Quantitation of skeletal alkaline phosphatase isoenzyme activity in canine serum.

Pursuing the hypothesis that quantitation of skeletal alkaline phosphatase (ALP) activity in canine serum would provide an index of the rate of bone formation, we compared three methods for isoenzyme-specific identification of skeletal ALP activity in canine serum: heat inactivation, wheat germ agglutinin (WGA) precipitation, and concanavalin A (ConA) precipitation. ALP isoenzyme activities were extracted from canine bone, intestine, and liver, diluted into heat-inactivated canine serum (i.e., serum without ALP activity), and used as calibrators of ALP isoenzyme activities. Differential sensitivity to inhibition by 10 mM L-homoarginine was used to distinguish intestinal ALP activity from hepatic and skeletal ALP activities (i.e., 9, 80, and 72% inhibition, respectively). To allow resolution of skeletal ALP activity from hepatic ALP activity, we tested two established methods (heat inactivation and WGA precipitation) and a novel method, ConA precipitation. The organ-derived skeletal and hepatic ALP isoenzyme activities were used to compare these three methods with respect to linearity, isoenzyme separation, and precision. All three methods were linear, but the WGA and ConA methods afforded greater isoenzyme separation and precision. The relative extent of isoenzyme separation (i.e., the difference in percentage remaining skeletal and hepatic ALP isoenzyme activities) averaged 23, 40, and 47% remaining ALP activity for the heat, WGA, and ConA methods, respectively. However, when these methods were applied to the quantitation of skeletal ALP activity in sera from 10 young and 10 adult beagles, the WGA method was found to be unacceptable because most of the results fell outside the range of the WGA assay calibrators (i.e., greater than 100% skeletal ALP activity). The heat and ConA methods showed that the amount of skeletal ALP activity in the beagle sera decreased with age, both as ALP activity per liter and as percentage of total serum ALP activity (p less than 0.001 for each). Skeletal ALP activity levels determined by ConA were correlated with values determined by heat inactivation (r = 0.87, p less than 0.001) but not with WGA-determined levels (r = 0.26). Intestinal ALP activity was detected in only 1 of these 20 sera. We conclude that ConA precipitation can be used for quantitation of skeletal ALP activity in beagle serum.     

Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells

Although quantitative measurement of skeletal alkaline phosphatase (sALP) activity in serum can provide an index of the rate of bone formation, the metabolic process that determines the release of sALP - from the surface of osteoblasts, into circulation-is unknown. The current studies were intended to examine the hypothesis that the release of sALP from human osteoblasts is a consequence of apoptotic cell death. We measured the release of sALP activity from human osteosarcoma (SaOS-2) cells and normal human bone cells, under basal conditions and in response to agents that increased apoptosis (TNF-a, okadiac acid) and agents that inhibit apoptosis (IGF-I, calpain, and caspase inhibitors). Apoptosis was determined by the presence of nucleosomes (histone-associated DNA) in the cytoplasm of the cells by using a commercial kit. The results of these studies showed that TNF-a and okadiac acid caused dose- and time-dependent increases in apoptosis in the SaOS-2 cells (r = 0.78 for doses of TNF-a and r = 0.93 for doses of okadiac acid, P <0.005 for each), with associated decreases in cell layer protein (P <0.05 for each) and concomitant increases in the release of sALP activity (e.g., r = 0.89 for TNF-a and r = 0.75 for okadiac acid, P <0.001 for each). In contrast, caspase and calpain inhibitors reduced apoptosis, increased cell layer protein, and decreased the release of sALP activity (P <0.05 for each). Exposure to IGF-I also decreased apoptosis, in a time- and dose-dependent manner (e.g., r = 0.93, P <0.001 for IGF-I doses), with associated proportional effects to increase cell layer protein (P <0.001) and decrease the release of sALP activity (P <0.001). IGF-I also inhibited the actions of TNF-a and okadiac acid to increase apoptosis and sALP release. The associations between apoptosis and sALP release were not unique to osteosarcoma (i.e., SaOS-2) cells, but also seen with osteoblast-line cells derived from normal human bone. Together, these data demonstrate that the release of sALP activity from human osteoblast-line cells in vitro is associated with, and may be a consequence of, apoptotic cell death. These findings are consistent with the general hypothesis that the appearance of sALP activity in serum may reflect the turnover of osteoblast-line cells.     

Skeletal Alkaline Phosphatase Activity Is Primarily Released from Human Osteoblasts in an Insoluble Form, and the Net Release Is Inhibited by Calcium and Skeletal Growth Factors

Skeletal alkaline phosphatase (ALP) is anchored to membrane inositol-phosphate on the outer surface of osteoblasts. Although skeletal ALP activity in serum is, essentially, all in an anchorless (soluble) form, in vitro studies indicate that ALP can be released in either an anchorless, soluble form (e.g., by a phospholipase) or an anchor-intact, insoluble form (e.g., by vesicle exocytosis). The current studies were intended to define the contributions of each of these putative processes of ALP release and to assess the significance of regulation by calcium (Ca) and skeletal effectors. ALP activity was measured in serum-free medium from replicate cultures of human osteosarcoma (SaOS-2) cells and normal human bone cells. Temperature-sensitive phase distribution (in Triton X-114) allowed separation of soluble from insoluble ALP activity. Our studies revealed that most of the ALP activity released from SaOS-2 cells was in an insoluble form (78% ± 8%), a percentage that was constant between 2 and 96 hours. A similar result was seen for normal human bone cells. Calcium had a negative, biphasic dose-dependent effect on net release of ALP activity: r=−0.85, P < 0.001 at 24 hours, with K_Iapparent values for biphasic inhibition of 20 and 300 μmol/l Ca. Of the skeletal effectors tested, insulin-like growth factor-II (IGF-II) had the greatest effect, decreasing the net release of ALP activity in a dose-dependent manner (r=−0.82, P < 0.005). Neither Ca nor IGF-II affected the distribution of soluble/insoluble ALP activity by more than 9%. IGF-II had no effect on extracellular ALP stability, but the addition of Ca to Ca-free cultures resulted in parallel losses of extracellular ALP activity and ALP immunoreactive protein (P < 0.001 for each). A similar effect was seen when Ca was added to Ca-free, cell-free, conditioned medium, but not when Ca was added to purified ALP, which is consistent with the general hypothesis that a Ca-dependent protease might be present in the cell-conditioned medium. Together, these data suggest that most of the ALP activity released from osteoblasts is insoluble (and, presumably, anchorless), net release of ALP activity is negatively regulated by Ca and skeletal growth factors, the effect of Ca may reflect Ca-dependent protease activity, and an exogenous (e.g., serum) phospholipase may be responsible for releasing ALP from its insoluble anchor. Received: 8 November 1997 / Accepted: 7 August 1997     

Differentiation-inducing factor-1 alters canonical Wnt signaling and suppresses alkaline phosphatase expression in osteoblast-like cell lines.

Because DIF-1 has been shown to affect Wnt/beta-catenin signaling pathway, the effects of DIF-1 on osteoblast-like cell lines, SaOS-2 and MC3T3-E1, were examined. We found that DIF-1 inhibited this pathway, resulting in the suppression of ALP promoter activity through the TCF/LEF binding site. INTRODUCTION: Differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium, inhibits cell proliferation and induces cell differentiation in several mammalian cells. Previous studies showed that DIF-1 activated glycogen synthase kinase-3beta, suggesting that this chemical could affect the Wnt/beta-catenin signaling pathway. This pathway has been shown to be involved in bone biology. MATERIALS AND METHODS: We studied the effects of DIF-1 on SaOS-2 and MC3T3-E1, osteosarcoma cell lines widely used as a model system for ostoblastic cells and murine osteoblast-like cell line, respectively. Reporter gene assays were also carried out to examine the effect of DIF-1 on the Wnt/beta-catenin signaling pathway. RESULTS: DIF-1 inhibited SaOS-2 proliferation and reduced alkaline phosphatase (ALP) activity in a concentration- and a time-dependent manner. The expression of ALP was markedly suppressed by DIF-1-treatment in protein and mRNA levels. DIF-1 also suppressed the expression of other osteoblast differentiation markers, including core binding factor alpha1, type I collagen, and osteocalcin, in protein and mRNA levels and inhibited osteoblast-mediated mineralization. Subsequently, we examined the effect of DIF-1 on the Wnt/beta-catenin signaling pathway. We found that DIF-1 suppressed the expression of beta-catenin protein and the activity of the reporter gene containing T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) consensus binding sites. We examined the effect of DIF-1 on a reporter gene driven by the human ALP promoter and found that DIF-1 significantly reduced the ALP reporter gene activity through the TCF/LEF binding site (-1023/-1017 bp). Furthermore, the effect of DIF-1 on MC3T3-E1, a murine osteoblast-like cell line, was examined, and it was found that DIF-1 suppressed ALP mRNA expression by the reduction of the ALP reporter gene activity through the TCF/LEF binding site. CONCLUSIONS: Our data suggest that DIF-1 inhibits Wnt/beta-catenin signaling, resulting in the suppression of ALP promoter activity. To our knowledge, this is the first report to analyze the role of the TCF/LEF binding site (-1023/-1017 bp) of the ALP gene promoter in osteoblast-like cell lines.     

Calcitonin acutely increases tyrosyl-phosphorylation of proteins in human osteosarcoma (SaOS-2) cells

In order to test the hypothesis that salmon calcitonin has direct effects to modulate tyrosyl-protein phosphorylation in human osteosarcoma cells, SaOS-2 cells (with very high steady-state levels of skeletal alkaline phosphatase) were exposed to calcitonin, in duplicate serum-free cultures, at concentrations ranging from 10^-13 to 10^-9 mol/liter, for 0–60 minutes at 37°C. Phospho-tyrosyl proteins were identified by autoradiography of Western blots after incubation with ¹²⁵I-labeled antiphosphotyrosine antibodies (or with unlabeled antibodies and ¹²⁵I-labeled protein A) and quantitated by laser densitometry. The results of these studies revealed (1) time-dependent effects of salmon calcitonin (sCt) (at 3×10^-12 mol/liter) to increase the level of tyrosyl-phosphorylation of at least six proteins, with apparent molecular weights of 20, 25, 27, 41, 48, and 135 kD (P<0.05 for each); and (2) dose-dependent effects of sCt (during 15 minutes of exposure) to increase the level of tyrosyl-phosphorylation of at least 10 proteins with apparent molecular weights of 19, 20, 27, 35, 41, 102, 135, 195, 220, and 244 kD (P<0.05 for each). A supplementary study of calcitonin effects on tyrosyl-protein phosphorylation in a subpopulation of SaOS-2 cells with very low steady-state levels of skeletal alkaline activity revealed similar responses—time and dose-dependent increases in the tyrosyl-phosphorylation of at least seven proteins with apparent molecular weights of 44, 48, 57, 62, 101, 244, and 280 kD (P<0.05 for each). Together, these studies demonstrate that sCt can have direct effects to modulate the level of tyrosyl-protein phosphorylation in human osteosarcoma cells, presumably by activation of tyrosyl-kinase activity and/or inhibition of phospho-tyrosyl-protein phosphatase activity.     

Monofluorophosphate is hydrolyzed by alkaline phosphatase and mimics the actions of NaF on skeletal tissues,In Vitro

Summary These studies were intended to assess the osteogenic activity of monofluorophosphate (MFP)in vitro, and to identify the enzyme(s) responsible for MFP hydrolysis—alkaline phosphatase (ALP) and/or acid phosphatase (AcP). ALP and AcP activities were determined by hydrolysis of p-nitrophenylphosphate (PNPP) at pH>8 and pH 5.5, respectively, and MFP hydrolysis was determined, between pH 5.5 and pH 9.0, from measurements of [fluoride ion], using an ion-specific electrode. We found (1) that MFP was an alternative substrate for purified ALP, but not for AcP; (2) that MFPase activity in the embryonic chick resembled ALP, but not AcP, with respect to pH-dependent hydrolysis, sensitivity to effectors (r=0.98,P<.001), and tissue distribution (r=0.96,P <.001); and (3) that intestinal MFPase activity in the embryonic chick co-purified with ALP activity (r=0.93,P<.01) and resembled ALP, but not AcP, in its distribution along the small intestine, being highest in the duodenum and lowest in the distal ileum (r=0.96,P<.001). We also found thatin vitro exposure to MFP increased (1) the proliferation rate of embryonic chick calvarial cells in serum-free monolayer cultures (i.e.,³[H]-thymidine incorporation into DNA,P<.001); (2) ALP activity in calvarial cells (P<.005) and in intact calvaria (P<.05); and (3) collagen production by intact calvaria (i.e.,³[H]-proline incorporation as³[H]-hydroxyproline,P<.05). Together these data indicate that ALP, and not AcP, is responsible for MFP hydrolysis, and that MFP can mimic the osteogenic actions of NaF,in vitro.     

Calcitonin has direct effects on3[H]-thymidine incorporation and alkaline phosphatase activity in human osteoblast-line cells

Summary Calcitonin had direct and dose-dependent actions on human osteoblast-line cells (in serum-free monolayer culture) to increase cell proliferation and alkaline phosphatase activity/mg cell protein. Salmon calcitonin increased (human osteosarcoma) SaOS-2 cell proliferation, as evidenced by dose-dependent increases in³[H]-thymidine incorporation into DNA (e.g., 153% of control after 20 h exposure at 0.1 nM,P<0.01), and MTT (thyzolyl blue) reduction/deposition (e.g., 161% of control after 72 h exposure at 0.03 nM). Continuous exposure was not required to elicit these proliferative responses. These effects were not unique to salmon calcitonin or to SaOS-2 cells. Similar effects were seen with human calcitonin (but not heat-inactivated human calcitonin) and with (human osteosarcoma) TE-85 cells and human osteoblast-line cells prepared from femoral heads. In addition to effects on cell proliferation, calcitonin also increased alkaline phosphatase-specific activity in SaOS-2 cells (e.g., 180% of control after 72 h of exposure to 0.1 nM salmon calcitonin,P<.005).     

The effects of prostaglandin E2, parathyroid hormone, 1,25 dihydroxycholecalciferol,and cyclic nucleotide analogs on alkaline phosphatase activity in osteoblastic cells

Summary Clone MC3T3-E1 cells isolated from newborn mouse calvaria had the same type of alkaline phosphatase (ALP) as that found in adult mouse calvaria (the liver-bone-kidney type), as judged by polyacrylamide gel electrophoresis as well as by heat lability and amino acid inactivation. The effects of prostaglandin E₂ (PGE₂), parathyroid hormone (PTH), 1,25 dihydroxycholecalciferol [1,25(OH)₂D₃], and adenosine-3′, 5′-cyclic monophosphate (cAMP) analogs on ALP were investigated. PGE₂ and 1,25(OH)₂D₃ increased ALP activity in dose-related manner with a maximal effect at concentrations of 10 ng/ml and 40 pg/ml, respectively. N⁶,O²-dibutyryl adenosine-3′, 5′-cyclic monophosphate (DBcAMP) also induced an increase in ALP activity in a dose-related fashion with a maximal effect at a concentration of 0.5 mM which was 2.2-fold over that of the controls. Induced ALP was of the “liver-bone-kidney” type. Antinomycin D and cycloheximide inhibited the increase in ALP activity induced by DBcAMP. The level of ALP was elevated by 8-bromo-adenosine-3′,5′-cyclic monophosphate and theophylline, but not by N⁶,O²-dibutyryl guanosine-3′,5′-cyclic monophosphate and sodium butyrate. Moreover, PGE₂ dramatically increased the level of cAMP in the cells with a maximal effect at a concentration of 10 ng/ml, indicating that PGE₂ and DBcAMP induce an increase of ALP activity in clone MC3T3-E1 cells by increasing the cAMP level; PTH did not affect enzyme activity and cAMP, level in the cells. These results suggest that PGE₂, DBcAMP, and 1,25(OH)₂D₃ are involved in bone formationin vivo as well.     

Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms

Three circulating human bone alkaline phosphatase (BALP) isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. The present study was designed to correlate differing glycosylation patterns of each BALP isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant isoforms of the tissue-nonspecific ALP (TNALP) isozyme, i.e., TNALP-flag, used extensively for mutation analysis of hypophosphatasia mutations and sALP-FcD₁₀, a chimeric enzyme recently used as therapeutic drug in a mouse model of infantile hypophosphatasia.The BALP isoforms were prepared from human osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-nitrophenylphosphate (pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic pyrophosphate (PP_i), pyridoxal 5′-phosphate (PLP), and phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by lectin binding and precipitation.The k_cat/K_M was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD₁₀ was similar to the activity of the human BALP isoforms. The BALP isoforms differed in their lectin binding properties and dose-dependent lectin precipitation, which also demonstrated differences between native and denatured BALP isoforms. The observed differences in lectin specificity were attributed to N-linked carbohydrates.In conclusion, we demonstrate significantly different catalytic properties among the BALP isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP isoforms or for the recombinant TNALP isoforms. The TNALP-flag and the sALP-FcD₁₀ isoforms faithfully mimic the biological properties of the human BALP isoforms in vivo validating the use of these recombinant enzymes in studies aimed at dissecting the pathophysiology and treating hypophosphatasia.     

Relationship between the uptake of calcium or phosphorus and alkaline phosphatase activity induced by certain modulators in rat organs

Summary The effects of phorbol ester or calmodulin on the calcium and phosphorus uptakes by rat tissues and their relationship to the alkaline phosphatase activity (ALP) were investigatedin vivo. In rat tissues, ALP activity and calcium uptake in the duodenum and liver were clearly induced by phorbol ester treatment, whereas in the calvarium and ileum they were decreased. Phosphorus uptake was increased by the administration of phorbol ester only in the calvarium. In rats pretreated with an injection of indomethacin as an inhibitor of prostaglandin-synthesizing enzyme, the selection uptake of calcium by phorbol ester was eliminated in the duodenum and liver, as was the ALP activity. In contrast, rats showed a marked increase in ALP activity in the ileum after calmodulin treatment. Moreover, the increased uptake of calcium after calmodulin treatment was clearly seen in the ileum, calvarium, the kidney, and an increased uptake in phosphorus was seen in the duodenum, ileum, and calvarium, but not in kidney. Furthermore, prior injection of W-7 or calmidazolium as an antagonist of calmodulin, reduced the increased ALP activities and the uptake of calcium in all organs tested, but did not reduce the increased phosphorus uptake by the calcarium. Consequently, it is suggested that calcium uptake under the above conditions correlated well with changes in the ALP activity; however, phosphorus uptake seemed to be less in accord with ALP activity. The amount of tested other mineral metabolic markers suggests that the Ca uptake and ALP activity induced by certain effectors regulate 1,25(OH)₂D₃ level by the modulation of 25(OH)D₃-1α-hydroxylase activity.     

Enzymatic characterization of the matrix vesicle alkaline phosphatase isolated from bovine fetal epiphyseal cartilage

Summary Purified matrix vesicle alkaline phosphatase from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and PP_i. Optimal activities forp-nitrophenyl phosphate, ATP, and PP_i are found at pH 10.5, 10.0, and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations.p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasing substrate concentration. Heat inactivation studies indicate that both phosphorohydrolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg²⁺ and Hg²⁺ ions inhibit thep-nitrophenyl phosphatase activity at pH 10.5 while Mn²⁺ ions show no effect. P_i, levamisole, CN⁻, Zn²⁺, Ca²⁺ ions, and L-phenylalanine are reversible inhibitors of the phosphomonoesterase activity. P_i is a linear noncompetitive inhibitor with a K_i of 8.0 mM. Levamisole and L-phenylalanine are uncompetitive inhibitors with inhibition constants of 0.02 and 39.4 mM, respectively. Ca²⁺ ions inhibit noncompetitively with a K_i of 9.3 mM. Zn²⁺ ion is a potent noncompetitive inhibitor with an inhibition constant of 0.026 mM. The enzyme is inhibited irreversibly by Be²⁺ ion, EDTA, EGTA, ethane-l-hydroxydiphosphonate, dichloromethanediphosphonate, L-cysteine, and N-ethylmaleimide. NaCl, KCl, and Na₂SO₄ at 0.5–1.0 M inhibit the enzyme. At pH 8.5, the cleavage of PP_i by the matrix vesicle enzyme is inhibited by Mg²⁺ and Ca²⁺ ions at concentrations greater than 0.5 mM. Mg²⁺ ions in the range of 0.1–4 mM stimulate the matrix vesicle ATPase whereas higher concentrations produce inhibition. Ca²⁺ ion does not affect the ATPase activity between 0.1 and 10 mM at either pH 7.5 or 10.0. With 5′-AMP as substrate, the 3.5-fold decrease in the maximum velocity of the matrix vesicle relative to the chondrocyte enzyme may be accounted for in terms of a small excess enthalpy of activation (1160 cal/mole) partially compensated by an increased entropy of activation. Deceased May 8, 1975.     

Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and alkaline phosphatase function: A unified model of the mechanisms of initiation of skeletal calcification

Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Alkaline phosphatase (TNAP) plays a crucial role promoting mineralization of the extracellular matrix by restricting the concentration of the calcification inhibitor inorganic pyrophosphate (PP_i). Mutations in the TNAP gene cause hypophosphatasia, a heritable form of rickets and osteomalacia. Here we show that PHOSPHO1, a phosphatase with specificity for phosphoethanolamine and phosphocholine, plays a functional role in the initiation of calcification and that ablation of PHOSPHO1 and TNAP function prevents skeletal mineralization. Phospho1^?/? mice display growth plate abnormalities, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis in early life. Primary cultures of Phospho1^?/? tibial growth plate chondrocytes and chondrocyte‐derived matrix vesicles (MVs) show reduced mineralizing ability, and plasma samples from Phospho1^?/? mice show reduced levels of TNAP and elevated plasma PP_i concentrations. However, transgenic overexpression of TNAP does not correct the bone phenotype in Phospho1^?/? mice despite normalization of their plasma PP_i levels. In contrast, double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality. We conclude that PHOSPHO1 has a nonredundant functional role during endochondral ossification, and based on these data and a review of the current literature, we propose an inclusive model of skeletal calcification that involves intravesicular PHOSPHO1 function and P_i influx into MVs in the initiation of mineralization and the functions of TNAP, nucleotide pyrophosphatase phosphodiesterase‐1, and collagen in the extravesicular progression of mineralization. © 2011 American Society for Bone and Mineral Research.     

Serum creatine kinase and alkaline phosphatase in experimental small bowel infarction

Serum creatine phosphokinase (CK) and alkaline phosphatase (ALP) rise after mesenteric infarction; it is not known which one rises earlier or which one has the greater elevation. This experiment compared and contrasted the elevations in both these enzyme systems after acute small bowel infarction. Isoenzymes of both systems were analyzed to determine if any qualitative changes occurred. After baseline blood samples had been drawn, 10 dogs had midline laparotomies under general anesthesia. Each was assigned to one of two groups according to a randomized block design. Controls (CON) were closed after exploration (N = 5). The infarction (INF) group had ligation and division of the arteries to the jejunum and ileum (N = 5). Blood samples were obtained from both groups at 3, 6, 9, 12, 24, and 27 hr after surgery. Sera were analyzed for total CK and ALP activity by automated spectrophotometry. Isoenzymes were determined by agarose gel electrophoresis. Serum CK rose faster and to a higher level than ALP after small bowel infarction (470 +/- 181 vs 196 + 28 IU/liter). CK-BB was a better marker of small bowel necrosis than was intestinal ALP. The elevation of both CK and ALP by 12 hr after infarction may be a diagnostic aid if similar changes occur in humans.     

乳酸对成骨样细胞成骨表型及碱性磷酸酶基因表达的影响研究

目的 明确聚乳酸降解终产物乳酸对细胞成骨表型和基因表达的影响机理，为聚乳酸支架的精确设计提供参考意义。方法 配制乳酸浓度为8、16、22、27 mmol/L的培养基，将不含乳酸培养基作为对照组，检测培养基的pH和渗透压。利用培养基培养大鼠成骨样细胞Ros17/2.8，观察细胞形态，测定细胞I型胶原、碱性磷酸酶（alkaline phosphatase，ALP）活性和骨钙素含量，盐酸四环素染色钙结节并定量分析。采用实时定量PCR法检测细胞ALP mRNA表达。结果 与对照组相比，随着乳酸浓度增加，培养基pH下降，渗透压升高，成骨样细胞形态改变，数量减少。低浓度乳酸组（8、16 mmol/L）的I型胶原量都显著高于对照组，其余组与对照组相比无显著差异。含乳酸组的细胞ALP活性都低于对照组，并且乳酸浓度越高，细胞ALP活性越低。含乳酸组的骨钙素量与对照组之间没有显著差异。随着乳酸浓度增加，含乳酸组细胞所形成的钙结节阳性面积减少，都低于对照组。低浓度乳酸组（8、16 mmol/L）的细胞ALP mRNA表达量都低于对照组。结论 聚乳酸降解终产物乳酸可抑制细胞ALP mRNA的表达，降低ALP活性。随着乳酸浓度增加，乳酸抑制作用增强，最终降低细胞的成骨矿化能力。此外，乳酸不会抑制细胞分泌I型胶原和骨钙素。     

Relationship of inorganic pyrophosphatase and alkaline phosphatase activities in hamster molars

1. Some properties of inorganic pyrophosphatase (PP_i-ase) and alkaline phosphatase (p-NPP-ase) in the molars of 3-day-old hamsters are described.
2. The pH optimum for inorganic pyrophosphatase is 8.7, for alkaline phosphatase 10.3.
3. The ratio of Mg²⁺: PP_i for optimal inorganic pyrophosphatase activity is 1∶1. There is no clear optimal ratio in the case of p-NPP-ase.
4. The ratio of the enzymic activity of the membrane bound fraction to the soluble fraction is 3.4∶1 (S.E. 0.37) for PP_i-ase and 3.2∶1 (S.E. 0.42) for p-NPP-ase activity.
5. A mutual substrate competition for the pyrophosphatase and alkaline phosphatase activities is demonstrated.
6. Both enzymatic activities have similar temperature-activity curves with the same maximum at 38°.
7. Microdissection of ameloblasts and stratum intermedium cells from lyophilized sections showed the same activity ratio for both enzyme activities: Stratum intermedium: ameloblasts for PP_i-ase 4.7 (S. E. 0.93) and for p-NPP-ase 4.2 (S.E. 0.79).
8. High-voltage, free-flowing electrophoresis of the homogenate gave equal distribution patterns for p-NPP-ase and PP_i-ase greatly different from the protein distribution pattern.
9. It is concluded that the two activities are due to the same enzyme.

     

Monolayer cultures of normal human bone cells contain multiple subpopulations of alkaline phosphatase positive cells

Summary Cytochemical staining of normal human bone cells in monolayer cultures for alkaline phosphatase (ALP) indicated that the cultures contained mixed-cell populations. Time course evaluations of the cytochemical staining revealed, in addition to the ALP-negative cell population, at least two subpopulations of ALP-positive human bone cells with different levels of ALP. A cytochemical method has been developed which separates the ALP-positive cells into high and intermediate ALP subpopulations. In this method, human bone cells were stained for ALP using an azo-dye method and incubating at 4°C for 10 and 30 minutes, respectively. We defined the cell population that stained positively for ALP at 10 minutes as strong ALP-positive cells, and both strong and intermediate cells were stained at 30 minutes. The intermediate cells were determined from the difference between the values at the two time points. The intra- and interassay variations of the assay, with the same investigator in blinded investigations, were both less than 10% and the interobserver variation was approximately 25%. Analysis of the distribution of ALP levels in cells with a laser densitometer confirmed the presence of at least three cell subpopulations. 1,25(OH)₂D₃ treatment increased the proportions of both ALP-positive cell populations, whereas TGF-beta treatment increased only the intermediate ALP-positive cell population. On the contrary, fluoride increased the proportion of the strong ALP cells, and IGF-1 had no effect on the proportions of either ALP-positive subpopulation. When the ALP-specific activity was compared with the percentage of each ALP-positive subpopulations for the cells treated with effectors, the ALP-specific activity correlated with the total ALP-positive and with the strong ALP-positive populations but not with the intermediate ALP-positive subpopulation. In summary, this study represents the first evidence that normal human bone cells in monolayer cultures contained at least two subpopulations of ALP-positive cells, and that bone cell effectors could have differential effects on each cell population.     

Calcitonin Increases the Concentration of Insulin-Like Growth Factors in Serum-Free Cultures of Human Osteoblast-Line Cells

The current studies were intended to determine whether the anabolic effects of calcitonin (CT) on human osteoblast-line cells were (1) unique to osteosarcoma cells or also evident in osteoblast-line cells derived from normal human bone; and/or (2) associated with effects on several insulin-like growth factor (IGF) system components. Preliminary studies identified several osteoblastic cell lines, derived from normal human bone, which showed calcitonindependent increases in cell proliferation, alkaline phosphatase activity, and/or ⁴⁵Ca uptake (P < 0.05–P < 0.001). Two of these cell lines—(human vertebrae) HBV-155 and HBV-163—were included with the human osteosarcoma cell line, SaOS-2, in most of our subsequent studies of calcitonin effects on selected IGF system components: IGF-II, IGF-I, and IGF binding proteins -3, -4, and -5. The results of those studies revealed that a 48 hour exposure to salmon CT caused a dose-dependent (0.03–3 mU/ml) increase in the net extracellular level of IGF-II (r = 0.96, P < 0.01) in serum-free cultures of SaOS-2 cells, with a maximal 60% increase at the highest tested dose (P < 0.02). Similar effects were seen with HBV-163 cells (r = 0.90, P < 0.01) and HBV-155 cells (r = 0.55, P < 0.02). The effect of calcitonin on the extracellular level of IGF-II was biphasic with respect to time: it decreased at 6 hours (P < 0.005 and P < 0.001, for SaOS-2 cells and HBV-163 cells, respectively) and increased at 24 hours (P < 0.02 and P < 0.05). These calcitonin-dependent increases in the extracellular level of IGF-II were associated with parallel increases in IGF-I (P < 0.005 for SaOS-2 cells and P < 0.03 for HBV-163 cells), but calcitonin did not affect the extracellular level of transforming growth factor (TGF)-β. The calcitonin-dependent changes in IGF-II were not associated with changes in the extracellular levels of IGF binding proteins -3, -4, or -5. Finally, our studies showed that two other members of the CT superfamily—CT gene-related peptide and amylin—did not mimic the effect of CT to increase the extracellular level of IGF-II. Together, these data demonstrate that human osteoblast-line cells derived from normal human bone can respond to CT, and that those responses can include CT dose- and time-dependent increases in the extracellular levels of IGF-I and IGF-II. Received: 10 September 1999 / Accepted: 15 February 2000     

Skeletal Response to Dietary Zinc in Adult Female Mice

The current studies were intended to assess dose- and time-dependent effects of dietary zinc (Zn) on alkaline phosphatase (ALP) activity and tartrate-resistant acid phosphatase (TRAP) activity in adult female mice. In the first study, mice were given 0, 1×, 2×, 3×, or 4× normal dietary Zn for 2 weeks, 4 weeks, or 6 weeks. In the second study, mice were given 0, 1×, 2×, 3×, 4×, and 5× normal dietary Zn for 4 weeks. Sera were collected for measurements of ALP and (in the second study) osteocalcin. Tibiae and calvaria were extracted for measurements of ALP, protein, and TRAP. The first study showed positive correlations between dietary Zn and serum ALP (4 and 6 weeks, P < 0.001), Zn and tibial ALP (2, 4, and 6 weeks, P < 0.03), and Zn and tibial protein (2, 4, and 6 weeks, P < 0.001), as well as a negative correlation between dietary Zn and tibial TRAP (2, 4, and 6 weeks, P < 0.001). Covariant analyses showed that serum ALP, tibial ALP, tibial protein, and tibial TRAP were affected by the dose of Zn (P < 0.005) and by the treatment time (P < 0.03). Supplemental studies showed that (1) the dose-dependent effect of dietary Zn on serum ALP (at 6 weeks) was proportional to the effects on tibial ALP and calvarial ALP, but not to the effects of Zn on renal, hepatic, or intestinal ALP; (2) 6 weeks of dietary Zn caused dose-dependent increases in ALP specific activity in the tibia, calvaria, and liver, but not kidneys or intestines; and (3) Zn increased ALP activity and cell layer protein and decreased TRAP activity in monolayer cultures of the murine osteoblastic cell line, MC3T3-E1. The second dietary study confirmed the results of the first: 4 weeks of treatment with Zn caused significant increases in serum ALP, calvarial ALP, and tibial ALP activities, and a significant decrease in tibial TRAP (P < 0.05–0.005 for each). This study also revealed an effect of Zn to increase serum osteocalcin (P < 0.03 at 2× normal Zn). Together, these data indicate that incremental increases in dietary Zn are associated with increases in ALP activity in serum and in bone. The effect of Zn to decrease TRAP activity in osteoblast-line cells precludes the interpretation of a Zn-dependent decrease in tibial TRAP activity as evidence of decreased bone resorption.