Plasma Membrane Alkaline Phosphatase Research Articles

Relation of placental alkaline phosphatase expression in human term placenta with maternal and offspring fat mass.

Alkaline phosphatase is implicated in intestinal lipid transport and in the development of obesity. Placental alkaline phosphatase is localised to the microvillous plasma membrane of the placental syncytiotrophoblast at the maternal-fetal interface, but its role is unclear. We investigated the relations of placental alkaline phosphatase activity and mRNA expression with maternal body composition and offspring fat mass in humans. Term human placentas from the UK Birthright cohort (n = 52) and the Southampton Women's Survey (SWS) (n = 95) were studied. In the Birthright cohort, alkaline phosphatase activity was measured in placental microvillous plasma membrane vesicles. In the SWS, alkaline phosphatase mRNA was measured using Nanostring. Alkaline phosphatase gene expression was compared to other lipid-related genes. In Birthright samples placental microvillous plasma membrane alkaline phosphatase activity was positively associated with maternal triceps skinfold thickness and BMI (β = 0.04 (95% CI: 0.01-0.06) and β = 0.02 (0.00-0.03) µmol/mg protein/min per SD, P = 0.002 and P = 0.05, respectively) after adjusting for potential confounders. In SWS samples placental alkaline phosphatase mRNA expression in term placenta was positively associated with maternal triceps skinfold (β = 0.24 (0.04, 0.44) SD/SD, P = 0.02), had no association with neonatal %fat mass (β = 0.01 (-0.20 to 0.21) SD/SD, P = 0.93) and was negatively correlated with %fat mass at ages 4 (β = -0.28 (-0.52 to -0.04) SD/SD, P = 0.02), 6-7 (β = -0.25 (-0.49 to -0.02) SD/SD, P = 0.03) years. When compared with placental expression of other genes, alkaline phosphatase expression was positively related to genes including the lysophosphatidylcholine transporter MFSD2A (major facilitator superfamily domain containing 2A, P < 0.001) and negatively related to genes including the fatty acid transport proteins 2 and 3 (P = 0.001, P < 0.001). Our findings suggest relationships between placental alkaline phosphatase and both maternal and childhood adiposity. The inverse relationship between placental alkaline phosphatase gene expression and childhood %fat mass suggests that placental alkaline phosphatase may help to protect the foetus from the adverse effects of maternal obesity.

Localization of hCAP-18 on the surface of chemoattractant-stimulated human granulocytes: analysis using two novel hCAP-18-specific monoclonal antibodies

The well-described antimicrobial and immunoregulatory properties of human cathelicidin antimicrobial protein 18 (hCAP-18) derive in part from the ability of its proteolytic fragment, LL-37 (a.k.a. CAP-37), to associate with activated immune and epithelial cells during inflammation. We now show a stable association between hCAP-18 and the cell surface of formyl-Met-Leu-Phe (fMLF)-stimulated neutrophils using two novel hCAP-18-specific mAb, H7 and N9, which recognize a single 16-kDa band, identified by N-terminal sequencing and mass spectrometry as hCAP-18. Phage display analysis of epitope-binding sites showed that both mAb probably recognize a similar five amino acid sequence near the C terminus of the prodomain. Immunoblot analysis of degranulated neutrophil supernatants resulted in mAb recognition of the 14-kDa prodomain of hCAP-18. Subcellular fractionation of unstimulated neutrophils on density gradients showed expected cosedimentation of hCAP-18 with specific granule lactoferrin (LF). fMLF stimulation resulted in an average 25% release of specific granule hCAP-18, with approximately 15% of the total cellular hCAP-18 recovered from culture media, and approximately 10% and approximately 75%, respectively, codistributing with plasma membrane alkaline phosphatase and specific granule LF. Surface association of hCAP-18 on fMLF-stimulated neutrophils was confirmed by immunofluorescence microscopy and flow cytometry analysis, which also suggested a significant up-regulation of surface hCAP-18 on cytochalasin B-pretreated, fully degranulated neutrophils. hCAP-18 surface association was labile to 10 mM NaOH treatment but resistant to 1 M NaCl and also partitioned into the detergent phase following Triton X-114 solubilization, possibly suggesting a stable association with one or more integral membrane proteins. We conclude that fMLF stimulation promotes redistribution of hCAP-18 to the surface of human neutrophils.

Ascorbic acid-induced chondrocyte terminal differentiation: the role of the extracellular matrix and 1,25-dihydroxyvitamin D

Chondrocyte terminal differentiation is associated with cellular hypertrophy increased activity of plasma membrane alkaline phosphatase and the synthesis of collagen type X. The hypertrophic phenotype of cultured chondrocytes can be stimulated by ascorbic acid but the underlying mechanisms for this phenotypic change are unclear. As ascorbic acid is central to many hydroxylation reactions, the possibility was examined that its pro-differentiating effects are mediated by its effects on collagen and vitamin D metabolite formation. In vitro studies indicated that ascorbic acid-induced chondrocyte alkaline phosphatase activity was inhibited by the addition of both collagen and proteoglycan synthesis inhibitors. The addition of arginine-glycine-aspartic acid (RGD)-containing peptides also resulted in lower alkaline phosphatase activity. Chicks supplemented with dietary ascorbic acid had higher concentrations of both collagen and proteoglycans within their growth plates but the chondrocyte maturation rate was unaltered. No evidence was obtained to suggest that ascorbic acid-induced collagen production was mediated by lipid peroxidation. In addition, supplementation with dietary ascorbic acid resulted in higher serum 1,25-dihydroxyvitamin D3 concentrations and increased chondrocyte vitamin D receptor number. Ascorbic acid-treated chondrocytes maintained in vitro also had increased vitamin D receptor numbers but chondrocyte receptor affinity for 1,25-dihydroxyvitamin D3 was unaltered. These results indicate that ascorbic acid promotes both chondrocyte matrix production and 1,25-dihydroxyvitamin D3 synthesis, accompanied by upregulation of the vitamin D receptor. Thus, ascorbic acid may be causing amplification of the vitamin D receptor-dependent genomic response to 1,25-dihydroxyvitamin D, resulting in promotion of terminal differentiation. Strong evidence is provided to support the hypothesis that ascorbic acid-induced chondrocyte terminal differentiation is mediated by interactions between integrins and RGD-containing cartilage matrix proteins.

Vitamin D metabolites regulate matrix vesicle metalloproteinase content in a cell maturation-dependent manner.

Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix. They contain enzymes that are associated with calcification and are regulated by vitamin D metabolites in a cell maturation-dependent manner. Matrix vesicles also contain metalloproteinases that degrade proteoglycans, macromolecules known to inhibit calcification in vitro, as well as plasminogen activator, a proteinase postulated to play a role in activation of latent TGF-beta. In the present study, we examined whether matrix vesicle metalloproteinase and plasminogen activator are regulated by 1, 25(OH)2D3 and 24,25(OH)2D3. Matrix vesicles and plasma membranes were isolated from fourth passage cultures of resting zone chondrocytes that had been incubated with 10(-10)-10(-7) M24, 25(OH)2D3 or growth zone chondrocytes incubated with 10(-11)-10(-8) M 1,25(OH)2D3, and their alkaline phosphatase, active and total neutral metalloproteinase, and plasminogen activator activities determined. 24,25(OH)2D3 increased alkaline phosphatase by 35-60%, decreased active and total metalloproteinase by 75%, and increased plasminogen activator by fivefold in matrix vesicles from resting zone chondrocyte cultures. No effect of vitamin D treatment was observed in plasma membranes isolated from these cultures. In contrast, 1,25(OH)2D3 increased alkaline phosphatase by 35-60%, but increased active and total metalloproteinase three- to fivefold and decreased plasminogen activator by as much as 75% in matrix vesicles isolated from growth zone chondrocyte cultures. Vitamin D treatment had no effect on plasma membrane alkaline phosphatase or metalloproteinase, but decreased plasminogen activator activity. The results demonstrate that neutral metalloproteinase and plasminogen activator activity in matrix vesicles are regulated by vitamin D metabolites in a cell maturation-specific manner. In addition, they support the hypothesis that 1,25(OH)2D3 regulation of matrix vesicle function facilitates calcification by increasing alkaline phosphatase and phospholipase A2 specific activities as well as metalloproteinases which degrade proteoglycans.

Osteosarcoma hybrids can preferentially target alkaline phosphatase activity to matrix vesicles: Evidence for independent membrane biogenesis

Alkaline phosphatase is the marker enzyme for matrix vesicles, extracellular organelles that play a major role in primary bone formation and calcification. Recently, we developed osteosarcoma x fibrosarcoma hybrids in which alkaline phosphatase expression was greatly reduced, a phenomenon known as extinction. In the present study, we used to cell hybrids, LTA-1 and LTA-5, constructed from a human osteoblast-like osteosarcoma. TE85, and a mouse fibrosarcoma, La-t-, to examine the differential distribution of alkaline phosphatase between matrix vesicles and the plasma membrane, postulated to be the parent membrane from which matrix vesicles are derived. While alkaline phosphatase in plasma membranes was extinguished, enzyme activity in matrix vesicles from LTA-1 hybrid cells was 34.2% of that present in matrix vesicles from the TE85 parent cells and 200 times that found in La-t- matrix vesicles. Matrix vesicles from LTA-5 had alkaline phosphatase levels similar to La-t-. When other membrane enzymes (phospholipase A2, 5'-nucleotidase, and Na+/K+ ATPase) were examined, hybrid matrix vesicle and plasma membrane levels were similar to those of TE85 and significantly higher than in La-t- membrane fractions. Northern analysis detected mRNA for alkaline phosphatase in TE85 cells, but not in the hybrids or La-t- cells. In contrast, reverse transcription-polymerase chain reaction (RT-PCR) revealed alkaline phosphatase mRNA in the hybrid cells, but at very low levels. Taken together, the data indicate that regulation of plasma membrane and matrix vesicle alkaline phosphatase is independent and suggest that matrix vesicle biogenesis is independent and distinct from that of plasma membrane biogenesis. Analysis of 1B- and 1L-type alkaline phosphatase mRNA by RT-PCR showed that alternate promoter usage of the alkaline phosphatase gene was not responsible for the differential localization of this enzyme in matrix vesicle. Thus, it is likely that matrix vesicle and plasma membrane alkaline phosphatase are regulated differently at a post-transcriptional level.

Initial Effects of Partially Purified Bone Morphogenetic Protein on the Expression of Glycosaminoglycan, Collagen, and Alkaline Phosphatase in Nonunion Cell Cultures

Bone morphogenetic protein (BMP) stimulates mesenchymal cells to differentiate, resulting in de novo endochondral ossification in vivo. The response of fibrocartilage and periosteal cells from human and canine nonunion tissues to partially purified BMP was examined in culture. Cells derived from neonatal rat muscle explants were used for comparison. Alkaline phosphatase activity and expression of alkaline phosphatase and Types I and II collagen mRNAs were compared to that of rat chondrocytes. Synthesis of Type II collagen by the muscle cells was verified by enzyme-linked immunosorbent assay (ELISA). Addition of BMP to the muscle cell and nonunion cell cultures resulted in a dose-dependent decrease in cell number. There was a decrease in matrix vesicle and plasma membrane alkaline phosphatase activity concomitant with an increase in mRNA levels for alkaline phosphatase and collagen genes. Synthesis of immunoreactive Type II collagen increased. These data indicate that neonatal rat muscle cells and nonunion cells may respond in a similar fashion to BMP. Bone morphogenetic protein stimulated hyaluronic acid synthesis at three days, but chondroitin sulfate synthesis did not increase until ten days exposure to BMP. These data, together with those summarized above, suggest that more than three days may be required for complete expression of the chondrocyte phenotype typical of endochondral ossification.

Expression of Plasma Membrane Alkaline Phosphatase in Normal and Regenerating Choriocapillaris in the Rabbit

Ultrastructural histochemistry for plasma membrane nonspecific alkaline phosphatase was performed on the normal and regenerating choriocapillaris (CC) of rabbits. In normal animals the CC endothelium expressed little or no staining, whereas in regenerating CC the endothelium exhibited staining. The staining was most intense at the unfenestrated plasma membrane. As the capillaries matured and the fenestrated plasma membrane became more extensive, the staining was reduced and eventually eliminated. Pericytes did not stain in normal or regenerating CC.

Induction of plasma membrane alkaline phosphatase in rat liver.

We have determined alkaline phosphatase activity in total liver plasma membrane fractions from rats subjected to a partial hepatectomy and sham operated with or without manipulation of the liver. In all these cases, an increase of the enzyme activity was observed. Kinetic studies of alkaline phosphatase activity performed on plasma membrane fractions from rats subjected to a partial hepatectomy suggest that alkaline phosphatase increase is produced by de novo biosynthesis of enzyme molecules. Determination of alkaline phosphatase activity in purified plasma membrane subfractions corresponding to each of the three functional regions of the hepatocyte surface (blood sinusoidal, lateral and bile canalicular), indicates that the increase of the enzyme activity observed after partial hepatectomy is selectively induced in the bile canalicular domain of the hepatocyte plasma membrane.

Distribution patterns of orthogonal arrays and alkaline phosphatase in plasma membranes of satellite cells in rat spinal ganglia.

The plasmalemmal structure of satellite cells in the lumbar spinal ganglia of rat was examined by freeze-fracture and by a cytochemical method for the demonstration of alkaline phosphatase activity. Plasma membranes of satellite cells are the only ones in the ganglia to contain, in addition to globular intramembrane particles, orthogonal arrays of particles 6-7 nm in diameter. The arrays are most concentrated in the portions of the membranes contacting the basal lamina, or outer membranes; they decrease considerably in number in lateral membranes, and are rare in the membrane regions adjacent to the neuronal perikaryon, or inner membranes. Such gradual decrease in array density in satellite cells suggests regional differences of plasma membrane properties within the same cell. Alkaline phosphatase, which was chosen as a cytochemical marker for membrane activity because of its relation to transport function, localizes to inner and lateral membranes, and not to outer membranes of satellite cells. The absence of correlation between localization of orthogonal arrays and such enzymatic activity suggests that the membranes provided with many arrays possess some characteristics different from other membranes that may exclude transport activity. The possible significance of orthogonal arrays and their close association with the basal lamina are discussed.

Inducing effect of clofibrate on alkaline phosphatase and histidine-glyoxylate aminotransferase in rat liver.

The activity of plasma membrane alkaline phosphatase and both mitochondrial and peroxisomal histidine-glyoxylate aminotransferase was significantly increased in the livers of male rats following treatment with the hypolipidemic drug clofibrate. Cycloheximide or puromycin administration to rats inhibited the effects of clofibrate.

Phosphorylated intermediates of CA 2+-ATPase and alkaline phosphatase in plasma membranes from rat duodenal epithelium

Purified basolateral membranes of rat duodenal epithelium were phosphorylated in the presence of [ γ- 32P]ATP and 1 μM free Ca 2+ under conditions favorable for phospho-intermediate formation. Analysis of 32P-labeled membrane proteins on SDS-acrylamide gel electrophoresis revealed the presence of three Ca 2+-sensitive phosphoproteins with molecular weights of 64 000 (protein I), 84 000 (II) and 115 000 (III). Both proteins I and II were identified as phospho-intermediate forms of alkaline phosphatase on the basis of the following criteria: these phosphoproteins were resistant to hydroxylamine and alkaline treatment, phosphate incorporation was stimulated by Zn 2+ but totally inhibited by excess β-glycerophosphate during phosphorylation. A similar labeling efficiency was obtained with inorganic 32P instead of [ γ- 32P]ATP. Protein III comigrated on SDS gels with the phosphorylated intermediate of Ca 2+-ATPase from rat heart sarcolemnal membranes and was identified as the intestinal form of Ca 2+-ATPase in view of the following observations: binding of 32P to this protein was hydroxylamine sensitive and alkaline labile, which is in agreement with an acylphosphate character of an ATPase intermediate, phospho-intermediate formation was dependent on low free Ca 2+ concentrations (1 μM) in the presence of 1 mM Mg 2+, phosphorylation was strongly inhibited in the presence of phenothiazines (0.1 mM chlorpromazine or trifluoperazine). The amount of 32P bound to Ca 2+-ATPase in basolateral membranes was only 6% of the amount incorporated into (Na + + K +)-ATPase. The turnover number of duodenal Ca 2+-ATPase estimated from this phosphorylation study is remarkably similar to the value for the red blood cell Ca 2+-ATPase. Phosphorylation experiments with purified leaky brush border membranes under similar conditions did not reveal a phospho-intermediate from of Ca 2+-ATPase as demonstrated in basolateral membranes. In contrast about 7-fold higher levels of 32P in phosphoprotein bands I and II were found. The 32P-labeling characteristics of these proteins were similar to those of alkaline phosphatase intermediates in the basolateral membrane. The results of this study are fully in support of an earlier study in which high affinity Ca 2+-ATPase was found exclusively in the basolateral membrane of duodenal epithelium.

Degradation of L-[3H]fucose-labeled plasma membrane components from regenerating rat liver.

The rates of degradation of liver cell plasma membranes from control and partially hepatectomized rats were investigated by a single isotope administration procedure utilizing L-[3H]fucose as a precursor. The label was incorporated maximally into normal plasma membranes at 2 to 4 h after intravenous injection. At 4 h, L-[3H]fucose activity was 8-fold more abundant in membrane protein than in total liver protein. Paper chromatography of neutral sugars liberated by acid hydrolysis of the plasma membranes indicated that greater than 90% of the recovered radioactivity was in the form of fucose. After partial hepatectomy, the wet weight of the right lateral liver lobe increased linearly during a 4-day postoperation interval. In addition, the specific activity of plasma membrane alkaline phosphatase was elevated to a level that remained 3 to 4 times higher than that of the control membranes. However, there was not much difference between the mean rates of degradation of the labeled plasma membrane components from the control and regenerating livers. The apparent half-lives were 33 and 40.8 h, respectively. This suggests that the dissociation of fucose-containing components from the plasma membrane is not markedly altered during the process of rapid compensatory liver growth.

Cell cycle variations in HeLa 65 plasma membrane alkaline phosphatase

HeLa plasma membranes from M, G 1, and S phase cells were isolated from growing synchronous cell cultures. It was found that the specific activity of plasma membrane alkaline phosphatase was over three times higher in the M phase cell than in the G 1 and S phase cell. However, sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis showed that the S phase plasma membrane contained 5.5 times more alkaline phosphatase protein than did the plasma membrane from mitotic cells, and 11.0 times more than the G 1 phase plasma membrane. This would indicate that the high specific activity in mitosis was due to modification of the alkaline phosphatase protein resulting in increased enzymatic activity.