A histochemical analysis of the enzymes of the enterocyte transport and bioenergetics in rat small intestine after vagotomy and dibunol administration

It was shown that the vagotomy leads to the elevation of the activity of alkaline phosphatase (AP) 7 days after the operation and to the decrease of its activity 30 days after the operation, and to the decrease of the activity of succinic dehydrogenase (SDG) 7 and 30 days after the operation. Introduction of dibunol leads to normalization of these disturbances of the activity of AP and SDG induced by vagotomy.     

Phosphatase activity in the larva of the euryhaline mosquito, Aëdes togoi Theobald,with special reference to sea-water adaptation

The alkaline and acid phosphatases in larvae of the euryhaline mosquito, Aëdes togoi Theobald, were measured and the distribution of alkaline phosphatase was examined histochemically. The optima pH for alkaline and acid phosphatases in the larvae were ≈9.0 and 3.2. respectively. The thorax region showed the highest activity of alkaline phosphatase. The enzyme activity of the thorax of seawater adapted larvae was about twice as high as that of freshwater larvae. When the larvae were transferred from fresh water to sea water, the alkaline phosphatase activity of the thorax increased greatly for 3 days, and thereafter decreased to the normal level of sea-water adapted larvae within seven days. In larvae transferred from sea water to fresh water, the activity of the thorax decreased gradually and after 7 days remained at the level of freshwater adapted larvae. No change in acid phosphatase activity was detected following transfer of the larvae from fresh water to sea water or vice versa. A strong alkaline phosphatase reaction was found only in the luminal border of the gastric caeca in the thorax region. The locality of this enzyme did not vary according to the salinity of environmental water.The activity change of alkaline phosphatase of the gastric caeca is discussed in relation to the absorption of the ingested medium from the gastric caeca.     

Sub-chronic diclofenac sodium induced alterations of alkaline phosphatase activity in serum and skeletal muscle of mice

The present study has been carried on changes in activity of alkaline phosphatase in serum and gastrocnemius muscle of mice after sub-chronic use of diclofenac. Mice in experimental group received diclofenac (10 mg/kg body wt /day) for 30 days while control group received normal saline. Alkaline phosphatase was assayed in muscle and serum and its activity was localized histochemically in muscle. Results showed that diclofenac induced changes in specific activity of alkaline phosphatase at different periods of treatment variably compared to control group. Specific activity of alkaline phosphatase decreased significantly in gastrocnemius initially (48.74%), increased thereafter (132.96%) and slight decrease (13.97%) was noticed after 30 days. In serum, the specific activity of alkaline phosphatase decreased slightly after 10 days (18.78%), increased in the middle of the treatment period (132.04%) as well as showed increase (109.09%) compared to control group after 30 days stage of investigation. These findings were also confirmed by electrophoretic studies in muscle.     

The activity of alkaline phosphatase in the process of regeneration of rat liver.

Using GOMORI'S technique, the present authors investigated the dynamics of the alkaline phosphatase activity in the process of liver regeneration after partial hepatectomy. In all 80 rats of the Wistar strain were subjected to experiment, 60 to 75% of the liver parenchyma being removed from each of them. In the course of regeneration a gradual increase in the enzyme activity was observed within the first 48 hours following the operation. This was succeeded by a slow decline of the activity, and after the 25th day after the operation the reaction intensity resembled that recorded for the control animals. It was also ascertained that the fatty degeneration of the liver noted in the initial period of regeneration does not inhibit the activity of alkaline phosphatase.     

Architecture of implanted bone matrix gelatin influences heterotopic calcification and new bone formation

Heterotopic bone formation in skeletal muscle induced by compacted demineralized bone matrix gelatin (BMG) was studied histologically and biochemically. BMG was obtained by dehydrating diaphyseal shafts of femora and tibiae of 4-week-old male Sprague-Dawley rats, cutting the bone into chips, and demineralizing and extracting the chips with various solutions. The BMG was treated with 4 M guanidine-HCl, and compacted BMG was prepared by centrifugation. The compacted BMG was implanted into the rectus abdominis muscle of 5-week-old male Sprague-Dawley rats. The resulting specimens were examined histologically, and their alkaline phosphatase activity and the calcium content of the tissues were measured 3, 5, 7, 10, and 15 days after implantation. The BMG (separated BMG) with 75- to 500-microns particle sizes were implanted into control rats. The results showed that calcification, alkaline phosphatase activity, and bone formation were suppressed by implantation of the compacted BMG and that scarcely any vascularization occurred. Calcification, vascularization, and alkaline phosphatase activity were related and were indispensable for bone formation. In the control group, bone formation was observed at sites of high activity of alkaline phosphatase and well-developed vascularization. These results suggested that compacting of BMG suppressed vascularization, decreased calcification, and consequently reduced the induction of bone formation.     

Fine structural localization of alkaline phosphatase in the granular pneumonocytes of hamster lung.

The fine structural localization of nonspecific alkaline phosphatase was studied in the granular pneumonocytes (type II alveolar epithelial cells) of hamster lung by incubating sections of glutaraldehyde-fixed tissues in a medium containing lead ions and sodium beta-glycerophosphate or alpha-naphthyl acid phosphate. The specificity of the reaction was tested by exposing the sections to inhibitors of alkaline phosphatase. The results showed that alkaline phosphatase activity was present in the inclusion bodies of granular pneumonocytes. The enzyme reaction was strong in the membrane lining the inclusion bodies and a weaker reaction was generally detectable in the inclusion contents. Although only a proportion of the inclusion bodies showed enzyme activity, there was no obvious correlation between the reactivity of the inclusions and their intracellular position or size. The other organelles were unreactive. The finding of alkaline phosphatase activity within the inclusion bodies of granular pneumonocytes is an enigma as these organelles are generally considered to be lyosomes.     

The relation of the soluble thiamine triphosphatase activity of various rat tissues to nonspecific phosphatases

Polyacrylamide gel electrophoresis was used to investigate the relation of the soluble thiamine triphosphatase activity of various rat tissues to other phosphatases. This technique separated the thiamine triphosphatase of rat brain, heart, kidney, liver, lung, muscle and spleen from alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and other nonspecific phosphatase activities. In contrast, the hydrolytic activity for thiamine triphosphate in rat intestine moved identically with alkaline phosphatase in gel electrophoresis. Thiamine triphosphatase from rat liver and brain was also separated from alkaline phosphatase and acid phosphatase by gel chromatography on Sephadex G-100. This gave an apparent molecular weight of about 30,000 and a Stokes radius of 2.5 nanometers for brain and liver thiamine triphosphatase. The intestinal thiamine triphosphatase activity of the rat was eluted from the Sephadex G-100 column as two separate peaks (with apparent molecular weights of over 200,000 and 123,000) which exactly corresponded to the peaks of alkaline phosphatase. The isoelectric point (pI) of the brain thiamine triphosphatase was 4.6 (4 degrees C). The partially purified thiamine triphosphatase from brain and liver was highly specific for thiamine triphosphate. The results suggest that, apart from the intestine, the rat tissues studied contain a specific enzyme, thiamine triphosphatase (EC 3.6.1.28). The specific enzyme is responsible for most of the thiamine triphosphatase activity in these tissues. Rat intestine contains a high thiamine triphosphatase activity but all of it appears to be due to alkaline phosphatase.     

5-Bromo-2'-deoxyuridine induces placental alkaline phosphatase biosynthesis in cultured choriocarcinoma cells

5-Bromo-2'-deoxyuridine (BrdUrd) stimulated the biosynthesis and hence increased the activity of placental alkaline phosphatase in choriocarcinoma cells. While BrdUrd had no effect on the rate of degradation or processing of placental alkaline phosphatase, it increased the rate of phosphatase synthesis. The stimulation of enzyme activity could be completely accounted for by the increase in alkaline phosphatase protein. Both control and BrdUrd-induced cells contained polypeptides of 61,500 and 64,500 Da, identified as the precursor and fully processed forms of placental alkaline phosphatase monomer. The half-life of this enzyme monomer in both control and BrdUrd-treated cells was estimated to be 36 h. BrdUrd induced a specific increase in the placental alkaline phosphatase mRNA leading to the observed enhancement of biosynthesis. The continued rise in alkaline phosphatase biosynthesis in BrdUrd-induced cells following BrdUrd removal indicated that this analog acted by incorporation into DNA.     

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.     

Mepe is expressed during skeletal development and regeneration

Matrix extracellular phosphoglycoprotein (Mepe) is a bone metabolism regulator that is expressed by osteocytes in normal adult bone. Here, we used an immunohistochemical approach to study whether Mepe has a role in murine long bone development and regeneration. Our data showed that Mepe protein was produced by osteoblasts and osteocytes during skeletogenesis, as early as 2 days postnatal. During the healing of non-stabilized tibial fractures, which occurs through endochondral ossification, Mepe expression was first detected in fibroblast-like cells within the callus by 6 days postfracture. By 10 and 14 days postfracture (the hard callus phase of repair), Mepe was expressed within late hypertrophic chondrocytes and osteocytes in the regenerating tissues. Mepe became externalized in osteocyte lacunae during this period. By 28 days postfracture (the remodeling phase of repair), Mepe continued to be robustly expressed in osteocytes of the regenerating bone. We compared the Mepe expression profile with that of alkaline phosphatase, a marker of bone mineralization. We found that both Mepe and alkaline phosphatase increased during the hard callus phase of repair. In the remodeling phase of repair, Mepe expression levels remained high while alkaline phosphatase activity decreased. We also examined Mepe expression during cortical bone defect healing, which occurs through intramembranous ossification. Mepe immunostaining was found within fibroblast-like cells, osteoblasts, and osteocytes in the regenerating bone, through 5 to 21 days postsurgery. Thus, Mepe appears to play a role in both long bone regeneration and the latter stages of skeletogenesis.     

Effect of starvation and sampling time on plasma alkaline phosphatase activity and calcium homeostasis in the rat

The effect of starvation and sampling time on plasma alkaline phosphatase activity, total plasma calcium concentration and whole blood ionized calcium concentration was determined in the rat. Starvation caused a significant fall in total and ionized calcium concentrations as well as in alkaline phosphatase activity. These changes were accompanied by a fall in whole blood pH and an increase in the anion gap and a decrease in urinary excretion of calcium. These indices were restored to normal following refeeding. There was no change in serum 25-OH vitamin D concentrations following starvation for 3 days. Alkaline phosphatase activity showed a pattern compatible with the presence of a circadian rhythm when sampling took place between 0800 and 1800 h. Total and ionized calcium concentrations did not show such a rhythm when animals were fed the present diet.     

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.     

The inducible alkaline phosphatase of rat heart. Some properties of the enzyme and factors influencing its activity

Subcutaneous injection of isoprenaline into rats produces a rapid rise in alkaline phosphatase activity which is largely confined to the right atrium of the heart. The phosphatase activity of extracts of atrial tissue from treated animals is on average four times that of controls. The rise in activity is largely suppressed by previously treating the animals with cycloheximide or actinomycin D. Comparison of the properties of the alkaline phosphatase in extracts of atria from isoprenaline-treated rats with control preparations shows the stimulated enzyme activity to be similar to that present in the resting state. In a number of respects, including substrate specificity, response to activators and inhibitors, rate of loss of activity on heating and electrophoretic mobility of the native and modified enzyme, the atrial phosphatase resembles alkaline phosphatases from other mammalian non-intestinal and non-placental tissues. These results suggest that the effect of isoprenaline is to increase the rate of synthesis of atrial alkaline phosphatase, and the ability of the tissues of the right atrium to respond in this way may be related to the rich sympathetic innervation of this part of the heart.     

Acid and alkaline phosphatase activities in the clam Scrobicularia plana: kinetic characteristics and effects of heavy metals

The acid and alkaline phosphatase activities of the clam Scrobicularia plana have been partially characterised in different organs and tissues (digestive gland, gills, foot, siphon and mantle) and the 'in vitro' effect of heavy metals on both types of enzymatic activity have been analysed. The optimal pH ranged between 4.0 and 5.5 for acid phosphatase activity and 8.5 and 9.5 for alkaline phosphatase activity. The apparent optimum temperature was in the 30-60 degrees range for acid phosphatase activity and in the 30-40 degrees C range for alkaline phosphatase activity. The effect of substrate concentration on enzymatic activities in the tissues showed a good fit to the Michaelis-Menten model. For both types of enzymatic activity, the highest values were found in the digestive gland. The effect of heavy metals was dependent on the tissue analysed. Mercury showed the highest inhibition in the organs/tissues and the parameters Km and Vmax were modified when the inhibitor concentration increased, thus indicating a mixed type of inhibition.     

Human alkaline phosphatase expression and secretion into chicken eggs after in vivo gene electroporation in the oviduct of laying hens

In vivo gene electroporation was used to examine whether or not a recombinant protein is synthesized in the chicken oviduct and subsequently secreted into eggs. A plasmid DNA containing a secretion form of the human alkaline phosphatase gene was injected into mucosa of the chicken magnum. Immediately, in vivo gene electroporation was conducted. The human alkaline phosphatase activity in the oviduct mucosa increased and reached its peak at 2 days posttransfection, followed by a sharp decrease to a negligible level at 4 days posttransfection. In the egg white, the alkaline phosphatase activity showed a similar change to that in the magnum mucosa except for a delay of 4 days. The present results imply that in vivo gene electroporation method in the oviduct may serve as a rapid production system of recombinant proteins into chicken eggs.     

Growth on type I collagen promotes expression of the osteoblastic phenotype in human osteosarcoma MG-63 cells.

Using MG-63 cells as a model system capable of partial osteoblastic differentiation, we have examined the effect of growth on extracellular matrix. MG-63 cell matrix and purified type I collagen induced a morphological change characterized by long cytoplasmic processes reminiscent of those seen in osteocytes. Concurrent biochemical changes involving bone marker proteins included increased specific activity of cell-associated alkaline phosphatase and increased secretion of osteonectin (up to 2.5-fold for each protein); all changes occurred without alterations in the growth kinetics of the MG-63 cells. The increase in alkaline phosphatase activity was maximal on days 6-8 following seeding; increased osteonectin secretion was most prominent immediately following seeding; all changes decreased as cells reached confluence. Growing cells on type I collagen resulted in an increased induction of alkaline phosphatase activity by 1,25(OH)2D3 (with little change in the 1,25(OH)2D3 induction of osteonectin and osteocalcin secretion), and increased TGF-beta induction of alkaline phosphatase activity as well (both TGF-beta 1 and TGF-beta 2). Both the 1,25(OH)2D3 and TGF-beta effects appeared to be synergistic with growth on type I collagen. These studies support the hypothesis that bone extracellular matrix may play an important role in osteoblastic differentiation and phenotypic expression.     

Presence and androgen control of an alkaline phosphatase in the nucleus of rat ventral prostate.

The presence of alkaline phosphatase (EC 3.1.3.1) activity has been demonstrated in nuclei of rat ventral prostate. This enzyme activity remained after washing of isolated nuclei with 0.5% Triton X-100; an acid phosphatase initially present with the nuclear fraction was removed by this treatment. The nuclear alkaline phosphatase, examined by utilizing p-nitrophenyl phosphate as substrate, had a pH optimum of 9.5-10.3, and a broad substrate specificity: p-nitrophenyl phosphate greater than phosphothreonine greater than beta-glycerophosphate greater than phosphoserine. The nuclear phosphatase was sensitive to denaturation by heat or urea treatments and was also inhibited by Pi, L-phenylalanine, homoarginine, dithiothreitol, and EDTA. The EDTA-inhibited enzyme was maximally reactivated by Zn2+, although Mg2+, or Ca2+ were also effective at somewhat higher concentrations. Orchiectomy of adult rats resulted in an increase in the nuclear alkaline phosphatase activity (2-3-fold at 24 or 48 h postorchiectomy). A decline in the protein: DNA ratio also occurred following orchiectomy, but the increase in phosphatase specific activity was evident whether expressed per unit of protein or per unit of DNA. Testosterone replacement following orchiectomy abolished the increase in nuclear phosphatase activity. The results suggest that the prostatic nuclear alkaline phosphatase may be involved in events related to inactivation of the prostate nucleus following androgen deprivation.     

Regenerative capacity of forelimb buds after amputation in mouse embryos at the early-organogenesis stage.

The ability of mouse forelimb buds at stage 1 (Wanek et al., '89a) of development to regenerate after amputation was investigated. The findings were as follows: 1. Outgrowths in the form of hillocks were found at the sites of amputation in 116 (95%) out of 122 embryos examined 24 hours after amputation. Examination of the amputated region after various intervals of time revealed that the outgrowths were established from flank tissues at the anterior and posterior borders of the wound. 2. Ectodermal thickening was found on the distal margin of the outgrowths in 21 (66%) out of 32 specimens examined. These thickenings were histologically similar to the apical ectodermal ridge (AER) present on the control limb buds. 3. Alkaline phosphatase activity was detected on the ectodermal thickening in 11 (79%) out of 14 experimental limb buds examined. The pattern of expression of alkaline phosphatase activity was similar to that observed in control limb buds. 4. There was no correlation between the size of the outgrowths and the presence of the ectodermal thickening or the enzymatic activity. The outgrowths developed despite the absence of ectodermal thickening and enzymatic activity, suggesting that the thickening and the presence of alkaline phosphatase are not crucial for the initiation and formation of the outgrowths. 5. Explants of the outgrowths, when grafted beneath adult kidney capsules, differentiated extensively into various tissues, which included bones, epiphyseal plates, skeletal muscles, and skin derivatives. Control explants also gave rise to the same spectrum of tissues. Hence, the flank tissues surrounding the site of amputation in E10 mouse embryos can regenerate to form a structure that is morphologically and histochemically similar to a limb bud and the mesenchyme within the structure is histogenetically competent to produce the variety of tissues that is normally found in the adult limb.     

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.     

Effect of fasting and refeeding on duodenal alkaline phosphatase activity in monosodium glutamate obese rats

In the present work the effects of fasting and refeeding on fat pad weight and alkaline phosphatase activity in the brush border of individual duodenal enterocytes have been evaluated in male Wistar rats with obesity induced by monosodium glutamate (MSG) treatment during the early postnatal period. Neonatal rats were treated subcutaneously with MSG (2 mg/g b.w.) or saline (controls) for 4 days after birth. At 4 months of age, two types of experiments were performed. In the first experiment rats, were submitted to 3 or 6 days lasting food deprivation. In the second experiment the rats were refed for 3 or 6 days ad libitum or restrictedly (60% of pre-fasting intake) after a 6 day-fasting period. Fasting and refeeding influenced the body fat and function of the duodenum in MSG-treated rats differently as compared to the controls. However, alkaline phosphatase activity and the weight of epididymal and retroperitoneal fat depots were significantly increased in MSG obese rats (P<0.001) during all the periods examined. While 3 days of food deprivation resulted in both groups in a similar loss of adipose tissue weight and alkaline phosphatase activity, the decrements of these parameters after 6 days of fasting were lower in obese rats suggesting that their capacity to spare body fat stores was enhanced. After 3 days of ad libitum refeeding, a more marked adaptational increase of food consumption and also a significantly increased alkaline phosphatase activity above the pre-fasting level (P<0.01) was observed in the MSG-treated rats. Consequently, a more rapid body fat restoration was demonstrated in these animals. Refeeding of rats at 60% of the pre-fasting intake level resulted in a significant increase of alkaline phosphatase activity in both the MSG and control group; moreover, as food restriction continued, MSG-treated rats tended to further increase the enzyme activity. Our results revealed that MSG treatment of neonatal rats may significantly change the intestinal functions. Permanently increased alkaline phosphatase activity observed in MSG obese rats during all investigated periods suggests that this functional alteration is probably not a consequence of actual nutritional variation but could be a component of regulatory mechanisms maintaining their obesity at critical values.