Ultrastructural localization of carbonic anhydrase activity in the rat choroid plexus epithelial cell

Summary Localization of carbonic anhydrase activity was studied electron microscopically on cells of the rat choroid plexus epithelium. For the ultracytochemical detection of these activities, Yokota's technique (1969), which is the modification of Hansson's method (1967) was employed. Numerous electron dense reaction products were observed in the microvilli of the choroidal epithelial cell. The reaction deposits were also remarkably present in the infoldings of the basal plasmalemma but to a lesser extent than in the microvilli. The localization sites were mainly on the plasma membrane, but some reaction products were also observed in the cytoplasm near the plasma membrane. Hardly any reaction product was found in the intracellular organelles except for the mitochondria in which reaction products were occasionally observed on the cristae. These activities were completely inhibited by acetazolamide. As the carbonic anhydrase activity was histochemically seen in the microvilli and the basal infoldings, it is likely that carbonic anhydrase is related to an active transport process in the secretion of cerebrospinal fluid as is Na⁺, K⁺-ATPase (Masuzawa et al. 1980).     

Histochemical and biochemical studies of carbonic anhydrase activity in the opercular epithelium of the euryhaline teleost, Fundulus heteroclitus

Carbonic anhydrase (CAH) activity was biochemically measured and histochemically localized (at both the light and electron microscope levels) in isolated opercular membranes from teleost fish, Fundulus heteroclitus, adapted to freshwater (FW), seawater (SW), and double-strength seawater (2 x SW). The normal morphology of this membrane showed that its epithelial portion consisted of five cell types: (1) chloride cells, which have been previously implicated as responsible for the active chloride transport across the epithelium; (2) mucous cells; (3) pavement cells, which formed the major portion of the free epithelial surface; (4) supportive cells, which had an abundance of intermediate (10 nm)-type filaments suggesting a structural role for these cells; and (5) vesicular cells, which were characterized by various types of membrane-bound vesicles, including lysosomes, and numerous free ribosomes. Vesicular cells may be stem cells and/or endocrine cells. Hansson's histochemical method for CAH revealed cobalt sulfide reaction product confined to the following structures in fish from each environment: (1) chloride cells: throughout the cytoplasm and some nuclear staining; (2) mucous cells: throughout the cytoplasm, some nuclear staining, and some in mucous granules; (3) vesicular cells: confined to lysosomes, some of the vesicles, and nucleoli; (4) a small portion of the intracellular space between adjacent vesicular cells and supportive cells; and (5) supportive cells: in nucleoli and occasionally in larger membrane-bound lysosomelike structures. Acetazolamide (10(-5) M) and potassium cyanate (KCNO) (10(-1) M) in Hansson's incubation medium completely inhibited the formation of reaction product. Biochemical determination of CAH activity on vascularly perfused, isolated opercular membranes showed no statistically significant difference in enzyme activity between environmental groups. The following units of activity/mg opercular membrane protein were measured: FW: 0.63 +/- 0.02; SW: 0.43 +/- 0.08; 2 x SW: 0.64 +/- 0.09.     

Histomorphometric identification of carbonic anhydrase in fetal rat bone embedded in glycolmethacrylate

Carbonic anhydrase was identified in bone-resorbing cells present in sections of fetal rat femur embedded in glycolmethacrylate. Using a slight modification of the Hansson's histochemical method, we demonstrated that most chondroclasts (91.8-95.4%) and osteoclasts (95.1-96.3%) display a positive histochemical reaction for carbonic anhydrase. This staining was consistently inhibited in the presence of very low concentrations (10(-6), 10(-7) M) of the specific inhibitor acetazolamide. The number of chondroclasts reacting for carbonic anhydrase was identical to the number of acid phosphatase-stained chondroclasts determined on adjacent sections. A large majority of osteoclasts (96.3%) stained for carbonic anhydrase and for acid phosphatase (97.2%), with more osteoclasts reacting for the latter enzyme than the former (76.8 +/- 8.5 (SD) vs 85.3 +/- 9.2 cells/mm2 of endosteal bone; p less than 0.01). The observation that acetazolamide at a concentration as low as 10(-7) M inhibited Hansson's reaction, together with our histomorphometric results, validates the use of histochemical staining for carbonic anhydrase to evaluate activity of bone-resorbing cells identified in plastic-embedded fetal bone tissue.     

Histochemical localization of calcium in the enamel organ of rat incisors in early-stage amelogenesis

The localization of calcium in the enamel organ of rapidly-frozen, freeze-substituted rat incisors in early-stage amelogenesis was examined by a histochemical calcium-staining method. In secretory ameloblasts, glyoxal bis(2-hydroxyanil) (GBHA) staining revealed intense red reactions in mitochondria and tubulovesicular structures located throughout the cytoplasm, while no reaction was seen in the nucleus and cytosol, nor along the plasma membranes of the respective cells. No significant GBHA reaction was observed in the intercellular compartment and other cells of the enamel organ. Some granular reactions were localized in the cells of the adjacent connective tissue. Control tests confirmed the specificity of GBHA reactions for calcium. Thus, the present observations provide histochemical evidence indicating an exclusive localization of calcium in mitochondria and tubulovesicular structures of the secretory ameloblast, and support their contributions to the translocation of calcium from the proximal to the distal pole of the cytoplasm.     

A modified semipermeable membrane technique for carbonic anhydrase histochemistry of the nervous system

We present a modification of Hansson's method for the demonstration of carbonic anhydrase activity. Using a semipermeable membrane together with a fluid incubation medium, frozen sections of aldehyde-fixed tissue were incubated without floating or dipping. Thin sections (thickness, 20-40 microns) were mounted on the outer surface of a tubular-shaped, semipermeable cellophane dialysis membrane containing the incubation fluid. After incubation for 25-30 min at room temperature, the sections were rinsed in buffer and treated with 0.5% (NH4)2S solution. The histochemical reaction was fully inhibited by 10(-4) M acetazolamide.     

Carbonic anhydrase is present in olfactory receptor cells

Summary A modification of Hansson's histochemical technique was used to reveal carbonic anhydrase activity in mounted cryostat sections of rat olfactory mucosa, after glutaraldehyde fixation. A positive reaction that could be inhibited by acetazolamide was found in a population of olfactory receptor cells, whereas the supporting cells were negative. Axons of receptor cells were also positive and could be traced through the cribriform plate to the olfactory bulb.     

Binding of lectin-fluorescein conjugates to intracellular compartments of growth-plate chondrocytes in situ

In this study, lectin-binding techniques are applied to growth-plate cartilage to analyze the intracellular localization of lectin-binding glycoconjugates of chondrocytes in situ. The binding of ten fluorescein-conjugated lectins is analyzed on 1-micron-thick Epon-embedded, nondecalcified sections of growth plates from Yucatan swine. Comparisons are made to intracellular binding in chondrocytes of tracheal, articular, and auricular cartilage. Ear epithelium, tracheal epithelium, and kidney are used as positive control tissues for the specificity of lectin binding. Only the mannose-binding lectins had affinity for the RER and nuclear envelope. Eight lectins reacted within the Golgi complex with characteristic patterns which ranged from localized fine linear strands to extensive vesicular accumulations. When cartilage slabs were exposed before embedment to the ionophore monensin to disrupt intracellular transport through the Golgi, it was possible to define differential subcompartments of the Golgi complex, based upon sites of sugar addition. Also, it was possible to characterize the cytoplasmic deposits of reserve-zone chondrocytes which were positive with concanavalin A as glycogen, based upon their sensitivity to amylase. This method allows resolution at the light-microscopic level of lectin-binding glycoconjugates with localization to specific organelles. Patterns of intracellular binding were consistent with biochemical data relating to the subcellular localization of processing steps of complex carbohydrates prior to secretion.     

Ultrastructural localization of calcium-activated adenosine triphosphatase (Ca2+-ATPase) in growth-plate cartilage

The electron-microscopic cytochemical localization of calcium-activated adenosine triphosphatase (Ca2+-ATPase) was determined in chick epiphyseal growth-plate cartilage. In the reserve zone, mitochondria and lysosomes contained substantial amounts of reaction product, while the plasma membrane and the Golgi complex showed very weak enzymatic activity, and matrix vesicle membranes did not exhibit the cytochemical reaction. As maturation proceeded, the plasma membrane, Golgi complex, and matrix vesicle membranes also stained and were most intense in the proliferative and early hypertrophic zones. From the hypertrophic to the calcifying zone, cytochemical staining decreased progressively in the plasma membrane, the Golgi complex, and lysosomes, while in some cases mitochondrial reaction product remained intense. Matrix vesicles lost their enzymatic activity at the same time that matrix vesicle calcification commenced. It is proposed that this event allows matrix vesicles to calcify, since efflux of calcium would no longer occur.     

Carbonic anhydrase activity in peripheral T-lymphocytes and appearance of the activity during their maturation in the thymus. A histochemical demonstration

Carbonic anhydrase (CA) activity is demonstrated in lymphoid tissue for the first time using the histochemical (Hansson's) method. A CA-positive reaction was seen in lymphocytes present in T-lymphocyte areas in both the lymph node and the spleen. The most intense staining was seen in the small T-lymphocytes, whereas the medium-sized T-lymphocytes were less markedly stained. The cortical lymphocytes in the thymus were completely devoid of staining, but the small medullary T-lymphocytes stained intensely. The results suggest that peripheral and thymic medullary T-lymphocytes contain CA activity, which appears in these cells during their maturation in the thymus.     

In situ localization of lectin-binding glycoconjugates in the matrix of growth-plate cartilage

In the distal hypertrophic zone of growth-plate cartilage, the pericellular matrix surrounding individual chondrocytes and the territorial matrix uniting chondrocytes into columnar groups are invaded by metaphyseal endothelial cells prior to osteogenesis. In the present study, lectin-binding glycoconjugates were analyzed in these two matrix compartments of growth-plate cartilage from Yucatan swine. Nine lectin-fluorescein conjugates were tested by a postembedment method on 1-micron-thick, nondecalcified, Epon-embedded sections. Chondrocytes in all cellular zones were surrounded by a pericellular matrix which showed positive binding for peanut agglutinin (PNA), ricin agglutinin (RCA-I), and soybean agglutinin (SBA). Binding by these lectins was sensitive to digestion with hyaluronidase, chondroitinase, and trypsin. Pericellular glyconconjugtes that bind RCA-I and concanvalin A (CONA) after periodic acid oxidation, and which were sensitive to trypsin but not to chondroitinase or hyaluronidase, were present in the hypertrophic cell zone. Within the territorial matrix, binding of lectins specific for galactose, N-acetylgalactosamine, and fucose showed gradients of intensity which became maximal at the last transverse septum. Lectin-binding histochemistry more precisely differentiated the microheterogeneity of glycoconjugate distribution within these two matrix compartments than has been possible with other histochemical techniques. Lectin-binding affinity is a potentially useful technique by which to isolate cartilage matrix macromolecules unique to specific cellular zones of the growth plate.     

CCN2: a master regulator of the genesis of bone and cartilage

CCN family member 2 (CCN2), also known as connective tissue growth factor (CTGF), has been suggested to be an endochondral ossification genetic factor that has been termed “ecogenin”, because in vitro studies revealed that CCN2 promotes the proliferation and differentiation of growth-plate chondrocytes, osteoblasts, and vascular endothelial cells, all of which play important roles in endochondral ossification. In addition to its action toward these three types of cells, CCN2 was recently found to promote the formation of osteoclasts in vitro, which cells play an important role in the replacement of cartilage by bone during endochondral ossification, thus strengthening the “ecogenin” hypothesis. For confirmation of this hypothesis, transgenic mice over-expressing CCN2 in cartilage were generated. The results proved the hypothesis; i.e., the over-expression of CCN2 in cartilage stimulated the proliferation and differentiation of growth-plate chondrocytes, resulting in the promotion of endochondral ossification. In addition to its “ecogenin” action, CCN2 had earlier been shown to promote the differentiation of various cartilage cells including articular cartilage cells. In accordance with these findings, cartilage-specific overexpression of CCN2 in the transgenic mice was shown to protect against the development of osteoarthritic changes in aging articular cartilage. Thus, CCN2 may also play a role as an anti-aging (chondroprotective) factor, stabilizing articular cartilage. CCN2 also had been shown to promote intramembranous ossification, regenerate cartilage and bone, and induce angiogenesis in vivo. For understanding of the molecular mechanism underlying such multifunctional actions, yeast two-hybrid analysis, protein array analysis, solid-phase binding assay, and surface plasmon resonance (SPR) analysis have been used to search for binding partners of CCN2. ECMs such as fibronectin and aggrecan, growth factors including BMPs and FGF2 and their receptors such as FGFR1 and 2 and RANK, as well as CCN family members themselves, were shown to bind to CCN2. Regarding the interaction of CCN2 with some of them, various binding modules in the CCN2 molecule have been identified. Therefore, the numerous biological actions of CCN2 would depend on what kinds of binding partners and what levels of them are present in the microenvironment of different types of cells, as well as on the state of differentiation of these cells. Through this mechanism, CCN2 would orchestrate various signaling pathways, acting as a signal conductor to promote harmonized skeletal growth and regeneration.     

Carbonic anhydrase activity in peripheral T-lymphocytes and appearance of the activity during their maturation in the thymus

Summary Carbonic anhydrase (CA) activity is demonstrated in lymphoid tissue for the first time using the histochemical (Hansson's) method. A CA-positive reaction was seen in lymphocytes present in T-lymphocyte areas in both the lymph node and the spleen. The most intense staining was seen in the small T-lymphocytes, whereas the mediumsized T-lymphocytes were less markedly stained. The cortical lymphocytes in the thymus were completely devoid of staining, but the small medullary T-lymphocytes stained intensely. The results suggest that peripheral and thymic medullary T-lymphocytes contain CA activity, which appears in these cells during their maturation in the thymus.     

Ruthenium hexaammine trichloride chemography for aggrecan mapping in cartilage is a sensitive indicator of matrix degradation.

We developed a new quantitative histochemical method for mapping aggrecan content in articular cartilage and applied it to models of cartilage degradation. Ruthenium hexaammine trichloride (RHT) forms co-precipitates with aggrecan, the main proteoglycan component of cartilage, and was previously found to be a good fixative in aiding the maintenance of chondrocyte morphology. We show that these RHT-aggrecan precipitates generate a positive chemographic signal on autoradiographic emulsions, in the absence of any radioactivity in the tissue section, via a process similar to the autometallographic process used previously for localization of trace metals ions in tissues. By exploiting the inherent depth-dependence of aggrecan concentration in adult articular cartilage, we demonstrated that the density of silver grains produced by RHT-derived chemography on autoradiographic emulsions correlated with locally measured aggrecan concentration as determined by the dimethylmethylene blue assay of microdissected tissue from these different depths of cartilage. To explore the benefits of this new method in monitoring tissue degradation, cartilage explants were degraded during culture using interleukin-1 (IL-1) or digested after culture using chondroitinase and keratinase. The RHT chemographic signal derived from these samples, compared to controls, showed sensitivity to loss of aggrecan and distinguished cell-mediated loss (IL-1) from degradation due to addition of exogenous enzymes. The RHT-derived chemographic grain density represents an interesting new quantitative tool for histological analysis of cartilage in physiology and in arthritis.     

The chondrocyte cytoskeleton in mature articular cartilage: structure and distribution of actin, tubulin, and vimentin filaments.

We investigated the structure of the chondrocyte cytoskeleton in intact tissue sections of mature bovine articular cartilage using confocal fluorescence microscopy complemented by protein extraction and immunoblotting analysis. Actin microfilaments were present inside the cell membrane as a predominantly cortical structure. Vimentin and tubulin spanned the cytoplasm from cell to nuclear membrane, the vimentin network appearing finer compared to tubulin. These cytoskeletal structures were present in chondrocytes from all depth zones of the articular cartilage. However, staining intensity varied from zone to zone, usually showing more intense staining for the filament systems at the articular surface compared to the deeper zones. These results obtained on fluorescently labeled sections were also corroborated by protein contents extracted and observed by immunoblotting. The observed cytoskeletal structures are compatible with some of the proposed cellular functions of these systems and support possible microenvironmental regulation of the cytoskeleton, including that due to physical forces from load-bearing, which are known to vary through the depth layers of articular cartilage.     

Replica immunoelectron microscopic study of the upper surface layer in rat mandibular condylar cartilage by a quick-freezing method

A quick-freezing and deep-etching method in combination with replica immunoelectron microscopy was applied for examining localization of hyaluronic acid and fibronectin on the upper surface layer of rat mandibular condylar cartilage. Rat temporomandibular joints were dissected with articular disks in order to leave the articular cartilage surface intact. The disks were slightly cut with razor blades for exposing the condylar articular cartilage surface. They were quickly frozen with the isopentane-propane cryogen (–193°C) and prepared for freeze-fracturing and deep-etching replica membranes. They were additionally treated with 5% SDS and 0.5% collagenase to keep some antigens attached on the replica membranes. After such a treatment, a routine immunogold method was applied for clarifying the localization of hyaluronic acid and fibronectin in the upper surface layer. Small immunogold particles for hyaluronic acid were mainly localized around upper filamentous networks covered with amorphous materials, but large immunogold ones for fibronectin were localized on deep thicker fibrils. We have revealed the native architecture of the upper surface layer of mandibular condylar cartilage on the replica membranes and also three-dimensional localization of hyaluronic acid and fibronectin by the immunogold method.     

Localization of acetylcholinesterase in normal human fibroblasts and in a human fibrosarcoma cell line

A modified histochemical procedure was used to detect specific acetylcholinesterase localization in cultured human cells. Enzymic activity was found in cytoplasm in the perinuclear zone and in the plasma membrane of 20% to 40% of HT-1080 human fibrosarcoma cells. The presence of pseudocholinesterase was excluded using specific substrates and inhibitors.     

The histochemistry of mucosaccharides in some organs of germfree rats.

In order to study the histochemical nature of mucosaccharides in germfree animals, the organs in natural contact with bacteria (stomach, small and large intestine) and those naturally remote from bacteria (tracheal and ear cartilage and aorta) were studied by means of light microscopic methods for mucosaccharides in germfree and conventional rats. In the stomach (surface and foveolar cells) of germfree rats the histochemical reactions for acid and neutral mucosaccharides were apparently less intense than in that of conventional rats, whereas in the small and large intestine (goblet cells) of germfree rats the reactions were significantly more intense than in those of conventional rats. In the cartilage (intercellular matrix, lacunar border and chondrocyte cytoplasm) and aorta (interelastic spaces) of germfree animals the reactions were less intense than in those of conventional animals. In addition, some differences in the histochemical nature of mucosaccharides between the organs of germfree and conventional rats were noted, as revealed by the effects of chemical modifications and digestions with enzymes upon the histochemical reactions studied.     

Localization of carbonic anhydrase in identified glial cells of the leech central nervous system: application of histochemical and immunocytochemical methods

We localized the enzyme carbonic anhydrase (CA) in frozen sections of the leech (Hirudo medicinalis) central nervous system by two histochemical techniques and the indirect immunofluorescence technique. Hansson's cobalt precipitation method and the use of 1-dimethylamino-naphthalene-5-sulfonamide (DNSA) to build a fluorescent enzyme-substrate complex showed that glial cells are the sites of CA activity in the leech. Neuropil and connective glial cells surrounding the axons had strong CA activity, whereas packet glial cells, which surround neuron cell bodies, and neurons themselves remained unstained. Glial cells reacted markedly with FITC-coupled antibodies against CA isoenzyme II, but experiments with antibodies against CA isoenzyme I showed no reaction.     

Complex carbohydrate histochemistry of the lateral prostate and seminal vesicle of the guinea pig.

A systematic histochemical study of the complex carbohydrates of the lateral prostate and seminal vesicle of the guinea pig has been made. The complex carbohydrates of the guinea pig male accessory sex glands were partially characterized by various conventional carbohydrate histochemical methods including periodic acid-Schiff, selective periodate oxidation-Schiff reaction, Alcian blue staining at pH 2.5 and 1.0, and high iron diamine. The results indicated that neutral glycoconjugates with 1,2-glycol groups and sialic acids were present in the luminal border and apical cytoplasm of the glandular cells, basement membrane and connective tissue in the lamina propria of the lateral prostate. Similar patterns were demonstrated in the seminal vesicle except that there were relatively fewer or no neutral carbohydrates in the apical cytoplasm of the vesicular epithelial cells. The epithelial basement membrane and connective tissue at the epithelial-stromal interface of both glands were rich in acidic and sulphated glycosaminoglycans. Partial characterization by bovine testicular hyaluronidase indicated the presence of chondroitin sulphates in the lamina propria of the glands.     

Carbonic anhydrase enzyme histochemistry of cranial nerve primary sensory afferent neurons in the rat

Summary Hansson's enzyme histochemical method for the demonstration of carbonic anhydrase has been used to examine primary sensory neurons of cranial nerves in the rat (cochlear ganglion cells excluded). Numerous carbonic anhydrase positive neurons were present in the trigeminal and geniculate ganglia as well as in the mecencephalic trigeminal nucleus. A few carbonic anhydrase positive ganglion cells were found in the nodose ganglion, but none in the petrosal and vestibular ganglia. However, in the latter ganglia, satellite cells surrounding the neurons frequently showed staining for carbonic anhydrase.