Immunocytochemical localization of cathepsins B, H, and L in the rat gastro-duodenal mucosa

Cathepsins B, H, and L are representative cysteine proteinases in lysosomes of a large variety of cells. Previous immunochemical studies indicated the presence of these enzymes also in the gastrointestinal wall. Using specific antisera, the cellular and subcellular distribution of cathepsins B, H, and L in rat gastric (oxyntic and pyloric part) and duodenal mucosa was investigated by light and electron microscopical immunocytochemistry. The subtypes of cathepsins were distributed differently in the cellular constituents of the epithelia: Cathepsin B was localized to lysosomes of all cells except goblet cells. Cathepsin H was found predominantly in gastric parietal cells (lysosomes) and in secretion granules of pyloric gastrin and duodenal cholecystokinin cells. Cathepsin L immunoreactivities were weak and restricted to a minority of cells (gastric mucous cells, enterocytes). Interstitial cells of the lamina propria immunoreactive for cathepsins H and L were identified as macrophages. The present findings suggest a dual function of cathepsins in the gastro-duodenal mucosa. They (1) cleave enzymatically proteins and peptides ingested in lysosomes, and (2) they may be involved in the processing of biologically active peptides (enteric hormones) from their precursor proteins.     

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.     

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.     

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.     

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 angiotensinogen and cathepsins B, H, and L in rat hepatocytes, with special reference to degradation of angiotensinogen in lysosomes after colchicine

We examined the effects of bilateral nephrectomy and colchicine treatment on localization and content of angiotensinogen and cathepsins B, H, and L in rat liver using immunohistochemistry, radioimmunoassay, and enzyme assay. Angiotensinogen content increased in the liver of colchicine-treated rats, whereas a clear-cut increase was not detected in the liver of nephrectomized rats. This tendency was consistent with the immunocytochemical results; only perivenous hepatocytes in control and nephrectomized rats were diffusely immunostained by anti-angiotensinogen, whereas perivenous and periportal hepatocytes of colchicine-treated rats were strongly immunostained. Enzyme assay revealed no significant change in activities of cathepsins B, H, and L in liver extracts under these experimental conditions. Immunocytochemical localization of these cysteine proteinases in hepatocytes after colchicine treatment was more widespread in the cytoplasm than that in the control hepatocytes. By electron microscopy, angiotensinogen was localized in smaller vesicles and some larger vesicles (lysosomes) of hepatocytes after colchicine treatment. Double immunostaining demonstrated co-localization of cathepsins B, H, and L with angiotensinogen in lysosomes. These results suggest that cathepsins B, H, and L play a role in the degradation of excess angiotensinogen in hepatocytes of rats after colchicine treatment.     

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, H, and their endogenous inhibitor, cystatin beta, in islet endocrine cells of rat pancreas

To determine the characteristics of lysosomes in rat islet endocrine cells, we examined the precise localization of cathepsins B, H, and L and their specific inhibitors, cystatins alpha and beta, using immunocytochemical techniques. By use of serial semi-thin sections, we detected immunoreactivity for cathepsin B in insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-positive (PP) cells. Strong immunoreactivity for cathepsin H was seen in A-cells and weak immunoreactivity in PP cells, but none in others. Immunodeposits for cystatin beta were demonstrated in B-cells. Brief dipping of thin sections in 1% sodium methoxide before the following immunocytochemical reaction enhanced specific deposits of immunogold particles on the target organelles. Use of a double-immunostaining technique showed co-localization of insulin with cystatin beta in many secretory granules. This suggests that cystatin beta may regulate converting enzymes participating in the maturation process of insulin. By use of an immunogold technique, heterogeneous localization of cathepsins B and H in lysosomes was also found among islet cells at the light microscopic level. This may be due to the difference in peptides degraded in lysosomes among the cells.     

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.     

Different immunolocalizations of cathepsins B, H, and L in the liver

Different localizations of cathepsin B, H, and L in normal rat liver were revealed immunohistochemically with anticathepsin Fab'-horseradish peroxidase conjugates. Staining of cathepsin B was strong in the periportal sinusoids, possibly in Kupffer cells; and weaker in panlobular hepatocytes. Staining of cathepsin H was strong in panlobular hepatocytes, especially in the periphery of the cytoplasm, possibly representing the peribiliary dense bodies; and weaker in periportal sinusoidal cells, possibly Kupffer cells. Staining of cathepsin L was strongest in centrilobular hepatocytes and weaker in periportal sinusoidal cells, possibly Kupffer cells. These findings, revealed for the first time in the present study, show that the histologic and intracellular localizations of the three cathepsins are different, suggesting that they have different roles in degradation of exogenous and endogenous proteins.     

Immunocytochemical localization of cathepsin H in rat kidney

Summary Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For ligh microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

Breast cancer cells express cathepsins B and L but not cathepsins K or H

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.     

Biosynthesis of lysosomal cathepsins B and H in cultured rat hepatocytes

The biosynthesis of lysosomal cysteine proteases, cathepsins B and H, was investigated by using pulse-chase experiments in vivo in primary cultures of rat hepatocytes. Cathepsins B and H were isolated from either total cell extracts or culture medium labeled with [35S]methionine by immunoprecipitation and analyzed for their molecular forms. Within 60 min of chase, cellular proforms of cathepsins B of 39 kDa and H of 41 kDa were converted to single-chain form cathepsins B of 29 kDa and H of 28 kDa, respectively, and persisted as these forms even after 12-h chase periods. The proforms of cathepsins B and H derived from pulse-labeling experiments showed complete susceptibility to endoglycosidase H treatment, indicating that these proenzymes bear high-mannose-type oligosaccharides at the stage of initial events of biosynthesis. In the presence of tunicamycin, unglycosylated proenzymes of cathepsins B of 35 kDa and H of 34 kDa were found to be secreted into the extracellular medium without undergoing proteolytic processing. Furthermore, in the presence of swainsonine, a potent inhibitor of Golgi mannosidase II, considerable amounts of the proenzymes were secreted and accumulated in the medium during chasing periods. These results suggest that the oligosaccharide moiety of these enzymes would be necessary for the intracellular sorting mechanism. In monensin-treated cells, the conversion of intracellular proenzymes to mature enzymes was significantly inhibited and the proenzymes were secreted into the medium. In the presence of chloroquine or ammonium chloride, proteolytic processing of the proenzymes was completely prevented and the enhanced secretion of proenzymes was observed. These results suggest that in the presence of lysosomotropic amines the intracellular sorting of proenzymes might not occur properly during biosynthesis.     

Effect of metabolic alterations on the density and the contents of cathepsins B, H and L of lysosomes in rat macrophages

Crude lysosomal preparations from non-cultured peritoneal rat macrophages were shown to separate into high-density fractions rich in cathepsin B and H and low-density fractions rich in cathepsin L when layered on Percoll density gradients. Morphologically, the heavy lysosome fractions were found to consist mainly of lysosomes labeled with gold particles for anti-(cathepsin B, H and L). The light lysosome fractions contained lysosomes labeled with anti-(cathepsin B, H and L) and many other contaminants. In addition, small vesicles labeled by anti-(cathepsin L) were detected in these fractions. Addition of calf serum to the cultured macrophages induced an increase in the density of lysosomes in both dose-dependent and time-dependent fashions. Cathepsins B, H and L all shifted to the heavy lysosome fractions following the addition of serum. Progressive increase in fluorescence-labeled calf IgG in the heavy lysosome fractions after its addition suggests that the continuous entrance of excess proteins to lysosomes causes an increase in their density. This idea is supported by the fact that the density of lysosomes increased in parallel with the accumulation of horseradish peroxidase taken up in the heavy lysosome fractions. Increase in the density of lysosomes after treatment with ethyl(2S,3S)-3[(S)-3-methyl-1-(3-methyl-butylcarbamoyl)]oxirane-2- carboxylate (E-64-d) was marked in the cells cultured with serum-containing medium but slight in serum-deprived cells. However, the level of pyruvate kinase, an autophagic sequestration marker in heavy autolysosomes from E-64-d-treated cells, was much higher in serum-deprived cells, indicating that the contribution of heterophagic sequestration towards an increase in the density of lysosomes is much greater than that of autophagy.     

Immunocytochemical localization of monoamine oxidase type B in rat liver

We used an immunohistochemical method to examine the localization of monoamine oxidase type B (MAOB) in rat liver. At the light microscopic level, MAOB was highly expressed in rat liver. It was intense around portal area, and weak around central area. All the hepatocytes examined had MAOB immunoreactivity. For the first time, using a double-labeling immunofluorescence histochemical method for laser microscopy, we report that no MAOB is found in endothelial cells, hepatic stellate cells, or Kupffer's cells. When examined under transmission electron microscopy, MAOB was localized to the mitochondrial outer membrane of hepatocytes. No apparent localization of MAOB was found in the rough endoplasmic reticulum, the crystal membrane of mitochondria, the nuclear envelope, or the plasma membrane.     

Localization of cathepsins B,D, and L in the rat osteoclast by immuno-light and -electron microscopy

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and-electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border. Cathepsin L may be more effective in the degradation of bone matrix than cathepsin B.     

2,5-Diaryloxadiazoles and their precursors as novel inhibitors of cathepsins B,H and L

High levels of cathepsins indicated in various pathological conditions like arthritis, cancer progressions, and atherosclerosis explains the need to explore potential inhibitors of these proteases which can be of great therapeutic significance. We, in the present work, report the synthesis of some 2,5-diaryloxadiazoles from N-subsitutedbenzylidenebenzohydrazides. The synthesized compounds were screened for their inhibitory potential on cathepsins B, H and L. Structure Activity Relationship studies show that 2,5-diaryloxadiazoles were less inhibitory than their precursors. 1i and 2k have been found to be most inhibitory to cathepsins B and L. Their K_i values have been calculated as 11.38 × 10⁻⁸ M and 66.4 × 10⁻⁸ M for cathepsin B and 4.2 × 10⁻⁹ M and 47.31 × 10⁻⁹ M for cathepsin L, respectively. However, cathepsin H activity was maximally inhibited by compounds, 1e and 2c with K_i values of 4.4 × 10⁻⁷ M and 5.6 × 10⁻⁷ M, respectively. Enzyme kinetic studies suggest that these compounds are competitive inhibitors to the enzymes. The results have been compared with docking results obtained using iGemDock.     

Active cathepsins B, H, K, L and S in human inflammatory bronchoalveolar lavage fluids

BACKGROUND INFORMATION: Chronic inflammation and tissue remodelling result from an imbalance between proteolytic enzymes and their inhibitors in the lungs in favour of proteolysis. While many studies have examined serine proteases (e.g. cathepsin G and neutrophil elastase) and matrix metalloproteases, little is known about the role of papain-like CPs (cysteine proteases). The present study focuses on the thiol-dependent cathepsins (CPs) and their specific cystatin-like inhibitors [CPIs (CP inhibitors)] in human inflammatory BALFs (BAL fluids, where BAL stands for broncho-alveolar lavage). RESULTS: Cathepsins B, K and S found were mostly zymogens, whereas cathepsins H and L were predominantly in their mature forms. Little immunoreactive cystatin C was found and the high- and low-molecular-mass ('weight') kininogens were extensively degraded. The BALF procathepsins B and L could be activated autocatalytically, indicating that alveolar fluid pro-CPs are reservoirs of mature enzymes. Hydrolysis patterns of 7-amino-4-methylcoumarin-derived peptide substrates showed that extracellular alveolar CPs remain proteolytically active, and that cathepsins B and L are the most abundant thiol-dependent endoproteases. The CP/CPI balance was significantly tipped in favour of cathepsins (3- or 5-fold), as confirmed by the extensive CP-dependent degradation of exogenous kininogens by BALFs. CONCLUSIONS: Although their importance for inflammation remains to be clarified, the presence of active cathepsins L, K and S suggests that they contribute to the extracellular breakdown of the extracellular matrix.