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Neuregulin-4 (Nrg4) is a newly discovered adipokine that is synthesized in many tissues and plays an important role in modulating systemic energy metabolism and in the development of metabolic disorders. However, little is known about the relationship between Nrg4 and coronary artery disease (CAD). In this study, we investigated the association between Nrg4 and the presence and severity of CAD.We enrolled 73 patients diagnosed by coronary angiography (CAG) as having CAD and 32 controls. The CAD group was divided into two subgroups according to their SYNTAX score. Plasma levels of Nrg4 were measured in all participants and compared among different groups. The relationship between Nrg4 and CAD was analyzed. Receiver operating characteristic (ROC) analysis was conducted to evaluate the usefulness Nrg4 in assessing the presence and severity of CAD.Nrg4 levels were negatively associated with the SYNTAX score (r = -0.401, P = 0.000). The patients with a higher SYNTAX score had significantly lower Nrg4 levels as compared with the low SYNTAX score subgroup and the controls (P < 0.05). The Nrg4 levels of the low SYNTAX score subgroup were much lower than controls (P < 0.05). Furthermore, an association between Nrg4 and CAD (odds ratio, 0.279; 95% confidence interval, 0.088-0.882) was observed. Nrg4 had 43.8% sensitivity and 96.9% specificity for identifying CAD, and 73.1% sensitivity and 87.3% specificity for identifying patients who had severe coronary artery lesions.Nrg4 levels were found to be inversely associated with the presence and severity of CAD.

To study whether urotensin II (UII), a potent vasoconstrictive peptide, is involved in the development of cardiac hypertrophy and fibrogenesis of rats induced by isoproterenol (ISO).Thirty male Wistar rats were randomly divided into 3 groups. Group 1 was the healthy control group, group 2 was the ISO group, and group 3 was the ISO+UII group. In groups 2 and 3, ISO (5 mg x kg(-1) x d(-1)) was given (sc) once daily for 7 d. Group 3 was also given UII in the first day [3 nmol/kg (5 microg/kg), iv], followed by sc (1.5 microg/kg) twice daily. Group 1 received 0.9% saline. UII receptor (UT) mRNA expression was determined by RT-PCR. The contents of UII and angiotensin II (Ang II) were determined by radioimmunoassay. In vitro, the effects of UII on DNA/collagen synthesis of cardiac fibroblasts were determined by [3H]thymidine/[3H]proline incorporation.The ratio of heart weight/body weight, plasma lactate dehydrogenase activity, myocardial malondialdehyde and hydroxyproline concentration increased significantly in the ISO group, as well as UT mRNA expression, plasma and cardiac UII and ventricular Ang II, compared with the control group (P0.01). ISO induced significant myocardial fibrogenesis. Moreover, UII+ISO co-treatment significantly increased the changes of biochemical markers of injury and the degree of cardiac hypertrophy and fibrosis. In vitro, 5 x 10(-9 )-5 x 10(-7 ) mol/L UII stimulated [3H]thymidine/[3H] proline incorporation into cardiac fibroblasts in a dose-dependent manner (P0.01).These results suggest that UII was involved in the development of cardiac fibrosis and hypertrophy by synergistic effects with ISO.

Aim: To determine the cardioprotective action of ghrelin and des-octanoyl ghrelin in rats with isoproterenol-induced myocardial injury. Methods: Rats were subcutaneously injected with isoproterenol (ISO; 20, 10, and 5 mg/kg) on d 1, 2 and 3, respectively, and then 3 mg/kg for the next 7 d with or without ghrelin or des-octanoyl-ghrelin (100 mug/kg, twice daily). Plasma ghrelin and growth hormone levels were assayed using radioimmunoassay methods. Growth hormone secretagogue receptor (GHSR) and ghrelin mRNA were determined using RT-PCR. The maximal binding capacity and the affinity for [ 3 H] ghrelin were determined by receptor binding assays. Results: Compared with controls, ISO-treated rats showed severe myocardial injury, cardiomegaly, infarction-like necrosis and massive fibrosis with increases in irradiated-ghrelin (ir-ghrelin) content in plasma by 67% and myocardia by 66% and in the mRNA level in the myocardia by 93% ( P P 3 H]ghrelin for myocardial sarcolemma was higher in ISO-treated rats than in controls. Ghrelin administration improved cardiac function and ameliorated cardiomegaly and attenuated myocardial lipid peroxidation injury and relieved cardiac fibrosis as compared with ISO treatment alone. Administration of des-octanoyl ghrelin effectively antagonized ISO-induced myocardial injury and improved all parameters measured. However, the therapeutic effect of des-octanoyl ghrelin was significantly weaker than that of ghrelin. The plasma growth hormone level increased markedly, by 1.5-fold ( P Conclusion: Myocardial ghrelin and GHSR were up-regulated during ISO-induced myocardial injury. The protective effect of ghrelin against ISO-induced cardiac function injury and fibrosis was more potent than that of des-octanoyl ghrelin, which suggests that ghrelin could be an endogenous cardioprotective factor in ischemic heart disease, and that its effects include growth hormone-dependent and -independent pathways.

To investigate the role of the endogenous cystathionine γ-synthase (CSE)/ hydrogen sulfide (H2S) pathway in vascular calcification in vivo. A rat vascular calcification model was established by administration of vitamin D3 plus nicotine (VDN). The amount of CSE and osteopontin (OPN) mRNA was determined by using semi-quantitative reverse-transcription polymerase chain reaction. The calcium content, 45Ca2+ accumulation and alkaline phosphatase (ALP) activity were measured. H2S production and CSE activity were measured. von Kossa staining produced strong positive black/brown staining in areas among the elastic fibers of the medial layer in the calcified aorta. The calcium content, 45Ca2+ accumulation and ALP activity in calcified arteries increased by 6.77-, 1.42-, and 1.87-fold, respectively, compared with controls. The expression of the OPN gene was upregulated (P< 0.01). Expression of the CSE gene was downregulated. However, calcium content, 45Ca2+ uptake and ALP activity in the VDN plus NaHS group was lower than that in the VDN group. The content of calcium and 45Ca2+ accumulation and activity of ALP in the aorta were 34.8%, 40.75% and 63.5%lower in the low-dosage NaHS group than in the VDN group, respectively (P< 0.01), and the calcium content and deposition of 45Ca2+ and activity of ALP was 83.9%, 37.8 % and 46.2% lower in the aorta in the high-dosage NaHS group than in the VDN group, respectively (P< 0.01). The expression of the OPN gene was downregulated. The production of H2S, and CSE activity were decreased and CSE gene expression was downregulated in rats with vascular calcification. H2S can ameliorate vascular calcification, suggesting that the H2S/ CSE pathway plays a regulatory role in the pathogenesis of vascular calcification.

Aim: To investigate effects of taurine on ischemia/reperfusion (I/R)-induced compartment syndrome in rabbit hind limbs. Methods: Rabbits underwent femoral artery occlusion after ligation of branches from terminal aorta to femoral artery. After a 7-h ischemia, reperfusion was established with the use of heparinized polyethylene shunts. Rabbits received taurine (1 g/kg) or normal saline (control) by iv infusion 10 min before shunt placement. During reperfusion, anterior compartment pressure (ACP) was monitored continuously in the left lower extremity. Gastrocnemius muscle triphenyltetrazolium chloride (TTC) level, taurine content and myeloperoxidase activity were assayed. Oxidative stress was induced in the in vitro gastrocnemius muscle slices by free radical generating systems (FRGS), and the malondialdehyde content was measured in presence or absence of taurine. Results: After 7 h of ischemia, none of the parameters that we measured were different from those before ischemia, except that TTC reduction decreased by 80%. In the control group, after 2 h of reperfusion, ACP increased 4.5-fold, and gastrocnemius muscle taurine content was reduced by 33%. In taurine-treated animals, at 2 h reperfusion, the mean arterial blood pressure and heart rate were increased, by 6% and 10%. ACP decreased by 39%, muscle edema decreased by 16%, TTC reduction increased by 150%, and lactate dehydrogenase decreased by 36% compared to control group. Plasma and muscle taurine content increased by 70% and 88%, respectively. In the taurine-treated group, at 2 h reperfusion, plasma malondialdehyde and conjugated diene content were decreased by 38% and 23%, respectively, and muscle malondialdehyde and conjugated diene content decreased by 22% and 30%, respectively compared to the control group. At 2 h reperfusion, myeloperoxidase activity was increased 3.5-fold in control animals. In the in vitro study, taurine decreased malondialdehyde content in muscle slices incubated with hypochlorous acid in a dose-dependent manner, but there was no change when incubated with hydrogen peroxide and xanthine oxidase. Conclusion: Treatment with taurine inhibited I/R-induced compartment syndrome by at least in part attenuating oxidative stress injury induced by I/R, suggesting clinical application of taurine might be a new strategy for the prevention and treatment of compartment syndrome.

Adrenomedullin (ADM) has the vasodilatory properties and involves in the pathogenesis of vascular calcification. ADM could be degraded into more than six fragments in the body, including ADM(27-52), and we suppose the degrading fragments from ADM do the same bioactivities as derived peptides from pro-adrenomedullin. The present study carries forward by assessing the effects on vascular calcification of the systemic administration of ADM(27-52). The rat vascular calcific model was replicated with vitamin D3 and nicotine. ADM or/and ADM(27-52) were systemically administrated with mini-osmotic pump beginning at seventh day after the model replication for 25 days. Vascular calcific nodules histomorphometry, vascular calcium content, vascular calcium uptake, alkaline phosphatase activity, and osteopontin-mRNA quantification in aorta were assessed. ADM limited 40.2% vascular calcific nodules (P0.01), did not effect on calcium content (P0.05), reduced 44.4% calcium uptake (P0.01), lowered 21.1% alkaline phosphatase activity (P0.01), and regulated 40.9% downwards osteopontin-mRNA expression (P0.01) in the aorta of rats with vascular calcification. ADM(27-52) receded 32.0% vascular calcific nodules (P0.01), taken from 55.5% calcium content (P0.01), did not affect calcium uptake (P0.05), inhibited 22.5% alkaline phosphatase activity (P0.01), and restrained 21.9% osteopontin-mRNA expression (P0.01) in the aorta of rats with vascular calcification. Both of ADM and ADM(27-52) did interact on vascular calcification each other. ADM could partially antagonize the effects of ADM(27-52) in taking from calcium content (17.5%, P0.01) and in receding vascular calcific nodules (18.6%, P0.01). ADM could obviously enhance the action of ADM(27-52) in inhibiting alkaline phosphatase activity (14.4%, P0.01) and in reducing calcium uptake (11.4%, P0.01). ADM(27-52) could partially antagonize the effects of ADM on regulating downwards osteopontin-mRNA expression (17.0%, P0.01). It is concluded that ADM(27-52) derived from ADM acts as an inhibitory agent on vascular calcification, with special mechanisms different from ADM derived from ADM progenitor molecule.

Objective Aldosterone is involved in cardiovascular diseases such as hypertension and heart failure by inducing sodium retention and vascular remodeling, which is characterized by fibroblast proliferation and migration in adventitia. It is well known that aldosterone stimulates vascular smooth muscle cells and fibroblasts to produce and secrete adrenomedullin (ADM), a multiple functional peptide with an important cytoprotective effect against cardiovascular damage. We examined the effect of aldosterone on ADM production and secretion and its mRNA expression in rat aortic adventitia to study the paracrine/autocrine interaction between endogenous ADM and aldosterone. Methods ADM produced and secreted from adventitia stimulated by aldosterone in the absence or presence of spironolactone, RU486 or spironolactone together with RU486 were detected by radioimmunoassay, proliferation in adventitia cells was evaluated by the level of [ 3 H]-thymine incorporation, and preproADM gene expression was measured by semi-quantitative reverse transcriptase polymerase chain reaction. Results Adventitial ADM secretion and mRNA expression stimulated by aldosterone were concentration-dependent as was the inhibitive effect of ADM on aldosterone-induced proliferation. The induction of aldosterone in ADM secretion was mediated by mineralocorticoid receptor. Antagonists of specific receptors of calcitonin gene-related peptide (CGRP) receptor type 1 and ADM both potentiated the proliferation effect-induced by aldosterone; and thiorphan, an inhibitor of the enzyme for ADM degradation, inhibited the adventitial [ 3 H]-thymine incorporation induced by aldosterone. ADM inhibited the activity of extracellular signal related kinase (ERK) stimulated by aldosterone. Conclusion Aldosterone stimulates adventitia to produce and secrete ADM, which in turn, antagonizes the aldosterone-induced proliferation in adventitia.

Previous work has shown that the endogenous cystathionine gamma-synthase (CSE)/hydrogen sulfide (H(2)S) pathway participates in the regulation of cardiac contraction. We hypothesized that the pathway might participate in the pathophysiological regulation of ischemic heart disease. Isoproterenol injection of rat hearts induced a myocardial ischemic injury model, with reduced myocardial and plasma H(2)S levels, decreased CSE activity, and upregulated CSE gene expression. Exogenous administration of the H(2)S donor NaHS reduced the mortality rate; increased left-ventricular pressure development and left-ventricular-end systolic pressure; and decreased left-ventricular-end diastolic pressure (LVEDP) and subendocardial necrosis, capillary dilatation, leukocytic infiltration, fibroblast swelling, and fibroblastic hyperplasia. As well, production of lipid peroxidation, including myocardial malondialdehyde (MDA), and plasma MDA and conjugated diene, was reduced. Oxidative stress injury is an important mechanism of isoproterenol-induced myocardial injury. In vitro experiments revealed that NaHS might antagonize myocyte MDA production by oxygen-free radicals and that NaHS directly scavenged hydrogen peroxide and superoxide anions. Our results suggest that the endogenous CSE/H(2)S pathway contributes to the pathogenesis of isoproterenol-induced myocardial injury. Administration of exogenous H(2)S effectively protects myocytes and contractile activity, at least by its direct scavenging of oxygen-free radicals and reducing the accumulation of lipid peroxidations.

We studied the effects of cobalt-60 Gamma-radiation on the gene expression and secretion of adrenomedullin (Adm) and endothelin (ET) in cultured rat vascular smooth muscle cells (VSMCs). Rat VSMCs cultured in Dulbecoo's modified Eagle's medium containing 10% FBS were radiated with cobalt-60 Gamma-radiation at doses of 1, 14, and 25 Gy, respectively. Then the mRNA of Adm and ET in VSMCs were detected by the reverse-transcriptative competitive polymerase chain reaction. Adm and ET levels in rat VSMCs were measured by radioimmunoassay. As compared with that of the control, the secretions of Adm in rat VSMCs radiated at doses of 14 and 25 Gy were increased by 270% (P0.05) and 233% (P0.05), respectively. The mRNA levels of Adm were increased by 82.4% (P0.01) and 101% (P0.01), respectively. Meanwhile, the secretions of ET were decreased by 27.3% (P0.01) and 58.0% (P0.01) in VSMCs radiated at doses of 14 and 25 Gy, respectively. In parallel, the mRNA levels of ET were decreased by 47.1% (P0.01) and 40.2% (P0.01), respectively. Radiation at a dose of 1 Gy had no significant effect on Adm and ET at the gene and protein levels. As compared with the control, the Adm/ET ratios in VSMCs increased by 65% (P0.05), 409% (P0.01), and 693% (P0.01), respectively, with radiation at doses of 1,14 and 25 Gy, respectively. The balance of Adm/ET in VSMCs could be changed by cobalt-60 Gamma-radiation, which might play an important role in the use of radiotherapy for restenosis.

We observed changes of endothelin content and endothelin mRNA in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells to explore the role of endothelin in vascular calcification. Calcification model in vivo was induced by administration of Vitamin D(3) plus nicotine. Calcification of vascular smooth muscle cells (VSMCs) was induced by beta-glycerophosphate. Endothelin content was measured by using radioimmunoassay. Endothelin mRNA amount was determined by using competitive quantitative RT-PCR. The results showed that calcium content, 45Ca(2+) uptake and alkaline phosphatase (ALP) activity were increased in calcified VSMCs, compared with controls, but were decreased, compared with calcified VSMCs plus BQ123 group. The endothelin content in the medium and endothelin mRNA in VSMCs were elevated by 35 and 120% (P

The effect of homocysteine (Hcy) on the synthesis of adrenomedullin (ADM) was examined in vascular fibroblasts of rats to explore the possible activity of ADM in cardiovascular diseases. ADM from cultured vascular fibroblasts was measured by Radioimmunoassay (RIA) and was characterized by reverse phase high performance liquid chromatography. ADM mRNA in the fibroblasts was quantitated by competitive RT-PCR. The synthesis of ADM from vascular fibroblasts was increased by Hcy in a concentration dependent manner. ADM content in cultured medium increased by 68%–150% (P < 0.01, as compared to the control) when the fibroblasts were incubated in 50 –200 μmol · L−1 Hey for 12 h, and the content increased by 55%–98% (P < 0.01) if the incubation lasted for 24 h. Hydrogen peroxide treatment also increased the synthesis of ADM and it promoted ADM synthesis synergetically with Hcy. The changes of ADM content were partially or completely inhibited by the antioxidant N-acetylcysteine (NAC) treatment. Hcy induced the increase of ADM mRNA and the amount of ADM mRNA was increased by 78% as compared with control (P < 0.05). NAC almost completely inhibited the increase of ADM mRNA induced by Hcy. In conclusion, Hcy treatment up-regulated the expression of ADM gene and increased the synthesis of ADM in vascular fibroblasts. Oxidative stress may be involved in the processes of the ADM changes induce by Hcy.

Adrenomedullin is a potent vasodilator peptide originally isolated from a pheochromocytoma. Recently, a novel adrenomedullin receptor has been identified as a complex of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 2 (RAMP2). To explore the pathophysiological roles of adrenomedullin and its receptor component RAMP2 in ischemic cardiovascular diseases, we studied the changes of adrenomedullin and RAMP2 mRNA in myocardium and aorta in rats with isoproterenol (ISO)-induced myocardial impairment. In ISO-treated rats, heart became enlarged markedly, the ratio of heart to body weight was increased by 54% (P0.01), and myocardial malondialdehyde content and plasma lactate dehydrogenase activity was elevated by 43% (P0.01) and 138% (P0.01), respectively. Immunoreactive adrenomedullin (ADM) in plasma, myocardium and aorta was augmented by 116.7% (P0.01), 50.8% (P0.01) and 12.5% (P0.05), respectively. ADM mRNA in myocardium and aorta was increased by 96.8% (P0.01) and 38.5% (P0.01), respectively. RAMP2 mRNA in myocardium and aorta was increased by 19.6% (P0.05) and 15.8% (P0.01), respectively. These results suggest that the increase of ADM level and the up-regulation of ADM and RAMP2 gene in myocardium and aorta may be significant in the pathogenesis of ischemic myocardiopathy.

Objective The aim of the present study was to investigate the effect of adrenomedullin (ADM) on vascular calcification. Methods The vascular calcification model was established in rats (VND group) by using vitamin D3 (300000IU/kg) and nicotine (25mg/kg, two doses). The effect of liposome-encapsulated ADM was observed. Vascular calcium content, alkaline phosphatase (ALP) activity, ADM in aortic tissue and plasma, binding ability of 125I-ADM for ADM receptor on vascular plasma membrane and content of cAMP in vessels were measured. Results Compared with control rats, the aortic calcium content and vascular ALP activity in rats of the VDN group was obviously increased; in addition ADM concentrations in plasma and vessels of rats in VDN group were increased. But the maximum binding sites of 125I-ADM for ADM receptor (Bmax) on vascular plasma membrane in rats of VDN group were significantly decreased compared with control rats. The affinity of 125I-ADM for the ADM receptor was reduced, as shown by the Kd value and vascular cAMP content being reduced in rats of the VDN group compared to the control group. The in vitro response of isolated vessels to ADM incubation was weakened. Administration of empty liposome had no effect on vascular calcification. But administration of ADM significantly decreased vascular calcium content and ALP activity. The Bmax of 125I-ADM for ADM receptors on vascular plasma membrane increased by 17.7% (p

Primary culture of vascular smooth muscle cells (VSMC) from rat aorta was used for the study of the effect of different peptides derived from proadrenomedullin on the expression of adrenomedullin (ADM) gene. ADM and preproADM(22-41) (PAMP) secreted by VSMC were measured by radioimmunoassay, and ADM mRNA in VSMC was determined by quantitative RT-PCR. After the incubation of VSMC in 10(-7)M ADM for 24h, PAMP in the medium and ADM mRNA in the VSMC were decreased by 34 and 41.3%, respectively, and cAMP concentration in the VSMC was increased by 385%. After the incubation of VSMC in 10(-7)M PAMP for 24h, ADM in the medium and ADM mRNA in the VSMC were decreased by 12.2 and 39.1%, respectively, and cAMP concentration in the VSMC was increased by 67%. The decreased ADM mRNA in VSMC induced by the ADM and PAMP treatment was completely reversed by the pre-treatment of the cells in 10(-7)M protein kinase inhibitor for 30 min. After the incubation of VSMC in 10(-7)M preproADM(153-185) (ADT) for 24h, however, ADM in the medium and ADM mRNA in the VSMC were increased by 21 and 35.2%. The increased ADM mRNA in VSMC induced by the ADT treatment was partially blocked by the co-incubation in ADM and ADT, and was totally blocked by the co-incubation in PAMP+ADM and ADT, but was not blocked by the co-incubation in PAMP and ADT. Our results suggest that the four peptides derived from proadrenomedullin may have different effects, possibly through a cAMP-dependent pathway, on the expression of ADM gene.

A rapidly emerging body of literature implicates a pivotal role for the Ca2+-calmodulin-dependent phosphatase, calcineurin, as a cellular target for a variety of Ca2+-dependent signaling pathways culminating in cardiac hypertrophy. The aim of the present study was to test whether calcineurin is involved in the signal transduction of angiotensin II (AngII)-induced cardiac myocyte hypertrophy and fibroblast hyperplasia. Firstly, we observed that calcineurin activity was significantly increased in AngII-stimulated cardiac myocytes as well as fibroblasts, but was markedly inhibited by Losartan (50 micromol/l), H7 (50 micromol/l), and Fura-2/AM (5 micromol/l). It is indicated that AngII-induced activation of calcineurin is through an ATI receptor, may be dependent on the sustained increases of [Ca2+]i, and be regulated by protein kinase C. In a second experiment, we found that cyclosporin (0.1-10micromol/l), a specific inhibitor of calcineurin, decreased the protein synthesis rate in AngII-stimulated cardiomyocytes and the DNA synthesis rate in AngII-treated fibroblasts in a dose-dependent manner. In the latter experiment, calcineurin inhibition reduced the mRNA level of the atrial natriuretic factor gene. These results indicate that calcineurin is involved in the signal transduction of AngII-induced cardiomyocyte hypertrophy and fibroblast hyperplasia.

Research has identified the vasoconstrictors endothelin-1 (ET-1) and urotensin-II (U-II) as having a role in the development of atherosclerotic cardiovascular disease. We aimed to observe alterations in plasma levels of both ET-1 and U-II in patients with coronary heart disease (CHD) undergoing percutaneous transluminal coronary angioplasty (PTCA) and stent therapy from November 2006 through May 2007. We examined plasma levels of ET-1 and U-II in 40 patients with CHD and 40 age-matched healthy subjects by radioimmunoassay (RIA). Chi-square test, Student's t-test, and one-way analysis of variance were used for statistical analyses. Correlations between variables were tested by simple linear regression analysis. Coronary heart disease patients had significantly higher ET-1 and UII levels than healthy controls (20.05 +/- 4.65 vs 8.16 +/- 3.38 and 71.90 +/- 11.61 vs 20.89 +/- 7.00 pg/ml, respectively, all P0.01). Importantly, plasma levels of U-II and ET-1 were correlated in patients with CHD (r = 0.64, P = 0.01). On day 1 after PTCA and stent therapy, plasma levels of ET-1 and U-II were significantly higher, by 99% and 25%, respectively, than those before therapy (all P0.01). On day 3 after therapy, ET-1 levels were higher by 25% (P0.01) than before therapy, and U-II levels decreased rapidly and were close to baseline levels (P0.05). On day 7 after therapy, CHD patients had significantly lower ET-1 and U-II levels than before therapy (all P0.01). Since ET-1 and U-II levels may be increased in plasma of patients with CHD, their activation might have clinical significance in terms of early intervention in patients with CHD, especially after PTCA and stent therapy.

Urotensin II (UII) is a potent vasoconstrictive peptide; however, its significance in vascular adventitia has not been clearly elucidated. In this study, rat aortic adventitia showed mRNA expression and immunoreactivity of UII and its receptor (UT). Moreover, radioligand-binding assay showed that maximum binding capacity (Bmax) of [(125)I]-UII was higher in adventitia than in media (28.60+/-1.94 vs. 20.21+/-1.11 fmol/mg, P0.01), with no difference in binding affinity (dissociation constant [Kd] 4.27+/-0.49 vs. 4.60+/-0.40 nM, P0.05). Furthermore, in cultured adventitial fibroblasts, UII stimulated DNA synthesis, collagen synthesis and secretion in a concentration-dependent manner. These effects were inhibited by the UII receptor antagonist urantide (10(-6) mol/l), Ca(2+) channel blocker nicardipine (10(-5) mol/l), protein kinase C inhibitor H7 (10(-6) mol/l), and mitogen-activated protein kinase inhibitor PD98059 (10(-6) mol/l) but not the phosphatidyl inositol-3 kinase inhibitor wortmannin (10(-7) mol/l). UII may act as an autocrine/paracrine factor through its receptor and the Ca(2+) channel, protein kinase C, and mitogen-activated protein kinase signal transduction pathways, in the pathogenesis of vascular remodeling by activating vascular adventitia.

As a novel vasoactive peptide, plasma coupling factor 6 (CF6) was shown to be elevated in patients with diabetes mellitus, yet the mechanism involved is unknown. We studied CF6 protein release and its potential mechanism in human umbilical vein endothelial cells (HUVECs) incubated with high glucose levels. High glucose level enhanced CF6 expression and peptide secretion in HUVECs in a time- and concentration-dependent manner, which was independent of increased osmolarity. PKC or p38 MAPK inhibition significantly suppressed high glucose-mediated CF6 release in HUVECs, and the inhibition rate was -45% and -30%, respectively. Also, high glucose-induced CF6 production was antagonized by insulin treatment. Hence, high glucose increases the expression and secretion of CF6 in endothelial cells and appears to be mediated by PKC and p38 MAPK activity.

Coupling factor 6 (CF6) is a novel endogenous inhibitor of prostacyclin. Plasma CF6 and 6-keto-PGF(1a) were measured by radioimmunoassay in 70 consecutively recruited patients with type 2 diabetes and in 56 healthy controls. A significantly increased plasma CF6 level was found in diabetics compared with controls. The CF6 level was inversely correlated with plasma 6-keto-PGF(1a) level and positively correlated with blood glucose and lipids. These results suggest that CF6 might be an obvious marker of impaired endothelium and might contribute to vascular damage in diabetes.

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.

Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family identified from human and other vertebrate tissues. Preprointermedin can generate various mature peptides by proteolytic cleavage. Amino acid sequence analysis showed cleavage sites located between two basic amino acids at Arg93-Arg94 resulting in the production of prepro-IMD(95-147), namely IMD(1-53). The present study was designed to determine the effects of the IMD(1-53) fragment in the central nervous system (CNS) on mean arterial blood pressure and heart rate in normal rats and its possible mechanism. Rats were given doses of adrenomedullin (ADM) or IMD(1-53), intracerebroventricularly or intravenously, respectively, with continuous blood pressure and heart rate monitoring for 45min. Analysis with CGRP receptor antagonist CGRP(8-37), ADM receptor antagonist ADM(22-52), and anti-prepro-IMD antibody showed that 0.1, 0.5, and 1.0 nmol/kg IMD(1-53), caused a dose-dependent elevation in blood pressure, which was more prominent than the increase with equivalent IMD(1-47) or ADM. As well, IMD(1-53) caused a persistent increase in heart rate. The CNS action of IMD(1-53) could be blocked by ADM(22-52), CGRP(8-37), or prepro-IMD antibody. In contrast to the CNS action, intravenous administration of IMD(1-53) induced a depressor effect. These results suggest that IMD(1-53) is an important regulatory factor in mean arterial blood pressure and heart rate through its central and peripheral bioaction.

Previous studies have shown that mitochondrial coupling factor 6 (CF6) is an endogenous peptide that inhibits prostacyclin (PGI2) synthesis in vascular endothelial cells. In this study, we measured the plasma CF6 level of patients with acute myocardial infarction (AMI) to observe dynamic changes of CF6. All patients showed elevated plasma CF6 levels upon admission for treatment of AMI. Their CF6 levels peaked approximately 72 h after the onset of AMI and remained high for 7 days. At 7 days, their CF6 levels decreased to the level seen upon admission, but not to within a normal range. Hyperlipidemic patients had significantly greater CF6 levels at 24 h after onset of AMI than patients with a normal lipid profile. On admission, the plasma CF6 level in patients with a cardiac function of Killip class ≥II was higher than that in patients with a Killip class I cardiac function. At 3 days after the onset of AMI, the plasma CF6 levels of patients with a creatinine kinase (CK) peak value ≥1,500 units/l were significantly higher than those of patients with a CK peak value

In this work, we aimed to observe the changes in adrenomedullin (ADM) and its receptor-calcitonin receptor-like receptor (CL), receptor activity-modifying protein (RAMP) 1, RAMP2 and RAMP3-in cardiac ventricles and aortas of hypertensive rats, and the responsiveness of injured cardiovascular tissue to ADM, then to illustrate the protective mechanism of ADM on the cardiovascular system. Male SD rats were subjected to treatment with chronic N(G)-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase. The ADM contents and cAMP production in myocardia and aortas were measured by RIA. The mRNA levels of ADM, CL, and RAMP1-3 were determined by RT-PCR. L-NNA induced severe hypertension and cardiomegaly. The ir-ADM content in plasma, ventricles and aortas in L-NNA-treated animals increased by 80%, 72% and 57% (all p

Adrenomedullin (ADM) is a multifunctional peptide with important roles in the cardiovascular system, especially in the adjustment of cardiovascular and renal homeostasis. ADM is present in plasma, organs and tissues, and its activity increases during hypertension. It remains unknown whether the clearance of this peptide is altered during hypertension. Neutral endopeptidase (NEP) is the major enzyme in ADM's degradation. We observed the activity and distribution of NEP and the expression of its mRNA in the plasma, cardiac ventricle, aorta, jejunum and kidney of spontaneously hypertensive rats (SHRs) in order to study the possible role of NEP in elevating tissue ADM concentrations during hypertension. ADM and NEP were diffuse in all tissues studied. The level of tissue ADM was generally higher in SHR tissues than in control tissues, except in the renal medulla, and its mRNA expression was higher in all tissues. Plasma NEP activity, general NEP activity and the expression of NEP mRNA in the left ventricle, aorta and jejunum in SHRs was lower than that of controls, and the level of ADM was inversely correlated with NEP activity. NEP activity and mRNA and protein expression in SHR kidneys were higher than in control kidneys; moreover, the ADM content was positively correlated with NEP activity in the renal cortex. NEP activity in the lung of SHRs did not differ from that of controls. Thus, in SHRs, the local concentration and action of ADM in the tissues may be differentially regulated by NEP.

Hydrogen sulfide (H2S), which was considered as a novel gasotransmitter, is produced endogenously from L-cysteine in mammalian brain and vessels, and might be a physiological function regulator to these organs. Here, we showed that mRNA for H2S producing enzyme, cystathionine gamma-lyase, was expressed in myocardial tissues and H2S could endogenously be produced in myocardial tissues. Negative inotropic effect of H2S was proved in present study in vitro and in vivo experiments, and the effect could partly be blocked by glibenclamide, a KATP channel blocker. An intravenous bolus injection of NaHS provoked a decrease in central venous pressure. The present findings suggested that H2S could be endogenously produced by heart tissues, as a physiological cardiac function regulator, mediated by KATP channel pathway.

Lysophosphatidic acid (LPA) is a bioactive phospholipid having growth factor-like activity on fibroblasts and is involved in cardiovascular diseases such as hypertension and heart failure by inducing vascular remodeling, characterized by fibroblast proliferation and migration in adventitia. Among various bioactive factors that LPA works with, adrenomedullin (ADM) is a multiple functional peptide with an important cytoprotective effect against cardiovascular damage. We studied rat aortic adventitia to explore the possible paracrine/autocrine interaction between endogenous ADM and LPA. LPA stimulation of the adventitia to secrete ADM and express its mRNA was concentration dependent. ADM inhibited LPA-induced proliferation in adventitial cells and attenuated the activity of mitogen-activated protein kinase (MAPK) stimulated by LPA. In contrast, treatment with specific antagonists of the ADM receptor potentiated the LPA-induced proliferation in adventitial cells. We concluded that LPA stimulates the adventitia to produce and secrete ADM, which in turn regulates the vascular biological effects of LPA.

Adrenomedullin (ADM), a multifunction peptide with important roles in regulating cardiovascular homeostasis, has the vasodilatory properties and is of particular interest in the pathophysiology of sepsis. ADM levels in plasma and tissues are regulated by the proteolysis of neutral endopeptidase (NEP), the major enzyme of ADM degradation. We observed the NEP activity in the plasma, the activity and distribution of NEP and its mRNA expression in the tissues of rats in septic shock to study the possible role of NEP in elevating tissue ADM concentration during sepsis. ADM level increases progressively during sepsis except in the jejunum. Rats in early phase of shock (ES) showed diverse changes in tissue NEP activity. Plasma NEP activity, tissue NEP activity and its protein and mRNA expression in the left ventricle, aorta, jejunum and lung in the late phase of shock (LS) rats were lower than those in ES and the control, but no statistical change of NEP activity in the kidney was observed. The level of ADM was inversely correlated with NEP activity in the plasma, ventricle and aorta and positively correlated with NEP activity in the jejunum. Thus, in sepsis, the local concentration and action of ADM in tissues may be differentially regulated by NEP.

Cultured rat cardiomyocytes were treated with 10, 20, and 30 mmol/L glucose and 30 mmol/L glucose plus protein kinase C (PKC) inhibitor, Chelerythrine. In the 20 and 30 mmol/L glucose-treated cells, taurine contents reduced by 15% and 27% (P < .05), respectively, taurine transporter (TAUT) mRNA levels reduced by 47% and 64% (P < .05), respectively, and cysteine sulfinate decarboxylase (CSD) mRNA reduced slightly, but not significantly. Time-dependent taurine uptakes reduced in the 10, 20, and 30 mmol/L glucose-treated cells, and time-dependent taurine release reduced in the 30 mmol/L glucose-treated cells. The Vmax of taurine transport decreased by 18%, 30%, and 35% (P < .05) in the 10, 20, and 30 mmol/L glucose-treated cells, respectively, while Km of taurine transport remained unchanged. When PKC inhibitor, Chelerythrine, combined with 30 mmol/L glucose was treated with the cells, the lowered taurine content, taurine uptake, taurine release, and Vmax of taurine transport caused by 30 mmol/L glucose were eliminated. These results demonstrate that high glucose considerably and specifically decreases intracellular taurine content, taurine transport activity, and TAUT mRNA, possibly through PKC-mediated transcriptional and posttranslational pathways.

The aim of the present study was to investigate the changes in heme-oxygenase (HO)-carbon monoxide (CO)-cyclic guanosine monophosphate (cGMP) pathway in clacified rat vascular smooth muscle cells (VSMCs).Calcification of cultured rat VSMCs was induced by incubation of VSMCs with beta-glycerophosphate. Cellular calcium content, ALP activities and (45)Ca uptake were measured. HO activity, HbCO formation and content of cGMP in VSMCs were determined. Immunocytochemistry for HO-1 expression was observed.In comparison of control VSMCs, the cellular calcium content, ALP activity and (45)Ca uptake in calcified VSMCs were obviously increased. Immunocytochemistry showed that HO-1 expression was weak and not well distributed in calcified cells as compared to non-calcified VSMCs, but interestingly, there was stronger staining in calcified nodules than in VSMCs. Compared with VSMCs, HO-1 activity in calcified cells decreased by 42.7% [36.4 +/- 2.8 pmol (mg Pr x h)(-1) vs 63.5 x 5.3 pmol (mg Pr x h)(-1), p0.01], and HbCO formation decreased by 39.2% (3.38 x 0.69 micromol/mg Pr vs 5.56 +/- 0.48 micromol/mg Pr, p0.05). The cGMP content in calcified VSMCs was 78.1% lower than that of non-calcified VSMCs (4.3 +/- 0.51 vs 19.6 +/- 1.2 pmol/mg, p0.01).The results showed that HO-CO-cGMP pathway in calcified vascular cells obviously changed, which might contribute to disturbance of vascular function.

The aim of this study was to observe the effects of adrenomedullin (ADM) on endothelin (ET) production induced by urotensin II (U II) in rat vascular smooth muscle cells (VSMCs). Cultured VSMCs which were incubated with U II (10−8 mol/L) and with various concentrations of ADM were used to measure the VSMCs 3H-TdR incorporation, the activity of extracellular signal-regulated kinase (ERK), the amount of ET mRNA and ET production in VSMCs. In this work we found that incubation with U II (10−8 mol/L) increased obviously the amount of ET mRNA in VSMCs and ET production in medium, however, co-incubation with ADM (10−10– 10−8 mol/L) and U II(10−8 mol/L) reduced the amount of ET mRNA by 15%, 24% and 45% (P 0.05), 32% (P 0.05), 32% (P < 0.05) and 36% (P < 0.05), respectively, in a concentration-dependent manner. The results show that in cultured VSMCs ADM inhibits ET mRNA expression, ET production and proliferation stimulated by U II, and that inhibitory effect of ADM on U II bioaction could be mediated through inhibiting MAPK pathway.