Identification of a novel adrenomedullin gene family in teleost fish

Adrenomedullin (AM) is a multifunctional peptide known to form a hormone family with calcitonin gene-related peptide (CGRP) and amylin. We have cloned five distinct AM cDNAs from the pufferfish, Takifugu rubripes, and named them TrAM-1, -2, -3, -4, and -5. Judging from the deduced precursor sequences and processing pattern of the C-terminal mature peptides, TrAMs may be divided into at least two groups; AM-2 and -3, and AM-1, -4, and possibly -5. Phylogenetic analysis of the mature peptides, exon-intron structure of their genes, and tissue distribution of their mRNA also support this classification. TrAM-1 and -4 were ubiquitously expressed in various tissues including the kidney and interrenal (adrenal homolog) as in the case of mammalian AM, while TrAM-2 and -3 were expressed most abundantly in the brain followed by the vascular tissues. Synteny of the genes around AM gene showed that TrAM-1 is the ortholog of mammalian AM. The presence of a PAMP-like sequence in the prosegment of TrAM-1 also supports this notion. Multiple AMs were also detected in another pufferfish, Tetraodon nigroviridis, and in zebrafish, Danio rerio. The present study shows for the first time the presence of a novel AM family in teleost fish that is independent from CGRP and amylin, which further suggests the possible existence of multiple AMs in mammals.     

Amylin or CGRP (8-37) fragments reverse amylin-induced inhibition of 14C-glycogen accumulation

The peptides amylin and calcitonin-gene related peptide (CGRP) have been shown to have similar effects on glycogen metabolism in vivo and in vitro. However, it is not clear whether they act via separate receptors. Peptide fragments based on the amino acid sequence of amylin or CGRP were evaluated for their ability to inhibit the action of the peptides in vitro. Insulin-stimulated glycogen turnover, as measured by 14C-glycogen accumulation, was inhibited about 70% by amylin (10nM) and 85% by CGRP (10nM). In the absence of exogenous peptide, peptide fragments based on the 8-37 and 10-37 amino acid sequences of rat amylin (10 uM) had no affect on 14C-glycogen accumulation. In the presence of amylin (10nM), the 8-37 and 10-37 fragments blocked amylin-induced inhibition of 14C-glycogen accumulation 100% and 11.4%, respectively. The 8-37 and 10-37 amylin fragments blocked CGRP inhibition of 14C-glycogen accumulation by 23.2% or 28.6%, respectively. The CGRP 8-37 fragment was equally effective as the amylin 8-37 reversing the effects of amylin than at reversing the effects of CGRP. These results demonstrate that amylin (8-37) completely antagonizes the effects of amylin with limited ability to block CGRP. Removing the eighth and ninth amino acids reduced the effectiveness of the inhibitor by about 90%.     

Intermedin/adrenomedullin-2 acts within central nervous system to elevate blood pressure and inhibit food and water intake

Intermedin (IMD)/adrenomedullin-2 (AM2) is a novel peptide that was independently discovered by two groups. The 47-amino acid peptide is homologous to adrenomedullin (AM) and can activate both the AM and calcitonin gene-related peptide (CGRP) receptors. IMD should therefore have actions similar to those of AM and CGRP. Indeed, like AM and CGRP, intravenous administration of IMD decreased blood pressure in rats and mice. We demonstrate here that immunoreactive IMD is present in plasma as well as heart, lung, stomach, kidney, pituitary, and brain. Because IMD is present in brain and both AM and CGRP have potent central nervous system (CNS) effects, we examined the ability of IMD within brain to regulate blood pressure and ingestive behaviors. Administration of IMD into the lateral cerebroventricle of rats caused significant, long-lasting elevations in mean arterial pressure and heart rate. These elevations are similar to the effects of CGRP and significantly greater than the effects of AM. IMD-induced elevations in mean arterial pressure were inhibited by intravenous administration of phentolamine, indicating that IMD activates the sympathetic nervous system. Intracerebroventricular administration of IMD also inhibited food and water intake in sated and in food- and water-restricted animals. The effects on feeding are likely related to activation of the CGRP receptor and are independent of the effects on water intake, which are likely through the AM receptor. Our data indicate that IMD has potent actions within the CNS that may be a result of the combined activation of both AM and CGRP receptors.     

Mechanism of action of amylin in bone.

Amylin is a 37 amino acid peptide produced mainly by beta-cells of the endocrine pancreas. Human amylin has 43% homology with human calcitonin gene-related peptide (CGRP) and 13% homology with human calcitonin (CT). Amylin and CGRP have been reported to have CT-like hypocalcemic activity in vivo. To investigate the role of amylin in bone, we examined the mechanisms of action of human amylin, CGRP, and CT in osteoclasts and osteoblasts. Both human amylin and CGRP inhibited 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]- induced bone resorption in an organ culture system, and the potencies of the two peptides were similarly approximately 60-fold lower than that of human CT. Using a recently developed procedure for preparing large numbers of osteoclast-like multinucleated cells (MNCs) formed in co-cultures of mouse osteoblasts and bone marrow cells in the presence of 1 alpha,25(OH)2D3, we found that both human amylin and CGRP stimulated cAMP production in osteoclast-like MNCs, but only at 60-fold higher concentrations than human CT. Specific binding of [125I]-human CT to osteoclast-like MNCs was detected (dissociation constant, 3 x 10(-8) M; binding sites, 3 x 10(7) per cell). To displace the bound [125I]-human CT from osteoclast-like MNCs, about 170-fold higher concentrations of human amylin and CGRP were required. No specific bindings of [125I]-amylin and [125I]-CGRP to osteoclast-like MNCs could be detected. Human CGRP stimulated cAMP production both in established mouse osteoblast-like cells (KS-4) and in mouse primary osteoblast-like cells. Amylin was a weak agonist for cAMP production in KS-4 cells. The increment in cAMP production induced by CGRP and amylin was abolished by the addition of human CGRP(8-37), a selective antagonist for CGRP receptors. CT did not stimulate cAMP production in KS-4 cells. Amylin, but not CT, displaced the bound [125I]-human CGRP from rat brain membranes. These results indicate that amylin binds not only to CT receptors in osteoclast-like MNCs but also to CGRP receptors in osteoblasts. The relative potencies of these compounds to induce cAMP production was CT greater than amylin not equal to CGRP in osteoclast-like MNCs and CGRP greater amylin much greater than CT in osteoblast-like cells.     

Vasorelaxant effect of glucagon-like peptide-(7-36)amide and amylin on the pulmonary circulation of the rat.

The gastrointestinal peptides glucagon-like peptide-1(7-36)amide (GLP-1) and amylin are currently being tested in clinical trials for the treatment of diabetes mellitus due to their effects in lowering blood glucose. Receptors for these polypeptides also exist in the lung and since polypeptides are known to modulate airway and pulmonary vascular tone, we investigated whether GLP-1 and amylin act similarly in the lung. We compared their effects with the well-known actions of calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP). Both GLP-1 and amylin induced a dose-dependent and time-reversible endothelial-dependent relaxation of preconstricted pulmonary artery rings. Amylin was approximately as strong as VIP and CGRP, GLP-1 however, was 2.3-fold less potent. GLP-1 as well as amylin also reduced the vascular tone in the isolated, perfused and ventilated rat lung. In contrast to their action on the pulmonary vasculature, neither GLP-1 nor amylin showed any effect on the tone of isolated preconstricted trachea rings. In conclusion, GLP-1 and amylin represent two additional peptides which may modulate pulmonary vascular tone.     

Comparisons between the effects of calcitonin receptor-stimulating peptide and intermedin and other peptides in the calcitonin family on bone resorption and osteoclastogenesis

Calcitonin receptor-stimulating peptide (CRSP) and intermedin (IMD) are two recently discovered peptides in the calcitonin (CT) family of peptides. CRSP and IMD, similar to CT, calcitonin gene-related peptide (CGRP), and amylin (AMY), but in contrast to adrenomedullin (ADM), inhibited bone resorption in mouse calvarial bones. CRSP and IMD, similar to CT, CGRP, AMY, but in contrast to ADM, decreased formation of osteoclasts and number of pits in bone marrow macrophage cultures stimulated by M-CSF and RANKL, with no effect on the expression of a number of genes associated with osteoclast progenitor cell differentiation. CRSP and IMD inhibited osteoclastogenesis at a late stage but had no effect on DC-STAMP mRNA. IMD, similar to CGRP, AMY, and ADM stimulated cyclic AMP formation in M-CSF expanded osteoclast progenitor cells lacking CT receptors (CTRs). RANKL induced CTRs and a cyclic AMP response also to CT and CRSP, and increased the cyclic AMP response to CGRP, AMY, and IMD but decreased the response to ADM. Our data demonstrates that CRSP and IMD share several functional properties of peptides in the CT family of peptides, including inhibition of bone resorption and osteoclast formation. The data also show that the reason why ADM does not inhibit osteoclast activity or formation is related to the fact that RANKL decreases ADM receptor signaling through the adenylate cyclase-cyclic AMP pathway. Finally, the findings indicate that activation by CGRP, AMY, and IMD may include activation of both CT and CT receptor-like receptors.     

Evolutionary history of the calcitonin gene-related peptide family in vertebrates revealed by comparative genomic analyses

The calcitonin gene-related peptide (CGRP) family is composed of CGRP, amylin and adrenomedullin (AM) in mammals. In teleost fish, AM forms an independent subfamily of five members (AM1–5), which inspired us to trace the evolutionary history of the CGRP family throughout vertebrates by comparative genomic approach. Linkage mapping and synteny analyses of the CGRP family genes in medaka, Oryzias latipes, revealed that AM1/CGRP, AM2/amylin, and AM5 genes were located on respective proto-chromosomes before the divergence of teleost lineage. In teleost fish, additional whole genome duplication generated AM1/4, CGRP1/2, AM2/3, but one of the duplicated amylin and AM5 genes was silenced. In mammals, the amylin or AM2 gene was translocated to different chromosomes, while the CGRP gene was multiplied in tandem to generate CGRP-,β, and recently identified calcitonin receptor-stimulating peptide genes. Based on these data, we identified a novel AM5 gene in several mammalian species as we previously did for AM2.     

Amylin-induced suppression of ANP secretion through receptors for CGRP1 and salmon calcitonin

Amylin cosecretes with insulin from pancreatic beta-cells and shows high sequence homology with CGRP, adrenomedullin, and salmon calcitonin. This study aimed to investigate the effect of amylin on the atrial hemodynamics and ANP release from rat atria and to identify its receptor subtypes. Isolated perfused left atria from either control or streptozotocin-treated rats were paced at 1.3 Hz. The concentration of ANP was measured by radioimmunoassay and the translocation of ECF was measured by [3H]-inulin clearance. Rat amylin increased atrial contractility and suppressed the release of ANP. Rat CGRP showed similar effects but was approximately 300-fold more potent than amylin. Pretreatment with receptor antagonist for CGRP1 [rat alpha-CGRP (8-37)] or salmon calcitonin [acetyl-(Asn30, Tyr32)-calcitonin(8-32), (AC 187)] blocked the suppressive effect of ANP release and the positive inotropic effect by rat amylin. However, receptor antagonists for amylin [amylin (8-37), acetyl-amylin] did not block those effects. Amylin (8-37), acetyl-amylin, or rat alpha-CGRP (8-37) alone accentuated the release of ANP with no changes in atrial contractility. The effect of rat amylin and rat amylin (8-37) on the ANP release was attenuated in streptozotocin-treated rats. We suggest that amylin suppressed ANP release with increased atrial contractility through receptors for CGRP1 and salmon calcitonin and the attenuation of amylin and its antagonist on ANP release from streptozotocin-treated rat atria may be due to the downregulation of amylin receptor.     

Receptor autoradiography as mean to explore the possible functional relevance of neuropeptides: focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides

Over the past 20 years, receptor autoradiography has proven most useful to provide clues as to the role of various families of peptides expressed in the brain. Early on, we used this method to investigate the possible roles of various brain peptides. Natriuretic peptide (NP), neuropeptide Y (NPY) and calcitonin (CT) peptide families are widely distributed in the peripheral and central nervous system and induced multiple biological effects by activating plasma membrane receptor proteins. The NP family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The NPY family is composed of at least three peptides NPY, peptide YY (PYY) and the pancreatic polypeptides (PPs). The CT family includes CT, calcitonin gene-related peptide (CGRP), amylin (AMY), adrenomedullin (AM) and two newly isolated peptides, intermedin and calcitonin receptor-stimulating peptide (CRSP). Using quantitative receptor autoradiography as well as selective agonists and antagonists for each peptide family, in vivo and in vitro assays revealed complex pharmacological responses and radioligand binding profile. The existence of heterogeneous populations of NP, NPY and CT/CGRP receptors has been confirmed by cloning. Three NP receptors have been cloned. One is a single-transmembrane clearance receptor (NPR-C) while the other two known as CG-A (or NPR-A) and CG-B (or NPR-B) are coupled to guanylate cyclase. Five NPY receptors have been cloned designated as Y(1), Y(2), Y(4), Y(5) and y(6). All NPY receptors belong to the seven-transmembrane G-protein coupled receptors family (GPCRs; subfamily type I). CGRP, AMY and AM receptors are complexes which include a GPCR (the CT receptor or CTR and calcitonin receptor-like receptor or CRLR) and a single-transmembrane domain protein known as receptor-activity-modifying-proteins (RAMPs) as well as an intracellular protein named receptor-component-protein (RCP). We review here tools that are currently available in order to target each NP, NPY and CT/CGRP receptor subtype and establish their respective pathophysiological relevance.     

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.     

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

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

Central amylin expression and its induction in rat dams

Amylin, or islet amyloid polypeptide, is known as a satiating signal expressed in pancreatic β‐cells but not in the brain. In this study, regulations of postpartum mRNA expressions were investigated in the preoptic area of the hypothalamus. mRNA levels of lactating dams and mothers whose pups were removed immediately after delivery were compared in microarray experiments. There were 20 genes identified with significantly increased and 14 with decreased expression 9 days postpartum. Amylin mRNA level demonstrated the largest change, a 25.7 times increase. Quantitative RT‐PCR measurements validated the increase in the mRNA level of amylin in the preoptic area of lactating dams while the expression level of other members of the calcitonin gene‐related peptide family did not change. In situ hybridization histochemistry for amylin further verified its induction in lactating mothers and demonstrated the distribution of amylin mRNA in the medial preoptic nucleus, parts of the medial preoptic area, and the ventral part of the bed nucleus of the stria terminalis but nowhere else in the rat brain. Immunolabeling verified the postpartum induction of amylin in the preoptic area at the peptide level, as well. The results suggest that amylin may play a part in maternal regulations.     

The role of calcitonin gene-related peptide (CGRP) in macrophages: the presence of functional receptors and effects on proliferation and differentiation into osteoclast-like cells

It has been shown that both calcitonin gene-related peptide (CGRP) and amylin bind weakly to calcitonin (CT) receptors in osteoclast-like cells formed in vitro and inhibit bone resorption by a cAMP-dependent mechanism. Osteoclasts are thought to be derived from cells of the monocyte macrophage lineage, in which CGRP, but not CT, induces cAMP production. In this study, we determined the presence of functional receptors for CGRP in mouse alveolar macrophages and the effects of this peptide on proliferation and osteoclastic differentiation in mouse alveolar and bone marrow-derived macrophages. Human CT did not stimulate cAMP production in macrophages. Human CGRP stimulated cAMP production in mouse alveolar macrophages and bone marrow-derived macrophages dose-dependently. Human amylin, which has 43% homology with human CGRP, also stimulated these macrophages to produce cAMP, but only at a 100-fold higher concentration. The increment in cAMP production induced by human CGRP and amylin was abolished by the addition of human CGRP(8–37), a selective antagonist for CGRP receptors. Specific binding of [¹²⁵I]human CGRP to alveolar macrophages was detected (dissociation constant, 2.5 × 10⁻⁸ M; binding sites, 1.4 × 10⁴/cell). Amylin, but not CT, displaced the bound [¹²⁵I]human CGRP from alveolar macrophages, but at a 100-fold higher concentration. No specific binding of [¹²⁵I]human CT and [¹²⁵I]human amylin to alveolar macrophages could be detected. Pretreatment with human CGRP for 24 h dose-dependently suppressed DNA synthesis in alveolar macrophages induced by granulocyte-macrophage colony-stimulating factor (GM-CSF). CGRP also suppressed the number of macrophage colonies formed from bone marrow cells induced by macrophage colony-stimulating factor (M-CSF). Pre-treatment of alveolar macrophages with CGRP inhibited differentiation into osteoclast-like cells in co-cultures with primary osteoblastic cells in the presence of 1α,25-dihydroxy vitamin D₃. These results indicate that specific receptors for CGRP are present in macrophages and that CGRP modulates proliferation and differentiation of macrophages into osteoclast-like cells by a receptor-mediated mechanism involving cAMP.     

Comparative affinities of adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) for [125I] AM and [125I] CGRP specific binding sites in porcine tissues.

We have investigated the binding characteristics of rat [125I] adrenomedullin (AM) and human [125I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [125I] rat AM and [125I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [125I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [125I] rat AM binding to various tissues were rat AM > or = human AM > or = human AM(22-52) > h alpha CGRP > or = h alpha CGRP(8-37) > sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20-300 fold more potent than hAM (22-52). When the same experiments were performed using [125I] h alpha CGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > h alpha CGRP > h alpha CGRP(8-37) in most of the tissues except in spleen and liver where h alpha CGRP was the most potent ligand. In lung, h alpha CGRP was almost as potent as rAM and hAM in displacing [125I] h alpha CGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.     

Organ distribution and characterization of porcine peptides (VIP, CGRP and PHI) that increase cAMP in rat platelets.

Using an assay for rat platelet cAMP, we investigated the organ distribution of peptides that increase cAMP in rat platelets in porcine tissues. Marked activity was observed in the duodenum, pancreas and brain. By analysis with reverse phase high performance liquid chromatography (HPLC), three major peaks of activity were observed in porcine tissues. The first peak was vasoactive intestinal polypeptide (VIP), and the second peak was calcitonin gene-related peptide (CGRP). The third peak of activity was isolated from porcine duodenum. By analysis with a gas phase sequencer and with an amino acid analyzer, this peptide was identified as peptide histidine isoleucine (PHI). In a glucagon-secretin family of neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP) significantly increased platelet cAMP levels in a dose-dependent manner; however, glucagon did not. These results suggest that not only VIP and CGRP but also PHI and PACAP act upon platelets, as well as vascular tissues.     

The effect of adrenomedullin,amylin fragment 8-37 and calcitonin gene-related peptide on contractile force,heart rate and coronary perfusion pressure in isolated rat hearts

The effect of human adrenomedullin, human amylin fragment 8-37 (amylin 8-37) and rat calcitonin gene-related peptide (CGRP) on contractile force, heart rate and coronary perfusion pressure has been investigated in the isolated perfused rat hearts. Adrenomedullin (2x10(-10), 2x10(-9) and 2x10(-8) M) produced a significant decrease in contractile force and perfusion pressure, but only the peptide caused a decline in heart rate at the highest dose. Amylin (10(-9), 10(-8) and 10(-7) M) significantly increased and then decreased contractile force. Two doses of amylin (10(-8) and 10(-7) M) induced a significant increase in heart rate, however amylin did not change perfusion pressure in all the doses used. Rat alpha CGRP (10(-8), 10(-7) and 10(-6) M) evoked a slight decline in contractile force following a significant increase in contractile force induced by the peptide. CGRP in all the doses raised heart rate and lowered perfusion pressure. Our results suggest that adrenomedullin has negative inotropic, negative chronotropic and coronary vasodilator actions. Amylin produces a biphasic inotropic effect and evokes a positive chronotropy. CGRP causes positive inotropic, positive chronotropic and vasodilatory effects in isolated rat hearts.     

Molecular characterization of the GHRH/GHRH-R and its effect on GH synthesis and release in orange-spotted grouper (Epinephelus coioides)

Growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide that stimulates growth hormone (GH) synthesis and secretion in the pituitary gland. In this paper, the full-length cDNAs of orange-spotted grouper GHRH and its receptor (GHRH-R) were cloned. The grouper GHRH cDNA is 713 bp in length and encodes a 141-aa precursor that includes an 18-aa signal peptide, a 27-aa mature GHRH mature peptide and a 47-aa carboxyl terminus. The grouper GHRH-R cDNA sequence is 1495 bp in length, encoding a 422-aa receptor with seven transmembrane domains. Tissue distribution analyses showed that both GHRH and GHRH-R mRNAs were predominantly expressed in the brain, while the GHRH-R mRNA was also abundantly detected in the pituitary gland. Both GHRH and GHRH-R mRNAs were expressed throughout embryonic development from the multi-cell stage to the newly hatched larvae stage, and the highest GHRH and GHRH-R expressions appeared at the brain vesicle stage and the heart stage, respectively. In vitro studies performed on the grouper pituitary primary cells showed that a synthetic grouper GHRH-NH(2) increased both GH mRNA expression and GH protein release in a dose-dependent manner. Together, these results suggest that the newly obtained grouper GHRH was able to stimulate GH synthesis and release, similar to its mammalian counterparts.     

Comparative Affinities of Adrenomedullin (AM) and Calcitonin Gene-Related Peptide (CGRP) for [125I] AM and [125I] CGRP Specific Binding Sites in Porcine Tissues

Abstract

We have investigated the binding characteristics of rat [¹²⁵I] adrenomedullin (AM) and human [¹²⁵I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [¹²⁵I] rat AM and [¹²⁵I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [¹²⁵I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [¹²⁵I] rat AM binding to various tissues were rat AM ≥ human AM ≥ human AM(22–52) > hαCGRP ≥ hαCGRP(8–37) <<<< sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20–300 fold more potent than hAM(22–52). When the same experiments were performed using [¹²⁵I] hαCGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > hαCGRP > hαCGRP(8–37) in most of the tissues except in spleen and liver. where hαCGRP was the most potent ligand. In lung, hαCGRP was almost as potent as rAM and hAM in displacing [¹²⁵I] hαCGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.