Studies on the effects of the N-terminal domain antibodies of calcitonin receptor-like receptor and receptor activity-modifying protein 1 on calcitonin gene-related peptide-induced vasorelaxation in rat uterine artery

The vascular relaxation sensitivity to calcitonin gene-related peptide (CGRP) is enhanced during pregnancy, compared with nonpregnant human and rat uterine arteries. In the rat uterine artery, two types of CGRP receptors have been shown to coexist, CGRP-A receptor, which is a complex of calcitonin receptor-like receptor (CRLR), and receptor activity-modifying protein (RAMP(1)) and CGRP-B receptor, which is different from CRLR. In the present study, we hypothesized that: 1) CGRP-induced vasorelaxation in rat uterine artery is mediated through CGRP-A receptor and 2) N-terminal (Nt) domain of CRLR (Nt-CRLR) has a major contribution in ligand binding and mediating CGRP- induced relaxation effects in rat uterine artery. Polyclonal antibodies against Nt-domain of CRLR and RAMP(1) (Nt-RAMP(1)) were raised in rabbits and characterized for their specificity and were used to inhibit CGRP-induced vasorelaxation in rat uterine artery. For vascular relaxation studies, uterine arteries from Day 18 pregnant rats were isolated, and responsiveness of the vessels to CGRP was examined with a small vessel myograph. CGRP (10(-10) to 10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in Day 18 pregnant rat uterine arteries. These effects were significantly (P < 0.05) inhibited when uterine arteries were incubated with the antibody raised against Nt-CRLR (PD(2) = 6.75 +/- 0.20) and were totally abolished in presence of antibodies for both Nt-CRLR and Nt-RAMP(1) (PD(2) = 6.14 +/- 0.35). In contrast, a monoclonal antibody for CGRP-B receptor had no effect on CGRP-induced rat uterine artery relaxation. These studies suggest that CGRP effects in rat uterine artery are mediated through CGRP-A receptor and that Nt-domain of CRLR may play a predominant role in CGRP binding and thus in causing CGRP-induced uterine artery relaxation.     

Progesterone upregulates calcitonin gene-related peptide and adrenomedullin receptor components and cyclic adenosine 3'5'-monophosphate generation in Eker rat uterine smooth muscle cell line

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM), two potent smooth-muscle relaxants, have been shown to cause uterine relaxation. Both CGRP- and AM-binding sites in the uterus increase during pregnancy and decrease at labor and postpartum. These changes in binding sites appear to be related to the changes in calcitonin receptor-like receptor (CRLR), receptor activity-modified protein 1 (RAMP1), RAMP2, and RAMP3 mRNA levels. It is not clear, however, whether the changes in the receptor components occur in the myometrial cells and whether the steroid hormones can directly alter these receptor components in the muscle cells. In addition, the mechanism of CGRP and AM signaling in the rat myometrium is not well understood. Therefore, we examined the mRNA expression of CGRP- and AM-receptor components, G protein Galphas, CGRP, and AM stimulation of cAMP and cGMP, and the effects of progesterone on these parameters in the Eker rat uterine myometrial smooth-muscle cell line (ELT3). ELT3 cells expressed CGRP- and AM-receptor components CRLR, RAMP1, RAMP2, and RAMP3. Expression of CRLR and RAMP1 mRNA increased with progesterone treatment and decreased with estradiol-17beta treatment. However, RAMP2 and RAMP3 mRNA expressions were unaltered by both progesterone and estradiol. Progesterone increased (P<0.05) Galphas expression and augmented CGRP- and AM-induced increases in cAMP levels. In uterine smooth-muscle cells, the antagonist to Galphas protein NF449 decreased basal as well as CGRP- and AM-stimulated cAMP levels. None of the cell treatments affected cyclic GMP production. Our results suggest that the progesterone-stimulated increases in CGRP and AM receptors, Galphas protein levels, and cAMP generation in the myometrial cells may be responsible for increased uterine relaxation sensitivity to CGRP and AM during pregnancy.     

Evidence for the existence of a new receptor for CGRP,which is not CRLR

Receptors for calcitonin gene-related peptide (CGRP), a neuropeptide known to be the most potent vasodilator, are abundantly expressed in cerebellum. A monoclonal antibody to cerebellar CGRP receptors specifically detects a 66 kDa protein from rat cerebellum and other rat and human tissues, but not from SK-N-MC cells which express calcitonin receptor-like receptor (CRLR), a recently described component of CGRP receptors. In contrast, mRNA expression for CRLR was abundant in SK-N-MC cells, but it was undetectable in rat cerebellum. Furthermore, the antibody could not detect any immunoreactive protein in HEK 293 cells transiently transfected with CRLR and receptor activity-modifying protein 1 (RAMP(1)) indicating the possible existence of another CGRP receptor, which does not involve CRLR. Due to the absence of biochemical or structural data on the existence of a CGRP(2) receptor and the new data provided in this paper, we suggest to identify the two CGRP receptors as CGRP-A and CGRP-B.     

Involvement of calcitonin gene-related peptide in control of human fetoplacental vascular tone

Calcitonin gene-related peptide (CGRP), one of the most potent endogenous vasodilators known, has been implicated in vascular adaptations and placental functions during pregnancy. The present study was designed to examine the existence of CGRP-A receptor components, the calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein 1 (RAMP1), in the human placenta and the vasoactivity of CGRP in the fetoplacental circulation. Immunofluorescent staining of the human placenta in term labor using polyclonal anti-CRLR and RAMP1 antibodies revealed that labeling specifically concentrated in the vascular endothelium and the underlying smooth muscle cells in the umbilical artery/vein, chorionic artery/vein, and stem villous vessels as well as in the trophoblast layer of the placental villi. In vitro isometric force measurement showed that CGRP dose dependently relaxes the umbilical artery/vein, chorionic artery/vein, and stem villous vessels. Furthermore, CGRP-induced relaxation of placental vessels are inhibited by a CGRP receptor antagonist (CGRP8-37), ATP-sensitive potassium (KATP) channel blocker (glybenclamide), and cAMP-dependent protein kinase A inhibitor (Rp-cAMPS) and partially inhibited by a nitric oxide inhibitor (Nomega-nitro-l-arginine methyl ester). We propose that CGRP may play a role in the control of human fetoplacental vascular tone, and the vascular dilations in response to CGRP may involve activation of KATP channels, cAMP, and a nitric oxide pathway.     

Mesenteric arterial relaxation to calcitonin gene-related peptide is increased during pregnancy and by sex steroid hormones

The present study investigated whether pregnancy and circulatory ovarian hormones increase the sensitivity of the mesenteric artery to calcitonin gene-related peptide (CGRP)-induced relaxation and possible mechanisms involved in this process. Mesenteric arteries from young adult male rats or female rats (during estrous cycle, after ovariectomy, at Day 20 of gestation, or Postpartum Day 2) were isolated, and the responsiveness of the vessels to CGRP was examined with a small vessel myograph. The CGRP (10(-10) to 10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in mesenteric arteries of all groups. Arterial relaxation sensitivity to CGRP was significantly (P < 0.05) greater in female rats compared with male rats. Pregnancy increased the sensitivity to CGRP significantly (P < 0.05) compared to ovariectomized and Postpartum Day 2 rats. In pregnant rats, CGRP-receptor antagonist, CGRP(8-37), inhibited the relaxation responses produced by CGRP. The CGRP-induced relaxation was not affected by N(G)-nitro-l-arginine methyl ester (nitric oxide inhibitor, 10(-4) M) but was significantly (P < 0.05) attenuated by an inhibitor of guanylate cyclase (1H-[1 , 2 , 4 ]oxadizaolo[4 , 3 -a]quinoxalin-1-one, 10(-5) M). Relaxation responses of CGRP on mesenteric arteries were blocked (P < 0.05) by a cAMP-dependent protein kinase A inhibitor, Rp-cAMPs (10(-5) M). The CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by calcium-dependent (tetraethylammonium, 10(-3) M), but not ATP-sensitive (glybenclamide, 10(-5) M), potassium channel blocker. Therefore, the results of the present study suggest that mesenteric vascular sensitivity to CGRP is higher during pregnancy and that cAMP, cGMP, and calcium-dependent potassium channels appear to be involved. Therefore, we propose that CGRP-mediated vasodilation may be important to maintain vascular adaptations during pregnancy.     

mRNA expression of novel CGRP1 receptors and their activity-modifying proteins in hypoxic rat lung

Calcitonin gene-related peptide (CGRP) is a potent vasodilator. Our group has reported that exogenous CGRP may prevent or reverse hypoxic pulmonary hypertension in rats. The vasodilatory action of CGRP is mediated primarily by CGRP1 receptors. The calcitonin receptor-like receptor (CRLR) and the orphan receptor RDC-1 have been proposed as CGRP1 receptors, and recent evidence suggests that CRLR can function as either a CGRP1 receptor or an adrenomedullin (ADM) receptor. Receptor activity-modifying proteins (RAMPs) determine the ligand specificity of CRLR: coexpression of CRLR and RAMP1 results in a CGRP1 receptor, whereas coexpression of CRLR and RAMP2 or -3 results in an ADM receptor. We used qualitative, semiquantitative, and real-time quantitative RT-PCR to detect and quantitate the relative expression of these agents in the lungs of rats exposed to normoxia (n = 3) and 1 and 2 wk of chronic hypobaric hypoxia (barometric pressure 380 mmHg, equivalent to an inspired O(2) level of 10%; n = 3/time period). Our results show upregulation of RDC-1, RAMP1, and RAMP3 mRNAs in hypoxic rat lung and no change in CRLR and RAMP2 mRNAs. These findings support a functional role for CGRP and ADM receptors in regulating the adult pulmonary circulation.     

Pregnancy-induced alterations of vascular function in mouse mesenteric and uterine arteries

Normal pregnancy involves dramatic changes to maternal vascular function, while abnormal vascular adaptations may contribute to pregnancy-associated diseases such as preeclampsia. Many genetic mouse models have recently emerged to study vascular pathologies of pregnancy. However, vascular adaptations to pregnancy in normal mice are not fully understood. Thus, we studied changes in vascular reactivity during normal mouse pregnancy. We hypothesized that pregnant mice will have enhanced endothelial-dependent vasodilation compared with nonpregnant mice, via an enhancement of the nitric oxide synthase (NOS) prostaglandin H synthase (PGHS), and other endothelial-derived hyperpolarizing pathways. Late pregnant (Day 17-18) C57BL/6J mice (n = 10) were compared with nonpregnant mice (n = 7). Uterine and mesenteric arteries were mounted on a wire myograph system and assessed for endothelium-dependent (methacholine) and -independent (sodium nitroprusside; SNP) relaxation responses. Endothelial-dependent relaxation was enhanced in pregnant uterine and mesenteric arteries, which was blunted after the addition of inhibitors of the PGHS or NOS pathways. In nonpregnant mice, these pathways had no effect in modulating relaxation in uterine arteries, whereas vasodilation in mesenteric arteries was reduced only by NOS inhibition. Both uterine and mesenteric vessels had nonnitric oxide- and nonprostaglandin-mediated relaxation, but this relaxation was not enhanced during pregnancy. Endothelial-independent relaxation was also enhanced in pregnant uterine but not mesenteric arteries. Our data indicate that uterine and mesenteric arteries from pregnant mice have enhanced vasodilation. Understanding vascular adaptations to normal mouse pregnancy is crucial for interpreting changes that may occur in genetic mouse models.     

Increased myocardial expression of RAMP1 and RAMP3 in rats with chronic heart failure

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) are potent vasodilators in humans and improved myocardial ischemia is observed after CGRP administration. Receptors for CGRP and ADM were already identified in heart. Receptor activity-modifying proteins (RAMPs) determine the ligand specificity of the calcitonin receptor-like receptor (CRLR); co-expression of RAMP1 and CRLR results in a CGRP receptor, whereas the association of RAMP2 or RAMP3 with CRLR gives an ADM receptor. As CGRP and ADM may play a beneficial role in heart failure, we investigated whether the CGRP and ADM receptors are upregulated in chronic heart failure. We have used semi-quantitative RT-PCR and Western-blot analysis to detect and quantify the mRNA and the protein of RAMP1 and RAMP3 in both atria and ventricles of failing hearts 6 months after aortic banding in rats. Our results showed for the first time an up-regulation of RAMP1 and RAMP3 mRNAs and proteins in this model of cardiac failure. No change was observed in mRNAs coding for CRLR, RAMP2, RDC1 (canine orphan receptor), and ADM. The present results suggested after congestive heart failure in adult rats, an up-regulation of the CGRP receptor (by an increase in RAMP1 that is associated with CRLR) in atria and ventricles and of ADM receptor (by increased RAMP3 expression that is associated with CRLR) in atria. These findings support a functional role for CGRP and ADM receptors to compensate the chronic heart failure in rats.     

Expression and regulation of calcitonin gene-related Peptide receptor in rat placentas

Calcitonin gene-related peptide (CGRP), one of the most potent vasodilators known, exerts its biological action by interacting with its receptors. Recent reports suggest the existence of two types of CGRP receptors, CGRP-A and CGRP-B. The current study was designed to examine whether CGRP-B receptors are present in the rat placenta, and if they are, whether they are modulated by gestational age and by sex-steroid hormones. Placentas were obtained from timed pregnant Sprague-Dawley rats that were killed on Days 17-21 and 22 before and during labor (n = 6 for each gestational age). In addition, placentas were also obtained from pregnant rats injected with progesterone (P(4); 4 mg per rat per day s.c. on Days 20-22), antiprogesterone RU-486 (10 mg/rat s.c. on Day 17), 17beta-estradiol (5 micro g/rat s.c. on Day 17), and antiestrogen ICI 182780 (0.3 micro g/rat s.c. on Day 17). Results showed that first, immunoflourescent staining of rat placentas using monoclonal anti-CGRP-B receptor antibody revealed the presence of CGRP-B receptors in the labyrinthine layer of the placenta, specifically to the trophoblast and blood vessel endothelium and underlying smooth muscle cells. The intensity of staining was lower in placentas obtained during labor. Second, a single band of 66 kDa, reactive to CGRP-B receptor antibody, was obtained in Western blotting of the rat placenta; third, densitometric analysis of protein bands showed that CGRP-B receptors were increased from Day 17 to Day 22, with maximal levels obtained on Day 22 before labor, which was 10 times higher than that of Day 17 (P < 0.01); fourth, expression of CGRP-B receptors in rat placenta decreased during labor (8% vs. 100% on Day 22 before labor, P < 0.01); fifth, P(4) given during Days 20-22 attenuated the fall in placental CGRP-B receptors at term labor; sixth, RU-486 given on Day 17 of gestation significantly decreased expression of placental CGRP-B receptors (18% vs. 100% in controls at 6 h, P < 0.01); seventh, a significant decrease in CGRP-B receptor expression was noted 48 h after estrogen administration; and eighth, ICI 182780 treatment on Day 17 increased placental CGRP-B receptors (152% vs. 100% in control at 48 h, P < 0.01). These results indicate that CGRP-B receptors are present in rat placenta and that receptor levels are higher with gestational age and lower at term labor. Progesterone stimulated and estrogen inhibited placental CGRP-B receptor expression. Thus, elevations in placental CGRP-B receptors in late pregnancy could play a role in increasing blood flow through the fetoplacental unit associated with rapid fetal growth during late gestation.     

The myocardial response to adrenomedullin involves increased cAMP generation as well as augmented Akt phosphorylation

In this work we aimed to observe (1) the changes in adrenomedullin (AM) and its receptor system - calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) - in myocardial ischemic injury and (2) the response of injuried myocardia to AM and the phosphorylation of Akt to illustrate the protective mechanism of AM in ischemic myocardia. Male SD rats were subcutaneously injected with isoproterenol (ISO) to induce myocardial ischemia. The mRNA levels of AM, CRLR, RAMP1, RAMP2 and RAMP3 were determined by RT-PCR. Protein levels of Akt, phosphor-Akt, CRLR, RAMP1, RAMP2 and RAMP3 were assayed by Western blot. Results showed that, compared with that of the controls, ISO-treated rats showed lower cardiac function and myocardial injury. The mRNA relative amount of AM, CRLR, RAMP1, RAMP2 and RAMP3 in the myocardia of ISO-treated rats was increased. The elevated mRNA levels of CRLR, RAMP1, RAMP2 and RAMP3 were positively correlated with AM content in injured myocardia. The protein levels of CRLR, RAMP1, RAMP2 and RAMP3 in injured myocardia were increased compared with that of control myocardia. AM-stimulated cAMP generation in myocardia was elevated in the ISO group, and was antagonized by AM(22-52) and CGRP(8-37). Western blot analyses revealed that AM significantly enhanced Akt phosphorylation in injured myocardia, which was blocked by pretreatment with AM(22-52) or CGRP(8-37). Ischemia-injured myocardia hyper-expressed AM and its receptors - CRLR, RAMP1, RAMP2 and RAMP3 - and the response of ischemic myocardia to AM was potentiated, and the level of Akt phosphorylation was also increased, which suggests that changes in cardiac AM/AM receptor might play an important role in the pathogenesis of myocardial ischemic injury.     

Effects of pregnancy and female sex steroid hormones on calcitonin gene-related peptide content of mesenteric artery in rats

Calcitonin gene-related peptide (CGRP) levels in plasma and the dorsal root ganglia (DRG) are increased during pregnancy and in ovariectomized rats injected with ovarian hormones. Vasodilatory responses to CGRP are also increased in these animals. In the present study, we hypothesized that pregnancy and ovarian hormones elevate the contents of CGRP in perivascular nerves. We assessed CGRP-dependent mesenteric vascular relaxation induced by electrical field stimulation (EFS) and arterial content of CGRP. Because the mesenteric artery represents resistance vessels, segments of mesenteric arteries collected from female rats at different stages of the estrous cycle, pregnancy, or postpartum and from male rats were used in this study. The EFS-induced relaxation in the presence and absence of CGRP(8-37), an antagonist of CGRP, was used to measure CGRP-dependent relaxation, and radioimmunoassay (RIA) of tissue homogenates was used to assess changes in CGRP content in mesenteric branch arteries. The results show that CGRP-dependent, EFS-induced relaxation response was lower in female rats at diestrus and proestrus than in male rats, and no statistically significant differences were observed between Gestational Day 20 and Postpartum Day 2. The RIA revealed significantly lower mesenteric artery CGRP levels in female rats at proestrus, gestation, and postpartum than in female rats at diestrus or in male rats. We conclude that no correlation exists between CGRP-dependent, EFS-induced relaxation and CGRP content in the mesenteric arteries of these animal groups. Because both CGRP levels in DRG and serum are reported to be elevated, the reduced CGRP content in the vasculature during pregnancy and proestrus implicate enhanced basal release of CGRP at the nerve terminal in these animals.     

Functional relevance of G-protein-coupled-receptor-associated proteins,exemplified by receptor-activity-modifying proteins (RAMPs)

The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.     

Increased expression in calcitonin-like receptor induced by aldosterone in cerebral arteries from spontaneously hypertensive rats does not correlate with functional role of CGRP receptor

OBJECTIVE: To analyze the effect of aldosterone on the expression of calcitonin gene-related peptide (CGRP) receptor components, calcitonin-like receptor (CL receptor) and receptor activity modifying protein 1 (RAMP1), as well as the effect of this mineralocorticoid on CGRP-mediated vasodilation in middle cerebral arteries from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). RESULTS: CGRP 0.1 nM-0.1 microM induced a concentration-dependent relaxation that was nitric oxide independent and higher in SHR middle cerebral arteries. CL receptor and RAMP1 expression were similar in both strains. The relaxation to CGRP was not modified by aldosterone 1 microM in either strain, although aldosterone 1 microM increased expression of CL receptor without modifying RAMP1 in segments from SHR rats. CONCLUSIONS: CGRP elicits greater vasodilation in middle cerebral arteries from SHR than WKY rats, that is nitric oxide independent, and by mechanism independent of CGRP receptor components expression. Although aldosterone increases the expression of CL receptor in SHR, it does not alter vasodilation to CGRP, since RAMP1 expression is not increased. These results indicate that the increase in CL receptor, without an increase in RAMP1, does not correlate with changes in functional role of the CGRP receptor.     

Receptor activity-modifying protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists

The heterodimeric CGRP receptor requires co-expression of calcitonin receptor-like receptor (CRLR) and an accessory protein called receptor activity-modifying protein (RAMP) 1 (McLatchie, L. M., Fraser, N. J., Main, M. J., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M. G., and Foord, S. M. (1998) Nature 393, 333-339). Several non-peptide CGRP receptor antagonists have been shown to exhibit marked species selectivity, with >100-fold higher affinities for the human CGRP receptor than for receptors from other species (Doods, H., Hallermayer, G., Wu, D., Entzeroth, M., Rudolf, K., Engel, W., and Eberlein, W. (2000) Br. J. Pharmacol. 129, 420-423; Edvinsson, L., Sams, A., Jansen-Olesen, I., Tajti, J., Kane, S. A., Rutledge, R. Z., Koblan, K. S., Hill, R. G., and Longmore, J. (2001) Eur. J. Pharmacol. 415, 39-44). This observation provided an opportunity to map the determinants of receptor affinity exhibited by BIBN4096BS and the truncated analogs, Compounds 1 and 2. All three compounds exhibited higher affinity for the human receptor, human CRLR/human RAMP1, than for the rat receptor, rat CRLR/rat RAMP1. We have now demonstrated that this species selectivity was directed exclusively by RAMP1. By generating recombinant human/rat CRLR/RAMP1 receptors, we demonstrated that co-expression of human CRLR with rat RAMP1 produced rat receptor pharmacology, and vice versa. Moreover, with rat/human RAMP1 chimeras and site-directed mutants, we have identified a single amino acid at position 74 of RAMP1 that modulates the affinity of small molecule antagonists for CRLR/RAMP1. Replacement of lysine 74 in rat RAMP1 with tryptophan (the homologous amino acid in the human receptor) resulted in a > or =100-fold increase in antagonist affinities, similar to the K(i) values for the human receptor. These observations suggest that important determinants of small molecule antagonist affinity for the CGRP receptor reside within the extracellular region of RAMP1 and provide evidence that this receptor accessory protein may participate in antagonist binding.     

Endogenous estrogen mediates vascular reactivity and distensibility in pregnant rat mesenteric arteries

The role of estrogen in the maternal systemic cardiovascular adaptations during pregnancy is still controversial. Female Sprague-Dawley rats were implanted at day 14 of pregnancy with either a 50-mg tamoxifen pellet (estrogen receptor blocker, n = 10) or placebo pellet (n = 10). Virgin female rats were a nonpregnant control (n = 7). At days 20-22 of pregnancy, resistance-sized mesenteric arteries were mounted onto a dual-chamber arteriograph system. Pregnancy significantly blunted the pressor response to phenylephrine [measurement of the effective concentration that yielded 50% maximum response (EC(50)) values were 1.5 +/- 0.22 vs. 0.69 +/- 0.16 microM (P < 0.05)] and enhanced vasodilation to ACh [EC(50) = 1.13 +/- 2.53 vs. 3.13 +/- 6.04 nM (P < 0.05)] compared with nonpregnant rats. However, tamoxifen treatment during pregnancy reversed these effects. Inhibition of nitric oxide (NO) synthase with N(G)-monomethyl-L-arginine (250 microM) shifted only the responses of the placebo-treated pregnant group to both phenylephrine and ACh. Arterial distensibility in the placebo-treated pregnant group was also significantly increased (P < 0.05) compared with nonpregnant and tamoxifen-treated pregnant animals. In summary, endogenous estrogen during pregnancy increases NO-dependent modulation of vessel tone and arterial distensibility.     

Aldosterone increases RAMP1 expression in mesenteric arteries from spontaneously hypertensive rats

OBJECTIVE: We analysed the effect of aldosterone on calcitonin gene-related peptide (CGRP) mediated vasodilation in noradrenaline precontracted endothelium denuded mesenteric arteries segments from Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) and the effect of aldosterone on calcitonin receptor-like receptor (CL receptor) and receptor activity modifying protein 1 (RAMP1) expression in endothelium-denuded mesenteric arteries from SHR rats. RESULTS: CGRP 0.1 nM-0.1 microM induced a concentration-dependent relaxation that was enhanced by aldosterone 1 microM in SHR only. Incubation with RU 486 10 microM significantly reduced the enhancement of CGRP-relaxation produced by aldosterone in SHR. CL receptor expression was not modified in either strain, while RAMP1 expression was enhanced in SHR by aldosterone 1 microM 120 min and 0.1 microM 120 min. This up-regulation of RAMP1 was prevented by RU 486 10 microM. CONCLUSIONS: Aldosterone, through glucocorticoid receptor activation, increases the vasodilatory effect of CGRP in SHR mesenteric arteries, which seems to be mediated by increased RAMP1 expression.