Brush Border Membrane Alkaline Phosphatase Research Articles

Structural changes induced by heating affect ovalbumin-antigen processing and reduce allergic response in mouse model of food allergy

Background Food allergy is a serious health concern affecting about 8% of young children and about 3% of adults. The egg protein ovalbumin belongs among six frequent food allergens. Our goal was to develop the mouse model of ovalbumin food allergy and investigate how thermal processing affects its ability to induce allergic responses. Methods Effect of thermal processing (70°C and 95°C) on an ovalbumin secondary structure was characterized by circular dichroism and by kinetics of pepsin digestion with subsequent HPLC analysis. BALB/c mice were sensitized intraperitoneally and challenged intragastrically with native form or thermal processed ovalbumin (70°C). Clinical symptoms (allergic diarrhea, weight and temperature) were observed during the mice challenging. Levels of allergen-specific serum antibodies were determined by ELISA (IgA, IgG1 and IgG2a) by beta-hexosaminidase release test (IgE). Cytokine production (IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-gamma) and mast cell protease-1 activity were assessed by ELISA. The activities of brush-border hydrolases were determined in the jejunum of treated mice. Results Heating of ovalbumin caused slight irreversible changes in secondary structure and significantly decreased clinical symptoms (allergic diarrhea). The thermal processing also decreased immune allergic response on the level of IgE, IL-4, IL-5, IL-13. Furthermore, thermal-processed ovalbumin induced lower activities of serum mast cell protease-1 and enterocyte brush border membrane alkaline phosphatase as compared to native ovalbumin. On the other hand, thermal-processed ovalbumin stimulated higher IgG2a antibodies in sera and IFN-gamma secretion by splenocytes. Conclusion In our mice model of food allergy, we show that even a slight change in the secondary structure of ovalbumin after thermal processing reduces its allergenicity and induces shift towards Th1 immune responses. At present, we utilize this model for studying of food allergy in germfree mice.

Heat-Induced Structural Changes Affect OVA-Antigen Processing and Reduce Allergic Response in Mouse Model of Food Allergy

Background and AimsThe egg protein ovalbumin (OVA) belongs to six most frequent food allergens. We investigated how thermal processing influences its ability to induce allergic symptoms and immune responses in mouse model of food allergy.Methodology/Principal FindingsEffect of increased temperature (70°C and 95°C) on OVA secondary structure was characterized by circular dichroism and by the kinetics of pepsin digestion with subsequent HPLC. BALB/c mice were sensitized intraperitoneally and challenged with repeated gavages of OVA or OVA heated to 70°C (h-OVA). Levels of allergen-specific serum antibodies were determined by ELISA (IgA and IgGs) or by β-hexosaminidase release test (IgE). Specific activities of digestive enzymes were determined in brush border membrane vesicles of jejunal enterocytes. Cytokine production and changes in regulatory T cells in mesenteric lymph nodes and spleen were assessed by ELISA and FACS. Heating of OVA to 70°C caused mild irreversible changes in secondary structure compared to boiling to 95°C (b-OVA), but both OVA treatments led to markedly different digestion kinetics and Tregs induction ability in vitro, compared to native OVA. Heating of OVA significantly decreased clinical symptoms (allergic diarrhea) and immune allergic response on the level of IgE, IL-4, IL-5, IL-13. Furthermore, h-OVA induced lower activities of serum mast cell protease-1 and enterocyte brush border membrane alkaline phosphatase as compared to native OVA. On the other hand h-OVA stimulated higher IgG2a in sera and IFN-γ secretion by splenocytes.ConclusionsMinor irreversible changes in OVA secondary structure caused by thermal processing changes both its digestion and antigenic epitopes formation, which leads to activation of different T cell subpopulations, induces shift towards Th1 response and ultimately reduces its allergenicity.

Ectopic expression of alkaline phosphatase in proximal tubular brush border membrane of human renal cell carcinoma

The present study was conducted to find out any alteration in the expression and activity of alkaline phosphatase in the brush border membrane (BBM) from renal cell carcinoma (RCC) in comparison to normal renal BBM. The specific activity of alkaline phosphatase was drastically reduced in homogenate as well as BBM from RCC kidney when compared to ALP activity in BBM of normal kidney. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (V(max)) and increase in affinity constant (K(m)) of the enzyme. SDS-PAGE studies showed that the BBM proteins having molecular weights ranging from 95 to 170 kDa were poorly expressed in RCC BBM in relative to normal kidney BBM. Incubation of SDS-PAGE gel with BCIP/NBT dye clearly showed that the expression of ALP in tumor renal BBM was markedly reduced as compared to normal kidney. Further, Western blot analysis using anti-alkaline phosphatase antibody also confirmed the reduced expression of ALP in tumor renal BBM. Lipid composition in reference to phospholipids, glycolipids and cholesterol in tumor renal BBM was altered to that of normal renal BBM, indicating alteration in membrane fluidity of tumor renal BBM.

Inhibition of human intestinal brush border membrane vesicle Na +-dependent phosphate uptake by phosphophloretin derivatives

Hyperphosphatemia and II o hyperparathyroidism are common and severe complications of chronic renal failure. Reduced dietary phosphorus has been shown to be an effective treatment in reducing serum phosphate and serum PTH. 2 ′-Phosphophloretin inhibited small intestine apical membrane Na +/phosphate cotransport and reduced serum phosphate in adult rats. 2 ′-PP and phosphoesters of phloretin were tested for inhibition of human small intestine brush border membrane alkaline phosphatase activity and for inhibition of Na +-dependent phosphate uptake. The IC 50’s for inhibition of alkaline phosphatase suggested an order of inhibitory potency of 4-PP > phloretin > 4 ′-PP > 2 ′-PP. Inhibition of Na +-dependent phosphate uptake followed the sequence 2 ′-PP ≫ 4 ′-PP > 4-PP > phloretin. These results are consistent with 2 ′-PP being a specific inhibitor of human intestinal brush border membrane Na +/phosphate cotransport.

Effect of Acetaminophen on Na+, K+ ATPase and Alkaline Phosphatase on Plasma Membranes of Renal Proximal Tubules

In previous work we reported that 1 h after acetaminophen (APAP) administration, tubular function remained at control values, while 16 h later a significant deterioration of tubular function was observed. The aim of the present work was to study if APAP induces its renal toxic effects by altering the normal activity of key tubular plasma membrane enzymes. We analyzed the effects of a nephrotoxic dose of APAP (1000 mg/kg b.wt., i.p.) on the activities of the brush-border membrane (BBM) enzyme, alkaline phosphatase, and the basolateral membrane (BLM) enzyme Na+, K+ ATPase 1 h (APAP1h) and 16 h (APAP16h) after dosing. Na+, K+ ATPase abundance in homogenates and each membrane domain were analyzed by Western blot. Cortical adenosine 5′ triphosphate (ATP) content was also evaluated. At each time studied, APAP promoted a diminution of alkaline phosphatase in BBM. Na+, K+ ATPase activity in BLM showed a biphasic response to APAP. One hour after APAP administration it was significantly increased, but it was decreased 16 h after dosing. Na+, K+ ATPase protein abundance was elevated in homogenates, BLM, and BBM after 1 h of APAP dosing. After 16 h, Na+, K+ ATPase abundance was increased in homogenates, while in BLM it was decreased. No differences were observed in cortical ATP content in each time studied. Our present results could contribute to the understanding of the molecular basis of the previously reported time course alteration in the fractional excretion of sodium promoted by a nephrotoxic dose of APAP.

Effect of fetal exposure to gentamicin on phosphate transport in young rat kidney

The renal phosphate (Pi)-transporting capacity normally increases, due to increased carrier system affinity, during the third postnatal week in rats. However, the tubular Pi reabsorption of rat pups born from gentamicin-treated mothers does not increase during this period. This study determines whether exposure to gentamicin in utero selectively alters the postnatal maturation of the carrier affinity for Pi. Pi and glucose transports by proximal tubule brush-border membrane (BBM) were studied. The maximal rate of uptake (Vmax) of Na-Pi cotransport was significantly lower (536 +/- 169 pmol.mg protein-1.10 s-1; n = 6, P < 0.01) in gentamicin-exposed rats than in controls (1,021 +/- 167 pmol.mg protein-1.10 s-1, n = 6), whereas the Michaelis constant (Km) values were the same. Gentamicin exposure had no effect on plasma parathyroid hormone concentration or on BBM glucose transport activity. The total phospholipid content of BBM, their phospholipid composition, cholesterol content, and cholesterol-to-total phospholipid mole ratio were unaltered, suggesting that membrane fluidity was unchanged. The Vmax of BBM alkaline phosphatase was lower in gentamicin-exposed rats than in controls.

Evidence for calcium mediated conformational changes in calbindin‐D28K (the vitamin D‐induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques

The role of the vitamin D-induced calcium binding protein termed calbindin-D (CaBP) in the biological response to 1,25-dihydroxyvitamin D3 was assessed by photoaffinity labeling techniques. The heterobifunctional cross-linking reagent methyl-4-azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin-D28K. Calcium-dependent interactions were evident with purified chick intestinal CaBP-immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca2+ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with M(R) = 60,000 and 130,000, which were photoaffinity cross-linked with CaBP in a calcium-dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol-specific phospholipase C (PI-PLC)-treated supernatant fractions from chick intestinal brush borders. PI-PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G-150 chromatography of Triton X-100 solubilized incubations between calbindin-D28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin-D28K undergoes calcium-dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium-binding proteins such as calmodulin or troponin C.

Phosphate transport and alkaline phosphatase in confluent MDCK cell monolayers.

Several studies have suggested that the brush border membrane alkaline phosphatase is involved in the renal reabsorption of inorganic phosphate along the proximal convoluted tubule. However, other studies on the influence of l(-)bromotetramisole, an inhibitor of alkaline phosphatase, upon phosphate transport into brush border membrane vesicles have failed to show an involvement of alkaline phosphatase. The present experiments were designed to demonstrate that the MDCK (Madin, Darby, canine, kidney) cell line can be used as an alternative model to study phosphate transport, to examine the effect of l(-)bromotetramisole and the role of alkaline phosphatase. MDCK cell monolayers were concomitantly used for alkaline phosphatase activity measurement and phosphate transport analysis. While alkaline phosphatase activity increases by 37-fold from day 2 to day 8 of culture, reaching a plateau at day 10, the sodium-dependent phosphate transport into the cell monolayers decreases by 2-fold during that same period. The phosphate transport was also studied in the presence of l(-)bromotetramisole at pH 8.5. The sodium-dependent phosphate uptake of inorganic phosphate is reduced by 43% in the presence of l(-)bromotetramisole at day 3 of culture but is not reduced in the 7-day-old culture. Our results suggest a participation of alkaline phosphatase upon phosphate transport in MDCK cell monolayers and indicate that this cell line constitutes a good model to study the relationship between alkaline phosphatase and phosphate transport.

Uptake of Prodrugs by Rat Intestinal Mucosal Cells: Mechanism and Pharmaceutical Implications

The in vitro intestinal ring uptake of prednisolone (11β, 17,21-trihydroxypregna-1,4-diene-3,20-dione), prednisolone 21-succinate, and prednisolone 21-phosphate was compared in rings prepared from rat jejunum and colon. An HPLC assay was developed to determine whether the drug or intact prodrug was taken up by the tissue. In jejunum and colon, the uptake of prednisolone was limited by its solubility (0.84mM, 37°C). The freely soluble succinate and phosphate esters were well absorbed in the jejunum. The only species detected in jejunal tissue after incubation with prednisolone 21-phosphate was prednisolone, indicating hydrolysis prior to absorption. This implication was verified by light microscopy. Incubation of tissue from the jejunum with prednisolone 21-succinate resulted in uptake of a mixture of prodrug and parent drug, with the latter form predominating. Prednisolone 21-succinate was also absorbed well in the colon, where the predominant species taken up by the tissue was the intact ester. The half-life of the succinate ester in the tissue was ∼1h postincubation, implicating enzyme mediation. Uptake of the phosphate ester by the colon was <20% of that observed in the jejunum, with the species absorbed still being primarily the parent alcohol. Light microscopy techniques confirmed that prednisolone 21-phosphate and hydrocorti-sone 21-phosphate are good substrates for brush border membrane alkaline phosphatase in the jejunum, and that lack of this enzyme in colon tissue was most likely responsible for poor uptake in the colon.

Effect of the Hyp mutation and diet-induced hyperparathyroidism on renal parathyroid hormone- and forskolin-stimulated adenosine 3',5'-monophosphate production and brush border membrane phosphate transport.

The present study was undertaken to investigate the effect of the Hyp mutation and diet-induced hyperparathyroidism on renal responsiveness to PTH and forskolin and to determine whether the renal brush border membrane phosphate transport defect is expressed in the vitamin D- and calcium-deficient Hyp mouse. Our results indicate that PTH is a potent activator of cAMP synthesis in renal slices obtained from vitamin D-replete normal mice and Hyp littermates. However, in the mutants, the amount of cAMP produced in response to similar concentrations of PTH (0.005-5 U/ml) is 55% of normal (P = 0.0076). PTH-dependent cAMP synthesis is significantly reduced in both normal and Hyp mice fed the vitamin D-deficient low calcium diet, and under these conditions, genotype differences are no longer apparent. In contrast, forskolin-stimulated cAMP production is identical in vitamin D-replete normal and mutant mice. Furthermore, cAMP accumulation in response to forskolin is not decreased by diet-induced hyperparathyroidism in either genotype. Vitamin D and calcium deprivation results in a significant decrease in renal brush border membrane Na+-dependent phosphate transport in both genotypes, and under these conditions, the phosphate transport defect persists in Hyp mice. Finally, vitamin D and calcium deprivation has no effect on renal brush border membrane alkaline phosphatase activity. The present results suggest that the catalytic subunit of adenylate cyclase is intact in the mutant strain and that the blunted renal response to PTH in vitamin D-replete Hyp mice as well as in vitamin D- and calcium-deficient mice may be due to uncoupling of the PTH-receptor-adenylate cyclase system. The data also suggest that the expression of the renal phosphate transport defect in Hyp mice is independent of PTH status and alkaline phosphatase activity.

Nucleotide inhibition of phosphate transport in the renal proximal tubule.

The observation that NAD inhibits sodium-dependent phosphate (P) uptake by the luminal brush border membrane (BBM) of the proximal tubule prompted us to examine the specificity and mechanism of this process. Addition of 10(-5) M NAD to the perfusate of isolated perfused rabbit proximal straight tubules inhibited lumen-to-bath P flux by approximately 50%. ADP-ribose had an identical effect, whereas nicotinamide had no effect. ADP and 5'-AMP (10(-5) M) also inhibited P flux. Na-dependent uptake of 32P by rabbit BBM vesicles was inhibited by 0.1-0.3 mM NAD, ADP-ribose, ADP, ATP, 5'-AMP, and GDP, which were preincubated with the vesicles for 30 min. The kinetics of inhibition showed an apparent increase in the Km for P but no change in Vmax. These findings are consistent with "competitive inhibition." The nucleotides inhibited P uptake even when BBM alkaline phosphatase was inhibited by 96% with 10 mM theophylline. Evidence of nonspecific phosphatase activity was present, since incubation of BBM with 0.1 mM solution of nucleotides for 30 min resulted in an elevation of free P in the medium of approximately 0.15-0.22 mM. Correction of 32P specific activity for this change resulted in values for Km and Vmax that were not significantly different from control. The "competitive inhibition" could thus be ascribed to an isotope-dilution effect. There was no evidence to suggest that NAD caused ADP-ribosylation of the luminal membrane. These studies indicate that adenine and guanine nucleotides do not inhibit P transport by a direct action on the luminal membrane of the proximal tubule but do inhibit lumen-to-bath P flux in isolated perfused proximal tubules at concentrations of 10(-5) M. Since there is no direct inhibitory effect of these compounds at the level of the BBM, it is possible that they inhibit P transport by altering some event subsequent to the transfer of P across the luminal membrane.