Differential effects of sodium butyrate and hyperosmolality on the modulation of alkaline phosphatases of LoVo cells

LoVo cells produce term-placental and intestinal alkaline phosphatases. Hyperosmolality and sodium butyrate increase the levels of both, but the effect of sodium butyrate is more pronounced on the intestinal enzyme. When applied together, induction of term-placental alkaline phosphatase is additive and that of the intestinal enzyme is synergistic. Induction by either stimulus or by their mixture is independent of cell density. However, whereas the effect of hyperosmolality is readily reversible, induction by sodium butyrate is not. No synergistic increase in intestinal alkaline phosphatase activity occurs when cells are sequentially treated with hyperosmolality and sodium butyrate or vice versa. This indicates that only when applied concurrently does one inducer amplify the effect of the other. Since the normal colonic mucosa produces intestinal alkaline phosphatase, its predominant induction by sodium butyrate in LoVo cells may reflect a more differentiated state.     

Modulation of alkaline phosphatases in LoVo, a human colon carcinoma cell line

LoVo, a continuous cell line derived from a human colon carcinoma produces two alkaline phosphatases: the heat-labile, L-homoarginine-insensitive, intestinal form, characteristic of its tissue of origin and the heat-stable, term-placental form, ectopically produced by a variety of tumors. Under basal conditions the activity levels of both enzymes are similar. Hyperosmolality and sodium butyrate induce increased levels of activity of the two alkaline phosphatases in a disparate fashion; whereas hyperosmolality augments the activity of both to the same extent, the effect of butyrate is more pronounced on the activity of the intestinal enzyme. When the two inducers are combined, induction of term-placental alkaline phosphatase is additive and that of the intestinal enzyme is synergistic. The effect of hyperosmolality is blocked by cycloheximide, and induction by sodium butyrate is inhibited by thymidine, cordycepin and cycloheximide. The known alkaline phosphatase inducer, prednisolone, has no effect on the enzymes of LoVo cells. Our results suggest that in these tumor cells the activity levels of the closely homologous term-placental and intestinal alkaline phosphatases appear to be independently controlled.     

Alkaline phosphatase protein increases in response to prednisolone in HeLa cells.

Quantification of term-placental alkaline phosphatase isoenzyme protein in HeLa TCRC-1 cells grown in the presence and absence of prednisolone indicates that there is a net increase in amount of enzyme-specific protein in prednisolone-stimulated cells. In a similar analysis of HeLa D98AH2 cells, prednisolone treatment causes the appearance of term-placental alkaline phosphatase protein and the loss of the intestinal isoenzyme protein. These results support the interpretation that the response of these cells to corticosteroids is the net accumulation of alkaline phosphatase protein rather than the modification of pre-existing enzyme to a more active state.     

Comparison of human alkaline phosphatase isoenzymes. Structural evidence for three protein classes.

The structural relationships among human alkaline phosphatase isoenzymes from placenta, bone, kidney, liver and intestine were investigated by using three criteria. 1. Immunochemical characterization by using monospecific antisera prepared against either the placental isoenzyme or the liver isoenzyme distinguishes two antigenic groups: bone, kidney and liver isoenzymes cross-react with anti-(liver isoenzyme) serum, and the intestinal and placental isoenzymes cross-react with the anti-(placental isoenzyme) antiserum. 2. High-resolution two-dimensional electrophoresis of the 32P-labelled denatured subunits of each enzyme distinguishes three groups of alkaline phosphatase: (a) the liver, bone and kidney isoenzymes, each with a unique isoelectric point in the native form, can be converted into a single form by treatment with neuraminidase; (b) the placental isoenzyme, whose position also shifts after removal of sialic acid; and (c) the intestinal isoenzyme, which is distinct from all other phosphatases and is unaffected by neuraminidase digestion. 3. Finally, we compare the primary structure of each enzyme by partial proteolytic-peptide 'mapping' in dodecyl sulphate/polyacrylamide gels. These results confirm the primary structural identity of liver and kidney isoenzymes and the non-identity of the placental and intestinal forms. These data provide direct experimental support for the existence of at least three alkaline phosphatase genes.     

The rabbit differs from other mammalian in the tissue distribution of alkaline phosphatase isoenzymes

There are only two gene loci code for alkaline phosphatase of mammalian other than human and great apes: one for the intestinal form and other for the liver/kidney/bone form. The former form is present only in the intestine and the latter form occurs in other tissues such as liver, kidney and bone. In the present study, the rabbit was found to be different from other mammalian in the tissue distribution of alkaline phosphatase isoenzymes: only in the rabbit, most of the enzyme in the kidney and liver was the third form which differs from the liver/kidney/bone form, and this form was enzymatically and immunologically similar to the intestinal form of ALPase.     

Comparative study of alkaline phosphatase isoenzymes,bone histology,and skeletal radiography in dialysis bone disease.

Liver, intestinal, and bone alkaline phosphatase isoenzymes were measured using heat stability and L-phenylalanine inhibition techniques in 78 patients on intermittent haemodialysis. Fifty-five patients had abnormalities in one or more of the isoenzymes. Changes in bone and intestinal alkaline phosphatase activities seemed to be related and raised liver isoenzyme activity was associated with the development of liver disease. Abnormal histological and radiological findings were better correlated with bone alkaline phosphatase levels than with total alkaline phosphatase, and serial estimations of bone isoenzyme activity were useful in assessing the response of renal osteodystrophy to treatment with a vitamin D analogue. Serum alkaline phosphatase isoenzyme measurement provides another useful and non-invasive index for monitoring metabolic bone disease in patients with chronic renal failure.     

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.     

The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum.

1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane.     

Levamisole inhibition of alkaline phosphatase and 5'-nucleotidase of bovine milk fat globule membranes

1. The effect of levamisole (LMS) on alkaline phosphatase (EC 3.1.3.1) and 5'-nucleotidase (EC 3.1.3.5) activities of bovine milk fat globule membranes (MFGM) was examined. 2. LMS inhibited MFGM alkaline phosphatase activity in a concentration-dependent manner with 50% inhibition produced by 49 +/- 23 microM LMS. 3. 5'-Nucleotidase was resistant to LMS inhibition with 30.9% inhibition produced by 10 mM LMS, the highest concentration tested. 4. LMS was an uncompetitive inhibitor of MFGM alkaline phosphatase with a Ki of 45 +/- 6 microM. 5. The extent of LMS inhibition of alkaline phosphatase was dependent on the substrate utilized in the assay. 6. The effect of LMS on bovine MFGM alkaline phosphatase was similar to LMS effects on other mammalian alkaline phosphatases of liver/kidney/bone/placental isoenzyme origin.     

Parathyroid hormone inhibition of induction of alkaline phosphatase activity in HeLa cells by 5-iodo-2′-deoxyuridine without increased adenylate cyclase activity or cellular cAMP concentration

Parathyroid extract (PTE) as well as purified parathyroid hormone (PTH) activators of adenylate cyclase in bone and kidney, produced dose-dependent decreases in the induction of alkaline phosphatase activity by 5-iodo-2′-deoxyuridine in HeLa cells. However, the combination of PTE and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor which also inhibits the induction of alkaline phosphatase activity, in most cases produced less than additive inhibition of enzyme induction. PTE or PTH in concentrations of up to 10 times greater than that necessary to have maximal effects on the induction of alkaline phosphatase activity produced no increase in adenylate cyclase activity, nor did they increase intracellular cAMP concentrations. In addition, PTE did not potentiate the increase in cAMP concentration produced by IBMX. It thus appears that the inhibition of alkaline phosphatase activity by PTH is not mediated by cAMP.     

Influence of Mg2+ ions on the activity measurement of isoenzymes of alkaline phosphatase

The activity of alkaline phosphatase isoenzymes from liver, bone and small intestine is differently influenced by Mg2+. The stimulation of isoenzymes from liver and bone is higher by Mg2+ ions than in the case of isoenzymes from small intestine. An obligatory preincubation of the serum sample in a buffer-Mg2+ mixture is necessary to avoid difficulties which may arise in the kinetic determination of alkaline phosphatase activity under extreme conditions, i.e. low Mg2+ concentration in serum, the necessity of dilution of the sample or the high isoenzyme content from liver or bone in the serum.     

Effects of combining transforming growth factor beta and 1,25-dihydroxyvitamin D3 on differentiation of a human osteosarcoma (MG-63).

Transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25D3), when added simultaneously to a human osteosarcoma cell line, MG-63, induce alkaline phosphatase activity 40-70-fold over basal levels, 6-7-fold over 1,25D3 treatment alone, and 15-20-fold over TGF beta treatment alone. TGF beta and 1,25D3 synergistically increased alkaline phosphatase specific activity in both matrix vesicles and plasma membrane isolated from the cultures, but the specific activity was greater in and targeted to the matrix vesicle fraction. Inhibitor and cleavage studies proved that the enzymatic activity was liver/bone/kidney alkaline phosphatase. Preincubation of MG-63 cells with TGF beta for 30 min before addition of 1,25D3 was sufficient for maximal induction of enzyme activity. Messenger RNA for liver/bone/kidney alkaline phosphatase was increased 2.1-fold with TGF beta, 1.7-fold with 1,25D3, and 4.8-fold with the combination at 72 h. Human alkaline phosphatase protein as detected by radioimmunoassay was stimulated only 6.3-fold over control levels with the combination. This combination of factors was tested for their effect on production of three other osteoblast cell proteins: collagen type I, osteocalcin, and fibronectin. TGF beta inhibited 1,25D3-induced osteocalcin production, whereas both factors were additive for fibronectin and collagen type I production. TGF beta appears to modulate the differentiation effects of 1,25D3 on this human osteoblast-like cell and thereby retain the cell in a non-fully differentiated state.     

Cytochemical detection and cytofluorimetric analysis of alkaline phosphatase in continuous lines of thymic lymphomas induced in mice by a radiation leukemia virus

Alkaline phosphatase was studied in cell lines established from Radiation Leukemia Virus induced thymic lymphomas of C57BL/Ka mice. The cytochemical staining techniques and flow cytofluorimetry analysis described by Dolbeare et al. (J. Histochem. Cytochem., 1980, 28, 419-426) were used. The alkaline phosphatase found in lymphoma cells was heat labile and L-homoarginine. L-phenylalanine and p-bromotetramisole sensitive and is probably similar to the isoenzyme present in mouse placenta, kidney and liver. Very little alkaline phosphatase activity was detected in normal thymus of adult mice, suggesting that the method used in the paper could be helpful for studying the emergence of the first neoplastic cells during the leukemogenic process.     

Induction of Rat Liver Alkaline Phosphatase by Bile Duct Ligation

Bile duct ligation causes a five- to sevenfold increase in the activity of rat liver alkaline phosphatase within 12 hours after ligation and a similar rise in the activity of alkaline phosphatase in serum. The increased serum activity is due entirely to the appearance of a new isoenzyme that has the properties of rat liver alkaline phosphatase. The increase in both serum and liver alkaline phosphatase is prevented by the prior administration of cycloheximide in a dose that inhibits protein synthesis by 70%. Rat liver alkaline phosphatase was then purified to homogeneity. Antibody was raised to purified rat liver alkaline phosphatase in rabbits. The antibody was coupled to sepharose 4B and affinity columns made. ³-H-leucine was then injected into the portal veins of sham operated rats and rats with bile duct ligation four hours after ligation. One hour after injection and five hours after ligation, animals were sacrificed. Liver alkaline phosphatase was purified by means of affinity chromatography and double immunoprecipitation with rabbit antibody to rat liver alkaline phosphatase and goat anti-rabbit gamma globulin. Bile duct ligation increased the incorporation of ³-H-leucine into liver alkaline phosphatase more than threefold compared with sham operated rats, 164 CPM/mg protein vs. 49 CPM/mg protein (p < .001). The data indicate that the increased activity of rat liver alkaline phosphatase after bile duct ligation is due to enzyme induction rather than to activation of a pre-existing, relatively inactive enzyme.     

Alkaline phosphatase in mitochondria

In electron microscope cytochemical studies alkaline phosphatase activity was present in the mitochondria of all liver cells and associated with the plasma membrane of the cells of bile canaliculi. The mitochondrial activity was partially inhibited by L-phenylalanine and Levamisole but the plasma membrane associated activity was completely inhibited by Levamisole. Biochemical assays have shown that a significant amount of the total mouse liver alkaline phosphatase activity was present in the mitochondrial fraction. Starch gel electrophoresis showed that this mitochondrial alkaline phosphatase had a characteristic isoenzyme pattern, consisting of 3 distinct bands which were not retarded by neuraminidase treatment. The enzyme in the mitochondria-free supernatant showed one wide band which was retarded by neuraminidase.     

Alkaline phosphatase and an acid arylamidase as marker enzymes for normal and transformed WI-38 cells

A survey of eleven enzyme activity levels in normal and SV40 transformed (VA-13) WI-38 cells revealed that the transformed cell enzymes differed by a quantitative and qualitative change of alkaline phosphatase and a quantitative loss of an arylamidase. Alkaline phosphatase activity was found to be elevated in the transformed cells at confluency but not in log phase cultures. This elevated activity was heat stable, L-homoarginine resistant and L-phenylalanine sensitive and is probably the term placental isoenzyme. In nontransformed WI-38 cells, the alkaline phosphatase was heat labile, L-homoarginine sensitive and L-phenylalanine resistant and so is probably the liver isoenzyme. While the arylamidase activity from both normal and transformed WI-38 cells had identical pH optima and Km values, the activity was approximately 20 times higher in confluent WI-38 cells than in confluent VA-13 cells. Cytochemical staining techniques for both activities are described that permit identification of fluorescent product within the cells, analysis of activity levels, and separation of cells with high and low activities. Mixtures of WI-38 cells and VA-13 cells separated by flow cytometry on the basis of arylamidase activity were subsequently evaluated for alkaline phosphatase isoenzyme and found to have been simultaneously separated into heat labile and heat stable samples.     

Differential theophylline inhibition of alkaline phosphatase and 5'-nucleotidase of bovine milk fat globule membranes

1. The effects of theophylline (1,3-dimethylxanthine) on alkaline phosphatase and 5'-nucleotidase activities of bovine milk fat globule membranes (MFGM) were examined. 2. Theophylline inhibited MFGM alkaline phosphatase in a concentration-dependent manner with 50% inhibition produced by 99 +/- 28 microM theophylline. 3. The 5'-nucleotidase activity was resistant to theophylline inhibition with 50% inhibition produced by 33.9 +/- 3.1 mM theophylline. 4. Theophylline was an uncompetitive inhibitor of MFGM alkaline phosphatase with a Ki of 126 +/- 15 microM. 5. The extent of theophylline inhibition of alkaline phosphatase activity was independent of the substrate utilized in the assay. 6. The effect of theophylline on bovine MFGM alkaline phosphatase was similar to theophylline effects on other mammalian alkaline phosphatases of liver/bone isoenzyme origin.