Calcitonin Gene Related Peptide, Adrenomedullin, and Adrenomedullin 2 Function in Uterine Artery During Human Pregnancy

Abstract

Rationale: Calcitonin gene-related peptide (CGRP) and its family members adrenomedullin (ADM) and adrenomedullin 2 (ADM2; also known as intermedin) support vascular adaptions in rat pregnancy.

Objective: This study aimed to assess the relaxation response of uterine artery (UA) for CGRP, ADM, and ADM2 in nonpregnant and pregnant women and identify the involved mechanisms.

Findings: (1) Segments of UA from nonpregnant women that were precontracted with U46619 (1μM) in vitro are insensitive to the hypotensive effects of CGRP, ADM, and ADM2; (2) CGRP, ADM, and ADM2 (0.1-100nM) dose dependently relax UA segments from pregnant women with efficacy for CGRP > ADM = ADM2; (3) the relaxation responses to CGRP, ADM, and ADM2 are differentially affected by the inhibitors of nitric oxide (NO) synthase (L-NAME), adenylyl cyclase (SQ22536), apamin, and charybdotoxin; (4) UA smooth muscle cells (UASMC) express mRNA for calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP)1 and RAMP2 but not RAMP3; (5) receptor heterodimer comprising CRLR/RAMP1 and CRLR/RAMP2 but not CRLR/RAMP3 is present in UA; (6) soluble fms-like tyrosine kinase (sFLT-1) and TNF-α treatment decrease the expression of RAMP1 mRNA (P < 0.05) in UASMC; and (7) sFLT-1 treatment impairs the association of CRLR with all 3 peptides while TNF-α inhibits the interaction of CGRP but not ADM or ADM2 with CRLR in UASMC (P < 0.05).

Conclusions: Relaxation sensitivity of UA for CGRP, ADM, and ADM2 is increased during pregnancy via peptide-specific involvement of NO system and endothelium-derived hyperpolarizing factors; vascular disruptors such as sFLT-1 and TNFα adversely impact their receptor system in UASMC.

Keywords: CGRP; adrenomedullin; adrenomedullin 2/intermedin; pregnancy; sFLT-1; uterine artery.

Figure 1.

Relaxation responses of uterine arteries to CGRP, ADM, and ADM2 in nonpregnant and pregnant women. A, Relaxation induced by Bradykinin in UA from nonpregnant (n = 4) and pregnant women (n = 8). Dose-response curve and representative tracings show increased sensitivity of UA in pregnant women for bradykinin compared with that in nonpregnant women (P < 0.05). B, Relaxation induced by CGRP in human UA from nonpregnant (n = 4) and pregnant (n = 8) women. Dose-response curve and representative tracings show that CGRP induced significant dose-dependent relaxations of UA in pregnant, but not in nonpregnant women (P < 0.05). C, Relaxation induced by ADM in human UA from nonpregnant (n = 4) and pregnant (n = 8) women. Dose-response curve and representative tracings show that ADM induced significant dose-dependent relaxations of UA in pregnant, but not in nonpregnant women (P < 0.05). D, Relaxation induced by ADM2 in human UA from nonpregnant (n = 4) and pregnant (n = 8) women. Dose response curve and representative tracings show that ADM2 induced significant dose-dependent relaxations of UA in pregnant, but not in nonpregnant women (P < 0.05). E, Relative vascular sensitivity for CGRP, ADM, and ADM2 in UA from pregnant women is CGRP > ADM = ADM2. Results are shown as mean ± SEM. Different letters indicate significant differences between groups (n = 8; P < 0.05).

Figure 2.

Mechanisms involved in the relaxant effects of CGRP, ADM, and ADM2 in uterine artery from pregnant women. All 3 peptides were added in cumulative doses either in presence or absence of inhibitors of cAMP (SQ), NO (L-NAME) or EDHF (apamin [APA] and charybdotoxin [CHX]) and E_max values were compared. A, Relaxations induced by CGRP are inhibited in the presence APA and CHX (P < 0.05), but not L-NAME or SQ (n = 8). B, Relaxations induced by ADM were attenuated in presence of SQ, L-NAME, as well as APA and CHX (P < 0.05, n = 8). C, Relaxations induced by ADM2 were also attenuated in presence of SQ, L-NAME, as well as APA and CHX (P < 0.05, n = 8). Results are shown as mean ± SEM. Different letters indicate significant differences between groups with P < 0.05.

Figure 3.

Expression of CRLR and RAMPs mRNA and their protein-protein interaction in uterine artery. A, Immunofluorescent staining with β-actin in isolated UASMC showing >98% purity of isolated cells used in the study (magnification 20×). B, Expression of CRLR and RAMPs mRNA in UASMC. The bar graphs represent PCR data for expression of CRLR and RAMPs in UASMC isolated from pregnant women. The mRNA levels were normalized to the mRNA expression of GAPDH. Data are presented as mean ± SEM. C, Proximity ligation assay (PLA) showing hetero-dimerization of CRLR with RAMPs in UASMC. Figure shows representative images of the association between CRLR and 3 RAMP proteins in UASMC. The fluorescent red signal is indicative of protein-protein interaction between the 2 target proteins. DAPI was used for nuclear staining and absence of probe served as the control (magnification = 20×, n = 3).

Figure 4.

Expression of CRLR and RAMPs mRNA and ligand receptor association in UASMC treated with sFLT-1 or TNF-α in UASMC: A, Real time PCR showing effect of sFLT-1 and TNF-α treatment on the expression of CRLR, RAMP1, and RAMP2 in UASMC. As shown, both sFLT-1 and TNF-α caused a significant decrease in the expression of RAMP1 mRNA without significant effect on the expression of CRLR or RAMP2 mRNA in UASMC. Asterisk indicates the significant differences between groups (P < 0.05). B, Proximity ligation assay (PLA) showing effect of sFLT-1 and TNF-α on the interaction of CRLR with CGRP, ADM, and ADM2 in UASMC. Representative images of associations between CRLR and its ligand peptides CGRP, ADM, or ADM2 are shown. The red fluorescent PLA signals are indicative of protein-protein interaction between the 2 target proteins that were counted and presented as bar graphs (± SEM) representing the respective protein-protein interaction. DAPI was used for nuclear staining, and absence of probe served as the control. Asterisks (*) indicate significant difference compared with the control. (P < 0.05; Magnification = 20×; n = 3).

Figure 4.

Expression of CRLR and RAMPs mRNA and ligand receptor association in UASMC treated with sFLT-1 or TNF-α in UASMC: A, Real time PCR showing effect of sFLT-1 and TNF-α treatment on the expression of CRLR, RAMP1, and RAMP2 in UASMC. As shown, both sFLT-1 and TNF-α caused a significant decrease in the expression of RAMP1 mRNA without significant effect on the expression of CRLR or RAMP2 mRNA in UASMC. Asterisk indicates the significant differences between groups (P < 0.05). B, Proximity ligation assay (PLA) showing effect of sFLT-1 and TNF-α on the interaction of CRLR with CGRP, ADM, and ADM2 in UASMC. Representative images of associations between CRLR and its ligand peptides CGRP, ADM, or ADM2 are shown. The red fluorescent PLA signals are indicative of protein-protein interaction between the 2 target proteins that were counted and presented as bar graphs (± SEM) representing the respective protein-protein interaction. DAPI was used for nuclear staining, and absence of probe served as the control. Asterisks (*) indicate significant difference compared with the control. (P < 0.05; Magnification = 20×; n = 3).