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.
The specific binding of ligand in cell membrane fractions from placenta and fetal membranes approached saturation in experiments with 125I-[Sar1-Ile5-Ile8]-Ang II and increasing concentrations of unlabeled [Sar1-Ile5-Ile8]-Ang II (Fig. 2a). The dissociation constant (Kd) determined by Scatchard plot (Fig. 2b) was 0.88 (0.32-1.51; n = 18) nM in the placentome. This value did not differ from those obtained in the intercotyledonary allantochorionic membrane (0.80 nM, range 0.40-1.24 nM; n = 18) and the allantoamnionic membrane (1.07 nM, range 0.27-1.56 nM; n = 11). buy antibiotics online
The proportions of the Ang II receptor types AT1 and AT2 were determined by displacement of the specific binding of 125I-[Sar1-Ile5-Ile8]-Ang II by the nonpeptide Ang II receptor antagonists losartan (DuP 753) and PD 123319 as shown in Figure 3. Losartan and PD 123319 are considered selective for the AT and AT2 receptors, respectively.
FIG. 1. a) Schematic drawing of a bovine fetus in utero. A, allantoam-nionic membrane; B, placentome; C, smooth part of the placenta. The white area represents the fetal membranes. IrC, intercotyledonary region. b) Detailed schematic drawing of a placentome (the box in a). The allan-tochorion (AC) is smooth at the IcR, which is apposed to the likewise smooth endometrium. In the placentome, the AC forms fetal villous trees (FV), cotyledons. The gray part represents the maternal compartment, which forms complementary crypt walls (CW), caruncles, in the placen-tomes. In the AC, a loose network of mesenchymal cells (MC) is covered with allantoic endoderm (AE) towards the allantoic cavity and with an intact trophoblast cell layer (TrC) apposing the uterine epithelium (UE). *Area of the placentome in which higher-order villi and crypts interdigi-tate, seen in Figures 7 and 8. a is reproduced from Hagemann et al., Clin Exp Pharmacol Physiol 1 993; 20:41-50, with permission. Drawings by Jesper Tom-Petersen.
FIG. 2. a) Specific binding of ligand in a cell membrane fraction, prepared from bovine placentome in the second part of gestation, obtained by using 125I-[Sar1-Ile5-Ile8]-Ang II and increasing concentrations of unlabeled [Sar1-Ile5-Ile8]-Ang II. b) Scatchard analysis of the same data. In this experiment, the Kd and Bmax were 0.82 nM and 81 pM, respectively.
FIG. 3. Representative displacement curves of the specific binding of 125I-[Sar1-Ile5-Ile8]-Ang II by unlabeled [Sar1-Ile5-Ile8]-Ang II (squares), PD 123319 (triangles), and losartan (circles) in a cell membrane fraction prepared from bovine placentome in the second part of gestation.