Ascorbic acid induces alkaline phosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. Chondrocyte hypertrophy and matrix mineralization are associated with expression of type X collagen and the induction of high levels of the bone/liver/kidney isozyme of alkaline phosphatase. To determine what role vitamin C plays in these processes, chondrocytes derived from the cephalic portion of 14-day chick embryo sternae were grown in the absence or presence of exogenous ascorbic acid. Control untreated cells displayed low levels of type X collagen and alkaline phosphatase activity throughout the culture period. However, cells grown in the presence of ascorbic acid produced increasing levels of alkaline phosphatase activity and type X collagen mRNA and protein. Both alkaline phosphatase activity and type X collagen mRNA levels began to increase within 24 h of ascorbate treatment; by 9 days, the levels of both alkaline phosphatase activity and type X collagen mRNA were 15-20-fold higher than in non-ascorbate-treated cells. Ascorbate treatment also increased calcium deposition in the cell layer and decreased the levels of types II and IX collagen mRNAs; these effects lagged significantly behind the elevation of alkaline phosphatase and type X collagen. Addition of beta-glycerophosphate to the medium increased calcium deposition in the presence of ascorbate but had no effect on levels of collagen mRNAs or alkaline phosphatase. The results suggest that vitamin C may play an important role in endochondral bone formation by modulating gene expression in hypertrophic chondrocytes.     

Is acid phosphatase activity present in bone matrix at sites of endochondral ossification in rabbit fracture callus?

It has been suggested that acid phosphatase activity is present in newly formed bone matrix at sites of endochondral ossification in rabbit fracture calluses. Because acid phosphatases are usually found intracellularly, it was decided to test this possibility more rigorously. Tissue from 10- and 14-day healing rabbit fractures was subjected to a series of critical tests for acid phosphatases with a pH optimum of 5.0. Fluoride, tartrate and molybdate were used as potential inhibitors of acid phosphatase activity. The effects of several counterstaining protocols were also investigated. A fluoride- and tartrate-resistant acid phosphatase is located in osteoclasts and mononuclear phagocytes. Diffuse staining of the bone matrix is seen, but it is dependent upon the length of incubation in the substrate medium and the distance from the acid phosphatase-reacting cells. It is concluded that the coloration of the bone matrix is probably caused by diffusion of the dye and reaction product and is, therefore, artifactual. © 1998 Chapman & Hall     

Chick osteoblasts contain fluoride-sensitive acid phosphatase activity

We used histological and biochemical methods to determine the cellular origin of bone matrix fluoride-sensitive acid phosphatase in chicken bone. Embryonic chicken calvariae were embedded in plastic and sections stained for acid phosphatase at various concentrations of substrate and fluoride. Acid phosphatase activity was observed in osteoblasts and osteoclasts but not in fibroblasts. Striking inhibition of osteoblastic acid phosphatase occurred at 100 microM fluoride, a concentration that had no apparent effect on osteoclastic acid phosphatase. Inhibition of osteoblastic and osteoclastic acid phosphatase by fluoride was also examined using extracts of embryonic chicken calvarial cells, mouse osteoblasts (MC3T3-El cell line), and purified chick osteoclasts, respectively. Fluoride is a partial competitive inhibitor of both chicken and mouse osteoblastic acid phosphatases, with apparent inhibition constants of 10-100 microM. These concentrations of fluoride correspond to those that increase bone formation in vitro and in vivo. In contrast, the apparent inhibition constant for fluoride of osteoclastic acid phosphatase was much higher (i.e., 0.5 mM). In summary, this study demonstrates that chicken osteoblasts contain an acid phosphatase that is sensitive to inhibition by low concentrations (i.e., microM) of fluoride.     

Different tartrate sensitivity and pH optimum for two isoenzymes of acid phosphatase in osteoclasts. An electron-microscopic enzyme-cytochemical study

Summary By differentiation of substrate specificity, pH optimum range, and sensitivity to various inhibitors, 2 isoenzymes of acid phosphatase in bone cells have been studied at the electron-microscopic level. When p-nitrophenyl phosphate was used for the substrate, the demonstrable enzyme activity was affected by neither tartrate nor sodium fluoride. The reaction product, when incubated at pH 5–6, was detected in all sites along the pathway for the biosynthesis of acid phosphatase in the osteoclast, including the perinuclear space, cisternae of the endoplasmic reticulum, Golgi complex, various vesicles, and vacuoles. In the osteoclasts attached to bone, the enzymatic activity was demonstrated at the extracellular ruffled border and on the eroded bone surface. Reaction products became confined to lysosomes and extracellular ruffled border when incubated at pH 6–7. Unattached osteoclasts showed a similar intracytoplasmic localization of enzyme as the attached ones, except for the absence of the extracellular enzyme activity. The mononuclear, immature type of osteoclast also resembled the mature osteoclast in terms of enzymatic localization. Except for the osteoclasts, the acid p-nitrophenyl phosphatase activity was restricted to lysosomal vesicles in various bone cells, monocytes, and macrophages. Such activity was inhibited by adding 50 mM tartrate to the p-nitrophenyl phosphate medium. When -glycerophosphate or p-nitrocatechol sulfate was the substrate, most of the reaction product was localized intracellularly. Unlike the acid p-nitrophenyl phosphatase, the acid -glycerophosphatase or arylsulfatase activity in osteoclasts and other bone cells was inhibited completely by 10 mM tartrate or 10 mM sodium fluoride. Even preincubation of 100 mM tartrate in the buffer inhibited -glycerophosphatase activity completely, but p-nitrophenyl phosphatase activity was inhibited incompletely. Consequently, our results suggest that acid p-nitrophenyl phosphatase is a useful cytochemical marker for identification of the osteoclast family at electron-microscopic levels of resolution.     

Activity of some enzymes during growth and sporulation of Clostridium botulinum type E

The activities of alkaline and acid phosphatases, glucose dehydrogenase and NADH oxidase were assayed in cell-free extracts of sporogenic and asporogenic mutants of Clostridium botulinum. During growth of both mutants, the activities of alkaline and acid phosphatases were relatively constant, but during sporulation of the sporogenic mutant, the alkaline phosphatase activity rose to a maximum of 70 mol/min·mg protein whereas the acid phosphatase decreased rapidly before it increased, indicating a possible role in sporogenesis. Glucose dehydrogenase activity was detected only in cell-free extracts of the sporogenic mutant and reached a maximum of 7 mol/min·mg protein during the endospore maturation stage. The NADH oxidase activity was detected in both mutants. The NADH oxidase seems to stimulate glucose oxidation in both mutants during growth and the dehydrogenation processes of the butyric type of fermentation during spore formation in the sporogenic mutant. The findings suggest that increased glucose dehydrogenase activity in C. botulinum, as in Bacillus species, may serve as a spore event marker and that alkaline and acid phosphatases may play a regulatory role in anaerobic sporulation metablolism.This work was supported by the Aquatic Biology Research Unit of the University of Manitoba from a Federal Fisheries Research Grant.     

Ultrastructural localization of alkaline phosphatase in the hypertrophic chondrocyte of the frog

Summary The ultrastructural localization of alkaline phosphatase was studied in the hypertrophic chondrocyte of the frog (Rana temporaria) by incubating sections of glutaraldehyde fixed tissue in a medium containing sodium glycerophosphate and calcium chloride. Control specimens were incubated in substrate free medium.Alkaline phosphatase (orthophosphoric monoester phosphohydrolase) is a hight molecular weight glycoprotein that hydrolyses phosphorylated metabolites much as acid phosphatase does except that its action is optimal at an alkaline pH.The results of this investigation showed that alkaline phosphatase activity was present within the cytoplasm and around the plasma membrane of frog hypertrophic chondrocytes. Although only a small proportion of frog hypertrophic chondrocytes demonstrated enzyme activity, there was evidence that this was concentrated within Golgi lamellae and vesicles leaving other organelles unreactive. The finding of alkaline phosphatase activity within Golgi lamellae of hypertrophic chondrocytes is regarded as unusual although positive reactions within chondrocyte lysosomes have previously been reported (Doty and Schofield, 1976).     

Cyclosporine A-induced toxicity in two renal cell culture models (LLC-PK1 and MDCK)

Renal damage caused by therapeutic treatment with cyclosporine A has been well documented. Clinical experiences have shown that cyclosporine A nephrotoxicity is determined by interstitial fibrosis with tubular atrophy. However, the exact mechanism by which this drug causes nephrotoxicity has not yet been clarified. This study used an in vitro model in an attempt to identify the cellular mechanisms underlying kidney cyclosporine A damage. We used two cell lines with the characteristics of proximal and distal tubule cells (pig kidney proximal tubular epithelial cell line [LLC-PK1] and Madin–Darby canine kidney cell line [MDCK]. The cell lines were treated with cyclosporine A for 24h. After the treatment, the cells were stained with Trypan Blue to estimate cell viability and processed by histochemical reactions to evaluate their cellular metabolism. Four enzymes (acid phosphatase, alkaline phosphatase, lactate dehydrogenase and succinate dehydrogenase) were considered. The cell viability assay showed that the LLC-PK1 cell line was more sensitive to cyclosporine A than MDCK. Remarkably, the LLC-PK1 cells disappeared with cyclosporine A treatment. As for the hydrolytic enzymes, only acid phosphatases showed an increased positivity in the treated LLC-PK1 cells. Similarly, lactate dehydrogenase showed a different activity histochemically. No statistically significant alterations were observed in the succinate dehydrogenase reaction.The cyclosporine A-treated MDCK cell lines did not show any difference in either their hydrolytic or succinate dehydrogenase enzyme positivity with respect to the control line. In contrast, there was a significant increase in lactate dehydrogenase activity. This study allowed the possible mechanism of cyclosporine A-induced damage in renal tubular cells to be evaluated. The enzymatic changes happened rapidly (during the 24h of treatment), suggesting that this alteration was one of the steps by which cyclosporine A induced toxicity. Moreover, since acid phosphatase is a marker of protein catabolism, the variation in the activity of this enzyme, in the LLC-PK1 line only, showed that cyclosporine can induce alterations leading to cellular toxicity. The modifications in lactate dehydrogenase activity, in both lines, suggested that this drug caused cell stress, inducing the production of lactic acid from glucose in the presence of oxygen. In conclusion, cyclosporine A treatment may force LLC-PK1 and MDCK cells to use anaerobic glycolysis preferentially. Further, these enzyme alterations may represent an epiphenomenon or a consequence of cyclosporine A toxicity.     

Acid and alkaline phosphatases activities in vascular smooth muscle: species differences and subcellular distribution

1. Acid and alkaline phosphatase activities were studied in rat and dog aortic muscle using p-nitrophenyl phosphate (p-NPP) as the substrate. Alkaline phosphatase activity was quite comparable to acid phosphatase activity in rat aortic microsomes as well as further purified plasma membranes, but considerably lower than acid phosphatase activity in dog aortic membranes. 2. Subcellular distribution of acid and alkaline phosphatase activities in these vascular muscles indicated that alkaline phosphatases and a large portion of acid phosphatase activities were primarily associated with plasma membranes and the distribution of acid phosphatase showed little resemblance to that of N-acetyl-beta-glucosaminidase, a lysosomal marker enzyme. 3. The rat aortic plasmalemmal acid and alkaline phosphatase activities responded very differently to magnesium, fluoride, vanadate and EDTA. The alkaline phosphatase was more susceptible to heat inactivation than acid phosphatase. 4. These results suggest that these two phosphatases are likely to be two different enzymes in the smooth muscle plasma membranes. The implication of the present findings is discussed in relation to the alteration of these phosphatases in hypertensive vascular diseases.     

Localization of phosphatases in young gametophytes ofPolypodium vulgare L.

Summary The distribution of activity of several phosphatases was investigated in filamentous gametophytes of the fernPolypodium vulgare L. High levels of acid phosphatase, alkaline phosphatase, adenosine triphosphatase, and 5-nucleotidase were found in the rhizoids, where they may be concerned in the uptake of substances by the rhizoids. Glucose-6-phosphatase was localized mainly at the base of the rhizoid where it may be involved in the transport of sugars from the protonema to the rhizoid. In the protonema acid and alkaline phosphatases were localized mainly along the transverse walls, particularly along the distal surface, and it is suggested that they may be concerned in movement of substances along the protonema.     

Enzymes involved in the metabolism of flavin nucleotides in Streptomyces olivaceus

The work was aimed at studying enzymes involved in the metabolism of flavin nucleotides, namely, riboflavin kinase (EC 2.7.1.26) and FAD pyrophosphorylase (EC 2.7.7.2), as well as flavin mononucleotide hydrolysis by acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) in Streptomyces olivaceus actively producing vitamin B12. No correlation could be established between changes in the activity of the above enzymes during the culture growth and the qualitative composition of flavins. The enzyme activity was assayed using, as an enzyme preparation, both intact cells and a cell-free extract obtained by disintegrating the mycelium with different techniques. The screening effect of phosphatases exerted when the activity of riboflavin kinase was assayed could be partly eliminated by adding sodium fluoride to the incubation medium. The localisation of the above enzymes in the cytoplasm is discussed.     

A quantitative cytochemical investigation of osteoclasts and multinucleate giant cells

Summary Quantitative cytochemical, immunocytochemical, autoradiographic and electron cytochemical investigations have been used to compare osteoclasts with multinucleate giant cells that had been freshly obtained from the same animal. The levels of -acid galactosidase activity, the DNA in individual nuclei and the cellular protein content were similar in both cell types. However, osteoclasts generally possessed greater acid phosphatase and NADH dehydrogenase activity but lower levels of fluoride-inhibited non-specific esterase activity than multinucleate giant cells. The acid phosphatase activity in multinucleate giant cells was completely inhibited by 100 mM tartrate, but in osteoclasts only a 20% reduction in activity was observed. Formation of multinucleate giant cells in a bone microenvironment (thin bone slices) did not increase their content of tartrate-resistant acid phosphatase activity. Moreover, in osteoclasts, endogenous peroxidase activity was undetectable but present in several granules within the cytoplasm of multinucleate giant cells. Osteoclasts and multinucleate giant cells displayed a similar microtubular distribution, but calcitonin, which induced rearrangement of microtubules and cellular contraction in osteoclasts, had no effect on multinucleate giant cells. Thus, these investigations reveal both similarities and differences between these two syncytia and support the hypothesis that osteoclasts and multinucleate giant cells are related. Possibly osteoclasts arise from monocyte progenitors before commitment to a macrophage lineage has occurred.     

Histochemical and Ultrastructural Characterization of Vacuoles and Spherosomes as Components of the Lytic System in Hyphae of the Fungus Botrytis cinerea

An integrated approach to acid phosphatase (EC 3.1.3.2) histochemistry by the azo-dye and lead-capture (Gomori) methods in phosphate-starved hyphae of the fungus Botrytis cinerea revealed strikingly different patterns of localization of activity staining. Reaction product formed with the azo-dye method was found in numerous small organelles (<;0.5µm diameter), which also accumulated the lipophilic dye Nile Red and mislocalized the formazan indicating mitochondrial succinate dehydrogenase activity. Such small organelles were stained only weakly and sporadically with the lead-capture method; instead, lead phosphate deposits were produced mainly in large vacuoles (up to 2.5µm diam.), similar to those accumulating the vital dye Neutral Red. Additionally, acid phosphatase activity was detected in apical secretory vesicles with the lead-capture method but not with the azo-dye method. Ultrastructural studies by transmission electron microscopy confirmed the presence of large vacuoles which showed evidence of autophagic activity, and of small moderately osmiophilic organelles. The latter are considered to be spherosomes rather than lysosomes because of their weak reaction with the lead-capture method and their high lipid content. It is suggested that their apparently strong reaction with the azo-dye method is caused partly by false localization due to the lipophilic nature of the reaction product.     

Fine structural localization of alkaline phosphatase in the fracture callus of the rat

Summary The fine structural localization of nonspecific alkaline phosphomonoesterase in the different cells constituting the fracture callus in the rat was studied by incubating sections of glutaraldehyde-fixed callus tissue of variable age in media containing -glycerophosphate and either lead or calcium ions. The specificity of the reactions were tested by exposing the tissues to inhibitors of alkaline phosphatase.The results showed presence of final product on the plasma membranes and associated structures (subplasmalemmal endocytotic vesicles) of fibroblasts, pre-osteoblasts, osteoblasts, and cartilaginous cells in the callus. With the calcium method, reaction product was demonstrated in vesicular elements of the Golgi apparatus in osteoblasts and chondrocytes. Precipitates indicating presence of alkaline phosphatase activity were also observed on the membranes bordering cytoplasmic projections and fragments of cytoplasm located adjacent to enzyme-containing cells. Furthermore, the globule-shaped bodies in the matrix (Bonucci-bodies) showed evidence of alkaline phosphatase activity.The evidence obtained supported the view that alkaline phosphatase plays a role in calcification. It is suggested that transfer of cellular alkaline phosphatase to the sites of initial calcification in the extracellular matrix occurs by way of pinched off vesicular fragments of the cytoplasm and plasma membrane of osteogenic enzyme-producing cells; these structures appear to move awy from their cells of origin to form the Bonucci bodies in the matrix.     

Bleach-stable, alkaline protease from Bacillus sp.

A bleach-stable, thermotolerant, alkaline protease for detergent formulation from a newly isolated Bacillus SB5 is reported. Most (85%) activity of the enzyme was retained in the presence of 10% (v/v) H2O2 and 1% SDS (w/v) at 40°C, after 1 h. The enzyme was optimal at pH 10 and 60°C to 70°C. Enzyme activity was enhanced 30 to 80% in presence of ionic and non-ionic detergents, surfactants and commercial detergents or bleach.     

Effects of Bone Morphogenetic Protein-2 and Transforming Growth Factor-β1 on Gene Expression of Decorin and Biglycan by Cultured Osteoblastic Cells

The influence of bone morphogenetic protein-2 (BMP-2) and transforming growth factor (TGF-) on the expression of small proteoglycans, decorin and biglycan was investigated in a clonal rat osteoblastic cell line, ROS-C26 (C26) cells, which is a potential osteoblast precursor cell line and capable of differentiating into mature osteoblasts after treatment with recombinant BMP-2 (rhBMP-2). Following the culture of C26 cells for 3, 6, and 9 days in the presence or absence of rhBMP-2, alkaline phosphatase activity increased in the rhBMP-2 treated cells in direct proportion to their differentiation into more mature osteoblastic cells, whereas decorin mRNA decreased in the cells, when compared to control cells without rhBMP-2 treatment. These results were evident 6 days after treatment. However, rhBMP-2 treatment had no effect on biglycan mRNA expression in the cells. Subsequently, after removal of rhBMP-2 from the culture media, the cells were further cultured for 24h with graded concentrations of TGF-1 (0, 0.1, 1.0, 5.0, and 10ng/ml). TGF-1 decreased decorin mRNA expression in the cells dose dependently, but did not affect their biglycan mRNA expression. Furthermore, either removal of rhBMP-2 from the culture media or addition of TGF-1 significantly decreased alkaline phosphatase activity of rhBMP-2-induced cells. These results indicate that osteoblastic differentiation is accompanied by increased alkaline phosphatase activity and decreased expression of decorin mRNA, but continuous expression of biglycan mRNA. Both rhBMP-2 and TGF-1 inhibit decorin mRNA expression in osteoblasts at varying stages of differentiation, but their effects on biglycan mRNA expression and alkaline phosphatase are different.     

The histochemical differentiation of various phosphatases in a population of osteoclasts by a simultaneous coupling method using different diazonium salts, with observations on the presence of inhibitors in stable diazonium salts

Synopsis The numerous osteoclasts in a giant cell tumour of bone were found to possess at least two distinct phosphatases capable of hydrolysing naphthol AS-TR phosphate. An acid phosphatase, with optimum activity about pH 4.7, could be demonstrated by simultaneous coupling with Fast Bordeaux OL or Red Violet LB, but not with Fast Red TR. The last-named salt, on the other hand, could be used for demonstrating a phosphatase with an optimum pH of activity about 7.3, showing some activity as an alkaline phosphatase at pH 8.3. This enzyme was markedly inhibited by zinc ions and could not be demonstrated by simultaneous coupling with diazonium salts stabilized with zinc chloride. The acid phosphatase was much less sensitive to zinc, but showed marked inhibition by aluminium, which had comparatively little effect on the other enzyme. Some discrepancies between the published formulae of stable diazonium salts and the substances found to be present in them are discussed.     

Studies on the ultrastructural localization of adenosine triphosphatase activity in fracture callus

Summary The fine structural localization of Mg⁺⁺-activated and Mg⁺⁺-independent neutral adenosine triphosphatase (ATPase) was studied in fracture callus of the rat using EDTA-decalcified DMSO-treated tissues incubated in Wachstein-Meisel type lead-containing media, and N-ethylmaleimide, NaF, EDTA and histidine as inhibitors to test the specificity of the reaction. Final product was found to be deposited on the plasma membranes and associated structures (subplasmalemmal vesicles and vacuoles) of phagocytic monocytoid cells, fibroblasts, osteoblasts and ruffled border regions of osteoclasts when Mg⁺⁺ was present in the incubation medium; the most abundant precipitate was noted on the plasma membranes of osteoblasts. When Mg⁺⁺ was omitted from the medium, the ruffled borders of osteoclasts were the only plasmalemmal sites showing conspicuous activity. This apparently Mg⁺⁺-independent ATPase was also demonstrated in the lysosomes of all the different cell types in the callus and in the vacuoles and specific granules located beneath the ruffled border of osteoclasts; lack of inhibition with NaF suggested that the enzyme was not a conventional nonspecific acid phosphatase. Neither the Mg⁺⁺-activated nor the Mg⁺⁺-resistant ATPase were inhibited by EDTA or histidine, indicating that they were unrelated to non-specific alkaline phosphatase. Deposition of final product did not occur on the plasma membranes of chondroblasts, chondrocytes of osteocytes.     

Lack of Types 1 and 2A Protein Serine(P)/Threonine(P) Phosphatase Activities in Chloroplasts

Protein phosphatase activity in crude leaf extracts and in purified intact chloroplasts of wheat (Triticum aestivum) and pea (Pisum sativum) was analyzed using exogenously supplied phosphoproteins or endogenous thylakoid proteins. Leaf extracts contain readily detectable amounts of protein phosphatase activity measured with either phosphohistone or phosphorylase a, substrates of mammalian protein phosphatases. No significant chloroplast protein phosphatase activity was detected using these exogenous phosphoproteins. The dephosphorylation of endogenous thylakoid light-harvesting chlorophyll a/b binding proteins in situ was inhibited by fluoride, but not by microcystin-LR or okadaic acid, diagnostic inhibitors of mammalian types 1 and 2A protein phosphatases. Additionally, no evidence for a pea chloroplast alkaline phosphatase activity was found using β-glycerolphosphate or 4-methylum-belliferyl phosphate as substrates. From these results, we conclude that phosphohistone and phosphorylase a are not useful substrates for chloroplast thylakoid protein phosphatase activity and that the chloroplast enzymes may not fit into one of the canonical classifications currently used for protein phosphatases.