Synthesis and secretion of alkaline phosphatase <i>in vitro</i> from first-trimester and term human placentas

An α2‐adrenoceptor antagonist, idazoxan, that binds to both α2‐adrenoceptors and to imidazoline sites (IR), has been used to characterize human placental IR. Human placenta is shown to be the richest source of IR (1800 ± 100 fmol mg−1 protein; Kd 38.9 ± 3.4 nm). Primary cells derived from human placenta and grown in monolayers, also displayed a high density of receptors (3209 ± 136 fmol mg−1 in cytotrophoblasts and 3642 ± 144 fmol mg−1 protein in syncytiotrophoblast enriched cell culture). [3H]‐idazoxan did not show binding characteristics of α2‐adrenoceptors in human placental membranes or human trophoblastic cells, thus making it a ligand of choice to study the imidazoline site. The tissue appeared to be lacking α2‐adrenoceptors in that other α2‐adrenoceptor ligands, [3H]‐rauwolscine and [3H]‐clonidine, do not bind to α2‐adrenoceptors in human placenta. IRs are localized on the cell surface, as determined by the release of bound [3H]‐idazoxan from cells, when washed with high ionic/acidic medium. Imidazoline receptors of human placenta display high affinity for amiloride (72 ± 27 nm). The high affinity was used as a criterion to classify IR to IRa subtype (placenta, rabbit kidney, rabbit liver and rabbit adipose cells) as opposed to the IRb subtype which display low affinity for amiloride (> 2 μm, in all the other tissues). Several novel ligands comprising a guanido functional group attached to an aromatic residue (e.g. benziliden‐amino‐guanidine (BAG), guanido pyrole) display pronounced selectivity for IR over the α2‐adrenoceptors as the affinity of BAG is about 40 fold higher (Kd = 18.9 ± 13.8 nm in human placenta), than the affinity for α2‐adrenoceptors (Kd = 768 ± 299 nm in human platelets). Imidazoline sites bind selectively BAG and other guanido ligands thus indicating a distinct structural requirement at its site of binding. K+ channel blockers and monovalent ions (e.g. Cs+ and NH4+) interfere with idazoxan binding to IR, indicating a possible involvement of IR in K+ transport.

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“…Placental alkaline phosphatase activity was determined at pH 10.5 with p-nitrophenyl phosphate as the substrate according to Galski et al (1981).…”

Section: Alkaline Phosphatase Activitymentioning

confidence: 99%

Imidazoline binding sites in human placenta: evidence for heterogeneity and a search for physiological function

Diamant

Eldar‐Geva

Atlas

1992

British J Pharmacology

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“…Similarly, secretion of alkaline phosphatase in high concentration by the trophoblastic cells were found in the maternal circuit, while only traces of the enzyme were present in the foetal perfusate. Although no data are available from an in-vitro perfusion system, Galski et al (1981), using tissue slices from both first trimester and term placenta, showed both synthesis and secretion of the enzyme to take place in the culture media. Moreover, clinical studies have also shown high concentrations of alkaline…”

Section: Discussionmentioning

confidence: 99%

Transfer of Heparin Across the Human Perfused Placental Lobule

Bajoria

Contractor

1992

Journal of Pharmacy and Pharmacology

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A system of dual perfusion of an isolated lobule of term human placenta was used as a model to study the transfer of heparin from maternal to foetal circulation. The metabolic viability of the system was assessed by measuring beta-HCG and alkaline phosphatase levels in both maternal and foetal perfusates. Creatinine and antipyrine were used as markers to determine juxtaposition of the maternal and foetal circulations. Results of this study indicate that following administration of a single bolus dose of heparin into the maternal circulation, its concentration declined slowly from 99.01 +/- 2.98 at 15 min to 97.23 +/- 4.12% and transfer of heparin in the foetal circulation was linear and increased from 0.10% +/- 0.05% at 15 min to 0.46 +/- 0.19% over a period of 120 min. The maternal (MAUC) and foetal (FAUC) concentration-time integrals were found to be 70160 +/- 1332 and 340 +/- 30 int. units min mL-1, respectively. Placental permeability of heparin and creatinine, calculated as the ratio of foetal concentration to the integral maternal-foetal concentration difference, was 8.65 x 10(-5) +/- 0.80 x 10(-5) and 0.033 +/- 0.006 mL min-1 g-1 of perfused placental weight, respectively. These data suggest that heparin was transferred from the maternal to the foetal circulation in small quantities.

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“…The phosphatase activity of the separated subunits, reconstituted enzyme, and the native enzyme was assayed using p ‐nitrophenylphosphate as the substrate [31].…”

Section: Methodsmentioning

confidence: 99%

A novel autophosphorylation mediated regulation of nitrite reductase in Candida utilis

1997

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The assimilatory nitrite reductase catalyses the conversion of nitrite to ammonia. The enzyme from Candida utilis has been previously purified to homogeneity and shown to be a heterodimer consisting of 58 kDa and 66 kDa subunits. The enzyme has also been shown to be induced by nitrate and repressed by ammonium ions. The levels of nitrite reductase mRNA, its protein and the enzyme activity were modulated together indicating that the primary level of regulation of this enzyme existed at the transcriptional level. Here we report that the 58 kDa and 66 kDa subunits of the enzyme were differentially phosphorylated under the induced and repressed conditions, indicating a second level of regulation. The highly phosphorylated 66 kDa subunit was shown to be dephosphorylated by calf intestinal alkaline phosphatase. The enzymatic activity associated with the native enzyme also decreased due to the dephosphorylation. Each of the subunits could undergo autophosphorylation at serine/threonine residues as demonstrated by thin layer chromatography and recognition by antibodies to phosphoamino acids. The presence of similar phosphorylated subunits under in vivo conditions has also been demonstrated. A model has been proposed to explain the posttranslational regulation of the enzyme.

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Synthesis and secretion of alkaline phosphatase in vitro from first-trimester and term human placentas

Cited by 14 publications

References 28 publications

Imidazoline binding sites in human placenta: evidence for heterogeneity and a search for physiological function

Imidazoline binding sites in human placenta: evidence for heterogeneity and a search for physiological function

Transfer of Heparin Across the Human Perfused Placental Lobule

A novel autophosphorylation mediated regulation of nitrite reductase in Candida utilis

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