Adrenomedullin Ameliorates Pulmonary Fibrosis by Regulating TGF-ß-Smads Signaling and Myofibroblast Differentiation.

Pulmonary fibrosis is an irreversible, potentially fatal disease. Adrenomedullin (AM) is a multifunctional peptide whose activity is regulated by receptor activity-modifying protein 2 (RAMP2). In the present study, we used the bleomycin (BLM)-induced mouse pulmonary fibrosis model to investigate the pathophysiological significance of the AM-RAMP2 system in the lung. In heterozygous AM knockout mice (AM+/-), hydroxyproline content and Ashcroft scores reflecting the fibrosis severity were significantly higher than in wild-type mice (WT). During the acute phase after BLM administration, FACS analysis showed significant increases in eosinophil, monocyte, and neutrophil infiltration into the lungs of AM+/-. During the chronic phase, fibrosis-related molecules were upregulated in AM+/-. Notably, nearly identical changes were observed in RAMP2+/-. AM administration reduced fibrosis severity. In the lungs of BLM-administered AM+/-, the activation level of Smad3, a receptor-activated Smad, was higher than in WT. In addition, Smad7, an antagonistic Smad, was downregulated and microRNA-21, which targets Smad7, was upregulated compared to WT. Isolated AM+/- lung fibroblasts showed less proliferation and migration capacity than WT fibroblasts. Stimulation with TGF-ß increased the numbers of α-SMA-positive myofibroblasts, which were more prominent among AM+/- cells. TGF-ß-stimulated AM+/- myofibroblasts were larger and exhibited greater contractility and extracellular matrix production than WT cells. These cells were α-SMA (+), F-actin (+), and Ki-67(-) and appeared to be nonproliferating myofibroblasts (non-p-MyoFbs), which contribute to the severity of fibrosis. Our findings suggest that in addition to suppressing inflammation, the AM-RAMP2 system ameliorates pulmonary fibrosis by suppressing TGF-ß-Smad3 signaling, microRNA-21 activity and differentiation into non-p-MyoFbs.

Anorexigenic effects of central adrenomedullin are associated with hypothalamic changes in juvenile Gallus gallus.

Adrenomedullin (AM), a 52 residue neuropeptide, is associated with anorexia in mammals and has a poorly understood central mechanism of action. Thus, this study focused on elucidating AM's central mechanism of action in an alternative vertebrate model, the chick (Gallus gallus). In Experiment 1, chicks centrally injected with AM dose-dependently reduced food but not water intake. In Experiment 2, those chicks that received central AM had increased c-Fos immunoreactivity in the magnocellular division of the paraventricular nucleus (PaMC), ventromedial hypothalamus (VMH) and doromedial hypothalamus (DM). The lateral hypothalamic area, parvocellular division of the paraventricular hypothalamus and the arcuate nucleus were not affected. In Experiment 3, antagonism of corticotrophin releasing factor (CRF) receptors did not affect AM-associated anorexia. In Experiment 4, a comprehensive behavior analysis was conducted and AM-treated chicks pecked less, moved more, jumped more and spent more time in deep rest. In conclusion, exogenous AM induced anorexia is associated with activation of the PaMC, VMH and DM of the hypothalamus, is not CRF dependent, and affects behaviors unrelated to food intake in chicks.

Involvement of PKA-dependent upregulation of nNOS-CGRP in adrenomedullin-initiated mechanistic pathway underlying CFA-induced response in rats.

We have previously shown that intrathecal administration of the adrenomedullin (AM) receptor antagonist AM(22-52) produces a long-lasting anti-hyperalgesia effect. This study examined the hypothesis that AM recruits other pronociceptive mediators in complete Freund's adjuvant (CFA)-induced inflammation. Injection of CFA in the hindpaw of rat produced an increase in the expression of nNOS in dorsal root ganglion (DRG) and the spinal dorsal horn. An intrathecal administration of AM(22-52), but not the CGRP antagonist BIBN4096BS, abolished the CFA-induced increase of nNOS. Moreover, AM-induced increase of CGRP was inhibited by the nNOS inhibitors L-NAME and 7-nitroindazole in cultured ganglion explants. Addition of AM to ganglion cultures induced an increase in nNOS protein, which was attenuated by the PKA inhibitor H-89. Treatment with AM also concentration-dependently increased cAMP content and pPKA protein level, but not its non-phosphorylated form, in cultured ganglia. In addition, nNOS was shown to be co-localized with the AM receptor components calcitonin receptor-like receptor and receptor activity-modifying protein 2- and 3 in DRG neurons. The present study suggests that the enhanced activity of nitric oxide (NO) mediates the biological action of AM at the spinal level and that AM recruits NO-CGRP via cAMP/PKA signaling in a mechanistic pathway underlying CFA-induced hyperalgesia.

Haemodynamic, endocrine and renal actions of adrenomedullin 5 in an ovine model of heart failure.

AM5 (adrenomedullin 5), a newly described member of the CGRP (calcitonin gene-related peptide) family, is reported to play a role in normal cardiovascular physiology. The effects of AM5 in HF (heart failure), however, have not been investigated. In the present study, we intravenously infused two incremental doses of AM5 (10 and 100 ng/min per kg of body weight each for 90 min) into eight sheep with pacing-induced HF. Compared with time-matched vehicle control infusions, AM5 produced progressive and dose-dependent increases in left ventricular dP/dt(max) [LD (low dose), +56 mmHg/s and HD (high dose), +152 mmHg/s] and cardiac output (+0.83 l/min and +1.81 l/min), together with decrements in calculated total peripheral resistance (-9.4 mmHg/min per litre and -14.7 mmHg/min per litre), mean arterial pressure (-2.8 mmHg and -8.4 mmHg) and LAP (left atrial pressure; -2.6 mmHg and -5.6 mmHg) (all P<0.001). HD AM5 significantly raised PRA (plasma renin activity) (3.5-fold increment, P<0.001), whereas plasma aldosterone levels were unchanged over the intra-infusion period and actually fell in the post-infusion period (70% decrement, P<0.01), resulting in a marked decrease in the aldosterone/PRA ratio (P<0.01). Despite falls in LAP, plasma atrial natriuretic peptide and B-type natriuretic peptide concentrations were maintained relative to controls. AM5 infusion also induced significant increases in urine volume (HD 2-fold increment, P<0.05) and urine sodium (2.7-fold increment, P<0.01), potassium (1.7-fold increment, P<0.05) and creatinine (1.4-fold increment, P<0.05) excretion and creatinine clearance (60% increment, P<0.05). In conclusion, AM5 has significant haemodynamic, endocrine and renal actions in experimental HF likely to be protective and compensatory in this setting. These results suggest that AM5 may have potential as a therapeutic agent in human HF.

[Effect of exogenous adrenomedullin on renal and hypothalamus adrenomedullin expression in rats early after mechanical renal trauma].

OBJECTIVE: To observe the effects of exogenous adrenomedullin (ADM) on endogenous expression of ADM in the kidney and hypothalamus of rats early after mechanical renal trauma. METHODS: Adult Wistar rats were randomized into 4 groups (n=32), namely the control group, renal impact trauma group, preventive ADM injection group, and therapeutic ADM injection group. In the latter two groups, ADM (0.1 nmol/kg) was administrated by intraperitoneal injection 10 min before and 10 min after renal trauma. The rats were executed at 1, 6, 12, and 24 h after the trauma to examine the expression of ADM in the kidney and hypothalamus. RESULTS: In preventive ADM injection group, the renal expression of ADM increased significantly at 1 h after the trauma (P<0.05) and tended to further increase with time till 24 h when its expression recovered the normal level. In the therapeutic ADM injection group, strong renal ADM positivity was found at 1 and 6 h after the injury (P<0.05) followed by gradual decrease till recovering the normal level at 24 h. Low renal ADM expression was detected, which was the strongest at 1 and 12 h (P<0.05) and became normal at 24 h. The time course of ADM expression in the hypothalamus was similar to that in the kidney in the therapeutic ADM injection group, and in the preventive injection group, the strongest ADM expression in the hypothalamus occurred at 6 and 24 h, and the lowest expression occurred at 12 h (P<0.05). The trauma group showed significantly decreased ADM expression in the hypothalamus compared with the control group (P<0.05). CONCLUSION: The hypothalamic ADM expression can upregulate renal ADM expression. ADM maintains the relative stability of the internal environment and physiological activity by local and systemic positive and negative feedback mechanisms.

Adrenomedullin improves cardiac expression of heat-shock protein 72 and tolerance against ischemia/reperfusion injury in insulin-resistant rats.

We recently reported that long-term treatment with pioglitazone restored cardiac Akt phosphorylation in response to hyperthermia (HT) and subsequent cardiac heat-shock protein 72 (HSP72) expression, in heredity insulin resistance rats via improvement of insulin sensitivity. Because adrenomedullin (AM) promotes Akt phosphorylation and attenuates myocardial ischemia/reperfusion injury, we tested the hypothesis that pretreatment with AM before HT could restore depressed Akt activation and cardiac HSP72 expression, thereby enhancing protection against ischemia/reperfusion injury in this model. At 16 wk of age, male insulin-resistant Otsuka Long-Evans Tokushima Fatty (OLETF) rats and control Long-Evans Tokushima Otsuka (LETO) rats were treated with AM (0.05 microg/kg . /min iv) or vehicle for 60 min. Thereafter, HT (43 C for 20 min) or normothermia (NT; 37 C for 20 min) was applied. The heart was isolated 1 and 24 h after HT. 1) Either AM or HT induced myocardial Akt phosphorylation in a phosphatidylinositol 3-kinase-dependent manner, which was augmented by their combination. 2) Akt phosphorylation induced by HT, or a combination of HT and AM, was attenuated in insulin-resistant OLETF rat hearts. 3) The levels of Akt phosphorylation in response to AM and/or HT correlated with reperfusion-induced left ventricular functional recovery and amount of released creatine kinase during reperfusion. 4) AM protected the hearts of OLETF rats and LETO rats. Our results suggest that AM pretreatment could enhance HT-induced myocardial Akt phosphorylation and subsequent HSP72 expression in a phosphatidylinositol 3-kinase-dependent manner, in association with tolerance against ischemia/reperfusion injury. This intervention was effective even in insulin-resistant hearts.

Adrenomedullin stimulates nitric oxide production from primary rat hypothalamic neurons: roles of calcium and phosphatases.

Adrenomedullin (ADM) in the brain plays important roles in the maintenance of homeostasis. Although in vivo evidence has suggested that nitric oxide (NO) mediates ADM's effects in the brain, mechanisms for ADM stimulation of NO production in neurons have not been identified. In the present study, primary hypothalamic neurons were used to characterize ADM-induced NO production and to study the underlying mechanisms. Using Calcium Orange/4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence live cell imaging, we found that ADM (1 or 10 nM, 5 min) significantly elevated [Ca(2+)](i) and NO production in a concentration-dependent manner. Ca(2+) and NO responses induced by 10 nM ADM were abolished by pretreatment with 50 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), an intracellular Ca(2+) chelator, or protein kinase A (PKA) inhibitors 5 microM N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and 50 microM Rp-cAMP. Furthermore, the ADM-induced NO production was significantly attenuated by a protein phosphatase 1/2A inhibitor, okadaic acid (OA; 0.1 microM), or calcineurin inhibitors, tacrolimus (FK506) (1 microM) and cyclosporin A (CsA; 0.1 microM). Using Western blotting, we found that ADM significantly decreased phosphorylation of neuronal nitric-oxide synthase (nNOS) at serine 847. This dephosphorylation was inhibited by 0.1 microM OA, 1 microM FK506, 0.1 microM CsA, or 5 microM H-89, and attenuated by 50 microM BAPTA-AM. These results suggest that, in hypothalamic neurons, ADM elevates [Ca(2+)](i) via PKA-associated mechanisms. The PKA/Ca(2+) cascade leads to protein phosphatase (PP) 1/PP2A- and calcineurin-mediated dephosphorylation of nNOS. We hypothesize that the Ca(2+) increase and nNOS dephosphorylation contribute to activation of nNOS and production of NO in hypothalamic neurons.

Adrenomedullin 2 (AM2)/intermedin is a more potent activator of hypothalamic oxytocin-secreting neurons than AM possibly through an unidentified receptor in rats.

Central administration of either adrenomedullin 2 (AM2) or adrenomedullin (AM) activates hypothalamic oxytocin (OXT)-secreting neurons in rats. We compared AM2 with AM, given intracerebroventricularly (icv), across multiple measures: (1) plasma OXT levels in conscious rats; (2) blood pressure, heart rate and circulating catecholamine levels in urethane-anesthetized rats; and (3) the expression of the c-fos gene in the supraoptic (SON) and the paraventricular nuclei (PVN). We also tested the effects of the AM receptor antagonist, AM(22-52) and calcitonin gene-related peptide (CGRP) antagonist, CGRP(8-37) on these measures. Plasma OXT levels at 10 min after icv injection of AM (1 nmol/rat) were increased (compared with vehicle), but OXT levels after AM2 (1 nmol/rat) were nearly double the levels seen after AM injection. OXT levels remained elevated at 30 min. Pretreatment with AM(22-52) (27 nmol/rat) and CGRP(8-37) (3 nmol/rat), nearly abolished the increase in plasma OXT level after AM injection, but partially blocked OXT level changes due to AM2. Increases in blood pressure, heart rate and circulating catecholamines were all greater in response to central AM2 than to AM at the same dose. In situ hybridization histochemistry showed that both AM2 and AM induced expression of the c-fos gene in the SON and the PVN, but AM(22-52)+CGRP(8-37) could only nearly abolish the effects of centrally administered AM. These results suggest that the more potent central effects of AM2 and only partial blockade by AM/CGRP receptor antagonists may result from its action on an additional, as yet unidentified, specific receptor in the central nervous system.

Adrenomedullin fails to reduce cadmium-induced oxidative damage in rat liver.

Recent studies have demonstrated that chronic cadmium administration induces oxidative stress. In the present study, we investigated the possible therapeutic effect of adrenomedullin, a potent antioxidant, in cadmium-induced morphological, ultrastructural and biochemical alterations. Two groups of rats were exposed to 100 ppm of CdCl(2) in drinking water for four weeks. One of these groups received 3000 ng/kg body weight of adrenomedullin (AdM) intraperitoneally during the last week. Hepatic oxidative stress markers were evaluated by changes in the amount of lipid peroxides and changes in the antioxidant enzyme activities, superoxide dismutase (SOD) and glutathione peroxidase (GPx) and glutathione reductase (GSH) levels. Hepatic damage score was significantly higher in Cd-administered rats than those of controls (p<0.005). Cd-induced ultrastructural changes in hepatocytes included focal parenchymal cell necrosis, dilatation of rough endoplasmic reticulum, proliferation of lysosomes and mitochondrial degeneration. Hepatic damage was accompanied by significant increase in tissue MDA level (p<0.05) and significant decrease in tissue GSH level (p<0.05), and SOD and GPx activities (p>0.05, p>0.005, respectively). Adrenomedullin failed to restore the light and electron microscopic, and biochemical changes. We conclude that although we administered a high dose of adrenomedullin, it failed to reduce cadmium-induced hepatic damage probably because of the irreversibility of Cd-induced hepatic injury.

Intermedin1-53 protects the heart against isoproterenol-induced ischemic injury in rats.

Intermedin is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family peptide, which has vasodilatory and hypotensive actions identical to those of adrenomedullin and CGRP. Cleavage sites located between 2 basic amino acids at Arg93-Arg94 result in the production of prepro-intermedin95-147, namely intermedin1-53. The bioactive action of intermedin1-53 and its physiological significance are unclear. In this work, we aimed to explore the effects of intermedin1-53 on acute myocardial injury induced by isoproterenol. Myocardial ischemia injury in rats was induced by subcutaneous injection of a high dose of isoproterenol, and the therapeutic effect of intermedin1-53 was observed. Plasma lactate dehydrogenase activity, myocardial and plasma malondialdehyde content were higher in the isoproterenol group than that in controls. Isoproterenol-treated rats showed lower maximal rate of increase and decrease of left-ventricle pressure development (+/-left-ventricle dp/dtmax) and higher left-ventricle end-diastolic pressure (all P<0.01), which suggested severe heart failure and myocardial injury. Semi-quantitative RT-PCR analysis showed that the gene expression of calcitonin receptor-like receptor and receptor-activity-modifying protein (RAMP)1, RAMP2 and RAMP3 in ventricular myocardia were up-regulated by 79% (P<0.01), 48% (P<0.01), 31% (P<0.05) and 130% (P<0.01), respectively, compared with controls. In myocardial sarcolemmal membranes, the maximum binding capacity for [125I]-intermedin1-53 was increased by 118% (P<0.01) in the isoproterenol group compared with controls. Rats treated with low dosage intermedin1-53 (5 nmol/kg/day, 2 days) showed 21% (P<0.05) higher myocardial cAMP content, 18% and 31% higher+left-ventricle dp/dtmax and -left-ventricle dp/dtmax respectively, 288% lower left-ventricle end-diastolic pressure (all P<0.01), and attenuated myocardial lactate dehydrogenase leakage and malondialdehyde formation (all P<0.01). Treatment with high dosage intermedin1-53 (20 nmol/kg/day, 2 days) gave better results than that with low dosage intermedin1-53. These results suggest that the intermedin receptor system was up-regulated in isoproterenol-induced myocardial ischemic injury and intermedin1-53 might play a pivotal cardioprotective role in such injury.

[Effect of intracerebroventricular injection of adrenomedullin on catecholaminergic neurons and expression of c-fos in the rat brain nuclei involved in cardiovascular regulation].

AIM AND METHODS: Using double immunohistochemical method for Fos and tyrosine hydroxylase(TH) to examine the effects of intracerebroventricular (icv) administration of adrenomedullin (AM) on catecholaminergic neurons and the expression of c-fos gene in rat brain nuclei involved in cardiovascular regulation in order to define whether the effects of central administration of adrenomedullin (AM) were induced by activating the catecholaminergic neurons. RESULTS: (1) Following icy administration of AM (3 nmol/kg), Fos-like immunoreactivity neurons were markedly increased in several brain areas of the rat, including the brainstem, the hypothalamus and the forebrain. (2) Following icy administration of AM (3 nmol/kg), double-labeled neurons for Fos and TH increased significantly in the area postrema (AP), the nucleus of the solitary tract (NTS), the nucleus paragigantocellularis lateralis (PGL) and the locus coeruleus (LC). (3) Pretreatment with calcitonin gene-related peptide receptor antagonism CGRP (8-37) (30 nmol/kg) significantly reduced the action of AM (3 nmol/kg) in the brain. CONCLUSION: AM activates the nuclei involved in cardiovascular regulation in the forebrain, the hypothalamus and the brainstem, and that the central actions of AM are induced by activating the catecholaminergic neurons of brainstem nuclei involved in cardiovascular regulation. CGRP receptor can mediate the effects of AM in brain.