Recognition of xenogeneic erythrocytes: the GalNAc/Gal-particle receptor of rat liver macrophages mediates or participates in recognition

We studied mechanisms that mediate recognition of human erythrocytes (HRBC) and sheep erythrocytes (SRBC) by rat liver macrophages. We used an in vitro cell binding assay that allows spontaneous formation of cell contacts. Binding of HRBC to rat macrophages shows the following characteristics: inhibition studies with several monosaccharides and oligosaccharides yield complete inhibition of cell contacts with saccharides, which block the GalNAc/Gal-particle receptor on rat liver macrophages. We found the inhibition pattern: N-acetyl-D-galactosamine, lactose greater than D-galactose, D-fucose greater than L-fucose much greater than N-acetyl-D-glucosamine. Cell binding is dependent on the presence of calcium ions, but not influenced by heat-aggregated IgG or gangliosides. The inhibition pattern was the same after treatment of HRBC with neuraminidase. Therefore, binding of HRBC, as well as binding of neuraminidase-treated HRBC, is mediated by the GalNAc/Gal-particle receptor. Binding of SRBC is partly inhibited by galactose-related saccharides. Binding is also partly inhibited by heat-aggregated IgG, gangliosides, and L-fucose. Complete inhibition of cell contacts with SRBC is achieved by combination of all inhibitors. We therefore conclude that binding of SRBC is mediated by several different mechanisms, including the GalNAc/Gal-particle receptor. Binding of neuraminidase-treated SRBC, however, was found to be completely inhibited by saccharides, which block the GalNAc/Gal-particle receptor. We conclude that the GalNAc/Gal-particle receptor mediates or participates in recognition of non-self structures.     

In vitro inhibition of alkaline phosphatase activities from intestine, bone, liver, and kidney by phenobarbital.

A kinetic study of the inhibition of several alkaline phosphatase (AP isoenzyme activities by phenobarbital was carried out using p-nitrophenylphosphate (10 mM) as a substrate at pH 9.8 in a 300-mM Hepes buffer. AP from bovine kidney, calf intestine, bovine liver, and rat bone was used. Over a phenobarbital concentration range of 20-400 mM, all these isoenzymes were inhibited in an uncompetitive manner with a Ki of 200 mM for intestinal AP, and in a linear mixed-type manner for all the other isoenzymes tested. The Ki values were 10, 40 and 55 mM for kidney, bone and liver AP, respectively. The use of 15 mM carbonate-bicarbonate or 400 mM diethanolamine buffer did not modify the degree of inhibition of intestinal AP activity. Dixon plots of the reciprocal of reaction velocity versus inhibitor concentration either at different substrate concentration or at different DEA concentration indicate uncompetitive inhibition for the intestinal enzyme. This in vitro inhibitory effect of phenobarbital is in contrast to its in vivo stimulating action on AP. However, in the whole animal, the effects of phenobarbital administration probably represent the sum of multiple effects.     

Membrane proteinases from normal and neoplastic tissues in man and the rat

Plasma membranes were purified from rat liver, muscle and sarcoma tissues and from human liver and hepatoma tissues. The plasma membranes all contained DFP-sensitive, neutral proteolytic activity. Plasma membranes from all normal tissues contained a single DFP-binding protein of apparent molecular weight 68,000. Only the plasma membranes from tumour tissue contained a plasminogen activator; the DFP-binding proteins from these membranes were more diverse than those from the normal samples. The rat liver plasma membrane proteinase was purified. It was a labile enzyme sensitive to inhibition by DFP and by calcium ions, and with a broad substrate specificity. A similar protein was the sole DFP-binding protein in rat liver microsomes. This and the properties of the enzyme suggested a possible role in the processing and secretion of newly-synthesized protein.     

Comparative antioxidant activity of individual herbal components used in Ayurvedic medicine

Four aqueous extracts from different parts of medicinal plants used in Ayurveda (an ancient Indian Medicine) viz., Momardica charantia Linn (AP1), Glycyrrhiza glabra (AP2), Acacia catechu (AP3), and Terminalia chebula (AP4) were examined for their potential as antioxidants. The antioxidant activity of these extracts was tested by studying the inhibition of radiation induced lipid peroxidation in rat liver microsomes at different doses in the range of 100-600 Gy as estimated by thiobarbituric acid reactive substances (TBARS). Of all these extracts, AP4 showed maximum inhibition in the TBARS formation and hence is considered the best antioxidant among these four extracts. The extracts were found to restore antioxidant enzyme superoxide dismutase (SOD) from the radiation induced damage. The antioxidant capacities were also evaluated in terms of ascorbate equivalents by different methods such as cyclic voltammetry, decay of ABTS(.-) radical by pulse radiolysis and decrease in the absorbance of DPPH radicals. The results were found to be in agreement with the lipid peroxidation data and AP4 showed maximum value of ascorbate equivalents. Therefore AP4, with high antioxidant activity, is considered as the best among these four extracts.     

The Role of DNase and EDTA on DNA Degradation in Formaldehyde Fixed Tissues

Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.     

In vivo dose response relationship between physostigmine and cholinesterase activity in RBC and tissues of rats

Dose response of physostigmine (Phy) was studied in rat using various doses (25-500 micrograms/kg i.m.). Rats were sacrificed 15 min after Phy administration. Blood and tissues were analyzed for ChE activity by radiometric method and Phy concentration by HPLC method. A comparison of ChE values in different tissues of rats indicated that ChE activity was highest in brain (7.11 mumol/min/g) and least in diaphragm (0.67 mumol/min/g). The enzyme activity was eleven times more in brain as compared to diaphragm. Phy produced a dose-dependent inhibition of ChE in RBC (18-42%), brain (23-35%) and diaphragm (25-35%) from 50 to 200 micrograms/kg, then ChE inhibition was plateaued from 200 to 500 micrograms/kg in these tissues. A dose related ChE inhibition was seen in heart (16-50%) and thigh muscle (8-53%) from 50 to 500 micrograms/kg. Phy concentration increased linearly from 50 to 400 micrograms/kg in plasma, brain, heart and thigh muscle. These results indicate that ChE inhibition is linear up to 200 micrograms/kg in RBC, 150 micrograms/kg in brain and 300 micrograms/kg in heart. This linearity is not consistent in other tissues.     

Demonstration of tartrate-resistant acid phosphatase in un-decalcified, glycolmethacrylate-embedded mouse bone: a possible marker for (pre)osteoclast identification

Fixed, undecalcified mouse long bones were embedded in glycol methacrylate (GMA), sectioned, and incubated for acid phosphatase in the presence or absence of tartrate, to investigate the feasibility of tartrate-resistant acid phosphatase as a histochemical marker for osteoclast identification. Naphthol AS-BI phosphate was used as the substrate and hexazonium pararosanaline as coupler. Cytocentrifuge preparations of mouse, rat, and quail bone marrow or frozen and GMA sections of mouse splenic tissue were used as controls to specify acid phosphatase activity. After adequate fixation, acid phosphatase activity sensitive to tartrate inhibition (TS-AP) was demonstrated in macrophages from spleen, bone marrow, and loose connective tissue surrounding bone rudiments. Acid phosphatase activity resistant to tartrate inhibition (TR-AP), was detected in multi-nuclear osteoclasts and in some mononuclear cells from bone marrow and periosteum. In cytocentrifuge preparations and frozen sections of mouse spleen, TR-AP was demonstrated after simultaneous incubation with substrate and tartrate. In GMA sections, however, TR-AP could only be demonstrated after pre-incubation with tartrate before application of substrate. We suggest that histochemical demonstration of TR-AP versus TS-AP on GMA-embedded bone sections by means of a pre-incubation method can be used as an identification marker of (pre)osteoclasts. Plastic embedding is recommended for its excellent preservation of morphology and enzyme activity.     

Lysosomal tartrate sensitive acid phosphatase deficiency in cells which contain lysosomal "high uptake forms"

The catalytic and immunological properties of acid phosphatases (EC 3.1.3.2.) in different tissues were studied. It was demonstrated that high uptake forms of lysosomal enzymes like beta-galactosidase isolated from human platelets and bovine testis are mature enzymes, which have not lost their mannose-6phosphate marker. The results presented indicate that this phenomenon is related to a low activity or the complete absence of the lysosomal tartrate sensitive acid phosphatase activity in the tissues concerned.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Patterns of proliferation of antibody-forming cells after an intravenous immunization with hamster erythrocytes (HRBC) were compared in groups of mice possessing different activities of thymus-derived lymphocytes (T cells). 1) Marked differences in the numbers of hemolysin plaque-forming cells (PFC) after HRBC injection were found among the low- and high-responder normal mice and those pretreated with HRBC in complete Freund's adjuvant (CFA) or incomplete adjuvant (IFA), and they appeared to depend primarily upon the different rates of proliferation of antibody-forming cells rather than on the numbers of antigen-specific lymphocytes initiating the antibody response. 2) The numbers of hemolytic foci were slightly larger in mice with large numbers of PFC (normal SL mice, the pretreated SL and C57BL/6 mice) than in those with small numbers of PFC (normal C57BL/6 mice). The numbers of hemolytic foci increased at almost the same rate from day 2 to day 3 in both groups, while the numbers of PFC increased more efficiently in mice with large numbers of PFC than in those with small numbers of PFC from day 2 to day 3. Individual hemolytic foci appeared to contain larger numbers of PFC in mice with large total numbers of PFC than in those with small total numbers of PFC. 3) The numbers of rosette-forming cells (RFC) were increased by pretreatment with HRBC in CFA and by pretreatment with HRBC in IFA to almost the same extent. Rates of increases in PFC were, however, larger by pretreatment with HRBC in CFA than with HRBC in IFA. These results suggested that the activity of the T cell determined not only the rates of proliferation of antibody-forming cells but also the antibody-producing capacity of each cell.     

胃癌细胞中视黄酸受体抑制AP-1活性的不同方式

 研究胃癌细胞中视黄酸受体RARα和RARβ抑制活化蛋白 1(activatorprotein 1,AP 1)活性的不同方式及其与全反式视黄酸 (ATRA)作用的相关性 .瞬时转染RARβ表达载体到MKN 4 5细胞后 ,佛波脂 (TPA)诱导的AP 1活性受到明显抑制 ,且与RARβ浓度正相关 ,与ATRA存在与否无关 ;相反 ,RARα转染细胞后 ,对TPA诱导的AP 1活性的抑制不仅与RARα的浓度相关 ,而且依赖于AT RA .凝胶阻抑测定表明 ,TPA可以显著加强AP 1结合活性 ,当ATRA处理不表达RARβ和低表达RARα的MKN 4 5细胞后 ,AP 1结合活性不受影响 ;然而 ,表达RARα和RARβ的BGC 82 3细胞经AT RA处理后 ,TPA诱导的AP 1结合活性则受到抑制 .另外 ,分析与抗AP 1活性相关的RARβ功能区表明 ,DNA结合区的缺失导致RARβ抑制AP 1活性作用的丧失 ,而配体结合区对于RARβ抑制AP 1活性则是非必需的 .以上结果证实 ,有胃癌细胞中 ,RARβ可能是AP 1活性的抑制因子 ,RARα则可能是ATRA作用的靶向 .尽管它们的作用方式有所不同 ,但最终都可以通过抑制AP 1活性来抑制胃癌细胞生长     

Cytochemistry and biochemistry of acid phosphatases. III. Inhibition experiments of lysosomal and secretory acid phosphatases of the rat ventral prostate

Biochemical and cytochemical inhibition experiments of rat prostatic acid phosphatase were performed using enzymes separated on isoelectric focusing (IEF) gels, and thin sections of the rat ventral prostate. Various inhibitors, including L (+) tartrate, mercuric ions and sodium fluoride were applied to electrofocused enzymes which were subsequently stained for acid phosphatase activity. Enzymes focused on IEF gels at pH 7.9 and 8.1, respectively, were inhibited with 1.8 x 10-3 M tartrate, while the enzyme activities with isoelectric points (pl) of 5.6 and 7.15, respectively, were only slightly inhibited by this compound. Using 10-3M mercuric ions, enzymes with pl of 5.6 and 7.15 were inhibited while the enzymes with pl of 7.9 and 8.1 were still active. The biochemical procedures were adapted to chopper sections of perfused-fixed ventral prostate of the rat. Preincubation of the sections with 2.4 x 10-3M mercuric chloride blocked the secretory enzyme and most of the lysosomal enzyme and resulted in an artificial staining of the Golgi apparatus and other cytoplasmic organelles. Nuclear precipitates however were prevented. L (+) tartrate could not be used at the ultrastructural level since it developed false positive results by the formation of lead tartrate. The results indicate that no selective inhibition of either secretory or lysosomal acid phosphatase can be achieved at the ultrastructural level using metal salts or tartrate, respectively.     

Inhibition of type I and type II iodothyronine deiodinase activity in rat liver, kidney and brain produced by selenium deficiency.

Selenium deficiency for periods of 5 or 6 weeks in rats produced an inhibition of tri-iodothyronine (T3) production from added thyroxine (T4) in brain, liver and kidney homogenate. This inhibition was reflected in plasma T4 and T3 concentrations, which were respectively increased and decreased in selenium-deficient animals. Although plasma T4 levels increased in selenium-deficient animals, this did not produce the normal feedback inhibition on thyrotropin release from the pituitary. Selenium deficiency was confirmed in the animals by decreased selenium-dependent glutathione peroxidase (Se-GSH-Px) activity in all of these tissues. Administration of selenium, as a single intraperitoneal injection of 200 micrograms of selenium (as Na2SeO3)/kg body weight completely reversed the effects of selenium deficiency on thyroid-hormone metabolism and partly restored the activity of Se-GSH-Px. Selenium administration at 10 micrograms/kg body weight had no significant effect on thyroid-hormone metabolism or on Se-GSH-Px activity in any of the tissues studied. The characteristic changes in plasma thyroid-hormone levels that occurred in selenium deficiency appeared not to be due to non-specific stress factors, since food restriction to 75% of normal intake or vitamin E deficiency produced no significant changes in plasma T4 or T3 concentration. These data are consistent with the view that the Type I and Type II iodothyronine deiodinase enzymes are seleno-enzymes or require selenium-containing cofactors for activity.     

Spontaneous rosette-forming lymphocytes in sheep lymph. A marker for antigen-binding cells

Type O Rh positive human red blood cells (HRBC), native or treated with one of three enzymes (papain, trypsin, or neuraminidase), were labeled with ⁵¹Cr and then sensitized with anti-Rh immune globulin. These cells served as targets in antibody-dependent cellular cytotoxicity (ADCC) for unfractionated human mononuclear cells (MC), MC depleted of monocytes by adhesion to plastic, and MC enriched for monocytes. Enzyme-treated HRBC were lysed with greater efficiency in ADCC than native HRBC. This was explained by the finding that the enzyme modified HRBC were lysed both by lymphocytes and monocytes, whereas native HRBC were lysed only by monocytes. The lysis of native HRBC was strongly inhibited by small amounts of human serum or free IgG. In contrast, the lysis of enzyme-treated HRBC was considerably more resistant to inhibition by human serum or free IgG. The enhanced lysis of enzyme-treated HRBC could not be the result of increased binding of antibody to the target cells, since augmented lysis was observed both for HRBC sensitized before neuraminidase treatment as well as for HRBC sensitized after neuraminidase treatment. These results suggest that the surface charge on target cells plays a critical role in determining which classes of leukocytic effector cells are active in ADCC systems.     

Rat alkaline phosphatase. I. Purification and characterization of the enzyme from osteosarcoma: generation of monoclonal and polyclonal antibodies

Alkaline phosphatase (AP) was purified to over 90% homogeneity from rat osteosarcoma by acetone precipitation followed by chromatography on DEAE-cellulose, Sephacryl S-200, and hydroxyapatite. The purified enzyme had a specific activity of 759 units/mg protein at its optimal pH (10.5), and a Km of 0.8 mM for p-nitrophenylphosphate. The enzyme's apparent subunit molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82,000 Da. The heat-inactivation profile and homoarginine inhibition were characteristic of the bone-liver-kidney AP isoenzyme. Monoclonal and polyclonal anti-AP antibodies were prepared and characterized. Polyclonal rabbit antiserum quantitatively precipitated the activity from purified AP preparations and tissue extracts but did not inhibit AP catalytic activity. This antiserum was almost 10-fold less active against heat-inactivated enzyme when tested in a competition assay using 125I-AP. Two distinct monoclonal antibodies were each partly effective in immunoprecipitating AP when tested individually; however, together they precipitated over 90% of the AP activity.     

Alkaline phosphatase in stallion semen: characterization and clinical applications

Significant amounts of alkaline phosphatase (AP) activity have been found in semen plasma from numerous species. In species in which the majority of semen plasma AP (SPAP) activity originates from the epididymis and testicle, SPAP activity can be used clinically as a marker to differentiate testicular origin azoospermia or oligospermia from ejaculatory failure. Information on SPAP activity in stallions to date has been limited. In this study, a standard clinical chemistry analyzer was used to determine AP activity in pre-ejaculatory fluid and ejaculates from groups of normal stallions. Additionally, accessory glands, epididymides, testicles and other components of the urogenital tract of normal stallions were assayed to determine which tissues contain SPAP activity. The results indicated that levels of AP activity are low in pre-ejaculatory fluid, but significantly higher in ejaculatory fluid from normal stallions. Spermatozoa were not a significant source of SPAP activity. High levels of SPAP activity were found in the testes and epididymides. These findings suggest that SPAP activity is a candidate for a sperm-independent marker for ejaculation in the stallion. Finally, AP activity was determined in ejaculatory fluid from a stallion with bilaterally blocked ampullae, both before and after relief of the blockage. While the blockage was present, AP activity in ejaculatory fluid was low. However, following relief of the blockage, AP activity in ejaculatory fluid rose dramatically, thus suggesting that AP activity will be useful as an inexpensive, simple clinical assay for differentiating ejaculatory failure or excurrent duct blockages from testicular origin azoospermia and oligospermia.     

Comparative immunochemical characteristics of different phosphodiesterases of cyclic nucleotides

Precipitating monospecific antibodies against purified bovine retinal rod outer segment phosphodiesterase (EC 3.1.4.17) were obtained from rabbit blood serum. These antibodies do not form precipitating complexes with phosphodiesterase isolated from rat or ox brain tissues or from the heart, lung, liver, kidney, testes and uterus of the rat. The antibodies inhibit the activity of retinal rod outer segment phosphodiesterase or that of rat brain, liver, heart and uterus enzyme (despite the lack of precipitation) but have no effect on the phosphodiesterase activity of preparations obtained from rat lungs, kidney or testes. The same effect on the phosphodiesterase activity of all these tissues is exerted by monovalent fragments of the antibodies. Using partially purified preparations of phosphodiesterase from retinal rod outer segments and brain of the ox and from human myometrium, the mechanisms of inhibition of the enzyme catalytic activity by the antibodies was studied. In the presence of the antibodies, the Km and V values appeared to be different, depending on the preparation. It was assumed that a certain site in the phosphodiesterase molecule is characterized by great structural rigidity. Taking into account the shifts in the Km values induced by the antibodies, the differences in the localization of the antigenic determinant in relation to the enzyme active center are discussed.     

Sublethal effects of an organophosphorus insecticide (RPR-II) on biochemical parameters of tilapia, Oreochromis mossambicus

The effect of exposure to sublethal concentrations (0.017 mg L(-1), 1/10 of LC50) of the novel organophosphate (OP) insecticide, 2-butenoic acid-3-(diethoxyphosphinothioyl) methyl ester (RPR-II) on biochemical parameters in Oreochromis mossambicus was studied during exposure for 3, 7, 15, 30 and its recovery response after seven days. Acetylcholinesterase (AChE) activity of brain, gill and muscle was inhibited by 67%, 77% and 73% respectively on day-30. The plasma and kidney alanine aminotransferase (AlaAT), and aspartate aminotransferase (AspAT) activity increased, while decreases were observed in gill and liver. Increases in acid phosphatase (AcP), and alkaline phosphatase (AP) activities were observed in plasma, gill, and kidney, and reductions of 20% and 61% in liver AcP and AP, respectively. Depletion of glycogen was observed in all tissues, an indication of typical stress related response of the fish with pesticide. Lactate dehydrogenase (LDH) activity decreased in liver and muscle, indicating tissue damage but a significant increase in LDH activity in gill and brain was observed. Depletion of glutathione (GSH) was observed in all tissues, thereby enhancing lipid peroxidation resulting in cell damage. The induction in hepatic glutathione-S-transferase (GST) levels indicates protection against the toxicity of xenobiotic-induced lipid peroxidation. There was a significant recovery in the above biochemical parameters, in all tissues of fish after a recovery period of seven days. These results revealed that the OP insecticide RPR-II is highly toxic and affects the intermediary metabolism of O. mossambicus.     

Immunocytochemical and biochemical demonstration of formaldhyde dehydrogenase (class III alcohol dehydrogenase) in the nucleus.

Alcohol dehydrogenase (ADH), the major enzyme catalyzing the biological oxidation of ethanol in mammals, includes four classes with very different capacities for ethanol oxidation. Class III ADH is present in all the tissues and is well conserved throughout evolution. This enzyme has a low activity with ethanol, is specific for the glutathione-dependent oxidation of formaldehyde, and is therefore a formaldehyde dehydrogenase (FALDH). Until now there have been few and conflicting studies concerning its intracellular distribution, which is important for the understanding of its role in cell function. In the present work we used biochemical and immunocytochemical methods to assess the distribution of FALDH in rat hepatocytes and astroglial cells. With the glutathione-dependent formaldehyde dehydrogenase assay, we found the highest activity in the cytosol of hepatocytes and brain cells (12 and 2.6 mU/mg protein, respectively), but nuclei also exhibited significant activity (1.16 and 2.1 mU/mg protein, respectively). The immunocytochemical results showed the presence of FALDH binding sites in both the cytoplasm and the nucleus of the different cell types studied. Whereas no specific gold particle labeling was seen associated with any cytoplasmic component, in the nucleus the particles were found mainly over condensed chromatin and interchromatin regions. Finally, the gold particle density over both the nucleus and cytoplasm was greater in differentiated than in proliferating astrocytes in primary culture. In contrast, class I ADH, primarily responsible for ethanol metabolism, was found only in the cytoplasm of hepatocytes. We propose that one of the functions of FALDH is to protect cell structures, including DNA, from the toxic effects of endogenous formaldehyde, which is an intermediate in many metabolic process.     

Atriopeptin biochemical pharmacology

Several low-molecular-weight peptides that possess potent natriuretic, diuretic, and vascular smooth muscle relaxant activity have been isolated from atrial extracts. Elucidation of their structure indicates that they consist of a 17-membered ring of amino acids formed by a cystine disulfide bond and that they differ only in the composition of the amino and carboxy termini. The 24-amino-acid peptide atriopeptin (AP) III was selected as the reference compound for structure-activity studies. Amino-terminal amino acid extensions on APIII markedly increase the natriuretic-diuretic but not the renal vasodilatory response in anesthetized dogs, which suggests a heterogeneity of AP receptors in renal tubular and vascular tissues. Radioligand (125I-labeled APIII) binding studies with fresh rat kidney slices indicate that the primary renal sites of specific AP binding are in the glomerulus and in the papillary segment of the medulla, thus implicating these structures in the natriuretic-diuretic effect. Data obtained from radioimmunoassay, chromatographic migration, vasorelaxant biological activity, and peptide sequence analysis indicate that Ser-Leu-Arg-Arg-APIII is the major circulating form of low-molecular-weight atrial peptide present in rat plasma. Circulating APs fulfill many of the criteria for involvement in the endocrine regulation of fluid and electrolyte homeostasis.     

Parathyroid hormone-induced changes in alkaline phosphatase expression in fetal calvarial osteoblasts: Differences between rat and mouse

We studied the effects of parathyroid hormone (PTH) on two markers of the osteoblast phenotype: alkaline phosphatase (AP) (activity and mRNA) and cyclic adenosine monophosphate (cAMP) accumulation. Osteoblast-like cells derived from fetal rat (ROB) and mouse (MOB) calvariae were isolated by collagenase treatment. Cells were cultured in α-Minimal Essential Medium (MEM) with 2% fetal calf serum (FCS) for 4 days. In ROB and MOB bPTH(1-34) induced a fast increase (up to 5 minutes) in cAMP accumulation. When equal amounts of cells were seeded, the cAMP accumulation was higher in MOB than in ROB. No difference in basal AP activity was observed between ROB and MOB. When bPTH(1-34) was added to ROB for the last 24 or 48 hr, AP activity decreased dose dependently. However, MOB treated with bPTH(1-34) for the last 24 or 48 hours showed an increase of AP activity. Basal AP activity was positively correlated with the seeding density of ROB and MOB cultures. Basal AP activity influenced the degree of inhibition (ROB) or stimulation (MOB) after incubation with bPTH(1-34). © 1993 Wiley-Liss, Inc.