Parvalbumin in non-muscle tissues of the rat. Quantitation and immunohistochemical localization

Parvalbumin, a high affinity Ca2+-binding protein, is known to be expressed only in muscles and brain in the rat. We have investigated its distribution and characteristics in other rat tissues by several biochemical and immunohistochemical methods. Evidence for the presence of parvalbumin in teeth, bone, skin, prostate, seminal vesicles, testes, and ovary is given by two-dimensional polyacrylamide gel electrophoresis, immunoblotting ("Western technique") of one-dimensional gels, and its concentration measured by reverse phase high performance liquid chromatography. The distribution within several parvalbumin-positive organs was monitored by the immunohistochemical peroxidase-antiperoxidase method. In teeth, only ameloblasts reacted with anti-rat parvalbumin serum and in bone the calcified extracellular cartilage was the target of the immunoreaction. The panniculus carnosus was the exclusive site of parvalbumin in the skin. Besides the already known parvalbumin distribution in the brain, parvalbumin is also expressed in distinct cell types of the peripheral nervous system. Leydig cells were found to be the only parvalbumin location in testes. These observations lead us to conclude that parvalbumin in contrast to the multifunctional and constitutive calmodulin must function in Ca2+-dependent processes related to specific cell types.     

Distribution of cathepsins B and H in rat tissues and peripheral blood cells

The concentrations of cathepsins B and H in various tissues and peripheral blood cells of rats were determined by means of sensitive immunoassays. The minimum detectable amounts of cathepsins B and H were 30 pg and 20 pg/assay, respectively, and the presence of endogenous thiol proteinase inhibitors did not interfere with the immunoassays. Cathepsin B was found at high levels in the kidney, vagina, spleen, and adrenal gland, and cathepsin H at high levels in the kidney, vagina, liver, lung, and spleen. Low levels of cathepsins B and H were present in the heart, skeletal muscle, and testis. The ratios of cathepsins B and H in various organs were different: the brain and adrenal gland contained much higher levels of cathepsin B than of cathepsin H, whereas the lung and liver contained higher levels of cathepsin H than of cathepsin B. In several organs such as the kidney, spleen, liver, and lungs, the level of cathepsins B plus H was much higher than that of thiol proteinase inhibitors (TPI-alpha + TPI-beta), whereas in tissues containing large amounts of TPI-alpha, such as the skin, esophagus and stomach, the level of inhibitors was higher than that of cathepsins B plus H. Of the peripheral blood cells tested, macrophages had the highest contents of cathepsins B and H, and so their level of cathepsins B plus H was much higher than that of TPI-alpha plus TPI-beta, whereas lymphocytes and neutrophils contained comparable amounts of proteinases and inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of cathepsins B,D and L in the rat osteoclast

Immunohistochemical localization of cathepsins B, D and L in the osteoclasts of rat alveolar and femoral bones was investigated by using the avidin-biotin-peroxidase complex method for semithin, 1-m-thick cryosections. Extracellular immunoreactivity for cathepsins B and L was clearly demonstrated along the bone resorption lacunae; the intensity of the extracellular immunoreactivity of cathepsin L was stronger than that of cathepsin B. However, the intracellular immunoreactivity of both cathepsins was weak compared with that of cathepsin D. The intracellular immunoreactivity of cathespin D in the osteoclasts was clearly observed in the granules and/or vacuoles, but extracellular cathepsin D immunoreactivity was either negligible or not detected along the resorption lacunae. In the adjacent sections stained with anti-cathepsin L or D, extensive extracellular deposition of cathepsin L was found along the bone resorption lacunae, with or without osteoclasts, although the intracellular reactivity of cathepsin L was weak. This is the first morphological study in which cathepsins B and L have been demonstrated to be produced in the osteoclasts and extensively secreted into resorption lacunae, and in which cathepsin D was found to be present in the cells but scantily secreted into the lacunae. These findings suggest that cathepsins B and L directly and effectively participate in the degradation of the bone matrix.     

Immunocytochemical localization of cathepsins B and H in rat liver

Summary Light and electron microscopic localization of cathepsins B and H in rat liver was investigated by immunoenzyme and protein A-gold techniques. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultrathin sections of the Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsins B and H were present in the cytoplasmic granules of parenchymal cells and endothelial cells, and Kupffer cells. The sinus-lining cells and the parenchymal cells showed the similar staining intensity. By EM, gold particles were present exclusively in lysosomes of all the cell types cited above. The same results were obtained from quantitative analysis. In addition, Golgi complexes themselves were mostly negative but some small vesicles on the trans side of them were labeled for these proteinases. The results indicate that cathepsins B and H are present in the lysosomes of rat liver and that these enzymes seem to be transported by small vesicles from endoplasmic reticulum to lysosomes via tubuloreticular network of the trans Golgi region.     

Immunocytochemical localization of cathepsins B and H in rat liver

Light and electron microscopic localization of cathepsins B and H in rat liver was investigated by immunoenzyme and protein A-gold techniques. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of the Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsins B and H were present in the cytoplasmic granules of parenchymal cells and endothelial cells, and Kupffer cells. The sinus-lining cells and the parenchymal cells showed the similar staining intensity. By EM, gold particles were present exclusively in lysosomes of all the cell types cited above. The same results were obtained from quantitative analysis. In addition, Golgi complexes themselves were mostly negative but some small vesicles on the trans side of them were labeled for these proteinases. The results indicate that cathepsins B and H are present in the lysosomes of rat liver and that these enzymes seem to be transported by small vesicles from endoplasmic reticulum to lysosomes via tubuloreticular network of the trans Golgi region.     

Immunoenzyme localization of cathepsins in the Golgi region of rat hepatocytes and renal tubule cells

Localization of carboxyl proteinase (cathepsin D) and cysteine proteinases (cathepsins B, H, and L) in Golgi region was studied using an immunoenzyme technique. Rat livers and kidneys were used. The results obtained from the livers were similar to those from the kidneys. All cathepsins were detected in lysosomal compartments such as secondary lysosomes, multivesicular bodies (endosomes), and autophagosomes. Rough endoplasmic reticulum (rER), including nuclear envelope was focally stained. Most of Golgi cisternae were negative, but sometimes only one cisterna or the terminal portion of the cisterna were stained focally. Rarely, the trans Golgi network (TGN) was positive for the proteinases. Among numerous Golgi vesicles, only a few of them were stained. The positive vesicles were divided into two groups, one had a bristle coat and heavily stained, and other were smaller than 40 nm in diameter and weakly stained. The small vesicles seemed to bud from the ER and to fuse with the Golgi cisternae, while the large clathrin-coated vesicles seem to bud from the TGN. The results suggests that cathepsins are transported by vesicular system from the rER to lysosomes via Golgi apparatus. In addition, it is suggested that the small vesicles transport the proteinases from the ER to the Golgi cisternae and the large clathrin-coated vesicles from the Golgi cisternae to the lysosomes.     

Immunoenzyme localization of cathepsins in the Golgi region of rat hepatocytes and renal tubule cells

Summary Localization of carboxyl proteinase (cathepsin D) and cysteine proteinases (cathepsins B, H, and L) in Golgi region was studied using an immunoenzyme technique. Rat livers and kidneys were used. The results obtained from the livers were similar to those from the kidneys. All cathepsins were detected in lysosomal compartments such as secondary lysosomes, multivesicular bodies (endosomes), and autophagosomes. Rough endoplasmic reticulum (rER), including nuclear envelope was focally stained. Most of Golgi cisternae were negative, but sometimes only one cisterna or the terminal portion of the cisterna were stained focally. Rarely, the trans Golgi network (TGN) was positive for the proteinases. Among numerous Golgi vesicles, only a few of them were stained. The positive vesicles were divided into two groups, one had a bristle coat and heavily stained, and other were smaller than 40 nm in diameter and weakly stained. The small vesicles seemed to bud from the ER and to fuse with the Golgi cisternae, while the large clathrin-coated vesicles seem to bud from the TGN. The results suggests that cathepsins are transported by vesicular system from the rER to lysosomes via Golgi apparatus. In addition, it is suggested that the small vesicles transport the proteinases from the ER to the Golgi cisternae and the large clathrin-coated vesicles from the Golgi cisternae to the lysosomes.     

Immunochemical and immunohistochemical localization of the G protein Gi1 in rat central nervous tissues

Antisera were raised in rabbits against purified alpha subunit of G protein Gi1 (Gi1 alpha) and also against a synthetic decapeptide corresponding to a sequence of Gi1 alpha. Antibodies in both antisera were purified with a Gi1-coupled Sepharose column, but purified anti-Gi1 alpha protein antibodies still reacted equally with both Gi1 alpha and Gi3 alpha, while anti-Gi1 alpha peptide antibodies reacted principally with Gi1 alpha. Using these antibodies, an enzyme immunoassay method for the quantification of Gi1 alpha was developed. The assay system consisted of polystyrene balls with immobilized anti-Gi1 alpha protein antibody F(ab')2 fragments and the anti-Gi1 alpha peptide antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the assay was 25 fmol of Gi1 alpha, and it did not cross-react with Gi2 alpha, Go alpha, or beta gamma. Samples from various regions of the rat central nervous system were homogenized in a 2% sodium cholate solution, and the concentration of Gi1 alpha in each extract was determined. Gi1 alpha was detected in all the regions, and the highest concentration was found in the olfactory bulb. Immunohistochemical study showed that Gi1 was mainly localized in the neuropil.     

Synthesis and intracellular distribution of cathepsins E and D in differentiating murine Friend erythroleukemia cells.

The synthesis, accumulation, and cellular distribution of cathepsins E and D during the dimethyl sulfoxide (DMSO)-induced differentiation of Friend erythroleukemia cells were investigated. The cellular levels of cathepsins E and D rapidly increased within 1 day of DMSO induction and then sharply decreased over the next 7 days. Since the cells during 1 day of differentiation were morphologically the same as uninduced cells, the results suggest the importance of these enzymes in more cellular proteolysis for the following committed differentiation. While cathepsin D was present mostly in the sedimentable fraction of cells throughout the differentiation period, the distribution of cathepsin E varied to the stage of differentiation. The ratio of the soluble/sedimentable cathepsin E content was 1.1, 1.4, 0.9, and 0.7 in cells after 0, 1, 4, and 7 days of DMSO treatment, respectively. The maturation of reticulocytes to erythrocytes was accompanied by complete loss of the soluble cathepsin E and of all of the cellular cathepsin D. Immunoblotting analyses revealed that both uninduced and induced cells contained two forms of cathepsin E; a high molecular weight form (82 kDa) which was mainly associated with the sedimentable fraction and a low molecular weight form (74 kDa) which was found largely in the soluble fraction. The distribution of these two forms was not significantly changed throughout the differentiation period, but the 74-kDa protein was completely eliminated with maturation of reticulocytes to erythrocytes. Cathepsin D also appeared in two forms in both uninduced and induced cells; a minor (46 kDa) and a major (42 kDa) form which appear to have a precursor-product relationship.     

Expression and immunohistochemical localization of galectin-3 in various mouse tissues

The expression and immunohistochemical localization of galectin-3, a beta-galactoside-binding protein, was studied in several mouse tissues. Galectin-3 expression was low in the cerebrum, heart, and pancreas, and moderate in the liver, ileum, kidney, and adrenal gland. High expression of galectin-3 was found in the lung, spleen, stomach, colon, uterus, and ovary. The results of Western blot analysis largely matched the immunohistochemical findings for galectin-3. These findings suggest that galectin-3 is differentially expressed in a variety of organs in the mouse. This study provides valuable information for research on galectin-3.     

Rho localization in cells and tissues

Rho family small GTPases regulate cytoskeletal organization. Although their spatiotemporal activities appear to be important for cellular morphogenesis, there has been little characterization of the localization of Rho family GTPases in cells and tissues. Here we show precise localization of Rho subfamily proteins in mammalian cultured cells and tissues through evaluation of anti-Rho antibodies and fixation protocols. Although Rho is not a structural protein but functions as a switching molecule, it often localizes at several distinct domains or structures of cells. In cultured epithelial cells, Rho was highly accumulated at lateral membranes. However, in fibroblastic cells, Rho appeared to be distributed evenly in the cytoplasm. Rho concentration at the cleavage furrow at cytokinesis was generally observed. In A431 cells, Rho translocation from the cytoplasm to elongating microvilli at the apical membrane within 30 s after EGF stimulation was clearly demonstrated. Also, Myc- or GFP-tagged RhoA did not always reflect the localization of endogenous Rho, indicating a drawback of protein-tagging methods for localization research. In mouse tissues, Rho localization differed depending on cell type, probably reflecting the functional differences of each cell type.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Summary Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cystine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cysteine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Histochemical and immunohistochemical localization of hexokinase isoenzymes in rat kidney

Summary Histochemical and immunohistochemical staining techniques have been used to investigate the localization of hexokinase isoenzymes within rat kidney tissue. Hexokinase type I was shown to be the major isoenzyme present. It was located mainly in the thin and thick limbs of loops of Henle, in distal tubules and in the transitional or dark cells in the initial portions of collecting ducts. The smooth muscle cells of arteries and arterioles, peripheral nerves and the transitional epithelial cells lining the renal pyramid also contained large amounts of the isoenzyme while smaller quantities were present in glomeruli and in collecting tubules near the papillary tip. The distribution pattern obtained in tubular epithelia agrees well with that demonstrated in earlier microdissection studies. It is also consistent with the suggestion that glycolysis provides the majority of the energy fuelling the sodium transport mechanisms which form such an essential feature of the countercurrent urine concentration system present within the renal medulla.     

An immunocytochemical study on distinct intracellular localization of cathepsin E and cathepsin D in human gastric cells and various rat cells.

Immunocytochemical localization of two distinct intracellular aspartic proteinases, cathepsins E and D, in human gastric mucosal cells and various rat cells was investigated by immunogold technique using discriminative antibodies specific for each enzyme. Cathepsin D was exclusively confined to primary or secondary lysosomes in almost all the cell types tested, whereas cathepsin E was not detected in the lysosomal system. The localization of cathepsin E varied with different cell types. Microvillous localization of cathepsin E was found in the intracellular canaliculi of human and rat gastric parietal cells, rat renal proximal tubule cells, and the bile canaliculi of rat hepatic cells. The immunolocalization of each enzyme in gastric cells were essentially the same in humans and rats. In the gastric feveolar epithelial cells and parietal cells, definite immunolabeling for cathepsin E was observed in the cytoplasmic matrix, the cisternae of the rough endoplasmic reticulum, and the dilated perinuclear envelope. In rat kidney, cathepsin E was detected only in the proximal tubule cells, while cathepsin D was found mainly in the lysosomes of the distal tubule cells but not in those of the proximal tubule cells. These results clearly indicate the distinct intracytoplasmic localization of cathepsins E and D and suggest the possible involvement of cathepsin E in extralysosomal proteolysis that is related to specialized functions of each cell type.     

Purification and immunohistochemical localization of rat liver glutathione peroxidase

Glutathione peroxidase was purified from the rat liver to give a single protein band in polyacrylamide gel electrophoresis. Rabbits were immunized with this purified enzyme, and a highly specific anti-glutathione peroxidase antiserum was obtained. Using this antibody, an immunohistochemical technique (the indirect method of peroxidase-labeled antibody) was applied to study the localization of the enzyme in the liver cells.On immunohistochemical observation, glutathione peroxidase was localized exclusively in the cytoplasm of hepatocytes, and a stronger ‘immuno-staining’ was exhibited in the peripheries of the hepatic lobules than in the central zone.     

Immunocytochemical localization of cathepsins B, H, L, and T4 in follicular cells of rat thyroid gland

To localize cathepsins B, H, and L in follicular cells of rat thyroid gland, we applied immunocytochemistry to the thyroid tissue using their respective monospecific antibodies. On serial semi-thin sections, cathepsins B, H, and L were localized in granules of various sizes located throughout the cytoplasm, whereas T4 was detected in larger granules located in the apical and supranuclear regions. By electron microscopy, cathepsins B, H, and L were localized in large less-dense granules (so-called colloid droplets) and in dense bodies of various sizes, whereas T4 was localized more intensely in large less-dense granules than in smaller dense bodies. By double immunostaining using an immunogold method, cathepsins H and B or L were co-localized in the same cytoplasmic granules. Moreover, immunoblotting demonstrated that proteins similar to cathepsins B, H, and L in the liver are present in the thyroid gland. These results suggest that cathepsins B, H, and L participate not only in degradation of thyroglobulin but in maturation of thyroid hormones, although it remains unknown whether all of them participate in the maturation process.     

Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues

Cathepsin X, a recently discovered lysosomal cysteine protease, shares common structural features and activity properties with cysteine protease cathepsin B. Based on its widespread mRNA distribution in primary tumors and tumor cell lines, a redundant function in tumor progression has been proposed. In this study, we have shown that these two related proteases exhibit different profiles with respect to their protein distribution in cells and tissues and to their possible roles in malignancy. Protein level of cathepsin X did not differ significantly between matched pairs of lung tumor and adjacent lung tissue obtained from patients with lung cancer whereas that of cathepsin B was 9.6-fold higher in tumor compared to adjacent lung tissue. Immunohistochemical analysis of lung tumor cathepsin X revealed very faint staining in tumor cells but positive staining in infiltrated histiocytes, alveolar macrophages, bronchial epithelial cells, and alveolar type II cells. Cathepsin X stained positive also in CD68+ cells in germinal centers of secondary follicles in lymph nodes, corresponding to tingible body macrophages. Two cell lines with proven invasive behavior, MCF-10A neoT and MDA-MB 231, showed positive staining for cathepsin B, but negative for cathepsin X. We showed that the invasive potential of MCF-10A neoT cells can be impaired by specific inhibitor of cathepsin B but not by that of cathepsin X. Cathepsin X was found in large amounts in the pro-monocytic U-937 cell line, in monocytes and in dendritic cells, generated from monocytes in vitro. Our results show that cathepsin X is not involved in degradation of extracellular matrix, a proteolytic event leading to tumor cell invasion and metastasis. Its expression, restricted to immune cells suggests a role in phagocytosis and the regulation of immune response.     

Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney

To examine the correlation of localization of prorenin, renin, and cathepsins B, H, and L, immunocytochemistry was applied to rat renal tissue, using a sequence-specific anti-body (anti-prorenin) that recognizes the COOH terminus of the rat renin prosegment. In serial semi-thin sections, immunodeposits for prorenin, renin, and cathepsins B, H, and L were localized in the same juxtaglomerular (JG) cells. Immunodeposits for renin were detected throughout the cytoplasm of the cells, whereas those for prorenin were detected in the perinuclear region. Immunoreactivity for cathepsin B was stronger than that for cathepsins H and L. By electron microscopy, prorenin was localized in small (immature) granules but not in large mature granules, whereas renin was localized mainly in mature granules. In serial thin sections, prorenin, renin, and cathepsin B were colocalized in the same immature granules containing heterogeneously dense material (intermediate granules). By double immunostaining, co-localization of renin with cathepsins B, H, or L was demonstrated in mature granules. The results suggest the possibility that processing of prorenin to renin occurs in immature granules of rat JG cells, and cathepsin B detected in JG cells may be a major candidate for the maturation of renin.