In this study, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the changes in mRNAs levels of preproadrenomedullin (ppADM) gene encoding adrenomedullin (ADM) and the essential receptor components of ADM, calcitonin receptor-like receptor (CRLR), and the receptor activity modifying protein 2 and 3 (RAMP2 and RAMP3) in the medulla oblongata, hypothalamus, midbrain, pituitary gland and adrenal gland of the stress-induced hypertensive rats. It was shown that chronic foot-shock and noise stress for 15 consecutive days induced a significant increase in systolic blood pressure (SBP) and unique changes in ppADM and its receptor components mRNAs in all areas studied. As compared with the control group, the level of ppADM mRNA, normalized against a glyceraldehydes-3-phosphate dehydrogenase (GAPDH) control, was up-regulated in the hypothalamus-pituitary-adrenal (HPA) axis, but down-regulated in the medulla oblongata and midbrain (P<0.01 and P<0.05, respectively). The relative amount of CRLR mRNA was higher in the hypothalamus than that in other areas. The level of CRLR mRNA expression was significantly increased in the medulla oblongata of the stress group (P<0.01), but decreased in the midbrain (P<0.01) as well as hypothalamus(P<0.05), as compared with that of the control group. Chronic stress for 15 consecutive days produced an increase in the level of RAMP2 mRNA expression in the medulla oblongata (P<0.01) and a decrease in the adrenal gland (P<0.01), as compared with the control. No significant stress-related changes in RAMP2 mRNA were observed in the midbrain, hypothalamus and pituitary gland. The amount of RAMP3 mRNA was relatively higher in the midbrain and hypothalamus than that in the medulla oblongata, adrenal gland and adrenal gland. Stress-induced hypertensive rats exhibited an increased RAMP3 mRNA expression in the hypothalamus and pituitary gland (P<0.01 and P<0.05, respectively) and a decrease in the adrenal gland and midbrain (P<0.05). No significant stress-related change in RAMP3 mRAN was observed in the medulla oblongata. Taken together, our results indicate that the significant changes in ppADM and its receptor components mRNAs expression in the HPA axis and autonomic centers may be related to the development of the stress-induced hypertension. Nevertheless, the pathophysiological significance of brain-derived ADM and its receptors in stress and blood pressure regulation and their roles in stress-induced hypertension still await further investigation.
Adrenomedullin ; Animals ; Brain Stem ; metabolism ; Hypertension ; etiology ; metabolism ; Hypothalamo-Hypophyseal System ; metabolism ; Male ; Peptides ; genetics ; metabolism ; Pituitary-Adrenal System ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenomedullin ; Receptors, Peptide ; biosynthesis ; genetics ; Stress, Physiological

OBJECTIVETo investigate the expression and role of adrenomedullin (ADM) and adrenomedullin receptor (ADMR) in patients with chronic obstructive pulmonary disease (COPD).

METHODSSmall pulmonary artery remodeling was observed using morphometric analysis. The expression of ADM and ADMR mRNA in lung tissue was calculated by in situ hybridization in 9 COPD cases. Cardiac catheterization was performed in 22 COPD cases to monitor changes of hemodynamic parameters and patients were divided into two groups based on mean pulmonary artery pressure (mPAP). The cases without pulmonary hypertension (PH) were placed in Group A (n = 12) and those with PH were placed in Group B (n = 10). The levels of pulmonary arterial plasma ADM were measured by radioimmunoassay. Blood gas analysis was also conducted.

RESULTSThe ratio of vascular wall thickness to external diameter (MT%) and the ratio of vascular wall area to total area (MA%) were higher in patients with COPD (P < 0.01). In situ hybridization showed that ADM mRNA and ADMR mRNA were expressed in the pulmonary artery walls of control subjects. The expression levels were significantly higher in those of COPD sufferers (P < 0.01). Statistically positive relationships were visible between ADM and ADMR, and the plasma ADM level of Group B was significantly higher than that of Group A (P < 0.05). The plasma ADM level had a significantly positive correlation to mPAP and pulmonary vascular resistance (PVR), while being negatively correlated to levels of PaO(2).

CONCLUSIONADM may play an extremely protective role as a local autocrine/paracrine factor in COPD.

Adrenomedullin ; Adult ; Aged ; Aged, 80 and over ; Female ; Hemodynamics ; Humans ; Male ; Middle Aged ; Oxygen ; blood ; Peptides ; blood ; genetics ; physiology ; Pulmonary Disease, Chronic Obstructive ; prevention & control ; RNA, Messenger ; analysis ; Receptors, Adrenomedullin ; Receptors, Peptide ; genetics ; physiology

OBJECTIVETo investigate the role of adrenomedullin (AM) in the development of hypoxic pulmonary hypertension (HPH), and to assess the expression of AM and adrenomedullin receptor (AMR) in the lungs of rats with HPH.

METHODSWe exposed 10 rats to normobaric hypoxic conditions for 3 weeks to establish rat model of pulmonary hypertension; and 10 other rats were used as normoxic controls. Mean pulmonary arterial pressure (mPAP) was measured by a right cardiac catheterization. The thickness of pulmonary arterioles was measured by a computerized image analyzer. We used the reverse transcription-polymerase chain reaction (RT-PCR) to assess the change of expression of AM and AMR in lung of HPH rat model.

RESULTSCompared with the control group, hypoxic rats developed remarkable pulmonary hypertension, increment in the thickness of pulmonary arterioles and right ventricular hypertrophy (P < 0.01). Chronic hypoxia elicited a considerable increment in expression of AM and AMR in the lungs of rats, and the ratio of AM/beta-actin and AMR/beta-actin in lungs of rats treated with hypoxia were significantly higher (P < 0.01).

CONCLUSIONSThe AM plays an important role in regulating pulmonary vascular tone and can ameliorate the development of hypoxic pulmonary hypertension in rats.

Adrenomedullin ; Animals ; Arterioles ; pathology ; Gene Expression ; Hypertension, Pulmonary ; metabolism ; pathology ; Hypertrophy, Right Ventricular ; etiology ; Hypoxia ; metabolism ; pathology ; Lung ; metabolism ; Male ; Peptides ; genetics ; Rats ; Rats, Wistar ; Receptors, Adrenomedullin ; Receptors, Peptide ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

The increase of pronociceptive mediators in the dorsal root ganglia (DRG) and spinal dorsal horn is an important mechanism in the pathogenesis of inflammatory pain and opioid tolerance. Adrenomedullin (AM) belongs to calcitonin gene-related peptide (CGRP) family and has been recently demonstrated to be a pain-related peptide. It has also been shown that the expression and release of AM are increased in the DRG and spinal dorsal horn during inflammation and repeated use of morphine. Intrathecal administration of the selective AM receptor antagonist AM22-52 abolishes inflammatory pain and morphine tolerance, suggesting that enhanced AM receptor signaling in the DRG and spinal dorsal horn contributes to the induction of inflammatory pain and morphine tolerance. The present review highlights the recent developments regarding the involvement of AM in these two disorders. The neurological mechanisms of AM's actions are also discussed.
Adrenomedullin ; pharmacology ; Animals ; Calcitonin Gene-Related Peptide ; Drug Tolerance ; Ganglia, Spinal ; drug effects ; Inflammation ; drug therapy ; metabolism ; Morphine ; pharmacology ; Pain ; drug therapy ; metabolism ; Peptide Fragments ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenomedullin ; metabolism

Intermedin/adrenomedullin-2 (IMD/AM2), a member of the calcitonin gene-related peptide/AM family, plays an important role in protecting the cardiovascular system. However, its role in the enhanced sympathoexcitation in obesity-related hypertension is unknown. In this study, we investigated the effects of IMD in the paraventricular nucleus (PVN) of the hypothalamus on sympathetic nerve activity (SNA), and lipopolysaccharide (LPS)-induced sympathetic activation in obesity-related hypertensive (OH) rats induced by a high-fat diet for 12 weeks. Acute experiments were performed under anesthesia. The dynamic alterations of sympathetic outflow were evaluated as changes in renal SNA and mean arterial pressure (MAP) in response to specific drugs. Male rats were fed a control diet (12% kcal as fat) or a high-fat diet (42% kcal as fat) for 12 weeks to induce OH. The results showed that IMD protein in the PVN was downregulated, but Toll-like receptor 4 (TLR4) and plasma norepinephrine (NE, indicating sympathetic hyperactivity) levels, and systolic blood pressure were increased in OH rats. LPS (0.5 µg/50 nL)-induced enhancement of renal SNA and MAP was greater in OH rats than in obese or control rats. Bilateral PVN microinjection of IMD (50 pmol) caused greater decreases in renal SNA and MAP in OH rats than in control rats, and inhibited LPS-induced sympathetic activation, and these were effectively prevented in OH rats by pretreatment with the AM receptor antagonist AM22-52. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) inhibitor U0126 in the PVN partially reversed the LPS-induced enhancement of SNA. However, IMD in the PVN decreased the LPS-induced ERK activation, which was also effectively prevented by AM22-52. Chronic IMD administration resulted in significant reductions in the plasma NE level and blood pressure in OH rats. Moreover, IMD lowered the TLR4 protein expression and ERK activation in the PVN, and decreased the LPS-induced sympathetic overactivity. These results indicate that IMD in the PVN attenuates SNA and hypertension, and decreases the ERK activation implicated in the LPS-induced enhancement of SNA in OH rats, and this is mediated by AM receptors.
Adrenomedullin ; metabolism ; Animals ; Blood Pressure ; drug effects ; physiology ; Hypertension ; etiology ; Lipopolysaccharides ; pharmacology ; Male ; Neuropeptides ; metabolism ; Obesity ; complications ; Rats, Sprague-Dawley ; Receptors, Adrenomedullin ; drug effects ; metabolism ; Sympathetic Nervous System ; drug effects ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVETo investigate the effects of TNF-alpha, TNF-beta and the acceptor expression about mechanical renal trauma with extraneous ADM.

METHODSThere were 104 healthy adult plain grade Wistar rat, randomly divided into four groups:8 in the group of control, 32 in the group of trauma, 32 in the group injected ADM before trauma, 32 in the group injected ADM post trauma. The experimental model of rat kidney with mechanical trauma was prepared by striking the area of rat skin reflecting by kidney with free dropping ferrous hammer in the last three groups. ADM (0.1 nmol/kg) administrated by intraperitoneal injection at 10 minutes before trauma or post trauma respectively in injected groups. All rats were executed by drawing-out all the blood in their hearts. Renal tissue was investigated to study positive expression of TNF-alpha, TNF-beta, TNFR after SABC stained.

RESULTSTNF-alpha expression:the TNF-alpha expression of trauma group was more positive than it of control group in the wound early time. The expression of group injected post trauma was less than it of trauma group at 1 h (P < 0.01). The expression of group injected before trauma was less than it of trauma group at 6 h (P < 0.05) TNF-beta expression: the TNF-beta expression of trauma group was less than it of control group at 1 h and 6 h (P < 0.05). The TNF-beta expression of group injected post trauma was more positive than it of trauma group at the same time of 1 h and 6 h (P < 0.01). TNFR expression: the TNFR expression of trauma group was less than it of control group at 6 h (P < 0.01). The TNFR expression of group injected before trauma was more positive than it of trauma group in the at the same time of 1 h and 6 h (P < 0.01).

CONCLUSIONSThe TNFR can regulate the TNF-alpha and the TNF-beta in dynamic balancing. The regulation of TNFR is main to TNF-alpha. What the TNF-beta participated in renal trauma mainly is the anti-damage process. ADM can reduce the expression of TNF-alpha. ADM increases the expression of TNF-beta and TNFR.

Adrenomedullin ; pharmacology ; Animals ; Disease Models, Animal ; Female ; Kidney ; injuries ; metabolism ; Lymphotoxin-alpha ; metabolism ; Male ; Rats ; Rats, Wistar ; Receptors, Tumor Necrosis Factor ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the expression of human adrenomedullin (ADM) and its receptor-receptor activity modifying protein 2/calcitonin receptor-like receptor (RAMP2/CRLR) mRNA in the tissues of normal adrenal medulla and pheochromocytoma.

METHODSTotal RNA was extracted from normal adrenal medulla and pheochromocytomas. The expression of ADM and RAMP2/CRLR mRNA were studied by reverse transcription-polymerase chain reaction. The ratios of ADM/GAPDH, RAMP2/ GAPDH, CRLR/GAPDH were used to evaluate the expression levels of ADM, RAMP2 and CRLR mRNA.

RESULTSExpressions of ADM and its receptor- RAMP2/CRLR mRNA were detected in normal adrenal medulla and pheochromocytoma tissues. ADM/GAPDH were 0.48+/-0.09 and 0.75+/-0.24, RAMP2/ GAPDH 0.79+/-0.12 and 1.29+/-0.30, CRLR/GAPDH 0.40+/-0.08 and 0.87+/-0.22 in normal adrenal medulla and pheochromocytomas, respectively (P < 0.05).

CONCLUSIONADM exerts a possible autocrine or paracrine effect in the adrenal. ADM may be involved in the pathogenesis of pheochromocytoma.

Adrenal Gland Neoplasms ; metabolism ; Adrenal Medulla ; metabolism ; Adrenomedullin ; Adult ; Calcitonin Gene-Related Peptide ; biosynthesis ; genetics ; Female ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Membrane Proteins ; biosynthesis ; genetics ; Middle Aged ; Peptides ; genetics ; metabolism ; Pheochromocytoma ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptor Activity-Modifying Protein 2 ; Receptor Activity-Modifying Proteins ; Receptors, Adrenomedullin ; Receptors, Calcitonin ; biosynthesis ; genetics ; Receptors, Peptide ; metabolism

The pain peptide adrenomedullin (AM) plays a pivotal role in pathological pain. The present study was designed to investigate the effect of blockade of AM receptor on bone cancer pain (BCP) and its mechanism. BCP was developed by inoculation of Walker 256 mammary gland carcinoma cells in the tibia medullary cavity of Sprague Dawley rats. The selective AM receptor antagonist AMwas administered intrathecally on 15 d after the inoculation. Quantitative real-time PCR was used to detect mRNA level of CC chemokine ligand 2 (CCL2) in dorsal root ganglion (DRG). Double immunofluorescence staining was used to analyze the localizations of CCL2 and AM in DRG of normal rats. The results showed that, from 6 to15 d after the inoculation, the animals showed significant reduction in the mechanical pain threshold in the ipsilateral hindpaw, companied by the decline in bone density of tibia bone. The expression of CCL2 mRNA in DRG of BCP rats was increased by 3 folds (P < 0.001 vs saline group). Intrathecal administration of AMabolished bone cancer-induced mechanical allodynia and increase of CCL2 mRNA level (P < 0.001). In normal rats, CCL2 was co-localized with AM in DRG neurons. These results suggest that AM may play a role in the pathogenesis of BCP. The increased AM bioactivity up-regulates CCL2 expression in DRG, which may contribute to the induction of pain hypersensitivity in bone cancer.
Adrenomedullin ; administration & dosage ; pharmacology ; Animals ; Bone Neoplasms ; drug therapy ; Chemokine CCL2 ; metabolism ; Ganglia, Spinal ; physiopathology ; Hyperalgesia ; drug therapy ; Pain ; drug therapy ; Pain Threshold ; Peptide Fragments ; administration & dosage ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptors, Adrenomedullin ; antagonists & inhibitors

The effects of adrenomedullin (ADM) on intracellular calcium concentration ([Ca(2+)](i)) were investigated in cultured hippocampal neurons. Changes in [Ca(2+)](i) were detected by laser scanning confocal microscopy using Fluo 3-AM as the calcium fluorescent probe. [Ca(2+)](i) was represented by relative fluorescent intensity. The results showed that: (1) ADM (0.01-1.0 micromol/L) decreased the resting [Ca(2+)](i) in a concentration-dependent manner. (2) Calcitonin gene-related peptide receptor antagonist CGRP(8-37) significantly inhibited the effects of ADM. (3) ADM significantly reduced the increase in [Ca(2+)](i) induced by high K(+). (4) ADM markedly inhibited the inositol 1,4,5-trisphosphate (IP(3))-induced increase in [Ca(2+)](i), while did not influence ryanodine-evoked increase in [Ca(2+)](i). These results suggest that ADM reduces [Ca(2+)](i) in cultured hippocampal neurons through suppressing Ca(2+) release from IP(3)-sensitive stores. Although ADM does not alter resting Ca(2+) influx, it significantly suppresses Ca(2+) influx activated by high K(+). These effects may be partly mediated by CGRP receptors. ADM in the CNS may act as a cytoprotective factor in ischemic/hypoxic conditions.
Adrenomedullin ; Animals ; Animals, Newborn ; Calcitonin Gene-Related Peptide ; metabolism ; Calcium ; metabolism ; Cells, Cultured ; Embryo, Mammalian ; Hippocampus ; cytology ; metabolism ; Inositol 1,4,5-Trisphosphate ; antagonists & inhibitors ; Neurons ; cytology ; metabolism ; Peptides ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Calcitonin Gene-Related Peptide ; antagonists & inhibitors ; metabolism

OBJECTIVEReceptor activity-modifying proteins (RAMPs) determine the ligand specificity of the calcitonin receptor-like receptor (CRLR); co-expression of RAMP1 and CRLR results in a calcitonin gene related peptide (CGRP) receptor, whereas the association of RAMP2 or RAMP3 with CRLR gives an adrenomedullin(ADM) receptor. As CGRP and ADM may play a beneficial role in heart failure, this study aimed at the question whether RAMPs mRNAs are changed in heart failure.

METHODSSemi-quantitative reverse transcription-PCR (RT-PCR) was used to detect and quantify the mRNAs of RAMP1 and RAMP3 in the atria of heart failing patients.

RESULTSIt was found that the expressions of RAMP1, RAMP2 and RAMP3 mRNAs increased with the worsening of heart function, but the expressions of RAMP1 and RAMP2 mRNA decreased at level IV of heart failure.

CONCLUSIONThe above results demonstrated in the atria of heart failure patients an up-regulation of CGRP receptor by an increase of RAMP1 in association with CRLR and an up-regulation of ADM receptor by an increase of RAMP2 expression in association with CRLR, thus suggesting that CGRP and ADM receptors be playing a functional role in compensating the chronic heart failure in human.

Adult ; Calcitonin Receptor-Like Protein ; Female ; Heart Atria ; metabolism ; Heart Failure ; genetics ; physiopathology ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; physiology ; Male ; Membrane Proteins ; genetics ; physiology ; Receptor Activity-Modifying Protein 1 ; Receptor Activity-Modifying Protein 2 ; Receptor Activity-Modifying Protein 3 ; Receptor Activity-Modifying Proteins ; Receptors, Adrenomedullin ; Receptors, Calcitonin ; genetics ; physiology ; Receptors, Calcitonin Gene-Related Peptide ; genetics ; physiology ; Receptors, Peptide ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction