Calcitonin/calcitonin Gene-related Peptide Research Articles

Adrenomedullin Triggers Migraine Attacks

Adrenomedullin is a peptide from the family of calcitonin/calcitonin gene-related peptide (CGRP) that may be involved in the regulation of the

Experimental study on renoprotective effect of intermedin on diabetic nephropathy

Intermedin(IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family that has anti-inflammatory, antioxidant and anti-apoptosis properties. This study aimed to evaluate the renoprotective effects of IMD on podocyte apoptotic loss and slit diaphragm protein deficiency the kidneys of rats with in streptozotocin (STZ) induced diabetes in high glucose-exposed podocytes. Our results showed that IMD significantly attenuated proteinuria, and alleviated the abnormal alterations in glomerular ultrastructure in vivo. IMD also improved the induction of slit diaphragm proteins, and restored the decreased Bcl-2 expression and suppressed Bax and caspase-3 induction in the diabetic glomeruli. In addition, IMD attenuated podocyte apoptosis and filamentous actin (F-actin) rearrangement in high glucose-exposed podocytes. Exposure to high glucose elevated the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress in renal podocytes, and IMD treatment blocked such ER stress responses pertinent to podocyte apoptosis and reduced synthesis of slit diaphragm proteins in vivo and in vitro. These observations demonstrate that targeting ER stress is an underlying mechanism of IMD-mediated amelioration of diabetes-associated podocyte injury and dysfunction.

Cardiac sympathetic afferent reflex response to intermedin microinjection into paraventricular nucleus is mediated by nitric oxide and γ-amino butyric acid in hypertensive rats.

Intermedin (IMD) is a member of calcitonin/calcitonin gene-related peptide (CGRP) and involves in the regulation of cardiovascular function in both peripheral tissues and central nervous system (CNS). Paraventricular nucleus (PVN) of hypothalamus is an important site in the control of cardiac sympathetic afferent reflex (CSAR) which participates in sympathetic over-excitation of hypertension. The aim of this study is to investigate whether IMD in the PVN is involved in the inhibition of CSAR and its related mechanism in hypertension. Rats were subjected to two-kidney one-clip (2K1C) surgery to induce renovascular hypertension or sham-operation (Sham). Acute experiments were carried out four weeks later under anesthesia. The CSAR was evaluated with the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to the epicardial application of capsaicin. The RSNA and MAP were recorded in sinoaortic-denervated, cervical-vagotomized and anesthetized rats. Bilateral PVN microinjection of IMD (25 pmol) caused greater decrease in the CSAR in 2K1C rats than in Sham rats, which was prevented by pretreatment with adrenomedullin (AM) receptor antagonist AM22-52, non-selective nitric oxide (NO) synthase (NOS) inhibitor L-NAME or γ-amino butyric acid (GABA)B receptor blocker CGP-35348. PVN pretreatment with CGRP receptor antagonist CGRP8-37 or GABA(A) receptor blocker gabazine had no significant effect on the CSAR response to IMD. AM22-52, L-NAME and CGP-35348 in the PVN could increase CSAR in Sham and 2K1C rats. These data indicate that IMD in the PVN inhibits CSAR via AM receptor, and both NO and GABA in the PVN involve in the effect of IMD on CSAR in Sham and renovascular hypertensive rats.

Intermedin in the Paraventricular Nucleus Attenuates Cardiac Sympathetic Afferent Reflex in Chronic Heart Failure Rats

Background and AimIntermedin (IMD) is a member of calcitonin/calcitonin gene-related peptide (CGRP) family together with adrenomedullin (AM) and amylin. It has a wide distribution in the central nervous system (CNS) especially in hypothalamic paraventricular nucleus (PVN). Cardiac sympathetic afferent reflex (CSAR) is enhanced in chronic heart failure (CHF) rats. The aim of this study is to determine the effect of IMD in the PVN on CSAR and its related mechanisms in CHF rats.Methodology/Principal FindingsRats were subjected to left descending coronary artery ligation to induce CHF or sham-operation (Sham). Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were recorded. CSAR was evaluated by the RSNA and MAP responses to epicardial application of capsaicin. Acute experiments were carried out 8 weeks after coronary ligation or sham surgery under anesthesia. IMD and angiotensin II (Ang II) levels in the PVN were up-regulated in CHF rats. Bilateral PVN microinjection of IMD caused greater decreases in CSAR and the baseline RSNA and MAP in CHF rats than those in Sham rats. The decrease of CSAR caused by IMD was prevented by pretreatment with AM receptor antagonist AM22-52, but not CGRP receptor antagonist CGRP8-37. Ang II in the PVN significantly enhanced CSAR and superoxide anions level, which was inhibited by PVN pretreatment with IMD or tempol (a superoxide anions scavenger) in Sham and CHF rats.ConclusionIMD in the PVN inhibits CSAR via AM receptor, and attenuates the effects of Ang II on CSAR and superoxide anions level in CHF rats. PVN superoxide anions involve in the effect of IMD on attenuating Ang II-induced CSAR response.

Correlation of Adrenomedullin gene expression in peripheral blood leukocytes with severity of ischemic stroke

Human adrenomedullin (ADM), a 52-amino acid peptide, belongs to the calcitonin/calcitonin gene-related peptide (CGRP)/amylin peptide family. ADM acts as a multifunctional regulatory peptide and is upregulated in response to hypoxia. Previous microarray studies have found increased ADM gene (ADM) expression in peripheral blood cells of patients with stroke, however, it is unknown if an increased ADM level is correlated with severity of human ischemic stroke. This study investigated ADM expression in peripheral blood leukocytes (PBL) of healthy controls and subjects at day 1, week 1 and week 3 postacute ischemic stroke using rtPCR methodology. We found that ADM expression was significantly upregulated on the first day of stroke compared to the healthy subjects and the disease controls; the levels remained elevated for up to week 3. Further, ADM expression at day 1 was correlated with stroke severity measured by the National Institute of Healthy Stroke Scale (NIHSS), the modified Barthel Index (mBI) and the modified Rankin Scale (mRS). This could indicate that ADM expression level is related to the severity of tissue damage. We suggest that increased ADM expression in PBL after acute ischemic stroke is most likely to indicate that these cells have been subjected to hypoxia and that the magnitude of expression is likely to be related to the volume of hypoxic tissue. Hypoxia can affect lymphocytes function and could affect the immune response to stroke. The correlation of ADM expression level with the measures of stroke severity implicates ADM –a potential blood bio-marker in studies of ischemic stroke.

Effects of intermedin1-53 on myocardial fibrosis

Intermedin (IMD) is a member of the calcitonin/calcitonin gene-related peptide (CGRP) family and has similar or more potent cardiovascular actions than adrenomedullin (ADM) and any other CGRP. The aim of the present work is to study the effects of IMD1-53 on cardiac fibroblast fibrosis in vivo and in vitro. Myocardial infarction model was prepared by ligating rats' left anterior descending coronary artery. Mesenchymal collagen contents in the left ventricle were accessed by Sirius-red stain. Heart functions were explored by hemodynamic changes. Expression of I and III type collagens, IMD1-53, receptor activity-modifying proteins (RAMP)1/2/3, and calcitonin receptor-like receptor (CRLR) in left ventricle were detected by western blot analysis. Cardiac fibroblasts (CFbs) fibrosis was induced by treating the cells with aldosterone (ALD). CFbs proliferation and the hydroxyproline contents in supernatants were determined by 3-[4,5-dimehyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide assay and enzyme-linked immunosorbent assay. Heart function was decreased in myocardial infarction model rats. Expression of type I and type III collagens in infarcted zone in myocardial rats was higher than those in the sham-operated group. IMD1-53, RAMP, and CRLR in left ventricle were also up-regulated. In vitro experiment showed that ALD was a powerful stimulator of CFbs activation. IMD1-53 decreased ALD-induced CFbs proliferation in a dose-dependent manner. Moreover, CGRP8-37 and ADM22-52 remarkably blocked the effect of IMD1-53 on ALD-induced myocardial cell fibrosis. IMD could be involved in the onset of cardiac fibrosis. Like ADM, IMD1-53 exerts an antifibrotic effect on CFbs, which might be mediated by CRLR/RAMP complex and ADM receptor.

Structural basis for the dual RNA-recognition modes of human Tra2-β RRM

Human Transformer2-β (hTra2-β) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-β specifically binds to two types of RNA sequences [the CAA and (GAA)2 sequences]. We determined the solution structure of the hTra2-β RRM (spanning residues Asn110–Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the β-sheet surface. We then solved the complex structure of the hTra2-β RRM with the (GAA)2 sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the β-sheet surface. Further NMR experiments revealed that the hTra2-β RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-β RRM recognizes two types of RNA sequences in different RNA binding modes.

Adrenomedullin 2/Intermedin in the Hypothalamo–Pituitary–Adrenal Axis

Adrenomedullin 2/intermedin (AM2/IMD) is a new member of the calcitonin/calcitonin gene-related peptide (CGRP) family. CGRP, adrenomedullin (AM), and AM2/IMD share the receptor system consisting of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP). The CRLR/RAMP2 or CRLR/RAMP3 complex forms the AM receptor, whereas the CRLR/RAMP1 forms the CGRP receptor. AM2/IMD binds non-selectively to all three CRLR/RAMP complexes. AM2/IMD has various actions, such as a potent vasodilator action and a protective action against oxidative stress, like AM and CGRP. When administered intracerebroventricularly, AM2/IMD stimulates the sympathetic nervous system and increases blood pressure. In human hypothalamus, AM2/IMD is expressed in the paraventricular and supraoptic nuclei and colocalized with arginine vasopressin. Anterior pituitary cells were diffusely immunostained for AM2/IMD. AM2/IMD stimulates the release of ACTH, prolactin, and oxytocin, but suppresses GH release. Some of these pituitary actions of AM2/IMD have been supposed to be mediated by an unidentified unique receptor for AM2/IMD. In the adrenal gland, immunoreactive (IR)-AM2/IMD and IR-AM were detected in the medulla, while the degree of IR-AM2/IMD and IR-AM in the cortex was relatively weak or undetectable. Furthermore, AM2/IMD and AM were expressed in adrenocortical tumors, such as aldosterone-secreting adenomas, and pheochromocytomas. CRLR and RAMPs are expressed in the hypothalamus, pituitaries, adrenal glands, and adrenal tumors. Thus, AM2/IMD is expressed in every endocrine organ of the hypothalamo-pituitary-adrenal axis together with its receptor. AM2/IMD may act as a neurotransmitter or modulator in the brain and as a paracrine/autocrine regulator in the hypothalamo-pituitary-adrenal axis.

Upregulated expression of intermedin and its receptor in the myocardium and aorta in spontaneously hypertensive rats

Intermedin (IMD), also called adrenomedullin 2 (ADM2), is a 47-amino acid peptide belonging to the calcitonin/calcitonin gene-related peptide (CGRP) family. IMD has similar or more potent vasodilatory and hypotensive actions compared with adrenomedullin (ADM) and CGRP. This study was designed to explore the role of IMD and its receptor in the pathogenesis of spontaneous hypertension and cardiac hypertrophy. Radioimmunoassay was employed to determine plasma immunoreactive IMD concentration and tissue immunoreactive IMD levels in the myocardium and aorta as well as cAMP concentration in the cardiovascular tissues in 13-week-old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). The mRNA expression of IMD, its receptor, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP)) were determined by semi-quantitative RT-PCR. Protein levels of CRLR and RAMPs were assayed by Western blotting. Our results showed that immunoreactive IMD concentration was enhanced in the SHR myocardium, aortas and plasma. Both the mRNA and protein levels of IMD, as well as those of CRLR and RAMP 1–3 were upregulated in SHRs. IMD affected cAMP generation in the myocardium and aorta, which were not attenuated by prior addition of either CGRP 8–37 or ADM 22–52 alone. These results indicate that the elevation of IMD and its receptor in the cardiovascular tissue may play an important role in the pathogenesis of spontaneous hypertension.

Genomic and Expression Analysis of Canine Calcitonin Receptor-stimulating Peptides and Calcitonin/Calcitonin Gene-related Peptide*

Calcitonin receptor-stimulating peptides (CRSPs) are new members of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family identified in pigs, dogs and other domestic animals, and CRSP-1 is an active ligand for the CT receptor (CT-R). We recently sequenced porcine CRSP genes (Crsps) and found similarity with the CT/CGRP gene (Ct/Cgrp) in sequence and genomic organization. In this study, we identified five Crsps, Crsp-1 to Crsp-5, in dogs. Crsp-1 has five exons with an exon-intron organization identical to that of porcine Crsp-1 or Crsp-2, while Crsp-2 and Crsp-3 have additional CT-2- and CT-3-coding exons like Ct/Cgrp. Crsp-2 was renamed as Ct-2/Crsp-2 because both CRSP-2 and CT-2 mRNAs were tissue-specifically expressed. Crsp-4 and Crsp-5 are presumably generated by retrotransposition. We postulate that Crsps were generated from the gene duplication of Ct/Cgrp, and gained their diversity during mammalian evolution. Among the canine CTs and CRSPs, CRSP-1, CT-1 and CT-2 are active ligands for the CT-R, but CRSP-2 and others are inactive. Canine CRSP-1 and CT-2 are expressed in the central and peripheral systems, while CT-1 is localized in the thyroid gland. These findings indicate that dogs can be used for an experimental model as analysing the physiological roles of the CT/CGRP/CRSP family.

Genomic organization, expression and evolution of porcine CRSP1, 2, and3

Recently we identified and characterized porcine calcitonin receptor-stimulating peptide (CRSP) 1, CRSP2 and CRSP3 as members of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family. In the present study, the genomic sequences and organization of CRSP1, 2, and 3 were determined, and the expression of the genes in the porcine brain was investigated using in situ hybridization. Analysis of 5′-upstream regions of the three CRSPs demonstrated that CRSP1 and CRSP2 have almost identical sequences (>98% similarity) and high sequence similarities including functional transcription binding sites with the corresponding region of human CALCA (CT/αCGRP), whereas CRSP3 retains less similarity with the above genes. RH mapping of CRSPs demonstrated that they resided in a region of swine chromosome 2 (SSC2). The arrangement of the genes in the region was found to be conserved in corresponding human and mouse regions. In situ hybridization demonstrated sense transcripts of the three genes in cerebrum, hippocampus, hypothalamus, pons/midbrain, and thalamus of 3-month-old pigs, and CRSP2 sense transcripts additionally in tractus opticus. The sense transcripts of αCGRP and CALCB (βCGRP) were detected in cerebrum, hippocampus, and pons/midbrain of newborn mice, and to a lesser extent in pons/midbrain of 8-week-old mice. These results taken together with the chromosomal conservation and phylogenetic clustering of CT/CGRP family indicate that CRSP1, 2, and 3 may be functionally different from αCGRP and βCGRP, though they are indicated to have a common progenitor gene.

Expression of adrenomedullin2/intermedin in human brain, heart, and kidney

Adrenomedullin2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family. In the present study, we developed a specific radioimmunoassay of human AM2/IMD. Expression of AM2/IMD was studied in the human brain, pituitary, heart and kidney obtained at autopsy by radioimmunoassay and immunocytochemistry. Immunoreactive-AM2/IMD was detected by radioimmunoassay in human brains (range; 0.163–1.495 pmol/g wet weight), pituitaries (4.46 ± 0.689 pmol/g wet weight, mean ± S.E.M, n = 3), left ventricles of hearts (0.251 ± 0.0321 pmol/g wet weight, n = 4), kidneys (3.49 ± 1.18 pmol/g wet weight, n = 5), and plasma obtained at healthy subjects (24.7 ± 1.78 pmol/l, n = 3). Reverse-phase high performance liquid chromatography showed that immunoreactive-AM2/IMD in human brain, kidney and plasma extracts were eluted in the position of authentic AM2/IMD. Additional peaks eluted earlier were found in the brain tissue and plasma. Immunocytochemistry showed that immunoreactive-AM2/IMD was localized in paraventricular and supraoptic nuclei of hypothalamus, anterior and posterior lobes of pituitary, cardiomyocytes, pericardial adipocytes, vascular endothelial cells of pericardial veins, and vascular smooth muscle cells of coronary arteries and renal arterioles as well as in renal tubular cells. The present study has shown expression of AM2/IMD in various types of cells in the central nervous system and the cardiovascular system, and suggested possible (patho)physiological roles of AM2/IMD in these systems.

Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing.

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.

Hu Proteins Regulate Polyadenylation by Blocking Sites Containing U-rich Sequences

A recent genome-wide bioinformatic analysis indicated that 54% of human genes undergo alternative polyadenylation. Although it is clear that differential selection of poly(A) sites can alter gene expression, resulting in significant biological consequences, the mechanisms that regulate polyadenylation are poorly understood. Here we report that the neuron-specific members of a family of RNA-binding proteins, Hu proteins, known to regulate mRNA stability and translation in the cytoplasm, play an important role in polyadenylation regulation. Hu proteins are homologs of the Drosophila embryonic lethal abnormal visual protein and contain three RNA recognition motifs. Using an in vitro polyadenylation assay with HeLa cell nuclear extract and recombinant Hu proteins, we have shown that Hu proteins selectively block both cleavage and poly(A) addition at sites containing U-rich sequences. Hu proteins have no effect on poly(A) sites that do not contain U-rich sequences or sites in which the U-rich sequences are mutated. All three RNA recognition motifs of Hu proteins are required for this activity. Overexpression of HuR in HeLa cells also blocks polyadenylation at a poly(A) signal that contains U-rich sequences. Hu proteins block the interaction between the polyadenylation cleavage stimulation factor 64-kDa subunit and RNA most likely through direct interaction with poly(A) cleavage stimulation factor 64-kDa subunit and cleavage and polyadenylation specificity factor 160-kDa subunit. These studies identify a novel group of mammalian polyadenylation regulators. Furthermore, they define a previously unknown nuclear function of Hu proteins.

Evolution of secretin family GPCR members in the metazoa

BackgroundComparative approaches using protostome and deuterostome data have greatly contributed to understanding gene function and organismal complexity. The family 2 G-protein coupled receptors (GPCRs) are one of the largest and best studied hormone and neuropeptide receptor families. They are suggested to have arisen from a single ancestral gene via duplication events. Despite the recent identification of receptor members in protostome and early deuterostome genomes, relatively little is known about their function or origin during metazoan divergence. In this study a comprehensive description of family 2 GPCR evolution is given based on in silico and expression analyses of the invertebrate receptor genes.ResultsFamily 2 GPCR members were identified in the invertebrate genomes of the nematodes C. elegans and C. briggsae, the arthropods D. melanogaster and A. gambiae (mosquito) and in the tunicate C. intestinalis. This suggests that they are of ancient origin and have evolved through gene/genome duplication events. Sequence comparisons and phylogenetic analyses have demonstrated that the immediate gene environment, with regard to gene content, is conserved between the protostome and deuterostome receptor genomic regions. Also that the protostome genes are more like the deuterostome Corticotrophin Releasing Factor (CRF) and Calcitonin/Calcitonin Gene-Related Peptide (CAL/CGRP) receptors members than the other family 2 GPCR members. The evolution of family 2 GPCRs in deuterostomes is characterised by acquisition of new family members, with SCT (Secretin) receptors only present in tetrapods. Gene structure is characterised by an increase in intron number with organismal complexity with the exception of the vertebrate CAL/CGRP receptors.ConclusionThe family 2 GPCR members provide a good example of gene duplication events occurring in tandem with increasing organismal complexity during metazoan evolution. The putative ancestral receptors are proposed to be more like the deuterostome CAL/CGRP and CRF receptors and this may be associated with their fundamental role in calcium regulation and the stress response, both of which are essential for survival.

A nuclear function of Hu proteins as neuron-specific alternative RNA processing regulators.

Recent advances in genome-wide analysis of alternative splicing indicate that extensive alternative RNA processing is associated with many proteins that play important roles in the nervous system. Although differential splicing and polyadenylation make significant contributions to the complexity of the nervous system, our understanding of the regulatory mechanisms underlying the neuron-specific pathways is very limited. Mammalian neuron-specific embryonic lethal abnormal visual-like Hu proteins (HuB, HuC, and HuD) are a family of RNA-binding proteins implicated in neuronal differentiation and maintenance. It has been established that Hu proteins increase expression of proteins associated with neuronal function by up-regulating mRNA stability and/or translation in the cytoplasm. We report here a novel function of these proteins as RNA processing regulators in the nucleus. We further elucidate the underlying mechanism of this regulation. We show that in neuron-like cells, Hu proteins block the activity of TIA-1/TIAR, two previously identified, ubiquitously expressed proteins that promote the nonneuronal pathway of calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA processing. These studies define not only the first neuron-specific regulator of the calcitonin/CGRP system but also the first nuclear function of Hu proteins.

Intermedin 1–53 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-intermedin 95–147, namely intermedin 1–53. The bioactive action of intermedin 1–53 and its physiological significance are unclear. In this work, we aimed to explore the effects of intermedin 1–53 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 intermedin 1–53 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 d p/d t max) 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]-intermedin 1–53 was increased by 118% ( P < 0.01) in the isoproterenol group compared with controls. Rats treated with low dosage intermedin 1–53 (5 nmol/kg/day, 2 days) showed 21% ( P < 0.05) higher myocardial cAMP content, 18% and 31% higher + left-ventricle d p/d t max and -left-ventricle d p/d t max 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 intermedin 1–53 (20 nmol/kg/day, 2 days) gave better results than that with low dosage intermedin 1–53. These results suggest that the intermedin receptor system was up-regulated in isoproterenol-induced myocardial ischemic injury and intermedin 1–53 might play a pivotal cardioprotective role in such injury.

Intermedin1-53 activates l-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas

Intermedin (IMD), a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family, has similar or more potent vasodilatory and hypotensive actions than adrenomedullin (ADM) and CGRP. The present study was designed to observe the effects of synthetic rat IMD1-53 on l-arginine ( l-Arg) cellular transport, nitric oxide synthase (NOS) activity, and nitric oxide (NO) production in the isolated rat aortic ring to illustrate its direct effect on the l-Arg/NOS/NO pathway in vasculature. IMD1-53 significantly increased NO production and cNOS activity in rat aortas and was more potent than equivalent ADM. But the peptides of both IMD and ADM had no effect on inducible NOS expression and activity. Otherwise, IMD1-53 induced a concentration-dependent increase in [ 3H] l-Arg transport and its effect was more potent than that of an equivalent concentration of ADM. Semiquantitative RT-PCR revealed that IMD1-53 significantly increased cationic amino acid transport (CAT)-1 and CAT-2B mRNA expression, and its effect was similar to that of ADM. All these results suggest that IMD1-53 might regulate vessel function homeostasis via upregulating the l-Arg/NOS/NO pathway.

Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney

Adrenomedullin 2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) peptide family. AM2/IMD has a vasodilator action, and antidiuretic and antinatriuretic effects in mice. The aim of the present study is to clarify immunolocalization of AM2/IMD in human hypothalamus, heart and kidney obtained at autopsy. Immunocytochemistry showed AM2/IMD-immunoreactive cell bodies in the paraventricular and supraoptic nuclei of human hypothalamus. Both parvocellular and magnocellular cells in the paravetricular nucleus are immunostained with AM2/IMD. Immunostaining of serial sections showed co-localization of AM2/IMD-like immunoreactivity and vasopressin in the paraventricular nucleus. Myocardial cells of the heart and renal tubular cells were positively immunostained with AM2/IMD, whereas neither renal glomeruli nor vasculature in the heart and kidney were immunostained. Reverse-transcriptase polymerase chain reaction confirmed expression of AM2/IMD mRNA in the brain, pituitary, heart and kidney. The present study has shown the wide expression of AM2/IMD in human hypothalamus, heart and kidney, raising the possibility that this novel peptide may be related to the central and peripheral regulation of the circulation and water–electrolyte metabolism.

Potential Role of Pancreatic and Enteric Hormones in Regulating Bone Turnover

Potential Role of Pancreatic and Enteric Hormones in Regulating Bone Turnover