Potent Hypotensive Peptide Research Articles

β-arrestins negatively control human adrenomedullin type 1-receptor internalization

Adrenomedullin (AM) is a potent hypotensive peptide that exerts a powerful variety of protective effects against multiorgan damage through the AM type 1 receptor (AM1 receptor), which consists of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2). Two β-arrestin (β-arr) isoforms, β-arr-1 and β-arr-2, play a central role in the agonist-induced internalization of many receptors for receptor resensitization. Notably, β-arr-biased agonists are now being tested in phase II clinical trials, targeting acute pain and acute heart failure. Here, we examined the effects of β-arr-1 and β-arr-2 on human AM1 receptor internalization. We constructed a V5-tagged chimera in which the cytoplasmic C-terminal tail (C-tail) of CLR was replaced with that of the β2-adrenergic receptor (β2-AR), and it was transiently transfected into HEK-293 cells that stably expressed RAMP2. The cell-surface expression and internalization of the wild-type or chimeric receptor were quantified by flow cytometric analysis. The [125I]AM binding and the AM-induced cAMP production of these receptors were also determined. Surprisingly, the coexpression of β-arr-1 or -2 resulted in significant decreases in AM1 receptor internalization without affecting AM binding and signaling prior to receptor internalization. Dominant-negative (DN) β-arr-1 or -2 also significantly decreased AM-induced AM1 receptor internalization. In contrast, the AM-induced internalization of the chimeric AM1 receptor was markedly augmented by the cotransfection of β-arr-1 or -2 and significantly reduced by the coexpression of DN-β-arr-1 or -2. These results were consistent with those seen for β2-AR. Thus, both β-arrs negatively control AM1 receptor internalization, which depends on the C-tail of CLR.

Functions of Third Extracellular Loop and Helix 8 of Family B GPCRs Complexed with RAMPs and Characteristics of their Receptor Trafficking

At least one of three receptor activity-modifying proteins (RAMP1, RAMP2 and RAMP3) can interact with 10 G protein-coupled receptors (GPCRs; nine Family B GPCRs and a Family C GPCR). All three RAMPs interact with the calcitonin (CT) receptor (CTR), the CTR-like receptor (CLR), the vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP) 1 (VPAC1) and the VPAC2 receptor, which are all Family B GPCRs. Three RAMPs enable CTR to function as three heterodimeric receptors for amylin, which is a feeding suppression peptide. These RAMPs also transport the CLR to the cell surface, where they function as a CT gene-related peptide (CGRP) receptor (CLR/RAMP1 heterodimer) and two adrenomedullin (AM) receptors (CLR/RAMP2 and CLR/RAMP3 heterodimers). CGRP and AM are potent hypotensive peptides that exert powerful protective effects against multi-organ damage. We recently reported that the third extracellular loop (ECL3) of CLR governs the activation of AM, but not CGRP, signaling in the three CLR/RAMP heterodimers. Furthermore, we showed that in the presence of RAMP2, the eighth helix (helix 8) in the proximal portion of the cytoplasmic C-terminal tail of the CLR, which is thought to be present in all family B GPCRs, participates in receptor signaling. In addition, we demonstrated that overexpression of GPCR kinase (GRK) 2, GRK3 and GRK4 enhances the AM-induced internalization of the CLR/RAMP2 heterodimer. In this review, we describe these studies and consider their implications for other Family B GPCRs that can interact with RAMPs.

955 NOVEL FUNCTIONS OF G PROTEIN-COUPLED RECEPTOR KINASES

Objectives: Adrenomedullin (AM), a potent hypotensive peptide, can powerfully protect against various cardiovascular diseases through the AM1 receptor, which consists of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2). It is well known that, of the seven types of G protein-coupled receptor kinases (GRKs), GRK4 and GRK5 promote the pathological conditions of hypertension and heart failure. Here, we examined the effects of non-retinal GRKs, GRK2 to GRK6, on the cell-surface expression and signaling of the human AM1 receptor. Design and methods: We constructed a set of V5-tagged mutants in which the cytoplasmic C-terminal tail (C-tail) of CLR was progressively truncated and transiently transfected these mutants into HEK-293 cells that stably expressed RAMP2. Results: The cell-surface expression of the wild-type or mutant receptors was quantified by flow cytometric analysis. The 125I-AM binding and AM-induced cAMP production of the receptors were also determined. Of the five GRKs tested, only GRK4 and GRK5 markedly reduced the translocation of AM1 receptors to the cell surface, thereby abolishing AM binding and signaling; in these cases, no receptor internalization was found. These novel functions of GRKs were abrogated by the complete loss of the C-tail of CLR or the substitution of the total C-tail of CLR with that of the β2-adrenergic receptor and, additionally, were critically dependent on the Ser-Phe-Ser-Asn-Ser sequence in the C-tail of CLR. Conclusions: The present findings suggest that the effects of AM may be impaired in circulatory diseases that are involved in the induction of GRK4 and GRK5 proteins.

Hemodynamic and hormonal effects of exogenous adrenomedullin administration in humans and relationship to insulin resistance

Although adrenomedullin (AM) is a potent hypotensive peptide that acts mainly as a vasodilative and proliferation inhibitory factor, there have been few hemodynamic studies on AM in humans, especially concerning arterial stiffness and hormonal effects. In addition, AM is a suppressive factor in insulin resistance, suggesting that the effects of AM in a state of insulin resistance are important. To evaluate the effects of AM in humans, 28 participants were intravenously administered AM (5 pmol min(-1) kg(-1)) for 90 min. They also received a representative vasodilator drug, nicardipine, as a reference drug. Blood pressure, heart rate, pulse wave velocity (PWV) and blood flow were monitored throughout the experiment. Hormonal changes were also monitored by blood tests. The effects of AM were compared with those of nicardipine. In addition, the effects of AM were re-evaluated against insulin resistance state. AM and nicardipine produced the same level of hypotension, but AM showed a more potent ability to increase heart rate, blood flow and cardiac output and reduce PWV. AM and nicardipine similarly stimulated plasma noradrenaline and renin activity. However, in the state of insulin resistance, favorable effects of AM on aortic stiffness were blunted and differences between AM and nicardipine disappeared. Furthermore, there was a significant correlation between maximum changes in the PWV induced by AM and the homeostasis model assessment of insulin resistance index (r=0.58, P=0.001). Our results suggest that AM may improve arterial stiffness and act as a compensatory factor against arterial sclerosis. Moreover, decreased reactivity of AM may participate in the progression of arterial sclerosis in insulin resistance.

A Potent Hypotensive Peptide, Novokinin, Induces Relaxation by AT2- and IP-Receptor-Dependent Mechanism in the Mesenteric Artery from SHRs

In this study, we found that novokinin (Arg-Pro-Leu-Lys-Pro-Trp), a potent hypotensive peptide acting through the AT(2) receptor, has vasorelaxing activity in the mesenteric artery isolated from spontaneously hypertensive rats. The vasorelaxing activity was significantly blocked by PD123319, indomethacin, and CAY10441, which are an AT(2) receptor antagonist, a cyclooxygenase inhibitor, and an IP receptor antagonist, respectively. N(G)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, did not block the vasorelaxing activity. These results suggest that the vasorelaxing activity of novokinin, which contributes to the hypotensive effect, is mainly mediated by prostaglandin I(2) (prostacyclin) and the IP receptor downstream of the AT(2) receptor.

Hypotensive activity of novokinin, a potent analogue of ovokinin(2–7), is mediated by angiotensin AT 2 receptor and prostaglandin IP receptor

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp) is a potent hypotensive peptide previously designed based on the structure of ovokinin(2–7) (Arg-Ala-Asp-His-Pro-Phe), a vasorelaxing and hypotensive peptide derived from ovalbumin. Novokinin exhibited an affinity for the angiotensin AT 2 receptor (Ki = 7.35 μM). Novokinin significantly lowered systolic blood pressure at a dose of 0.03 and 0.1 mg/kg after intravenous and oral administration, respectively, in spontaneously hypertensive rats (SHRs), and the hypotensive activity was blocked by PD123319, an antagonist of the AT 2 receptor. Novokinin lowered blood pressure in C57BL/6J mice after oral administration at a dose of 50 mg/kg. However, in AT 2 receptor-deficient mice, novokinin did not reduce blood pressure. These results demonstrate that the hypotensive activity of novokinin is mediated by the AT 2 receptor. The hypotensive activity of novokinin in SHRs was completely blocked by indomethacin and CAY10441, an inhibitor of cyclooxygenase and an antagonist of the prostaglandin IP receptor, respectively. These suggest that the hypotensive activity is mediated by prostacyclin and the IP receptor downstream of the AT 2 receptor.

The Proinflammatory Cytokine Interleukin 1β and Hypoxia Cooperatively Induce the Expression of Adrenomedullin in Ovarian Carcinoma Cells through Hypoxia Inducible Factor 1 Activation

Adrenomedullin (ADM) is a potent hypotensive peptide produced by macrophages and endothelial cells during ischemia and sepsis. The molecular mechanisms that control ADM gene expression in tumor cells are still poorly defined. It is known, however, that hypoxia potently increases ADM expression by activation of the transcription factor complex hypoxia inducible factor 1 (HIF-1). Proinflammatory cytokines produced by tumor invading macrophages likewise activate expression of ADM. Herein, we show that apart from hypoxia, the proinflammatory cytokine interleukin 1beta (IL-1beta) induced the expression of ADM mRNA through activation of HIF-1 under normoxic conditions and enhanced the hypoxia-induced expression in the human ovarian carcinoma cell line OVCAR-3. IL-1beta significantly increased accumulation and nuclear translocation of HIF-1alpha under normoxic conditions and amplified hypoxic HIF-1 activation. IL-1beta treatment affected neither HIF-1alpha mRNA levels nor the hydroxylation status of HIF-1alpha and, thus, stability of the protein. Instead cycloheximide effectively prevented the increase in HIF-1alpha protein, indicating a stimulatory effect of IL-1beta on HIF-1alpha translation. Finally, treatment of HIF-1alpha with short interfering RNA revealed a significant role for HIF-1 in the IL-1beta-dependent stimulation of ADM expression.

Angiotensin II Stimulates Cardiac Adrenomedullin Production and Causes Accumulation of Mature Adrenomedullin Independently of Hemodynamic Stressin Vivo

Adrenomedullin is a potent hypotensive peptide that may act on myocytes to inhibit hypertrophy and on fibroblasts to inhibit growth in vitro induced by mechanical stretching and angiotensin II. Adrenomedullin is processed from the inactive intermediate adrenomedullin precursor with a glycine extension, which is subsequently converted to biologically active mature adrenomedullin by enzymatic amidation. Total adrenomedullin is the sum of intermediate and mature adrenomedullin. We examined the effect of a subpressor dose of angiotensin II on the production of left ventricular adrenomedullin and on protein levels of mature adrenomedullin in the left ventricle in vivo. We also investigated whether the effect is mediated by the angiotensin II type 1 receptor. Concentrations of total and mature adrenomedullin in the left ventricle and mature adrenomedullin-to-intermediate adrenomedullin ratio were significantly increased by angiotensin II infusion, regardless of pressure overload. Total and mature adrenomedullin concentrations significantly correlated with the weight of the left ventricle. Furthermore, increased adrenomedullin gene expression and protein levels were completely suppressed by a subdepressor dose of angiotensin II type 1 receptor blocker. In conclusion, angiotensin II stimulates the production of cardiac adrenomedullin and accumulates mature adrenomedullin in the left ventricle independently of hemodynamic stress. These processes are partially regulated through the angiotensin II type 1 receptor in vivo.

Adrenomedullin, ANP and BNP are colocalized in a subset of endocrine cells in the rat heart

We investigated by immunocytochemistry (ICC) the distribution in the rat heart of adrenomedullin (AM), a potent and long-lasting hypotensive peptide which is expressed in the cardiovascular system, where it is known to play a major regulatory role. Hearts were collected from adult male Sprague-Dawley rats, and were perfused for 20 min, according to the Langendorff technique, with endothelin-1 (ET-1) or the mast cell-degranulator compound 48/80. Hearts were frozen, and ICC was performed using standard techniques and a specific anti-rat AM1-50 antibody. We confirmed the presence of a low AM-immunoreactivity in cardiomyocytes and cardiac fibroblasts, as well as in endothelial and smooth muscle cells of coronary vessels. Moreover, we provided evidence of the presence in both atria and ventricles of sparse voluminous AM-positive cells, mainly located near coronary vessels. These cells had the same juxtavascular location of toluidine blue-positive mast cells and their number decreased upon acute exposure to the 48/80 compound. However, ICC showed that in these cells AM was always colocalized with atrial and brain natriuretic peptides. Moreover, AM-storing cells were also positive to MyHC-Apla2, indicating that they share some phenotypic features with immature smooth muscle cells. The number of AM-storing cells underwent a dramatic decrease in response to the potent vasoconstrictor ET-1, suggesting an acute release of stored vasodilatory AM aimed at counteracting coronary constriction. Taken together, our present findings support the hypothesis that these cells may represent a novel subset of endocrine cells, strategically located near blood vessels in the mammalian heart, where they can release vasoactive peptides.

Acute Hyperinsulinemia is Associated with Increased Plasma Adrenomedullin Concentrations in Uncomplicated Obesity

Adrenomedullin (AM) is a potent hypotensive peptide which may be implicated in the insulin regulatory system. Acute hyperinsulinemia exerts no influence on plasma AM in normal subjects while no data on obese subjects has been reported. The aim of the study was to investigate the effect of acute hyperinsulinemia on the plasma AM concentration in patients with uncomplicated obesity. We measured the plasma AM levels in 23 obese subjects (BMI 41.9 +/- 9.8 kg/m2), 21 females and 2 males (mean age 31 +/- 7.2 years), before and during a euglycemic hyperinsulinemic clamp. The control group consisted of 43 healthy subjects (HS) (22 males and 21 females; mean age 38 +/- 12 years; BMI 23.3 +/- 3.2 kg/m2). Baseline plasma AM was found to be higher in obese subjects (20.4 +/- 8.4 pg/ml) than in normal subjects (11.3 +/- 0.8 pg/ml) (p < 0.001). A significant increase in the plasma AM levels was observed in obese subjects during acute hyperinsulinemia (from 20.4 +/- 8.4 pg/ml at 0 min to 26 +/- 8.9 pg/ml at 120 min, p < 0.02). Plasma AM concentrations were significantly correlated with insulin levels at 30 min (r = 0.44; p = 0.04) and 120 min (r = 0.40, p = 0.05) during the clamp. In conclusion, acute hyperinsulinemia induced a significant increase in the plasma levels of AM in uncomplicated obese subjects. Hyperinsulinemia may, at least in part, regulate levels of AM in obesity, explaining the high levels of the peptide in these subjects.

Acute effect of ischemia on adrenomedullin immunoreactivity in the rat heart: An immunocytochemical study

We investigated by immunocytochemistry (ICC) the acute effects of ischemia on the distribution in the rat heart of adrenomedullin (AM), a potent hypotensive peptide which is expressed in the cardiovascular system, where it is known to play a major regulatory and protective role. Hearts, collected from adult male Sprague-Dawley rats, were perfused with the Langendorff technique, and "global" ischemia was obtained by stopping perfusion for 20 min. Hearts were frozen, and ICC was performed using a specific anti-rat AM1-50 antibody and secondary peroxidase-conjugated antibodies. ICC demonstrated AM-immunoreactivity (IR) in cardiomyocytes and especially in the wall of coronary vessels. Quantitative densitometry showed that acute ischemia significantly decreased AM-IR in coronary arterioles, thereby suggesting that it markedly stimulates AM release. The conclusion is drawn that acute ischemia and ensuing hypoxia activate in the rat heart the release of AM, which by its coronarodilatory action may enhance heart blood flow.

Optimal designing of β-conglycinin to genetically incorporate RPLKPW, a potent anti-hypertensive peptide

Previously, we introduced the RPLKPW sequence, a highly potent hypotensive peptide designed based on ovokinin (2–7), into three homologous sites in the soybean β-conglycinin α′ subunit by site-directed mutagenesis. The modified protein expressed in Escherichia coli reduced blood pressure of spontaneously hypertensive rats (SHRs) after oral administration at a dose of 10 mg/kg, which suggested about 30% of the introduced peptide was released in vivo. In this study amino acid residues around the RPLKPW sequence were optimized with a use of synthetic peptides to facilitate release of RPLKPW by gastrointestinal proteases. Then, fourth RPLKPW was also introduced into the extension domain of the protein. The newly modified protein, which was produced in E. coli, significantly lowered blood pressure in SHRs at a dose of 2.5 mg/kg 4 h after oral administration. Furthermore, we produced an extension domain that corresponds to residues 1–143 of the modified α′ subunit containing four RPLKPW sequences by introducing a termination codon. The minimum effective dose of the modified extension domain was 1.0 mg/kg, which is 1/2000 that of ovalbumin.

Development of an ultrasensitive enzyme immunoassay for human proadrenomedullin N-terminal 20 peptide and direct measurement of two molecular forms of PAMP in plasma from healthy subjects and patients with cardiovascular disease

Objective: Proadrenomedullin N-terminal 20 peptide (PAMP) processed from an adrenomedullin precursor is a potent hypotensive peptide. It was anticipated that a mature form of PAMP (m-PAMP) and an intermediate PAMP-gly existed together in the blood. To measure concentrations of PAMPs in human plasma directly, we have developed a highly sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay, ICT-EIA). Design and methods: PAMP was reacted simultaneously with 2,4-dinitrophenyl (DNP)-biotinyl-bovine serum albumin (BSA)-anti-PAMP Fab' conjugate and anti-PAMP Fab'-β- d-galactosidase conjugate. The immune complex that was formed was initially trapped onto a polystyrene bead coated with anti-DNP IgG, and then transferred onto a second polystyrene bead coated with streptavidin. The resulting three-component complex was then assayed fluorometrically. Results: The detection limits of ICT-EIA for both m-PAMP and PAMP-gly were 0.1 pmol/l with as little as 10 μl of plasma, and were a hundred times higher than with conventional radioimmunoassay (RIA). Using ICT-EIA, we determined that the plasma concentrations of m-PAMP and PAMP-gly in 51 healthy volunteers were 0.51 ± 0.19 and 1.15 ± 0.38 pmol/l (mean ± SD), respectively. Both plasma m-PAMP and PAMP-gly concentrations in patients with a variety of diseases, including hypertension, heart failure, chronic renal failure, and hemodialysis, were significantly higher than those in healthy subjects. In addition, both plasma m-PAMP and PAMP-gly concentrations in patients with New York Heart Association (NYHA) class I–IV heart failure were increased in proportion to clinical severity. Conclusions: These sensitive and specific ICT-EIAs may be used as a powerful tool for investigating the cardiovascular system in patients with heart failure.

Adrenomedullin (11–26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla

Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from pheochromocytoma tissue. Both the ring structure and the C-terminal amide structure of AM are essential for its hypotensive activity. We have developed an RIA which recognizes the ring structure of human AM. Using this RIA, we have characterized the molecular form of AM in bovine adrenal medulla. Gel filtration chromatography revealed that three major peaks of immunoreactive AM existed in the adrenal medulla. The peptide corresponding to Mr 1500 Da was further purified to homogeneity. The peptide was determined to be AM (11–26) which has one intramolecular disulfide bond. Amino acid sequences of bovine AM and its precursor were deduced from the analyses of cDNA encoding bovine AM precursor. The synthetic AM (11–26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. The hypertensive activity lasted about one minute, and a dose dependent increase in heart rate was also observed. The present data indicate that AM (11–26) is a major component of immunoreactive AM in bovine adrenal medulla and shows pressor activity.

Calcitonin gene-related peptide (CGRP), acting via CGRP type 1 receptors, inhibits potassium-stimulated aldosterone secretion and enhances basal catecholamine secretion from rat adrenal gland.

Calcitonin gene-related peptide (CGRP) is a potent hypotensive peptide, that acts via two main subtypes of receptors, named CGRP1 and CGRP2. CGRP belongs to a regulatory-peptide family, that includes adrenomedullin (ADM) whose aldosterone antisecretagogue and catecholamine secretagogue actions are well demonstrated. Quantitative autoradiography showed the presence of [125I]CGRP binding sites in both rat adrenal zona glomerulosa (ZG) and medulla. Binding was displaced by the CGRP1-receptor antagonist CGRP(8-37), but not by the CGRP2-receptor agonist [cys(Et)2,7]-alphaCGRP (CGRP2-A). CGRP concentration-dependently inhibited 10 mM-stimulated (but not basal) aldosterone secretion from dispersed rat ZG cells, and enhanced basal catecholamine secretion from rat adrenomedullary fragments. The responses to the maximal effective concentration of CGRP (10-8 M) were blocked by 10-7 M CGRP(8-37). CGRP2-A (10-7 M) neither altered aldosterone response to 10 mM K+ nor enhanced basal catecholamine secretion. The conclusion is drawn that CGRP, like ADM, inhibits agonist-stimulated aldosterone secretion and stimulates basal catecholamine release in the rat, exclusively acting via CGRP1 receptors.

Distribution and molecular forms of adrenomedullin and proadrenomedullin N-terminal 20 peptide in the porcine gastrointestinal tract.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are potent hypotensive peptides that are generated from the same precursor and expressed in a variety of mammalian tissues. The purpose of this study was to elucidate the distribution and endogenous molecular forms of AM and PAMP. To this end, we used new radioimmunoassays that recognize the ring structure of AM and the C-terminal region of PAMP to compare the distribution of AM and PAMP in porcine gastrointestinal tissues. Immunoreactive AM was abundant in the duodenum (0.334+/-0.132 fmol/mg) and ileum (0.439+/-0.235 fmol/mg), and ubiquitous in other gastrointestinal tissues. The duodenal and ileal concentrations of AM were about 4 to 14 times higher than those in other gastrointestinal tissues. Similarly, immunoreactive PAMP was abundant in the duodenum (0.577+/-0.417 fmol/mg) and ileum (1.575+/-1.445 fmol/mg) and ubiquitous in other gastrointestinal tissues. The concentrations of immunoreactive AM and PAMP were highest in the ileum. Characterization of these peptides in the ileum, using high-performance liquid chromatography, showed that they were authentic. Furthermore, the concentrations of immunoreactive AM and PAMP in the mucosa and submucosa of the ileum were significantly higher than those in whole ileum. These results suggest that AM and PAMP play physiological roles in the porcine small intestine.

Coordinated Up-regulation by Hypoxia of Adrenomedullin and One of Its Putative Receptors (RDC-1) in Cells of the Rat Blood-Brain Barrier

Adrenomedullin (ADM) is a potent hypotensive peptide, which is produced during sepsis and ischemia. We demonstrate here that hypoxia induced a time-dependent increase of both ADM mRNA and protein expressions in cultured astrocytes and endothelial cells from rat brain microvessels. Gene reporter analyses showed a 2-fold increase in ADM gene transcription which was suppressed when the ADM promoter was deleted of its hypoxia responsive element. Hypoxia increased 7-fold the stability of pre-formed ADM mRNAs. Rat brain microvessels expressed mRNAs coding for the different putative ADM receptors but they did not respond to exogenous ADM and calcitonin gene-related peptide by the formation of cAMP. In contrast, ADM and calcitonin gene-related peptide increased the formation of cAMP in astrocytes and their actions were potentiated about 2-fold after hypoxia. Messenger RNA species coding for three putative ADM receptors (the L1 orphan receptor, RDC-1, and calcitonin receptor-like receptor) and accessory proteins (receptor-activity modifying proteins) were present in astrocytes. Hypoxia selectively up-regulated expression of RDC-1 receptor mRNAs. The results indicate that ADM and RDC-1 are hypoxia-sensitive genes and that RDC-1 receptors may mediate some actions of ADM in hypoxic astrocytes.

Expression of adrenomedullin in the human corpus luteum

Objective: Adrenomedullin (AM) is a potent hypotensive peptide found in human pheochromocytoma tissue. In the present study, the expression of AM mRNA in the human ovary was examined. Design: Ovarian mRNA was analyzed in the follicle, the corpus luteum of mid-luteal phase, and early pregnancy. Setting: Gunma University School of Medicine, Gunma, Japan. Patient(s): Premenopausal women with histologically normal ovary who were undergoing salpingoophorectomy. Intervention(s): The dominant follicle and corpora lutea were isolated and total RNA was extracted from these tissues. Main Outcome Measure(s): Northern blot analysis of AM, receptor activity–modifying protein 2 (RAMP2), and LH/hCG receptor mRNA in human samples. Result(s): An AM mRNA transcript of 1.6 kilobases (kb) was detected in corpus luteum tissue; this transcript was identical to that which has been detected in placenta and fetal membrane. The AM and LH/hCG receptor mRNA levels were low in the mature follicle but increased in the corpus luteum of the mid-luteal phase and were maintained during early pregnancy. A single transcript of 0.8 kb for RAMP2 was also seen in the follicle and corpus luteum, the level of RAMP2 mRNA was relatively high in the preovulatory follicle and RAMP2 was present in the corpus luteum. Conclusion(s): The expression of AM, its receptor, and LH/hCG receptor may be an important component in the process of development and differentiation of the corpus luteum.

A physiological role for adrenomedullin in rats; a potent hypotensive peptide in the hypothalamo-neurohypophysial system.

Adrenomedullin, a potent hypotensive peptide, was originally isolated from human phaeochromocytoma. Adrenomedullin immunoreactivity and gene expression are found not only in peripheral organs but also in the central nervous system. Adrenomedullin labelled cells were localised in the hypothalamus, including in the paraventricular and supraoptic nuclei, in rats. Abundant adrenomedullin-immunoreactive fibres and varicosities were found in the hypothalamo-neurohypophysial tract and the internal zone of the median eminence in colchicine-treated and hypophysectomized rats, whereas in control rats few adrenomedullin-labelled fibres were observed. We examined the effects of intracerebroventricular administration of adrenomedullin on neurosecretory cells in the paraventricular and supraoptic nuclei of rats, using immunohistochemistry for Fos protein and in situ hybridisation histochemistry for c-fos mRNA. Intracerebroventricular administration of adrenomedullin caused a marked induction of Fos-like immunoreactivity in the paraventricular nucleus and the dorsal part of the supraoptic nucleus. In the paraventricular and supraoptic nuclei, nuclear Fos-like immunoreactivity was predominantly in oxytocin-immunoreactive cells rather than vasopressin-immunoreactive cells. The induction of c-fos mRNA in the paraventricular and supraoptic nuclei was increased in a dose-related manner 30 min after intracerebroventricular administration of adrenomedullin. This induction was reduced by pre-treatment with the adrenomedullin receptor antagonist, human adrenomedullin-(22-52)-NH2. Intracerebroventricular administration of adrenomedullin also caused a marked increase in the plasma concentration of oxytocin. Extracellular recordings from magnocellular neurosecretory cells in the paraventricular nucleus revealed that putative oxytocin-secreting cells were activated by intracerebroventricular administration of adrenomedullin. These results suggest that central adrenomedullin preferentially stimulates the secretion of oxytocin by activating hypothalamic oxytocin-secreting cells and may have an important role in salt appetite and body fluid homeostasis in rats.

Adrenomedullin: Is There Physiological Relevance in the Pathology and Pharmacology?

The adrenomedullin gene encodes two potent hypotensive peptides, adrenomedullin and proadrenomedullin NH(2)-terminal 20 peptide. As with other vasoactive peptides, the most difficult challenge is to prove the physiological relevance of their recognized pathological and pharmacological actions and to establish, against that physiological background, their therapeutic potential.