Requirement of serum pretreatment for induction of alkaline phosphatase activity with prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCl in human liver cells continuously cultured in serum-free medium

A cell line (HuL-1) derived from normal fetal human liver was adapted to grow continuously in a modified Eagle's minimum essential medium without serum or hormones. The population doubling time of this adapted cell line (HuL-1-317) was about 72 h and the modal number of chromosomes was 54. The morphology of HuL-1-317 cells was round in the absence of serum, but at 37 degrees C with the addition of serum (1-10%), the cells flattened. HuL-1-317 cells had a low level of alkaline phosphatase activity. However the enzyme activity was slightly enhanced by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and a hypertonic concentration of NaCl after 3 days of incubation at 37 degrees C. The increase in alkaline phosphatase activity with the four agents was further amplified dose-dependently by the pretreatment of the cells with serum. The stimulatory effect of the serum was evident at concentrations as low as 1%, and was maximal at 20%. The half life of the effect of serum on alkaline phosphatase induction was 48 h at 37 degrees C. Serum alone could not enhance the enzyme activity without the four agents. The present results indicate that serum contributes to the regulation of alkaline phosphatase induction by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCl in fetal human liver cells (HuL-1-317).     

Effects of dibutyryl adenosine 3':5'-cyclic monophosphate and other agents on induction of alkaline phosphatase activity in monkey kidney cells

The induction of alkaline phosphatase (ALP) by dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) was investigated in strain JTC-12 . P3 cells derived from monkey (Maccaca irus) kidney cortex. ALP activity was increased by Bt2cAMP in a dose-dependent manner, reaching a plateau at concentrations higher than 5 mM with the activity being about 4 times that of the controls. The concentration of Bt2cAMP required for half-maximal induction of ALP activity was about 0.8 mM. ALP activity was increased rapidly by Bt2cAMP for the first 5 days and then continued to increase gradually towards a plateau level. Removal of Bt2cAMP from the medium caused a rapid decrease in the activity, suggesting that the induction of ALP activity by Bt2cAMP is reversible. ALP activity was induced synergistically in the presence of 1 mM sodium butyrate together with Bt2cAMP at concentrations from 0.01 to 1 mM. It was also found that in the presence of 1 mM Bt2cAMP, sodium butyrate increased ALP activity in the same manner as Bt2cAMP did in the presence of 1 mM sodium butyrate. Although dexamethasone, a potent glucocorticoid, had no effect on ALP activity in control cells, the hormone suppressed the ALP activity induced by Bt2cAMP in a dose-dependent manner. At concentrations above 0.2 mM, two xanthine derivatives, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX), also inhibited the induction of ALP activity by 1 mM Bt2cAMP. Inhibitors of protein synthesis, cycloheximide (1.5 micrograms/ml) and pactamycin (10 micrograms/ml), as well as inhibitors of RNA synthesis, actinomycin D (2 micrograms/ml) and alpha-amanitin (50 micrograms/ml), suppressed the induction of ALP activity.     

The effect of bromodeoxyuridine and dibutyryl cyclic AMP on the induction of placental alkaline phosphate in human choriocarcinoma cells.

The effect of 5-bromo-2'-deoxyuridine (BrdUrd) and dibutyryl cyclic AMP (Bt2cAMP) on the expression of the placental isoenzyme of human alkaline phosphatase was examined in BeWo choriocarcinoma cells. By using a combination of specific immunoprecipitation and polyacrylamide-gel electrophoresis of cells labelled either metabolically with [35S]methionine or cell-surface-labelled with 125I, both BrdUrd (5 micrograms/ml) and 1 mM-Bt2cAMP were shown to result in the enhanced accumulation of a specific protein. This protein has immunochemical identity and co-electrophoreses with placental alkaline phosphatase in two-dimensional gels. These results clearly demonstrate that the induction of placental alkaline phosphatase activity in choriocarcinoma cells treated with these agents is a consequence of the accumulation of specific enzyme protein rather than of altered catalytic activity.     

Induction of alkaline phosphatase activity by synergistic action of dibutyryl cyclic adenosine monophosphate, prednisolone, butyrate and sodium chloride in cultured cells

Human urinary bladder carcinoma cells (JTC-32) retain a low alkaline phosphatase activity. Prednisolone or a hypertonic concentration of NaCl caused a moderate increase in the activity (10- to 15-fold of control), but dibutyryl cAMP or butyrate did not. Examination of the combined effect of these four agents revealed that they acted synergistically in any combination. When the cells were incubated with the four agents together, the enzyme activity increased 60- to 250-fold. Serum also contributed to this synergistic increase. These agents slightly inhibited cell growth and protein synthesis. The enzyme induction was completely inhibited by cycloheximide or actinomycin D. The synergistic effect of the four agents on the enzyme activity was also observed in other strains of carcinoma cells, human urinary bladder carcinoma cells (JTC-30) and monkey hepatocarcinoma cells (NCLP-6E). Thus, it is concluded that the coexistence of the four agents provides general and superior conditions for the induction of alkaline phosphatase in cultured carcinoma cells.     

Parathyroid hormone inhibition of induction of alkaline phosphatase activity in HeLa cells by 5-iodo-2′-deoxyuridine without increased adenylate cyclase activity or cellular cAMP concentration

Parathyroid extract (PTE) as well as purified parathyroid hormone (PTH) activators of adenylate cyclase in bone and kidney, produced dose-dependent decreases in the induction of alkaline phosphatase activity by 5-iodo-2′-deoxyuridine in HeLa cells. However, the combination of PTE and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor which also inhibits the induction of alkaline phosphatase activity, in most cases produced less than additive inhibition of enzyme induction. PTE or PTH in concentrations of up to 10 times greater than that necessary to have maximal effects on the induction of alkaline phosphatase activity produced no increase in adenylate cyclase activity, nor did they increase intracellular cAMP concentrations. In addition, PTE did not potentiate the increase in cAMP concentration produced by IBMX. It thus appears that the inhibition of alkaline phosphatase activity by PTH is not mediated by cAMP.     

Activation of alkaline phosphatase with Mg2+ and Zn2+ in rat hepatoma cells. Accumulation of apoenzyme

In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis.     

Parathyroid hormone stimulation of alkaline phosphatase activity in cultured neonatal mouse calvarial bone cells: involvement of cyclic AMP and calcium

The involvement of cAMP and calcium in the rise in alkaline phosphatase (AP) activity observed when confluent, serum-free primary cultures of neonatal mouse calvarial cells are treated with parathyroid hormone (PTH) has been studied. Synthetic bovine PTH [bPTH-(1-34)] increased cellular cAMP at concentrations (10(-9) to 10(-7) M) previously found to elevate AP activity. Other substances that increase cAMP in these cells (forskolin, prostaglandin E2, 8-bromoadenosine cAMP and 3-isobutyl-1-methylxanthine) also increased enzyme activity. By comparison, increasing the concentration of calcium in the culture medium from 1.8 to 3.8 or 5.8 mM lowered the magnitude of the maximal AP response. In addition, treatment of cultures with the divalent cation ionophore A23187 caused a significant decrease in AP activity. These results suggest that: 1) cAMP mediates the rise in the specific activity of AP in cultured neonatal mouse calvarial cells treated with bPTH-(1-34) and 2) the concentration of calcium in the environment significantly influences the responsivity of bone cells to the hormone.     

Evidence for tight coupling of thyrotropin-releasing hormone receptors to stimulated inositol trisphosphate formation in rat pituitary cells

The effect of decreasing the concentration of receptors for thyrotropin-releasing hormone (TRH) on the surface of cloned rat pituitary (GH3) cells on TRH-stimulated inositol trisphosphate (Ins-P3) formation was investigated. Incubation of cells with dibutyryl cAMP (Bt2cAMP) for 16 h caused a decrease in [3H] TRH binding to intact cells to a minimum level 37 +/- 9.1% of control. Scatchard analysis of the concentration dependency of [3H]TRH binding showed that the effect of Bt2cAMP was to lower the receptor concentration without affecting its affinity for TRH. Similar decreases in [3H]TRH binding were found in cells incubated with 8-bromo-cAMP, cholera toxin, and sodium butyrate and, as shown previously, with TRH. In cells incubated with 1 mM Bt2cAMP for 16 h, but not for 1 h, the maximum TRH-induced increase in Ins-P3 was inhibited to 25 +/- 3.2% of that in control cells. Inhibition of TRH-induced Ins-P3 formation was also observed in cells treated with 8-bromo-cAMP, cholera toxin, and sodium butyrate for 16 h, and with TRH for 48 h. Inhibition of TRH-induced Ins-P3 formation and lowering of TRH receptor concentration caused by Bt2cAMP occurred in parallel with increasing doses of Bt2cAMP; at 16 h of exposure, half-maximal effects occurred with 0.3 mM Bt2cAMP. The concentration dependency of TRH-induced Ins-P3 formation was the same in control and Bt2cAMP-treated cells; half-maximal effects occurred with 10 nM TRH. These data demonstrate that decreases in TRH receptor concentration caused by several agents that act via different mechanisms are associated with reduced stimulation of Ins-P3 formation and suggest that the TRH receptor is tightly coupled to stimulation of hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phospholipase C.     

Regulation of Glycerol Phosphate Dehydrogenase and Lactate Dehydrogenase Activity by Forskolin and Dibutyryl Cyclic AMP in the C6 Glial Cells

We have compared the effects of norepinephrine, forskolin, and dibutyryl cyclic AMP (Bt2cAMP) on the regulation of the cytosolic enzyme glycerol phosphate dehydrogenase (GPDH) in the C6 rat glioma cell line. Forskolin and Bt2cAMP elicit a dose-dependent increase in the levels of the enzyme that was, however, unaffected by norepinephrine. The half-maximal effect of forskolin was obtained at 7-8 microM, and the effect was maximal at 30 microM. Dexamethasone at a 50 nM concentration produced a two- to sixfold induction of GPDH after 48 h. The combination of dexamethasone with forskolin or Bt2cAMP leads to an elevation in GPDH levels that is higher than that produced by one of the compounds alone. This potentiation is found when both agents are added together with or after the glucocorticoid. The increase in uninduced and dexamethasone-induced GPDH activity was blocked by cycloheximide and actinomycin D, indicating that de novo protein and RNA synthesis are required. The activity of cytosolic lactate dehydrogenase activity did not change after incubation with dexamethasone, but increased with forskolin or Bt2cAMP.     

ATP-induced cell contraction in dermal fibroblasts: effects of cAMP and myosin light-chain kinase

When 1 mM ATP is added to human dermal fibroblasts (DF) in monolayer culture permeabilized by glycerol, they undergo a rapid reduction in length and their intracellular actin filaments aggregate. This process is referred to as cell contraction. Treating glycerol-permeabilized DF with alkaline phosphatase before adding 1 mM ATP should cause dephosphorylation. Dephosphorylated preparations do not undergo cell contraction initiated by ATP. When myosin light-chain kinase (MLCK) isolated from turkey gizzard is added with cofactors to cells dephosphorylated by alkaline phosphatase treatment, contraction is restored. DF incubated for 24 h with db cAMP or cholera toxin show elevated intracellular concentrations of cAMP and little cell contraction. Contraction is reestablished when MLCK with cofactors is incubated with these preparations before ATP is added. Fibroblasts from Epidermolysis Bullosa dystrophica recessive patients produce excess cAMP. Those cells show minimal contraction, however; treating them with MLCK and cofactors renews contraction brought about by ATP. When DF are incubated with trifluoperazine to block calmodulin-dependent enzyme reactions, cell contraction is inhibited. Adding cytochalasin B disrupts microfilaments and also inhibits contraction. This work supports the idea that myosin ATPase is critical to cell contraction. Myosin ATPase is dependent on the phosphorylation of the regulatory peptide, myosin light chain. Elevating intracellular concentrations of cAMP or treatment of permeabilized cell preparations with alkaline phosphatase may inhibit myosin ATPase activity. The restoration of phosphorylation by adding MLCK with cofactors served to reestablish cell contraction.     

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.     

Induction of the messenger ribonucleic acid coding for phosphoenolpyruvate carboxykinase in H4-II-E cells. Evidence for a nuclear effect of cyclic AMP

The effect of N6,O2'-dibutyryl cyclic adenosine monophosphate (Bt2cAMP) on the induction of the mRNA coding for the enzyme phosphoenolpyruvate carboxykinase was examined in H4-II-E cells. this mRNA comprised about 0.1% of total cellular poly(A)+RNA activity in uninduced cells and was increased 5- to 7-fold by the cyclic nucleotide. The maximal level was reached 3 h after addition of the nucleotide to the cell culture. This induction is attributed to cAMP since the nonmetabolizable analogs 8-bromocAMP and 8-(4-chlorophenylthio)cAMP produce inductions comparable to Bt2cAMP while sodium butyrate and dibutyryl cyclic GMP had little effect. The increased translational activity correlated well with a proportionate increase in the amount of phosphoenolpyruvate carboxykinase (P-enolpyruvate carboxykinase) mRNA sequences which were hybridizable to a specific cDNA probe. Blot hybridization of total nuclear RNA isolated from uninduced H4-II-E cells revealed eight P-enolpyruvate carboxykinase RNA sequence species ranging in size from 1.8 to 6.9 kilobases. Treatment with Bt2cAMP increased the amount of all eight of these forms. This increase became maximal by 45-60 min and was maintained for at least 1 h. In contrast, analysis of cytoplasmic RNA showed a single 3.2-kilobase (23 S) band, which was still increasing in amount 2 h after Bt2cAMP treatment. Thus, Bt2cAMP resulted in a sequential induction of nuclear P-enolpyruvate carboxykinase RNA sequences followed by an increase in cytoplasmic phosphoenolpyruvate carboxykinase mRNA. We conclude that cyclic AMP exerts its main effect on P-enolpyruvate carboxykinase induction at the nuclear level.     

Regulation of alkaline phosphatase activity in rat hepatoma cells. Effects of serum proteins, cycloheximide, actinomycin D, chloroquine, dinitrophenol and potassium cyanide

Alkaline phosphatase activity in rat hepatoma cells (R-Y121B) cultured in a monolayer at 0.5% serum was enhanced by serum, bovine serum albumin, casein and gamma-globulin, but ovalbumin, polyvinylpyrrolidone, dexamethasone, insulin and dibutyrylcyclic AMP showed little effect on alkaline phosphatase activity. In addition, cycloheximide, actinomycin D, chloroquine, dinitrophenol and potassium cyanide also increased the enzyme activity, although the incorporation of [14C]leucine into cellular proteins was almost completely inhibited in the presence of these cytotoxic substances. When R-Y121B cell homogenates were incubated at 37 degrees C, alkaline phosphatase activity increased in a pH-dependent manner: the maximal increase was observed at pH 7.1. The magnitudes of the increase differed among cell homogenates and a 4- to 10-fold increase was observed. Alkaline phosphatase in R-Y121B cells was apparently heat-stable, but that in the cells obtained from various treatments was heat labile and the latter activity decreased to less than 50% of the initial activity after 15 min of incubation at 56 degrees C. Alkaline phosphatase in the control and also in the treated cells was more sensitive to L-homoarginine than L-phenylalanine. The Lineweaver-Burk plot showed that the increases in the enzyme activity were accompanied by changes not only in V but also in Km for alkaline phosphatase reaction. Finally, it has been suggested that the increases in alkaline phosphatase activity under various conditions are due to the conversion of the molecule with a low enzyme activity to the molecule with a high enzyme activity in R-Y121B cells.     

Metabolic activity of cholesteryl esters in aortic smooth muscle cells is altered by prostaglandins I2 and E2

Among the biochemical processes associated with the atherogenic process are increased aortic cholesteryl ester (CE) accumulation and altered prostaglandin (PG) production. The precise physiological role of PG, particularly prostacyclin (PGI2), in the control of CE metabolism in intact aortic smooth muscle cells remains to be fully elucidated. We report here that cytosolic neutral cholesteryl ester hydrolytic activity (NCEH) in intact cultured aortic smooth muscle cells is significantly increased by 75-250 nM PGI2 at the end of a 2-hr incubation period. The effect was mediated by increased intracellular cAMP levels since the effect of PGI2 on NCEH activity was abolished in the presence of an inhibitor of adenylate cyclase activity, viz., dideoxyadenosine (DDA0. Although the addition of 20-100 microM dibutyryl cAMP (Bt2cAMP) and 50-100 microM sodium arachidonate also increased NCEH activity twofold, 6-keto PGF1 alpha, PGE1, and PGE2 did not increase the activity of this enzyme. In contrast to these findings, 75-250 nM PGE2 significantly inhibited CE synthetic activity (ACAT) approximately 60%. Arachidonate or Bt2cAMP did not affect ACAT activity. This decrease in ACAT activity induced by PGE2 does not appear to be mediated by cAMP. Taken together, these findings suggest that PGI2, a well known potent vasodilator and inhibitor of platelet aggregation, and PGE2 may have an important regulatory role in aortic CE metabolism.     

1,25-Dihydroxyvitamin D3 increases bone alkaline phosphatase isoenzyme levels in human osteogenic sarcoma cells

The specific activity of alkaline phosphatase was increased in two human osteogenic sarcoma cell lines, SAOS and TE85, after treatment with 1,25 dihydroxy-vitamin D3 (1,25(OH)2D3). Enzyme activity increased when the cells were incubated with concentrations of 1,25(OH)2D3 between 10(-9) and 10(-7) M and cell growth was not inhibited at these concentrations. The specific activity of alkaline phosphatase was 4- to 7-fold higher than that in the control cells after 5 to 7 days of continuous exposure to 1,25(OH)2D3. Immunochemical studies demonstrated that the enzyme from both control and 1,25(OH)2D3-treated cultures cross-reacted with antisera specific for the phosphatase isoenzyme produced by normal human bone, and did not cross-react with antisera specific for the placental alkaline phosphatase isoenzyme. The increased enzyme activity in cultures induced with 1,25(OH)2D3 correlated with an absolute increase in the number of bone-specific phosphatase molecules, as determined by radioimmunoassay. No effect on alkaline phosphatase activity was observed when the cells were treated with other vitamin D metabolites or with 5-bromo-2'-deoxyuridine. Comparative studies demonstrated that hydrocortisone, another steroid hormone, increased the phosphatase activity with a different time course than did 1,25(OH)2D3. High affinity cytoplasmic receptors for 1,25(OH)2D3 and hydrocortisone were found in the SAOS and TE85 cells.     

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.     

Impact of transgenic cotton varieties on activity of enzymes in their rhizosphere

The impact of five Bacillus thuringiensis (Bt) cotton varieties and their respective isogenic non-Bt(NBt) isolines (ANKUR-2534, MECH-6304, RCH-317, ANKUR-651 and MECH-6301) was assessed on the key soil enzymes i.e., dehydrogenase, alkaline phosphatase and urease in their rhizosphere at four growth stages of the crop, namely vegetative, flowering, bolling and harvesting. These varieties were grown on farmer's field in villages 22 miles and 24 miles of Ganganagar District of Rajasthan State in India. Results showed that dehydrogenase, alkaline phosphatase and urease activities were higher in rhizosphere of Bt isolines as compared to NBt isolines of all the varieties. Except phosphatase, differences in dehydrogenase and urease activities in rhizosphere of Bt and NBt isolines of all five varieties were significant (P < 0.05). Maximum enhancement in the three enzymes activities was observed in MECH-6304 Bt isoline rhizosphere. Maximum and minimum activities of dehydrogenase and urease were observed in MECH-6304 and RCH-317 Bt isolines, respectively, whereas phosphatase activity was maximum and minimum in MECH-6304 and ANKUR-651 Bt isolines, respectively. Maximum dehydrogenase and urease activities were observed at boll formation and minimum at flowering and harvesting stage, respectively, while maximum phosphatase activity was observed at vegetative stage and minimum at harvesting stage. In conclusion, all the studied Bt isolines of cotton varieties showed no adverse effect on dehydrogenase, alkaline phosphatase and urease activities in the rhizosphere.     

Alkaline phosphatase activity in whitefly salivary glands and saliva

Alkaline phosphatase activity was histochemically localized in adult whiteflies (Bemisia tabaci B biotype, syn. B. argentifolii) with a chromogenic substrate (5-bromo-4-chloro-3-indolylphosphate) and a fluorogenic substrate (ELF-97). The greatest amount of staining was in the basal regions of adult salivary glands with additional activity traced into the connecting salivary ducts. Other tissues that had alkaline phosphatase activity were the accessory salivary glands, the midgut, the portion of the ovariole surrounding the terminal oocyte, and the colleterial gland. Whitefly nymphs had activity in salivary ducts, whereas activity was not detected in two aphid species (Rhodobium porosum and Aphis gossypii). Whitefly diet (15% sucrose) was collected from whitefly feeding chambers and found to have alkaline phosphatase activity, indicating the enzyme was secreted in saliva. Further studies with salivary alkaline phosphatase collected from diet indicated that the enzyme had a pH optimum of 10.4 and was inhibited by 1 mM cysteine and to a lesser extent 1 mM histidine. Dithiothreitol, inorganic phosphate, and ethylenediaminetetraacetic acid (EDTA) also inhibited activity, whereas levamisole only partially inhibited salivary alkaline phosphatase. The enzyme was heat tolerant and retained approximately 50% activity after a 1-h treatment at 65 degrees C. The amount of alkaline phosphatase activity secreted by whiteflies increased under conditions that stimulate increased feeding. These observations indicate alkaline phosphatase may play a role during whitefly feeding.     

Inductive effects of prostaglandins on alkaline phosphatase in osteoblastic cells, clone MC3T3-E1

The effects of prostaglandins (PGs) on the induction of alkaline phosphatase (ALP) were investigated in osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. Prostaglandin E2 (PGE2) stimulated ALP activity in the cells in a dose-dependent fashion with a maximal effect which was about twice that in the control cells at concentrations of 100-500 ng/ml. Actinomycin D and cycloheximide inhibited the stimulative effect of PGE2 on ALP activity in the cells. PGE2-induced and native ALPs in the cells were of the same type as that in adult mouse calvaria, being heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive. Isobutyl methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, stimulated the inductive effect of PGE2 on ALP activity at 0.1 mM, at which concentration IBMX alone had little effect on the activity. PGE2 also increased the intracellular cAMP content in a dose-dependent fashion with a maximal effect at 100 ng/ml. PGE1, PGF1 alpha, and PGF2 alpha (primary PGs like PGE2) increased the activity. Our present results suggest that PGs stimulate the differentiation of osteoblasts and are involved in bone formation in vivo, as well as in bone resorption.