Abstract Background Adrenomedullin (ADM), a novel vasorelaxant peptide, was found in human/rat ovaries and uteri. Plasma ADM level increases in pregnant women and pregnant rats. Methods The gene expression levels of Adm and its receptor components - Crlr , Ramp1 , Ramp2 and Ramp3 , the ADM peptide concentration and localization in the rat female reproductive system during gestation were studied by real-time RT-PCR, EIA and immunohistochemical techniques. Results The mRNAs of Adm and its receptor component and ADM were differentially distributed between implantation sites and inter-implantation sites of the pregnant uterus. The day on which vaginal sperm were found was taken to be pregnancy day 1. The Adm mRNA levels in the implantation sites of the uteri in mid- (day 12) and late pregnancy (day 17) were more than 10-fold higher than those in nonpregnancy, pre-implantation (day 3) or early (day 7) pregnancy. ADM was localized in the endometrial stroma with increased immunoreactivity from nonpregnancy to pregnancy. The ADM level and the mRNA levels of Adm , Crlr , Ramp2 and Ramp3 in the corpus luteum all increased in late pregnancy compared with early pregnancy. The gene expression of Adm and it receptor components and intense immunostaining of ADM were also found in the oviduct during pregnancy. Conclusions The gene expressions levels of Adm and its receptor components - Crlr, Ramp1, Ramp2 and Ramp3 , and ADM peptide concentration exhibited a spatio-temporal pattern in the rat female reproductive system during gestation and this suggests that ADM may play important roles in gestation.
Adrenomedullin (ADM) is found in male accessory sex glands and is part of the seminal secretion. It plays an important role in protecting the sperm in the female reproductive tract. In this study, we investigated the roles of ADM in inflammation and oxidative stress in the endometrium and in leukocyte and macrophage infiltration in the endometrial stroma. The expression of the ADM gene in the ventral prostate, coagulating gland, and seminal vesicle was determined by real time PCR. The peptide levels in the tissue and secretion were measured using an EIA Kit. The highest ADM mRNA and peptide levels were found in the ventral prostate. Most of the ADM in the seminal vesicle was stored in the tissue while little was secreted. The expression of the IL-1β gene and the secretion of TNFα and IL-6 in uterine tissue decreased significantly after treatment with ADM for 4 hours. Using an immunostaining method, the levels of leukocyte and macrophage infiltration were found to be lower at 24 hours post coitus than 1.5 hours post coitus. The infusion of ADM receptor antagonist reduced the infiltration of leukocyte and macrophages in the endometrial stroma at 24 hours post coitus. As to the anti-oxidative effect of ADM in the female tract, the reactive oxygen species (ROS) level in isolated endometrial epithelial cells was significantly decreased after treatment with ADM or seminal fluid. Our findings demonstrated that ADM in the seminal secretion may modify the inflammatory responses, play an anti-oxidative role, and increase leukocyte and macrophage infiltration in the uterus.
SUMMARY 1. Alterations of mRNA levels of ai‐adrenoceptor subtypes during maturation and ageing were determined by reverse trans‐cription‐polymerase chain reaction (RT‐PCR) in aortae and renal, pulmonary and mesenteric arteries isolated from 3,12 and 24‐month‐old rats. 2. The steady state levels for α 1A ‐, α 1B ‐ and α 1D ‐adrenoceptors in aorta declined with maturation and ageing. In renal artery there was a decrease in mRNA for the α 1B ‐adrenoceptor in aged rats. However, in mesenteric and pulmonary arteries there were no changes in mRNA levels for the three subtypes of α 1 ‐adrenoceptors as a result of maturation and ageing. 3. The results suggest that expression of α 1 ‐adrenoceptors is changed heterogeneously in different blood vessels during maturation and ageing in rats.
Adrenomedullin (ADM), a novel vasorelaxant peptide, was found in human/rat ovaries and uteri. Plasma ADM level increases in pregnant women and pregnant rats. Our previous study in Sprague-Dawley rats showed that large antral follicles (diameter above 900 µm) and newly formed corpus luteum (CL) expressed higher levels of Adm and one of its receptor activity-modifying protein (Ramp2) than small antral follicles (diameter of 200-400 µm). EDN is luteolytic and is known to inhibit progesterone production. The aims of this study was to study the interaction of ADM and endothelin 1 (EDN1) in follicles and newly formed CL and the action of ADM on progesterone production in CL during pregnancy. Adult Sprague-Dawley rats were induced to ovulate by administration of eCG followed by hCG injection. Isolated large follicles were incubated with ADM/EDN1 for 6 h; newly formed corpora lutea were also incubated with ADM/EDN1 for 6 h with/without hCG. For the effects of EDN1, the gene expression of Adm and its receptor components in the large antral follicles and CL were measured by real-time RT-PCR. For the effects of ADM, the gene expression of Edn1 and endothelin receptor B (Ednrb) was measured. The effects of the ADM and CGRP receptor antagonists (hADM22-25 and hCGRP8-37) were also tested. CL were isolated from early (7-day), mid (12-day), and late (17-day) pregnant rats and were incubated for 6 h with ADM with or without an ADM or a receptor antagonist. Progesterone secretion from CL was measured by Enzyme Immunoassay. EDN1 was found to reduce the gene expression of Adm and Ramp1 in large antral follicles after 6 h incubation with ADM. ADM treatment upregulated Edn1 expression levels in large antral follicles. EDN1 also reduced the gene expression of Adm in the CL with/without hCG. ADM treatment increased Edn1 expression. In the presence of hCG, ADM treatment increased both Edn1 and also Ednrb expression. ADM suppressed progesterone production from the CL in early and late pregnancy while enhancing progesterone production in mid-pregnancy, which correlated well with the greater progesterone production at mid-pregnancy. The effects of ADM on Edn1 gene expression and progesterone production were blocked by the CGRP receptor antagonist. One of the roles of ADM in CL during pregnancy might be the regulation of progesterone production and this may be achieved via the interaction with END1. Acknowledgement: This work was substantially supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (HKU7736/07M). (poster)
The [ 125 I]‐iodomelatonin binding sites in chicken brain membrane preparations were studied. The binding of [ 125 I]‐iodomelatonin to the membrane preparations of chicken brain was rapid, stable, saturable, and reversible. The order of pharmacological affinities of [ 125 I]‐iodomelatonin binding sites in the chicken brain membrane preparations was: melatonin > 6‐chloromelatonin > N‐acetylserotonin > 5‐hydroxytryptamine > tryptamine > 5‐methoxytryptophol, >> 1‐acetylindole‐3‐carboxaldehyde, 5‐hydroxyindole‐3‐acetic acid, L‐tryptophan, 5‐hydroxytryptophan, 3‐acetylindole. Compounds known to act on the receptor of norepinephrine or acetylcholine were inactive as compared to melatonin. Among the various brain regions studied, melatonin binding had maximal level in the hypothalamus, intermediate levels in the mid‐brain, ponsmedulla, and telencephalon, and minimum level in the cerebellum. Subcellular fraction studies indicated that 40% of the binding was located in the mitochondrial fraction, 27% in the nuclear, 26% in the microsomal, and 6% in the cytosol fraction. Scatchard analysis of the membrane preparations revealed a dissociation constant (Kd) of 199.6 ± 17 pM and a total number of binding sites (Bmax) of 16.6 ± 0.75 fmol/mg protein at midlight. Thus, our results showed the presence of specific melatonin binding sites in the chicken brain membrane preparations. Saturation studies demonstrated that [ 125 I]‐iodomelatonin binding capacity in chicken brain membrane preparations were 40% greater at midlight (16.6 ± 0.75 fmol/mg protein) than at middark (10.6 ± 0.56 fmol/mg protein), with no significant variation in their binding affinities.
Abstract Background Adrenomedullin (ADM), a novel vasorelaxant peptide, was found in human/rat ovaries. The present study investigated the interaction of ADM and endothelin-1 (ET-1) in follicles and newly formed corpora lutea (CL) and the actions of ADM on progesterone production in CL during pregnancy. Methods The peptide and gene expression level of adrenomedullin in small antral follicles, large antral follicles and CL was studied by real-time RT-PCR and EIA. The effect of ADM treatment on oestradiol production in 5-day follicular culture and on progesterone production from CL of different pregnant stages was measured by EIA. The interaction of ADM and ET-1 in follicles and CL at their gene expression level was studied by real-time RT-PCR. Results In the rat ovary, the gene expression of Adm increased during development from small antral follicles to large antral follicles and CL. In vitro treatment of preantral follicular culture for 5 days with ADM increased oestradiol production but did not affect follicular growth or ovulation rate. The regulation of progesterone production by ADM in CL in culture was pregnancy-stage dependent, inhibitory at early and late pregnancy but stimulatory at mid-pregnancy, which might contribute to the high progesterone production rate of the CL at mid-pregnancy. Moreover, the interaction between ADM and ET-1 at both the production and functional levels indicates that these two vasoactive peptides may form an important local, fine-tuning regulatory system together with LH and prolactin for progesterone production in rat CL. Conclusions As the CL is the major source of progesterone production even after the formation of placenta in rats, ADM may be an important regulator in progesterone production to meet the requirement of pregnancy.
Plasma volume expansion stimulates cardiac secretion of atrial natriuretic factor (ANF) and also increases the ANF concentration in cerebrospinal fluid. In order to determine whether brain ANF is involved in the compensatory response to hypervolemia or the regulation of cardiac secretion of ANF, we have studied the integrated hemodynamic, renal, and hormonal response to acute volume expansion (15 ml/kg Dextran over 30 min) in five sheep given nonimmune serum (control) and ANF antiserum by intracerebroventricular (icv) injections on separate days. Dextran loading caused similar decreases in hematocrit and increases in central venous and mean arterial pressures on both study days. Heart rate was higher after antiserum injections (P less than 0.05). Dextran loading increased plasma ANF on the control (20 pmol/liter maximal mean increment above baseline) but not on the antiserum day (P less than 0.01). The diuresis (P less than 0.01) and natriuresis (P less than 0.05) observed on the control day was inhibited by icv antiserum. Plasma aldosterone and cortisol levels showed similar falls in response to the dextran load on both days. These experiments show that icv ANF antiserum inhibits both the increase in cardiac secretion of ANF and the renal response to plasma volume expansion without affecting hemodynamic status. These data support the hypothesis that the brain ANF system is important in the systemic responses to volume loading.
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