Altered endothelin receptor binding in response to nitric oxide synthase inhibition in the pregnant rat

Abstract

The authors evaluate the expression of endothelin-1 (ET-1) and its receptors in the uterus and placenta during maternal nitric oxide synthase (NOS) inhibition. Timed-pregnant rats received L-NAME (2.5 mg/kg/h) or saline from day 14 to 21 of gestation. Uterine and placental tissues collected on day 21 were assayed for preproET-1, ET( A), and ET(B) mRNA expression; localization and expression of ET-1 and receptor proteins; and receptor activity. NOS inhibition did not affect preproET-1 mRNA expression in the placenta or uterus. ET(A) expression decreased in the uterine free wall, but no other changes in receptor mRNA expression were observed in the uterus or placenta. ET-1 and receptor proteins were unchanged. Placental ET(A) and ET(B) receptor binding decreased. Uterine ET(A) receptor binding decreased in the placental bed. ET-1, a prominent mediator during NOS inhibition, is not of uterine or placental origin. Reduced receptor binding activity is the primary means by which these tissues regulate their response to ET-1 in the setting of NOS inhibition.

Figure 1

Expression of endothelin-1 (ET-1) as well as ET_A and ET_B receptor mRNA in rat placentas (P), uterine placental beds (UPB), and the uterine free wall (UFW). Comparative mRNA expression was determined by real-time quantitative reverse transcription polymerase chain reaction in tissues from rats treated with N^G-nitro-L-arginine-methyl ester (L-NAME; 2.5 mg/kg/h, gestational days 14-21, n = 6) or with saline vehicle (n = 6). Results are expressed as a percentage of the internal control gene, cytoplasmic β-actin, and each value represents the mean ± SEM. *P < .05 versus vehicle-treated control, by ANOVA.

Figure 2

Protein expression of ET_A (48 kDa, left) and ET_B (49 kDa, right) receptors in pregnant rats. Western blots were performed on membranes isolated from placental (P), uterine placental bed (UPB), and uterine free wall (UFW) homogenates from N^G-nitro-L-arginine-methyl ester (L-NAME)–treated rats (L, gestational days 14-21, n = 6) and vehicle-treated controls (V, n = 6) pregnant rats. All gels included control membranes (CM) from a rat brain, and the densitometric results are expressed as the mean ± SEM of the ratio of reproductive tissue and control receptor band densities. Na⁺/K⁺ transporting ATPase (112 kDa) was used as a control membrane protein to verify loading of equal amounts of protein into each well.

Figure 3

Immunolocalization of endothelin-1 (ET-1), ET_A, and ET_B in uterine vessels and placental labyrinth from pregnant rats treated with saline vehicle or with 2.5 mg/kg/h N^G-nitro-L-arginine-methyl ester (L-NAME) from gestational day 14 to 21. Immunostaining patterns and intensities were similar between the 2 treatment groups. ET-1 localized to the uterine vascular walls in both the vehicle-treated group (A) and the L-NAME–treated group (C). A negative control (omission of primary antibody) from the vehicle-treated group indicates no binding of the secondary antibody-chromogen complex in the absence of primary antibody (B). Placental ET-1 localized primarily to the membranes of the labyrinth in both groups; a representative photomicrograph from the vehicle-treated group is shown (D). ET_A and ET_B receptors in the uterus (E and F, respectively) were localized in the vascular wall in both vehicle and L-NAME–treated groups. In the placenta, these receptors were distributed along the labyrinthine membranes, with ET_A appearing in patches (G) and ET_B having a more uniform distribution (H). Light methyl green counterstain. Bar = 100 μm (A-C and E-H) and 200 μm (D).

Figure 4

Homologous competitive binding of endothelin-1 (ET-1) and ET-3 by ETA and ETB receptors in rat placenta (P), uterine placental bed (UPB), and uterine free wall (UFW). Placental membranes were prepared from vehicle-treated (n = 6) and L-NAME-treated (2.5 mg/kg/h, gestational days 14-21, n = 6) pregnant rats. (A, C, E) Representative binding curves for P, UPB, and UFW membrane preparations, respectively, from vehicle-treated rats. (B, D, F) Representative binding curves for P, UPB, and UFW membrane preparations, respectively, from L-NAME-treated rats. Each experiment was performed in triplicate. (G) The maximum binding coefficient, Bmax, for both receptors is expressed as fmoles endothelin/μg protein, and each value represents the mean ± SEM. ET in captions and axis labels refers to both ET-1 and ET-3. **P < .01 versus vehicle-treated control by ANOVA.