Category Archives: Placenta : Part 2

Autoradiographic Localization and Characterization of Angiotensin II Receptors: RESULTS(2)

RESULTS(2)

Concentrations of losartan from 10~6 to 105 M fully displace 125I-[Sar1-Ile5-Ile8]-Ang II from the AT1 receptor, whereas binding to the AT2 receptor is only slightly affected. At higher concentrations, 125I-[Sar1-Ile5-Ile8]-Ang II is also displaced from the AT2 receptor. Concentrations of PD 123319 from 10~6 to 105 M fully displace 125I-[Sar1-Ile5-Ile8]-Ang II from the AT2 receptor and very slightly affect the binding to the AT1 receptor.

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Autoradiographic Localization and Characterization of Angiotensin II Receptors: RESULTS(1)

Characterization of Angiotensin II Receptors

Specific binding of 125I-[Sar1-Ile5-Ile8]-Ang II was found in all cell membrane fractions of placenta and fetal membranes from all 23 animals examined. The nonspecific binding was low and did not change during incubation for 5 h (n = 4). The time course of the specific binding in the placenta and fetal membranes indicated that equilibrium was reached after 45-60 min (n = 4). Accordingly, incubation for 60 min at 37°C was used in order to obtain equilibrium.

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Autoradiographic Localization and Characterization of Angiotensin II Receptors: MATERIALS AND METHODS(5)

MATERIALS AND METHODS(5)

Autoradiography

For in situ autoradiographic studies, the tissue was serial-sectioned (10 ^m) in a cryostat at -16°C, and thaw-mounted on Polysine microscope slides (Menzel-Glaser, Braunschweig, Germany). The tissue sections were dried overnight at – 2°C and used immediately or stored at – 20°C until later use. buy flovent inhaler

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Autoradiographic Localization and Characterization of Angiotensin II Receptors: MATERIALS AND METHODS(4)

The dissociation constant (Kd) and the maximal binding capacity of ligand (Bmax) were determined by Scatchard analysis of data obtained with unlabeled [Sar1-Ile5-Ile8]-Ang II by using the RADLIG program for the analysis of radioligand binding experiments (Version 4; Biosoft, Cambridge, UK). The binding data were best fitted with a one-site fit. The Ang II receptor density was calculated by using the Bmax and the protein concentration, assuming binding of one molecule of ligand to each receptor. In some cell membrane fractions, the Ang II receptor density was calculated by using the bound/free value, assuming binding characteristics (Kd) similar to those previously determined. buy asthma inhalers

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Autoradiographic Localization and Characterization of Angiotensin II Receptors: MATERIALS AND METHODS(3)

MATERIALS AND METHODS(3)

The final concentrations of 125I-[Sar1-Ile5-Ile8]-Ang II were 18-52 pM. For characterization of the receptor type, 25 ^l of varying concentrations of losartan (DuP 753; Du Pont Merck Pharmaceutical Company, Wilmington, DE), or PD 123319 ((S)-1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(di-phenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid, ditrifluoroacetate, monohydrate; Parke-Davis, Ann Arbor, MI) in 0.2 M Tris-HCl (pH 7.5) containing 2 g/L human serum albumin were used as ligands. In this way, the relative amounts of the Ang II receptors that were AT1 or AT2 receptors were determined by displacement curves obtained with losartan and PD 123319 in a number of animals in each group. buy ortho tri-cyclen online

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Autoradiographic Localization and Characterization of Angiotensin II Receptors: MATERIALS AND METHODS(2)

The pellet was suspended in 10 mM sodium phosphate (pH 7.4) containing 120 mM sodium chloride, 1 mM EDTA (Merck), and 1 mM PMSF, centrifuged at 45 000 X g for 25 min at 4°C, and suspended in the same buffer with 5 mM MgCl2. The cell membrane fractions were frozen at -20°C for later analysis. flovent inhaler

For measurement of renin concentrations, 100 mg of tissue was homogenized at 0°C in 0.5 ml of 10 mM sodium phosphate (pH 7.5) containing 140 mM sodium chloride, 10 mM EDTA, 10 mM A/-ethylmaleimide (Sigma), 2 mM 8-hydroxyquinoline (Merck), and 0.3 mM sodium azide. The Ultra Turrax T8 was used for 30 sec followed by the Potter-Elvehjem homogenizer for 3 min at 250 rpm. The homogenate was centrifuged at 1850 X g for 15 min at 4°C. The supernatant was stored at -20°C for later analysis.

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Autoradiographic Localization and Characterization of Angiotensin II Receptors: MATERIALS AND METHODS(1)

MATERIALS AND METHODS(1)

Collection of Tissues

Bovine placentae and fetal membranes were obtained at an abattoir 20-30 min after death. The stage of gestation was estimated by the fetal crown-rump length. The animals used for Ang II receptor binding studies (n = 23) were grouped according to 3 stages of gestation: Days 195 (7 animals; median 75 days, range 40-92 days), Days 96-190 (9 animals; median 131 days, range 108-190 days), and Days 191-290 (7 animals; median 252 days, range 191-275 days). Tissue samples for Ang II receptor binding studies were transported to the laboratory on ice (1-2 h) and stored at -20°C. Tissue samples for autoradiography were collected from animals (n = 16) at different days of gestation ranging from Day 50 to 252, snap-frozen in liquid nitrogen, wrapped in Parafilm (American Can Company, Greenwich, CT) to prevent dehydration, transported on dry ice, and stored at -80°C. buy asthma inhaler

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Autoradiographic Localization and Characterization of Angiotensin II Receptors in the Bovine Placenta and Fetal Membranes(2)

The physiological effects of the AT2 receptor are presently much less characterized. It has been suggested, however, that the AT2 receptor participates in the control of cell proliferation and/or differentiation. The identity and roles of non-AT:/non-AT2 Ang II binding sites are unclarified. Species differences in Ang II receptor expression in the uteroplacental unit were previously demonstrated (for review see Nielsen et al. ). Recently, we demonstrated a predominance of AT2 receptors in the porcine placenta and fetal membranes.

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Autoradiographic Localization and Characterization of Angiotensin II Receptors in the Bovine Placenta and Fetal Membranes(1)

Fetal Membranes(1)

Angiotensin (Ang) II exerts a variety of physiological effects in the cardiovascular, endocrine, and nervous system when its specific receptors are stimulated. A number of studies have provided evidence for the presence of all components of local tissue renin-angiotensin systems (RAS) in the uteroplacental unit in humans as well as in many other species. These studies strongly suggest that these local RAS have regulatory functions in the placenta. buy antibiotics online

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