stainability of the nuclear areas is very weak; in the perikarya of the NSO a few labeled granules can be found, whereas the perikarya of the NPV often display only a labeled Golgi area, the cytoplasm being devoid of granules. At the end of the first (NSO) and the second (NPV) postnatal weeks, the filling of the neurosecretory granules with vasopressin is inhomogeneous with irregular spots of reaction product distributed on the granules. This feature is less obvious during the following week and has nearly disappeared after the third and fourth postnatal weeks. Already in the neonate two types of vasopressin-positive fibers are observed in the median eminence, characterized by the different diameters of their granules and by their typical location in the internal and the external pericapillary contact zone. Especially in one and two week-old animals, in the internal zone of the median eminence and, to a lesser degree in the neural lobe, the immunocytochemical reaction product is deposited on an axonal tubular network. Judging from the presence of very few vasopressin-negative fibers in the neural lobe of the neonate, the development of the oxytocin system appears to be delayed. A characteristic relationship between pituicytes and the neurosecretory fibers can be observed during the first two postnatal weeks. After the third postnatal week the immunocytochemical features of the vasopressin system correspond approximately to that in adult rats.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and Stiftung Volkswagenwerk     

Electron-cytochemical study of ATP-ase activity in the brain after death]

The lead method was applied to determine the localization of the ATP-asic activity in the rat and human brains at different periods after death. This activity was revealed in the cytoplasm of the cells, chromatin and the nucleolus, and also in the synaptic terminals. In the vascular capillaries the product of reaction was localized in the basal layer and on the cytomembrane of the endothelial cells. The results obtained pointed to a good preservation of the ATP-asic activity in the postmortem brain.     

Ultrastructural localization of neuropeptide FF, a new neuropeptide in the brain and pituitary of rats

The octapeptide FLFQPQRF-NH2 or neuropeptide FF ('F8Famide'; FMRFamide-like peptide'; 'morphine-modulating peptide') has been isolated from the bovine brain. In this study, the ultrastructural localization of neuropeptide FF-like immunoreactivity was examined with pre-embedding immuno-electron microscopy in the nucleus of the solitary tract and in the posterior lobe of the pituitary gland of an adult rat. Neuropeptide FF-like immunoreactivity was detected only in neuronal structures of the medial and commissural nuclei of the solitary tract and in the neurohypophysis. In the medulla, the peroxidase-antiperoxidase reaction product was localized in large (100 nm) dense-cored vesicles and in the cytoplasm of the neuronal perikarya, dendrites and axon terminals. In the labeled terminals, small (50 nm) clear vesicles rimmed with the peroxidase-antiperoxidase reaction product were seen. Synaptic contacts of labeled perikarya and dendrites with unlabeled axon terminals were observed. Labeled axon terminals formed contacts with unlabeled dendrites and perikarya. In the posterior lobe of the pituitary gland, neuropeptide FF-like immunoreactivity was localized in nerve terminals frequently associated with blood vessels. The results suggest that neuropeptide FF-like peptides are localized exclusively in neuronal structures and that they are synthesized in cell somata and released from axon terminals. In the brain, neuropeptide FF-like peptides may act as neuromodulators involved in the regulation of autonomic functions. The localization of neuropeptide FF-like immunoreactivity in the neurohypophysis suggests endocrine regulatory functions of these peptides.     

Quantitative histochemical study of acid phosphatase activity in rat liver using a semipermeable membrane technique

Acid phosphatase activity has been demonstrated in rat liver with the semipermeable membrane technique using naphthol AS-BI phosphate as substrate and hexazotized pararosaniline (HPRA) as simultaneous coupling agent. With this method the final reaction product (FRP) appeared in rat liver as intensely colored red granules in liver parenchymal cells and in Küpffer cells. The absorbance spectrum of the FRP peaks between 510 and 550 nm. A nonspecific reaction product, as has been found in skeletal muscle, did not occur in rat liver. A substrate concentration of 5 mM and a HPRA concentration of 10 mM result in optimum localization and activity. We concluded from the results with different enzyme inhibitors that lysosomal acid phosphatase was demonstrated. The mean absorbance of the FRP increased linearly with incubation time (15-60 min). Furthermore, we found a linear increase of the FRP with increasing section thickness (4-10 micron). When the simultaneous coupling method was replaced by a post-coupling technique, the colored reaction product was diffusely located throughout the cytoplasm. In conclusion, the simultaneous coupling technique in combination with the semipermeable membrane method is a valuable tool for detecting and quantifying lysosomal acid phosphatase activity in rat liver. We demonstrated that acid phosphatase activity is 1.2 times higher periportally than pericentrally in rat liver, and that 24 hr fasting before the experiments did not change the acid phosphatase activity.     

Localization of acetylcholinesterase in dissociated cell cultures of the carotid body of the rat

Summary The localization of acetylcholinesterase (AChE) was investigated at the cellular and subcellular levels in dissociated cell cultures of the carotid body of the neonatal rat, prepared by the methods of Fishman and Schaffner (1984). In the presence of iso-OMPA, which blocks non-specific cholinesterase, staining was confined almost exclusively to glomus-cell clusters and occasional isolated cells. These clusters grow as discrete islands scattered throughout the culture and display typical catecholamine (CA) fluorescence as in vivo. AChE staining was abolished or reduced by the cholinesterase inhibitors eserine (30–100 M), or (the poorly lipid soluble) echothiophate (8 (M). Processing of the same culture sequentially for the demonstration of both AChE and CA revealed that glomus-cell clusters and individual glomus cells were consistently positive for both. In electron micrographs AChE reaction product was associated intracellularly with the nuclear envelope and cytoplasm of glomus cells (identified by their characteristic dense cored granules), as well as extracellularly with the boundaries of contiguous glomus cells. Significantly, reaction product occurred in some glomus cell profiles that had both dense-cored and clear (cholinergic-like) vesicles. These findings are discussed in the context of a possible dual (adrenergic/cholinergic) function status of glomus cells in the rat's carotid body.     

Morphological aspects of Culex quinquefasciatus salivary glands

The salivary glands of Culex quinquefasciatus female mosquitoes are paired organs composed of two lateral lobes with proximal and distal secretory portions, and a medial lobe. All portions comprise a simple epithelium that surrounds a salivary duct. In the apical portion of the medial lobe, non-secretory cells strongly resemble cells involved in ion and water transport. The general architecture of the secretory portions is similar between lobes. The appearance of the secretory material and the morphological aspect of the apical cell membrane are the most distinctive features among the three secretory portions. Cells in the lateral proximal lobe display thin membrane projections extending into a translucent and finely filamentous secretory product. At the lateral distal portion, the apical cell membrane forms an intricate meshwork that encloses a dark secretory product. Medial lobe secretory cells also contain secretory cavities surrounded by intracytoplasmic vesicles, all containing a very dark and uniform product. Scattered cells holding numerous vacuoles, some of them containing a small and electron-dense granule eccentrically located and resembling those of the diffuse endocrine system, are frequently observed in the periphery of all secretory portions. Immunofluorescence assays revealed that the distal portion of the lateral lobes contains apyrase, an enzyme putatively responsible for platelet aggregation inhibition, diffusely distributed in the cell cytoplasm.     

A comparison between osmiophilic reagents and the Gomori lead method for the electron cytochemical demonstration of two lysosomal enzymes in oral epithelium

Synopsis Osmiophilic reagents were used to study the histochemical localization of acid phosphatase and non-specific esterase in the keratinized oral mucosa of rat. The reaction product from both enzymes was found in the epithelium and in cells of the corium as discrete granules, suggestive of a lysosomal localization. Treatment with E-600 before incubation for non-specific esterase did not change this localization. The osmium black end-product, due to acid phosphatase activity, was examined with the electron microscope and compared with the localization obtained by the Gomori lead phosphate technique. Both methods produced a reaction product in membrane-bounded bodies resembling lysosomes, as described in other tissues. These organelles were present in the basal prickle and granular cell layers of the epithelium. In the keratinized layer the reaction product was localized between the cell membranes of the deeper cells and no deposits were present in the cells. It is suggested that the osmiophilic reagents provide a good alternative to the Gomori method for the localization of lysosomal acid phosphatase at both the light and electron microscope levels.     

COMBINED CYTOCHEMICAL AND ELECTRON MICROSCOPIC DEMONSTRATION OF β-GLUCURONIDASE ACTIVITY IN RAT LIVER WITH THE USE OF A SIMULTANEOUS COUPLING AZO DYE TECHNIQUE

Rat liver was fixed in formal-cacodylate-sucrose and frozen sections were incubated in a simultaneous-coupling medium containing naphthol AS-BI glucuronide as substrate and hexazonium pararosanilin as the diazo reagent. By light microscopy, the sections demonstrated β-glucuronidase activity as red discrete granules in the pericanalicular cytoplasm and as a generalized cytoplasmic stain in the parenchymal cells. Brief treatment of sections in cold ethanol prior to incubation markedly enhanced the staining for the enzyme and made it possible to demonstrate sufficient amounts of the reaction product in sections embedded in epoxy resin following dehydration and propylene oxide treatment. Electron microscopy revealed that the reaction product was moderately electron opaque and deposited in greater amounts in the vacuolated dense bodies and occasionally in the dense bodies which did not show obvious vacuoles. In each dense body, the deposits occurred preferentially at the edge as well as in the area surrounding the vacuoles in the matrix. Control sections incubated in the presence of glucosaccharo-1:4-lactone were devoid of the reaction product. No deposits of the reaction product were found in the nucleus, mitochondria, or microbodies. The limitations of the present cytochemical technique for use in electron microscopy are briefly discussed.     

Ultrastructural patterns of the alpha-naphthyl butyrate esterase activity in normal and leukaemic peripheral blood leucocytes

Summary The activity of -naphthyl butyrate esterase was investigated at the ultrastructural level in normal human peripheral blood and in a few cases of hairy cell leukaemia, B-chronic lymphocytic leukaemia and acute monocytic leukaemia. A membrane reactivity was detected in most normal monocytes and lymphocytes. The activity in monocytes was very strong and was inhibited by NaF. It was NaF-resistant and less intense in lymphocytes. The reaction product was localized in the cytoplasm only in a small percentage of lymphocytes.In lymphocytes and monoblasts from pathological samples the pattern of reactivity was similar to that found in their normal counterparts, except for a lower intensity. The hairy cells showed a discrete distribution of the NaF-resistant reaction product on their cell surface.The different patterns of enzyme distribution are discussed critically.     

Localization of Glucose-6-phosphate Dehydrogenase Activity on Ribosomes of Granular Endoplasmic Reticulum, in Peroxisomes and Peripheral Cytoplasm of Rat Liver Parenchymal Cells

Glucose-6-phosphate dehydrogenase activity has been localized ultrastructurally in fixed tissues. Activity was found in particular in association with ribosomes of granular endoplasmatic reticulum. Biochemical studies indicated that glucose-6-phosphate dehydrogenase activity is also present in the cytoplasm and in peroxisomes. Fixation may be held responsible for selective inactivation of part of glucose-6-phosphate dehydrogenase activity. In the present study, we applied the ferricyanide method for the demonstration of glucose-6-phosphate dehydrogenase activity in unfixed cryostat sections of rat liver in combination with the semipermeable membrane technique and in isolated rat liver parenchymal cells. Isolated liver parenchymal cells were permeabilized with 0.025% glutaraldehyde after NADP⁺ protection of the active site of glucose-6-phosphate dehydrogenase. This treatment resulted in only slight inactivation of glucose-6-phosphate dehydrogenase activity. The composition of the incubation medium was optimized on the basis of rapid light microscopical analysis of the formation of reddish-brown final reaction product in sections. With the optimized method, electron dense reaction product was observed in cryostat sections on granular endoplasmic reticulum, in mitochondria and at the cell border. However, the ultrastructural morphology was rather poor. In contrast, the morphology of incubated isolated cells was preserved much better. Electron dense precipitate was found on ribosomes of the granular endoplasmic reticulum, in peroxisomes and the cytoplasm, particularly at the periphery of cells. In conclusion, our ultrastructural study clearly demonstrates that it is essential to use mildly-fixed cells to allow detection of glucose-6-phosphate dehydrogenase activity in all cellular compartments where activity is present.     

Endorphins are located in the intermediate and anterior lobes of the pituitary gland, not in the neurohypophysis.

Immunocytofluorescence techniques with well characterized anti-sera to α-endorphin and β-endorphin show presence of these two peptides in all cellular elements of the pars intermedia of the rat hypophysis, and in discrete cells of the pars distalis (adenohypophysis) at the complete exclusion of the neurohypophysis (pars nervosa, posterior lobe).     

Localization of xanthine oxidase in crystalline cores of peroxisomes. A cytochemical and biochemical study

The ultrastructural cytochemical localization of xanthine oxidase activity in rat liver was investigated by the cerium technique. The reaction product was found in the cytoplasm of endothelial cells in liver sinusoids and, in addition, in crystalline cores of peroxisomes of liver parenchymal cells. Xanthine oxidase was also present in peroxisomal cores of beef liver and kidney, but not in rat kidney peroxisomes, which lack crystalline cores. The localization in peroxisomal cores of rat liver was confirmed also biochemically using highly purified peroxisomal fractions and subfractions containing exclusively the crystalline cores. Moreover, high levels of molybdenum were found in isolated peroxisomal cores by atomic absorption spectroscopy, thus corroborating the association of the molybdenum-containing enzyme with the cores. Since urate oxidase is also present within the same compartment of peroxisomes, it is possible that the crystalline cores harbor a complex of several enzymes involved in the purine metabolism.     

Three-dimensional organization of the cytoplasmic neuroglobin-immunopositive structures in the rat medulla oblongata neurons

Neuroglobin is an iron-containing protein, most abundant in the vertebrate nervous system. Since neuroglobin is able to bind oxygen reversibly owing to the heme prosthetic group, it was believed that its function is an intercellular transport of oxygen in the nervous system and accumulation of oxygen for energy supply of cells in hypoxic conditions. In this work, a three-dimensional reconstruction of the neuroglobin distribution in large neurons of the rat medulla oblongata was carried out by means of immunocytochemistry and confocal laser microscopy. Positive neuroglobin immunocytochemical reaction was observed mainly in the perinuclear areas of large nerve cells exhibiting a discrete staining of the cytoplasm. Examination under the microscope at a high magnification revealed some neuroglobin-immunopositive granules, ring-like objects 1–2 μm in diameter, as well as linear and branched structures in neuronal cytoplasm and, occasionally, in the proximal segments of neuronal processes. Three-dimensional reconstruction of the neuroglobin-immunopositive structures showed that they mainly have the form of continuous lines and curves interlaced in some sites, about 1.0–1.5 μm thick, forming a complex network in the cytoplasm. The neuroglobin-immunopositive complexes found for the first time in neuronal cytoplasm are not identical to any known cytoplasmic compartments of nerve cells, but the diameter of their elements, as well as the shape and location suggest a possible link of neuroglobin with a mitochondrial network.     

Uptake of microperoxidase by segmenting rat ova in vitro

Summary Uptake and incidence of microperoxidase (Sigma) in the rat ova during cleavage at the 1-, 2-, 8-cell and blastocyst stages were studied after 30 min incubation with the enzyme (molecular weight of 1,900 and size of molecule of 2 nm).Evidence was furnished of the presence of a small amount of reaction product of microperoxidase in the zona pellucida and its local concentration in some parts of the perivitelline space. The larger part of surface of the ovum, however, was free of microperoxidase. In the cytoplasm microperoxidase was found in pinocytotic vesicles, less frequently in large vacuoles and starting with the eight-cell stage in secondary lysosomes. The largest amount of microperoxidase was ascertained at the stage of blastocyst, chiefly in the cells of the trophoblast. In the cells of the embryoblast, on the contrary, microperoxidase was found but occasionally. The reaction product of microperoxidase was also present in the intercellular space of ova in negligible amount, and likewise on the side of blastocysts' cavity. In comparison with the ingestion of horseradish peroxidase the incidence of microperoxidase in the segmenting rat ova was less frequent.