Effects of adrenomedullin on acute ischaemia-induced collateral development and mobilization of bone-marrow-derived cells.

Adrenomedullin exerts not only vasodilatory effects, but also angiogenic effects. In the present study, we investigated the effects of adrenomedullin on collateral formation and circulating bone-marrow-derived cells after acute tissue ischaemia. Bone marrow of 8-10-week-old female C57BL/6J mice was replaced with that from GFP (green fluorescent protein) transgenic mice (GFP mice). At 8 weeks after transplantation, hindlimb ischaemia was induced by resecting the right femoral artery and a plasmid expressing human adrenomedullin (50 mug) was injected into the ischaemic muscle, followed by in vivo electroporation on a weekly basis. Overexpression of adrenomedullin significantly enhanced the blood flow recovery compared with controls (blood flow ratio, 1.0+/-0.2 compared with 0.6+/-0.3 respectively, at week 4; P<0.05) and increased capillary density in the ischaemic leg as determined by anti-CD31 immunostaining of the ischaemic muscle (567+/-40 compared with 338+/-65 capillaries/mm(2) respectively, at week 5; P<0.05). There were more GFP-positive cells in the thigh muscle of the mice injected with adrenomedullin than in that of the control mice (29.6+/-4.5 compared with 16.5+/-3.3 capillaries/mm(2) respectively, at week 5; P<0.05). We repeated the same experiments using LacZ-knock-in mice instead of GFP mice, and obtained similar results. These findings suggest that adrenomedullin may augment ischaemia-induced collateral formation with some effects on circulating bone-marrow-derived cells.

Bone morphogenetic protein-3 family members and their biological functions.

Bone morphogenetic protein-3 and 3b (BMP-3 and BMP-3b) together represent a unique subgroup of the BMP family. BMP-3b shares 82% amino acid identity with BMP-3 in the mature region (ligand domain), but only 37% in the pro-region (pro-domain). In osteoblasts, BMP-3 and 3b have similar antagonistic activity against BMP-2, but they are differentially regulated. In developing embryos, BMP-3 and 3b have different dorsalizing activities. BMP-3b triggers secondary head formation in an autonomous manner, whereas BMP-3 induces aberrant tail formation. Loss-of-function analysis demonstrates that coordinated activity of xBMP-3b and cerberus, a head inducer, are required for head formation in Xenopus embryos. At the molecular level, BMP-3b antagonizes both nodal-like proteins (Xnr1 and derriere) and ventralizing BMPs (BMP-2 and ADMP), whereas BMP-3 only antagonizes ventralizing BMPs. Moreover, BMP-3b, but not BMP-3, associates with the monomeric form of Xnr1, a nodal-like protein. These molecular features of BMP-3 and 3b are due to their distinct pro-regions. These findings suggest that the processing of precursor regions and assembly of BMP-3 and 3b are important in various developmental processes and organogenesis.

Coordination of BMP-3b and cerberus is required for head formation of Xenopus embryos.

Bone morphogenetic proteins (BMPs) and their antagonists are involved in the axial patterning of vertebrate embryos. We report that both BMP-3b and BMP-3 dorsalize Xenopus embryos, but act as dissimilar antagonists within the BMP family. BMP-3b injected into Xenopus embryos triggered secondary head formation in an autonomous manner, whereas BMP-3 induced aberrant tail formation. At the molecular level, BMP-3b antagonized nodal-like proteins and ventralizing BMPs, whereas BMP-3 antagonized only the latter. These differences are due to divergence of their pro-domains. Less BMP-3b than BMP-3 precursor is proteolytically processed in embryos. BMP-3b protein associated with a monomeric form of Xnrl, a nodal-like protein, whereas BMP-3 did not. These molecular features are consistent with their expression profiles during Xenopus development. XBMP-3b is expressed in the prechordal plate, while xBMP-3 is expressed in the notochord. Using antisense morpholino oligonucleotides, we found that the depletion of both xBMP-3b and cerberus, a head inducer, caused headless Xenopus embryos, whereas the depletion of both xBMP-3 and cerberus affected the size of the somite. These results revealed that xBMP-3b and cerberus are essential for head formation regulated by the Spemann organizer, and that xBMP-3b and perhaps xBMP-3 are involved in the axial patterning of Xenopus embryos.

Adrenomedullin augments collateral development in response to acute ischemia.

Expression of adrenomedullin, discovered as a vasodilatory peptide, is markedly up-regulated under pathological conditions such as tissue ischemia and inflammation, which are associated with neovascularization. Here, we tested the hypothesis that overly expressed adrenomedullin may augment collateral flow to ischemic tissues. We induced hindlimb ischemia in wild-type mice and injected a naked plasmid expressing human adrenomedullin or an empty vector into the ischemic muscle, followed by in vivo electroporation. Adrenomedullin markedly enhanced blood flow recovery as determined by Laser Doppler imaging. The mice treated with an empty vector suffered frequent autoamputation of the ischemic toe, which was completely prevented by adrenomedullin. Anti-CD31 immunostaining revealed that adrenomedullin significantly increased capillary density. The angiogenic effect of adrenomedullin was abrogated in endothelial nitric oxide synthase (eNOS)-deficient mice. These results indicate that adrenomedullin may promote collateral growth in response to ischemia through activation of eNOS.

Endothelial responses of the aorta from adrenomedullin transgenic mice and knockout mice.

Adrenomedullin (AM) is a potent vascular wall-derived vasorelaxing peptide which induces the release of nitric oxide (NO). To explore the role of endogenous AM in vascular function, we examined the effects of acetylcholine (ACh), AM, and AM receptor antagonists [AM (22-52), and calcitonin gene-related peptide (CGRP) (8-37)] on the isometric tension of aortic rings isolated from AM transgenic (TG) and knockout (KO) mice and wild type littermates (WT). ACh and AM caused a dose-dependent reduction of the isometric tension of aortic rings, but the degree of vasodilatation was smaller in TG than in KO or WT (% delta tension [10(-6) mol/l ACh]: KO -69 +/- 10%, WT -39 +/- 8%, TG -29 +/- 1%, p < 0.01). On the other hand, N(G)-nitro-L-arginine methyl ester, an NO synthase inhibitor, induced greater vasoconstriction in TG (% delta tension 10(-5)mol/l: KO +78 +/- 16%, WT +99 +/- 27%, TG +184 +/- 20%, p < 0.01), whereas E-4021, a cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase inhibitor, caused greater vasodilation in TG mice. Both AM antagonists increased tension in TG to a greater extent than in KO or WT mice (% delta tension [10(-6) mol/l CGRP (8-37)]: KO +24 +/- 5%, WT +51 +/- 6%, TG +75 +/- 7%, p < 0.01). Endothelial denudation of the aorta diminished the vasoconstriction caused by the AM antagonists. In conclusion, the amounts of AM expressed in the aortic endothelium influenced baseline NO release. AM antagonists increased vascular tone in WT as well as in TG, suggesting that endogenous AM plays a physiological role in the regulation of aortic tone.

Somatostatin suppresses ghrelin secretion from the rat stomach.

Ghrelin is an acylated peptide that stimulates food intake and the secretion of growth hormone. While ghrelin is predominantly synthesized in a subset of endocrine cells in the oxyntic gland of the human and rat stomach, the mechanism regulating ghrelin secretion remains unknown. Somatostatin, a peptide produced in the gastric oxyntic mucosa, is known to suppress secretion of several gastrointestinal peptides in a paracrine fashion. By double immunohistochemistry, we demonstrated that somatostatin-immunoreactive cells contact ghrelin-immunoreactive cells. A single intravenous injection of somatostatin reduced the systemic plasma concentration of ghrelin in rats. Continuous infusion of somatostatin into the gastric artery of the vascularly perfused rat stomach suppressed ghrelin secretion in both dose- and time-dependent manner. These findings indicate that ghrelin secretion from the stomach is regulated by gastric somatostatin.

Calcitonin receptor-stimulating peptide, a new member of the calcitonin gene-related peptide family. Its isolation from porcine brain, structure, tissue distribution, and biological activity.

We isolated a novel biologically active peptide, designated calcitonin receptor-stimulating peptide (CRSP), from the acid extract of the porcine brain by monitoring cAMP production in the porcine kidney cell line LLC-PK(1). Determination of the amino acid sequence and cDNA analysis encoding a CRSP precursor showed that this peptide has approximately 60% identity in the amino acid sequence with human calcitonin gene-related peptide type-alpha (alphaCGRP), type-beta (betaCGRP), and porcine CGRP. Northern blot analysis and radioimmunoassay demonstrated that CRSP is expressed mainly in the thyroid gland and the central nervous system, in which the calcitonin receptor was abundantly expressed. Synthetic CRSP elicited a potent stimulatory effect on the cAMP production in LLC-PK(1) cells. Although it shows significant sequence similarity with CGRPs, this peptide did not elicit cAMP elevation in cells that endogenously expressed a CGRP receptor or an adrenomedullin receptor or were transfected with either of these recombinant receptors. Administration of CRSP into anesthetized rats did not alter the blood pressure but induced a transient decrease in the plasma calcium concentration. In fact, this peptide potently increased the intracellular cAMP concentration in COS-7 cells that expressed the recombinant calcitonin receptor. These unique properties indicate that CRSP is not a porcine counterpart of betaCGRP and probably elicits its biological effects via the calcitonin receptor.

The effect of pituitary adenylate cyclase activating polypeptide on cultured rat cardiocytes as a cardioprotective factor.

In the cardiovascular system, pituitary adenylate cyclase activating polypeptide (PACAP) exhibits not only vasodilation but also positive inotropic action by increasing cardiac output. Then the effect of PACAP in cultured cardiovascular cells was examined. In neonatal rat myocytes, PACAP evoked concentration-dependent increase in intracellular cyclic AMP content more potently than vasoactive intestinal polypeptide (VIP). However, in neonatal rat nonmyocytes, PACAP and VIP showed equal potency. The characterization of the subtype of PACAP/VIP receptors by RT-PCR analysis revealed that PAC1 receptor mRNA is dominantly present in the myocytes, but VPAC2 receptor mRNA is abundant in the nonmyocytes. In the myocytes, PACAP did not change the protein synthesis stimulated by endothelin or by itself. However, PACAP moderately stimulated the secretion of atrial natriuretic polypeptide (ANP). On the other hand, PACAP inhibited the protein synthesis and DNA synthesis of the nonmyocytes. These indicate that PACAP might be involved in the regulation of cardiac hypertrophy and fibrosis as a cardioprotective factor.

The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats.

BACKGROUND & AIMS: Visceral sensory information is transmitted to the brain through the afferent vagus nerve. Ghrelin, a peptide primarily produced in the stomach, stimulates both feeding and growth hormone (GH) secretion. How stomach-derived ghrelin exerts these central actions is still unknown. Here we determined the role of the gastric afferent vagal nerve in ghrelin's functions. METHODS: Food intake and GH secretion were examined after an administration of ghrelin intravenously (IV) to rats with vagotomy or perivagal application of capsaicin, a specific afferent neurotoxin. We investigated Fos expression in neuropeptide Y (NPY)-producing and growth hormone-releasing hormone (GHRH)-producing neurons by immunohistochemistry after administration IV of ghrelin to these rats. The presence of the ghrelin receptor in vagal afferent neurons was assessed by using reverse-transcription polymerase chain reaction and in situ hybridization histochemistry. A binding study on the vagus nerve by (125)I-ghrelin was performed to determine the transport of the ghrelin receptor from vagus afferent neurons to the periphery. We recorded the electric discharge of gastric vagal afferent induced by ghrelin and compared it with that by cholecystokinin (CCK), an anorectic gut peptide. RESULTS: Blockade of the gastric vagal afferent abolished ghrelin-induced feeding, GH secretion, and activation of NPY-producing and GHRH-producing neurons. Ghrelin receptors were synthesized in vagal afferent neurons and transported to the afferent terminals. Ghrelin suppressed firing of the vagal afferent, whereas CCK stimulated it. CONCLUSIONS: This study indicated that the gastric vagal afferent is the major pathway conveying ghrelin's signals for starvation and GH secretion to the brain.

Role of endogenous adrenomedullin in the regulation of vascular tone and ischemic renal injury: studies on transgenic/knockout mice of adrenomedullin gene.

Adrenomedullin (AM) is a potent depressor peptide whose vascular action is suggested to involve nitric oxide (NO) release. To explore the role of endogenous AM in vascular and renal function, we examined the effects of acetylcholine (ACh), AM, and AM receptor antagonists AM(22-52) and CGRP(8-37) on the renal perfusion pressure (RPP) of kidneys isolated from AM transgenic (TG)/heterozygote knockout (KO) mice and wild-type littermates (WT). Furthermore, we evaluated the renal function and histology 24 hours after bilateral renal artery clamp for 45 minutes in TG, KO, and WT mice. Baseline RPP was significantly lower in TG than in KO and WT mice (KO 93.4+/-4.6, WT 85.8+/-4.2, TG 72.4+/-2.4 mm Hg [mean+/-SE], P<0.01). ACh and AM caused a dose-related reduction in RPP, but the degree of vasodilatation was smaller in TG than that in KO and WT (%DeltaRPP 10(-7) mol/L ACh: KO -48.1+/-3.9%, WT -57.5+/-5.6%, TG -22.8+/-4.8%, P<0.01), whereas N(G)-nitro-L-arginine methyl ester (L-NAME) caused greater vasoconstriction in TG (%DeltaRPP 10(-4) mol/L: KO 33.1+/-3.3%, WT 55.5+/-7.2%, TG 152.6+/-21.2%, P<0.01). Both AM antagonists increased RPP in TG to a greater extent compared with KO and WT mice (%DeltaRPP 10(-6) mol/L CGRP(8-37): KO 12.8+/-2.6%, WT 19.4+/-3.6%, TG 41.8+/-8.7%, P<0.01). In mice with ischemic kidneys, serum levels of urea nitrogen and renal damage scores showed smaller values in TG and greater values in KO mice (urea nitrogen: KO 104+/-5>WT 98+/-15>TG 38+/-7 mg/dL, P<0.05 each). Renal NO synthase activity was also greater in TG mice. However, the differences in serum urea nitrogen and renal damage scores among the 3 groups of mice were not observed in mice pretreated with L-NAME. In conclusion, AM antagonists increased renal vascular tone in WT as well as in TG, suggesting that endogenous AM plays a role in the physiological regulation of the vascular tone. AM is likely to protect renal tissues from ischemia/reperfusion injury through its NO releasing activity.

Possible involvement of oxidative stress in hypoxia-induced adrenomedullin secretion in cultured rat cardiomyocytes.

Although hypoxia induces adrenomedullin gene expression in cultured rat cardiac myocytes, it is still unknown whether oxidative stress is involved in the hypoxia-induced adrenomedullin production. We investigated whether oxidative stress might participate in hypoxia-induced adrenomedullin secretion and whether adrenomedullin might have a protective effect on damaged myocytes. Hypoxia increased adrenomedullin secretion and its gene expression in cardiac myocytes, but not in nonmyocytes. Furthermore, oxidative stress (hydrogen peroxide) also increased adrenomedullin secretion from myocytes. N-acetyl-L-cysteine, a free radical scavenger, completely inhibited the stimulation of adrenomedullin secretion by hydrogen peroxide, and this agent reduced the stimulation of adrenomedullin secretion by hypoxia. Lactate dehydrogenase leakage, a marker of cell injury, was significantly increased with the exposure to hydrogen peroxide and adrenomedullin significantly reduced this leakage. These findings suggest that an oxidative stress may be involved, in part, in the increased adrenomedullin secretion from cardiac myocytes under hypoxic condition. Adrenomedullin secreted from myocytes may play a cell protective role in an autocrine manner.

Ghrelin is present in pancreatic alpha-cells of humans and rats and stimulates insulin secretion.

Ghrelin, a novel growth hormone-releasing peptide isolated from human and rat stomach, is a 28-amino acid peptide with a posttranslational acylation modification that is indispensable for stimulating growth hormone secretion by increasing intracellular Ca(2+) concentration. It also functions in the regulation of feeding behavior, energy metabolism, and gastric acid secretion and motility. Using two different antibodies against the NH(2)- and COOH-terminal regions of ghrelin, we studied its localization in human and rat pancreas by immunohistochemistry. Ghrelin-immunoreactive cells were identified at the periphery of pancreatic islets in both species. Ghrelin co-localized exclusively with glucagon in rat islets, indicating that it is produced in alpha-cells. We identified ghrelin and des-acyl ghrelin in the rat pancreas using reverse-phase high-performance liquid chromatography combined with two radioimmunoassays. We also detected mRNA encoding ghrelin and its receptor in the rat pancreatic islets. Ghrelin increased the cytosolic free Ca(2+) concentration in beta-cells and stimulated insulin secretion when it was added to isolated rat pancreatic islets. These findings indicate that ghrelin may regulate islet function in an endocrine and/or paracrine manner.

Plasma atrial natriuretic peptide concentration inversely correlates with left atrial collagen volume fraction in patients with atrial fibrillation: plasma ANP as a possible biochemical marker to predict the outcome of the maze procedure.

OBJECTIVES: We hypothesized that the plasma atrial natriuretic peptide (ANP) level reflects atrial degenerative change and may predict the outcome of the maze procedure. BACKGROUND: Although a larger preoperative left atrial dimension and longer duration of atrial fibrillation (AF) have been reported in patients with persistent AF than in those with sinus rhythm (SR), these individual factors were not enough to predict the outcome of the maze procedure. METHODS: Preoperative plasma ANP levels were measured in consecutive 62 patients who underwent the Kosakai's modified maze procedure. Moreover, we performed histological and molecular biological examinations in the resected left atrial tissues. RESULTS: The preoperative plasma ANP was lower in the AF group (n = 13) than it was in the SR group (n = 49) (p < 0.001). Multiple logistic regression analysis revealed that duration of AF and plasma ANP were independently associated with postoperative cardiac rhythm. Among 41 patients with a higher plasma ANP or shorter duration of AF than the median value, SR was restored in 95% of patients. In contrast, in 21 patients with a lower plasma ANP and a longer duration of AF than the median value, SR was restored only in 48% of patients. Histological examination revealed that the collagen volume in the left atrial tissue was higher in AF than it was in SR and inversely correlated with plasma ANP. In addition, the messenger RNA expressions of ANP, collagen type I and type III were lower in AF than they were in SR. CONCLUSIONS: These results suggest that a combination of plasma ANP and/or duration of AF may predict the success rate for the maze operation. Advanced atrial degenerative change may result in a decrease of atrial ANP secretion.