Colchicine increases hepatic alkaline phosphatase activity

In vivo administration of colchicine increases the activity of alkaline phosphatase significantly in the livers of rats. Prior treatment with cycloheximide prevented the induction of the enzyme by colchicine suggesting that de novo protein synthesis was essential for the effect of colchicine on alkaline phosphatase activity. Bilateral adrenalectomy did not affect the response of alkaline phosphatase following the administration of colchicine. This indicates that the rise in the level of alkaline phosphatase in liver caused by colchicine is not secondary to the release of glucocorticoids.     

Measurement of serum bone-specific alkaline phosphatase activity in cynomolgus macaques

Serum levels of bone-specific alkaline phosphatase activity (B-ALP) in cynomolgus monkeys were evaluated as an index of elevated bone turnover following ovariectomy. The enzyme immunoassay 96-well microtiter plate B-ALP assay, developed by Metra Biosystems (Mountain View, CA) for human use, was employed and compared with a standard automated assay measuring total serum levels of alkaline phosphatase activity (T-ALP). The B-ALP assay was first validated for use in these monkeys. Ovariectomy led to increased bone turnover as indicated by approximately 2-fold higher activity in both assays and this elevation was inhibited by daily estradiol administration. Although both assays provided generally similar results, several monkeys were observed to have greatly elevated values of T-ALP but not B-ALP. This discrepancy is believed to result from high levels of the liver isoform of alkaline phosphatase in monkeys with hepatic dysfunction, which are not detected by the B-ALP assay.     

Hepatic alkaline phosphatase isoenzymes: isolation, characterization and differential alteration.

Although it is generally believed that hepatic alkaline phosphatase is localized to liver plasma membranes, 63% is present in the cytosol fraction after ultracentrifugation of rat liver homogenates. Divalent cation requirements, heat inactivation, pH optima, Km and chemical inhibition characteristics of partially purified alkaline phosphatase enzymes prepared from membrane and cytosol fractions suggested different structural forms. Furthermore, bile duct obstruction and ethinyl estradiol administration preferentially increased membrane-bound alkaline phosphatase activity, while cytosol activity was unaltered. In contrast, phenobarbital treatment decreased membrane-bound alkaline phosphatase and increased cytosol activity. These studies support the presence of two forms of hepatic alkaline phosphatase in rat liver which are regulated by different control mechanisms.     

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.     

EFFECT OF THYROID DEFICIENCY ON THE LEVELS OF SEVERAL ENZYMES IN RAT BRAIN

—Activities of acid phosphatase, alkaline phosphatase and β-glucuronidase have been estimated in the brain tissues, using various subcellular particles, in growing thyroidectomized rats and also using cytoplasmic extracts free from debris and nuclear fraction in young hypothyroid animals. Hepatic glucose-6-phosphate dehydrogenase activity was markedly reduced after thyroidectomy but the enzyme was brought back to normal levels by thyroxine treatment. There was no change, however, in the activity of neural glucose-6-phosphate dehydrogenase after thyroidectomy. In the thyroidectomized animals an increase only in the free acid phosphatase activity in the neural synaptosomes was found and this increase in activity was not counteracted by administration of thyroxine. In the hypothyroid young animal β-glucuronidase, acid phosphatase and alkaline phosphatase activities were found to be affected during development.     

Interaction of H2-receptor antagonists, cimetidine and ranitidine with microsomal drug metabolizing and other systems in liver.

Cimetidine has been demonstrated to impair microsomal oxidative drug metabolizing and other enzyme systems in mouse liver. The inhibition is rapid, occurring after a single administration and also found to be dose-dependent. It is more significant after daily administration for 15 days. Enzyme inhibition by ranitidine, another H2-receptor antagonist was comparatively less at all the concentrations of the drug tested. An increased activity of alkaline phosphatase, glutamate-pyruvate and glutamate-oxaloacetate transaminase was observed in liver with cimetidine administration, whereas that of lactate and succinate dehydrogenase was inhibited only after administration of 2000 mg cimetidine per kg body weight. Except alkaline phosphatase other enzymes were unaffected after ranitidine administration. Analysis of lipid classes in liver showed that phospholipid, triglycerides and free fatty acid contents were significantly decreased in drug administration while cholesterol level showed very little or no change. Microsomal and soluble protein contents were significantly increased which probably indicate that the inhibition in the enzyme activity by histamine H2-receptor antagonists may be a lipid mediated process and not resulted from the reduced availability of the enzyme protein.     

Zinc metabolism in testicular feminization and surgical cryptorchid testes in rats

Rats with testicular feminization (Tfm) had been reported to have a testis specific zinc deficiency. In this report it is demonstrated that this organ specific zinc deficiency was not corrected by intraperitoneal zinc administration. Normal littermates on the other hand showed a positive testicular response to zinc administration. The increased testicular zinc level in control animals returned to normal 1 week after the zinc treatment probably due to the fast turnover of this element in the testis. Not only surgically induced cryptorchidism but also surgical cryptorchidism and epididymodeferentectomy (to simulate Tfm conditions in normal adult rats) caused a drastic reduction in testicular zinc level. Unlike in Tfm rats, however, the decrease in zinc content in operated animals was not accompanied by a corresponding decrease in alkaline phosphatase activity. Zinc concentration and alkaline phosphatase activity in plasma or other organs were not affected by the surgical procedure. The testicular copper content in the operated animals was higher than that of the unoperated controls.     

Independence of 1,25-dihydroxyvitamin D3-mediated calcium transport from de novo RNA and protein synthesis

The administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to rachitic chicks produces an increase in (a) RNA and protein synthesis, (b) calcium binding protein (CaBP) concentration, and (c) alkaline phosphatase activity in the duodenum. These events occur concomitantly with enhanced calcium transport. We inhibited RNA and protein synthesis in richitic chicks and measured the subsequent response to 1,25(OH)2D3. Actinomycin D, injected prior to and following 1,25(OH)2D3 administration, inhibited intestinal RNA polymerase activity, blocked the rise in serum calcium, reduced the amount of CaBP, and increased alkaline phosphatase activity. Cycloheximide injected in similar fashion, inhibited the 1,25(OH)2D3-mediated increase in intestinal protein synthesis, serum calcium, CaBP, and alkaline phosphatase activity. Neither inhibitor blocked the ability of 1,25(OH)2D3 to stimulate calcium transport as measured in isolated duodenal loops in vivo. The ability of either inhibitor to block 1,25(OH)2D3-mediated calcium transport despite inhibition of CaBP production and alkaline phosphatase activity (by cycloheximide) indicates that de novo RNA and protein synthesis, and in particular CaBP and alkaline phosphatase, are not required for the 1,25(OH)2D3 stimulation of calcium transport.     

Effect of estrogen administration on activities of testosterone 5alpha-reductase, alkaline phosphatase and arginase in the ventral and the dorsolateral prostates of rats.

Daily treatment with 5 or 500 mug of estradiol benzoate of male adult rats for 7 days did not change the activity of testosterone 5alpha-reductase in the ventral prostate, while the activity decreased slightly in the dorsolateral prostate. The activity of alkaline phosphatase was increased by 60% over the respective control in the ventral prostate from rats treated with the larger dose of estrogen, but the estrogen treatment did not affect the alkaline phosphatase activity in the dorsolateral prostate. On the contrary, the estrogen treatment evoked three-fold elevation in the arginase activity of the dorsolateral prostate in contrast to the decreased arginase activity in the ventral prostate following estrogen administration. From these results, it was concluded that the alterations in some enzyme activity of the ventral and the dorsolateral prostates evoked by estrogen treatment were different from those observed in the respective lobes from castrated animals, although both estrogen administration and castration induced atrophy of the tissue. Furthermore, it might be also worth-while to mention that the ventral and the dorsolateral prostates of rats responded in a different manner to estrogen administered.     

Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases

Purified alkaline phosphatase and plasma membranes from human liver were shown to dephosphorylate phosphohistones and plasma membrane phosphoproteins. The protein phosphatase activity of the liver plasma membranes was inhibited by levamisole, a specific inhibitor of alkaline phosphatase, and by phenyl phosphonate and orthovanadate, but was relatively insensitive to fluoride (50 mM). Endogenous membrane protein phosphatase activity was optimal at pH 8.0, compared to pH 7.8 for purified liver alkaline phosphatase. Plasma membranes also exhibited protein kinase activity using exogenous histone or endogenous membrane proteins (autophosphorylation) as substrates; this activity was cAMP-dependent. Autophosphorylation of plasma membrane proteins was apparently enhanced by phenyl phosphonate, levamisole, or orthovanadate. The dephosphorylation of phosphohistones by protein phosphatase 1 was not inhibited by levamisole but was inhibited by fluoride. Inhibition of endogenous protein phosphatase activity by orthovanadate during autophosphorylation of plasma membranes could be reversed by complexation of the inhibitor with (R)-(-)-epinephrine, and the dephosphorylation that followed was levamisole-sensitive. Neither plasma membranes nor purified liver alkaline phosphatase dephosphorylated glycogen phosphorylase a. These results suggest that the increased [32P]phosphate incorporation by endogenous protein kinases into the membrane proteins is due to inhibition of alkaline phosphatase and that the major protein phosphatase of these plasma membranes is alkaline phosphatase.     

Absence of increase of serum alkaline phosphatase activity with parenteral nutrition-associated cholestasis: possible consequence of hypozincemia and hypomagnesemia

We reviewed charts of 38 Patients who developed cholestasis whilst receiving total parenteral nutrition (TPN). A standard protocol was followed for administration of TPN and included crystalline amino acid solution with lipid emulsion and dextrose as calorie sources. 5 of the 38 patients did not exhibit an increase in alkaline phosphatase. This may be related to the concomitant low levels of either serum zinc or magnesium or both. We also obtained a positive correlation between serum levels of zinc and magnesium and the peak activity of alkaline phosphatase (r = 0.49, p less than 0.01, r-0.55, p less than 0.01, respectively) associated with cholestasis. We hypothesize that the reason for this association is related to both zinc and magnesium being activators of alkaline phosphatase activity.     

A highly sensitive assay method for human placental alkaline phosphatase involving a monoclonal antibody bound to a paper disk

A monoclonal antibody which is specific for human placental alkaline phosphatase and does not cross-react at all with intestinal alkaline phosphatase was prepared, and a procedure for the determination of placental alkaline phosphatase activity in serum was developed involving this monoclonal antibody bound to a paper disk. The minimum amount of placental alkaline phosphatase detectable by this method is 0.0025 King-Armstrong unit. Good correlation with the heat-treatment method was obtained. Therefore this proposed method can be used as a routine clinical test for the determination of serum placental alkaline phosphatase.     

In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.     

Alkaline phosphatase in the lactating bovine mammary gland and the milk fat globule membrane. Release by phosphatidylinositol-specific phospholipase C.

1. Alkaline phosphatase is covalently bound to bovine mammary microsomal membranes and milk fat globule membranes through linkage to phosphatidylinositol as demonstrated by the release of alkaline phosphatase following treatment with phosphatidylinositol-specific phospholipase C. 2. The release of alkaline phosphatase from the pellet to the supernatant was demonstrated by enzyme assays and electrophoresis. 3. Electrophoresis of the solubilized enzymes showed that the alkaline phosphatase of the microsomal membranes contained several isozymes, while only one band with alkaline phosphatase activity was seen in the fat globule membrane. 4. Levamisole and homoarginine were potent inhibitors of the alkaline phosphatase activities in both membrane preparations and in bovine liver alkaline phosphatase, but not in calf intestinal alkaline phosphatase.     

Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum

Summary Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.     

Alkaline phosphatase activation and collagen synthesis in human skin fibroblasts in culture

It has been noted in regenerating wounds that alkaline phosphatase activity in fibroblasts reaches a maximum when the collagen production is greatest. Tissue culture studies were carried out to show that prednisolone phosphate, while increasing the specific activity of alkaline phosphatase in human diploid skin fibroblasts, did not affect accumulation of collagen-hydroxyproline in monolayers or media. Addition of sodium ascorbate, resulted in rapid accumulation of hydroxyproline in the culture over a 13-day interval, while alkaline phosphatase activity increased only slightly over the last 6 days. When prednisolone and ascorbate were added in combination, alkaline phosphatase activity was significantly increased: but accumulation of hydroxyproline was no greater than in cultures to which ascorbate alone was added. Activation of alkaline phosphatase induced by prednisolone phosphate does not appear to be directly related to the biosynthesis of collagen in human skin fibroblasts in tissue culture.     

Treatment of immature mice with gonadotrophins. Effects on some enzymic activities of unfractionated ovarian homogenates

Treatment of immature mice with both follicle-stimulating hormone and human chorionic gonadotrophin in vivo resulted in large increases in the specific activities of ovarian alkaline phosphatase and alkaline nucleotidase. The specific activities of other ovarian enzymes studied were not altered by gonadotrophin treatment. A simultaneous change in the Michaelis constant of ovarian alkaline phosphatase accompanied the increase in specific activity. These changes commenced 6-8h after injection of human chorionic gonadotrophin plus follicle-stimulating hormone. Injection of human chorionic gonadotrophin induced the change in Michaelis constant and increased ovarian alkaline phosphatase activity. Treatment with follicle-stimulating hormone had no effect on ovarian alkaline phosphatase. However, follicle-stimulating hormone synergistically augmented the response to human chorionic gonadotrophin. A latent period of about 24h elapsed before this augmentation was expressed. Augmentation of ovarian alkaline phosphatase was directly related to the dose of follicle-stimulating hormone at a fixed dose of chorionic gonadotrophin. No response of ovarian alkaline phosphatase was observed after treatment of immature mice in vivo with oestrogens, progesterone, growth hormone or prolactin. Unlike chorionic gonadotrophin, sheep luteinizing hormone over a wide dose range induced no response within 24h. However, a response in ovarian alkaline phosphatase was observed when sheep luteinizing hormone was administered in combination with follicle-stimulating hormone. The specific activity and K(m) of ovarian alkaline phosphatase increased during normal maturation. The Michaelis constant ceased to increase as sexual maturity was reached. The changes in alkaline phosphatase activity were of a similar magnitude to those induced by gonadotrophin treatment. It is concluded that the changes induced acutely by treatment in vivo with unphysiological doses of gonadotrophins occur in the maturing mouse under the influence of endogenous, homologous gonadotrophins at physiological concentrations.     

dl-Buthionine-S,R-sulfoximine affects intestinal alkaline phosphatase activity

The susceptibility of intestinal alkaline phosphatase to dl-buthionine-S,R-sulfoximine was investigated in chicks fed a commercial diet. The results show that dl-buthionine-S,R-sulfoximine produced inhibition of intestinal alkaline phosphatase activity. This effect showed dose- and time-dependency and it was caused by either in vivo dl-buthionine-S,R- sulfoximine administration or in vitro dl-buthionine-S,R-sulfoximine incubation with villus tip enterocytes. dl-Buthionine-S,R-sulfoximine did not act directly on intestinal alkaline phosphatase but it provoked glutathione depletion which led to changes in the redox state of the enterocyte as shown by the production of free hydroxyl radicals and an incremental increase in the carbonyl content of proteins. The reversibility of the buthionine sulfoximine effect on intestinal alkaline phosphatase was proved by addition of glutathione monoester to the duodenal loop.     

Species and organ dependence of alkaline phosphatase activity in lymphatic tissues. A histochemical, biochemical and electrophoretic study

Synopsis Alkaline phosphatase activity in lymphatic tissues of guineapig, cat, cow, dog, rabbit, sheep, rat, mouse, hamster, chicken and man was studied with histochemical, biochemical and electrophoretic techniques. The thymus showed decreasing alkaline phosphatase activity from species to species in the order just given. Activity of alkaline phosphatase in other lymphatic tissues did not show such clear species and organ dependence. Spleens of the cat, cow and rabbit and lymph nodes of the cow and sheep gave, however, very characteristic patterns of alkaline phosphatase activity. In the chicken there was no difference between the alkaline phosphatase content of the thymus and that of the bursa of Fabricius. The lymphatic follicles of human tonsils and appendix and in the appendix of the rabbit exhibited alkaline phosphatase activity in the circular cell layer. This was also seen in some follicles in the lymph nodes of certain species. Electrophoretically, the main alkaline phosphatase fraction of the lymphatic tissues closely resembled the main fraction of blood, though it is probably not identical with it. Although the biological function of alkaline phosphatase is unknown, the greatly varying alkaline phosphatase content in different lymphatic organs of different species indicates that immunological studies with one species or with cells derived from a certain lymphatic tissue or with both are probably not directly comparable with studies using other species or cells from other lymphatic tissues.