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Vascular calcification (VC) is thought to be an independent predictor of cardiovascular morbidity and mortality. IntermedinApoEAs compared to the high-fat diet alone, Hcy treatment significantly increased atherosclerotic lesion areas and the number of calcified nodules in aortic roots and was reduced by IMD infusion or 4-phenylbutyric acid (PBA) treatment. In vitro, as compared to calcifying medium alone, Hcy treatment further increased alkaline phosphatase activity, calcium content, and calcium nodule number in human aorta vascular smooth muscle cells (HA-VSMCs), all blocked by IMD or PBA pretreatment. Mechanistically, IMD or PBA significantly alleviated endoplasmic reticulum stress (ERS) activation compared with Hcy treatment. In parallel, IMD or PBA attenuated the messenger RNA levels of osteogenic markers and inflammatory cytokines in aortas and their protein levels in lesions of aortic roots. In vitro, Hcy treatment significantly increased the protein levels of osteoblast-like cell markers in primary rat VSMCs and inflammation markers in mouse peritoneal macrophages, all decreased with IMD or PBA pretreatment. IntermedinIntermedin

Vascular calcification is a common phenomenon in older adults. Intermedin (IMD) is a cardiovascular bioactive peptide inhibiting vascular calcification. In this study, we aimed to investigate whether IMD1-53 attenuates aging-associated vascular calcification. Vascular calcification was induced by vitamin D3 plus nicotine (VDN) in young and old rats. The calcification in aortas was more severe in old rats treated with VDN than young control rats, and IMD expression was lower. Exogenous administration of IMD1-53 significantly inhibited the calcium deposition in aortas and the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) in VDN-treated old rats. Moreover, levels of aging-related p16, p21 and β-galactosidase were all greatly decreased by IMD1-53. These results were further confirmed in rat and human VSMCs in vitro. In addition, IMD-deficient mouse VSMCs showed senescence features coinciding with osteogenic transition as compared with wild-type mouse VSMCs. Mechanistically, IMD1-53 significantly increased the expression of the anti-aging factor sirtuin 1 (sirt1); the inhibitory effects of IMD1-53 on calcification and senescence were blocked by sirt1 knockdown. Furthermore, preincubation with inhibitors of PI3K, AMPK or PKA efficiently blunted the upregulatory effect of IMD1-53 on sirt1. Consequently, IMD1-53 could attenuate aging-associated vascular calcification by upregulating sirt1 via activating PI3K/Akt, AMPK and cAMP/PKA signaling.

Our previous work reported that endoplasmic reticulum stress (ERS)-mediated apoptosis was activated during vascular calcification (VC). Activating transcription factor 4 (ATF4) is a critical transcription factor in osteoblastogenesis and ERS-induced apoptosis. However, whether ATF4 is involved in ERS-mediated apoptosis contributing to VC remains unclear. In the present study, in vivo VC was induced in rats by administering vitamin D3 plus nicotine. Vascular smooth muscle cell (VSMC) calcification in vitro was induced by incubation in calcifying media containing β-glycerophosphate and CaCl2. ERS inhibitors taurine or 4-phenylbutyric acid attenuated ERS and VSMC apoptosis in calcified rat arteries, reduced calcification and retarded the VSMC contractile phenotype transforming into an osteoblast-like phenotype in vivo. Inhibition of ERS retarded the VSMC phenotypic transition into an osteoblast-like cell phenotype and reduced VSMC calcification and apoptosis in vitro. Interestingly, ATF4 was activated in calcified aortas and calcified VSMCs in vitro. ATF4 knockdown attenuated ERS-induced apoptosis in calcified VSMCs. ATF4 deficiency blocked VSMC calcification and negatively regulated the osteoblast phenotypic transition of VSMCs in vitro. Our results demonstrate that ATF4 was involved at least in part in the process of ERS-mediated apoptosis contributing to VC.

Intermedin (IMD) plays an important regulatory role in cardiovascular function. We aimed to explore the protein expression of IMD and its receptors, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs), and the role of endogenous IMD in myocardial ischemia/reperfusion (I/R) injury in rats. The rat model of I/R was created by ligating cardiac left anterior descending artery. Western blot was used to determine protein expression of CRLR and RAMPs, and radioimmunoassay was used to detect IMD content. Compared with control, protein levels of CRLR and RAMPs in both ischemic and nonischemic region were upregulated at different stages of reperfusion. IMD protein content in nonischemic area myocardium also increased. However, IMD protein content in ischemic area downregulated at 3-, 6-, and 12-h reperfusion. In hypoxia/reoxygenation model of neonatal cardiomyocytes, IMD attenuated myocyte injury, and IMD receptor antagonist IMD17-47 aggravated myocyte impairment by blocking endogenous IMD. In conclusion, the downregulation of IMD at early stage of reperfusion might augment myocardium injury.

The main objective of this study is to test the hypothesis that N-methyl-D-aspartate (NMDA) receptors within the rostral ventrolateral medulla (RVLM) are involved in the inhibition of clonidine on the RVLM presympathetic neurons. Totally, 22 presympathetic neurons were recorded in anesthetized and paralyzed rats. The majority of these neurons (n=16 of 22) were significantly inhibited by iontophoretic (30 nA) clonidine, the other 6 neurons were insensitive to clonidine. In seven clonidine-sensitive neurons, iontophoretic clonidine (30 nA) antagonized the neuronal excitation of iontophoretic NMDA receptor agonist NMDA (20 nA). In remaining nine clonidine-sensitive neurons, iontophoretic NMDA receptor antagonist MK801 (60 nA) significantly attenuated the neuronal inhibition of iontophoretic (30 nA) clonidine. In conclusion, these results suggest that NMDA receptors contribute to the inhibition of clonidine on the RVLM presympathetic neurons.

Although it is recognized that imidazoline receptors play an important role in the central regulation of cardiovascular activities, little is known about their role in the caudal ventrolateral medulla. In male Sprague-Dawley rats anesthetized with urethane, we used antagonists of I1-imidazoline receptor or alpha2-adrenoceptor to assess the function of these receptors in the caudal ventrolateral medulla in controlling the cardiovascular effects of clonidine. Unilateral microinjection of clonidine (6 nmol/50 nl) into the caudal ventrolateral medulla significantly (P

To observe the vasodilating effect of adrenomedullin 2 (ADM2) by antagonizing angiotensin 1 (Ang II), and to explore its mechanism.Eighteen male, 180-200 g SD rats were randomly divided into 3 groups (n = 6): control group, Ang II (150 ng/(kg x min)) group and Ang II (150 ng/(kg x min)) + ADM2(500 ng/(kg x h)) group. Mini-osmotic pumps filled with peptide were implanted in the back of rats subcutaneously. After two weeks, the blood pressure was measured by the way of carotid intubation. The plasma was collected for the detection of nitric oxide (NO) content and the activity of endothelial nitric oxide synthase (eNOS). The in situ oxidation of fluorescent dye dihydroethidium (DHE) was used for detecting superoxide in rat arteries. The rat isolated arterial rings were made for studying the vasodilating effect of ADM2. Human umbilical vein endothelial cell line EA. hy 926 cells were cultured and their intracellular reactive oxygen species (ROS) were evaluated by probe DCFH-DA.ADM2 dramatically decreased the blood pressure in angiotensin II-induced hypertension rat model, enhanced plasma NO content and the activity of eNOS and reduced superoxide formation in vessel walls. ADM2 also induced relaxation of the vascular rings preconstricted by Ang II in a concentration-dependent and endothelium-dependent manner. In cultured vascular endothelium, ADM2 ameliorated the ROS generation induced by Ang II.Adrenomedullin 2 relaxed blood vessels by antagonizing angiotensin II-induced oxidative stress and improving the vascular endothelial function.

Skeletal muscle has recently been recognized as an endocrine organ that can express, synthesize and secrete a variety of bioactive molecules which exert significant regulatory effects. Hydrogen sulfide (H2S) is endogenously produced in mammalian tissues and participates in a number of physiological and pathophysiological processes. We aimed to verify whether H2S could be endogenously generated and released by rat skeletal muscle, and determine the biological effects of H2S in rat skeletal muscle.The study was divided into two parts: detection of endogenous H2S generation and release in rat skeletal muscle and determination of antioxidative activity of skeletal muscle-derived H2S. H2S content and production in tissues were detected by sensitive sulfur electrode method. The expressions of H2S producing enzymes cystathionine β-synthase, cystathionine γ-lyase and mercaptopyruvate sulfurtransferase were detected by real-time PCR and western blotting and their tissue distributions were observed by immunohistochemical and immunofluorescent analysis. Rat skeletal muscular ischemia-reperfusion (I-R) injury model was created and evaluated by histological analysis under microscope. The malondialdehyde (MDA) contents, hydrogen peroxide levels, superoxide anion and superoxide dismutase (SOD) activities were detected using spectrophotometer.H2S could be endogenously generated and released by skeletal muscle of Sprague-Dawley rats (H2S content: (2.06 ± 0.43) nmol/mg; H2S production: (0.17 ± 0.06) nmol×min(-1)×mg(-1)). Gene and protein expressions of the three H2S producing enzymes were detected in skeletal muscle, as well as the liver and kidney. Endogenous H2S content and production were decreased in skeletal muscles of rats with I-R skeletal muscle injury (P0.05). Furthermore, H2S significantly protected rat skeletal muscle against I-R injury and resulted in decreased MDA content, reduced hydrogen peroxide and superoxide anion levels, but increased SOD activity and protein expression in skeletal muscles (all P0.01).H2S generation pathway exists in rat skeletal muscle and it acts as an antioxidant in skeletal muscle.

In past decade, hydrogen sulfide (H₂S) as a novel gasotransmitter, covered many fields in biological and medical research. However, there is no effective, convenient and high-throughput method for determination of circulatory H₂S until now. Here, we aim to develop an easy method for measurement of circulatory H₂S by modified methylene blue method. In the present study, we added Zn²⁺ to plasma sample to deposit H₂S, HS⁻ and S²⁻, as well as plasma protein, then used NaOH to re-dissolve plasma protein. ZnS deposition was re-dissolved by the addition of N, N-dimethyl-p-phenylenediamine, and the remnant protein was deposited by trichloroacetic acid. After centrifugation, ferriammonium sulfate was added to the supernatant fluid to generate methylene blue, which was analyzed by spectrophotometer at 665 nm. Using the present method, we found that most ions including sulfate and thiosulfate did not affect detection of H₂S concentration, but albumin (physiological concentration) reduced the detection value, which suggested the binding of serum albumin and a certain amount of H₂S. The relative recovery ratio of present method is 81.9%, which implies that the method is relative accurate for the determination of H₂S concentration in plasma or serum. H₂S levels of frozen plasma samples from 65 healthy volunteers detected by the present method were (13.93 ± 4.98) µmol/L. There was no obvious difference between the detection values of fresh and frozen samples from the same SD rats. These results suggest the modified methylene blue assay is stable, sensitive, convenient and high-throughput. The method can be used to analyze the circulatory H₂S in clinical and basic research.

Endogenous hydrogen sulfide is a new neuromodulator which takes part in the regulation of central nervous system physiology and diseases. Whether endogenous hydrogen sulfide in the central nervous system regulates cardiovascular activity is not known. In the present study, we observed the hemodynamic changes of hydrogen sulfide or its precursor by intracerebroventricular injection, and investigate the possible roles of endogenous digitalis like factors and sympathetic activity in the regulation.Ninety-four Sprague-Dawley rats underwent a right cerebroventricular puncture, then the hydrogen sulfide saturation buffer or its precursor injected by intrcerebroventricular catheter. A heperin-filled catheter was inserted into the right femoral artery or into the left ventricle, and changes of blood pressure or cardiac function recorded by a Powerlab/4S instrument. Phentolamine or metoprolol were pre-injected to observe the possible role in autonomic nerve activity. After rats were sacrificed, plasma was collected and endogenous digitalis-like factors were measured with a commercial radioimmunoassay kit. The aortic, cardiac sarcolemmal vesicles were isolated and the activity of Na(+)-K(+)-ATPase was measured as ouabain-sensitive ATP hydrolysis under maximal velocity conditions by measuring the release of inorganic phosphate from ATP. Unpaired Student's t test for two groups or analysis of variances (ANOVA) for multiple groups were used to compare the differences of the changes.Intracerebroventricular injection of hydrogen sulfide induced a transient hypotension, then dramatic hypertenive effects in a dose-dependent manner. Bolus injection of L-cysteine or beta- mercaptopyruvate also increased mean arterial pressure (P0.01), whereas hydroxylamine-a cystathionine beta synthase inhibitor decreased the arterial pressure (P0.01). Hydrogen sulfide and L-cysteine increased mean arterial pressure, left ventricular develop pressure and left-ventricle maximal rate of systolic and diastolic pressure; these functions were decreased by hydroxylamine (P0.01). Glibenclamide (a K(ATP) channel blocker) blocked the transient hypotensive effect, phentolamine (an alpha-adrenergic receptor blocker) blocked the hypertensive effect, and metoprolol (a selective beta 1 receptor blocker) blocked the positive inoptropic effect of central nervous system hydrogen sulfide. The endogenous digitalis-like factors in plasma were elevated (P0.01) after treatment with L-cysteine, association with decreasing Na(+)-K(+)-ATPase activity in cardiac or aortic sarcolemmal vesicles (P0.01). Hydroxylamine injection reduced the endogenous digitalis-like factors level in plasma association with increasing Na(+)-K(+)-ATPase activity in cardiac and aortic sarcolemmal vesicles.Central nervous system endogenous hydrogen sulfide upregulated mean arterial pressure and cardiac systolic function by activation of sympathetic nerves or release of endogenous digitalis-like factors.

Osteogenic differentiation of vascular smooth muscle cells (VSMCs) results in medial artery calcification, which is common in diabetes, but the pathogenesis is poorly understood. We aimed to explore the pathophysiological roles of insulin resistance (IR) on medial artery calcification in rats with 10% fructose in drinking water. After 12 weeks of fructose feeding, rats showed severe IR, with increased levels of fasting blood glucose, serum insulin and oral glucose tolerance test (OGTT). Fructose-fed rats showed aortic calcification, increased aortic calcium deposition and irregular elastic fibers in the medial layer of the vessel wall. Moreover, plasma phosphorus concentration, calcium × phosphorus product and alkaline phosphatase (ALP) activity, and aortic calcium content and ALP activity were significantly increased. Fructose feeding increased mRNA levels of osteopontin, type III sodium-dependent phosphate co-transporter, bone morphogenetic protein-2 and the key transcription factor core binding factor alpha 1 in aortic tissue and downregulated mRNA levels of osteoprotegerin and matrix γ-carboxyglutamic acid protein. Fructose feeding decreased protein levels of smooth-muscle lineage markers and induced severe lipid peroxidation injury. IR induced by high fructose feeding could evoke osteogenic transdifferentiation of VSMCs and promote vascular calcification.

Angiotensin II (Ang II) is an important regulator of cardiac function and injury in hypertension. The novel Ang IV peptide/AT4 receptor system has been implicated in several physiological functions and has some effects opposite to those of Ang II. However, little is known about the role of this system in Ang II-induced cardiac injury. Here we studied the effect of Ang IV on Ang II-induced cardiac dysfunction and injury using isolated rat hearts, neonatal cardiomyocytes and cardiac fibroblasts. We found that Ang IV significantly improved Ang II-induced cardiac dysfunction and injury in the isolated heart in response to ischemia/reperfusion (I/R). Moreover, Ang IV inhibited Ang II-induced cardiac cell apoptosis, cardiomyocyte hypertrophy, and proliferation and collagen synthesis of cardiac fibroblasts; these effects were mediated through the AT4 receptor as confirmed by siRNA knockdown. These findings suggest that Ang IV may have a protective effect on Ang II-induced cardiac injury and dysfunction and may be a novel therapeutic target for hypertensive heart disease.

1. Sulfur dioxide (SO(2) ) has recently been found to have various biological effects on the cardiovascular system. The present study was designed to explore the effects of SO(2) derivatives on the L-type calcium current (I (Ca, L) ) in isolated rat ventricular cardiomyocytes. 2. A Langendorf system was used to dissociate single ventricular cells. SO(2) derivatives from 5 to 1000 μmol/L were incubated with cardiomyocytes. The whole-cell patch-clamp technique was used to record I (Ca, L) . The effect of SO(2) derivatives on intracellular calcium concentration ([Ca(2+) ](i) ) was detected by confocal microscopy. 3. Concentrations of 5 or 10 μmol/L SO(2) derivatives could not change I (Ca, L) evoked by a single pulse from -40 to 0 mV for 200 ms in rat ventricular cardiomyocytes; however, 50, 100, 500 or 1000 μmol/L SO(2) derivatives could depress the peak amplitudes of calcium currents in 6 min, and the I (Ca, L) was attenuated by 13.19%, 16.59%, 21.23% and 24.72%, respectively, as compared with corresponding controls (P0.05). The 50, 100, 500 or 1000 μmol/L SO(2) derivatives also depressed the peak I-V curves, without altering the reversal potential and the voltage dependence of the peak I (Ca, L) . Therefore, 1000 μmol/L SO(2) derivatives could reduce [Ca(2+) ](i) in cardiomyocytes. 4. The results of the present study suggest that SO(2) derivatives can depress I (Ca, L) in cardiomyocytes, which might have a protective effect in cardiovascular diseases.

To study human urotensin II (hUII) expression in intrapulmonary arteries of rats with pulmonary hypertension induced by high pulmonary blood flow and explore the role of hU II in the development of pulmonary hypertension induced by left to right shunt.Aortocaval shunting was produced for 11 weeks in rats. Pulmonary artery mean pressure (PAMP) of each rat was evaluated using right cardiac catheterization. The pulmonary vascular structural changes, including the percentage of muscularized arteries of small pulmonary vessels and relative medial thickness of intra-acinar pulmonary arteries were examined. Meanwhile, the expression of hU II by pulmonary arteries was detected by immunohistochemistry.After 11-week aortocaval shunting, PAMP was significantly increased. The percentage of muscularized arteries of small pulmonary vessels and relative medial thickness of pulmonary arteries were obviously increased in shunting rats compared with controls (P0.01, respectively). Meanwhile, hU II expression by pulmonary artery endothelial cells and smooth muscle cells was significantly augmented in rats of shunt group, which was positively correlated with PAMP and the structural changes in pulmonary arteries.The up-regulation of hU II in pulmonary arteries might be involved in the development of pulmonary vascular structural remodeling and pulmonary hypertension induced by high pulmonary blood flow.

To investigate the changes and probable roles of adrenomedullin2/intermedin (AIDM2/IMD), a novel micromolecular bioactive peptide, in the lungs of rats with chronic hypoxic pulmonary hypertension.Twenty male SD rats were randomly divided into normal control group (NC) and normobaric hypoxia group (4H). The protein levels of ADM and ADM2/IMD) in the plasma and lung were measured by radioimmunoassay and immunohistochemistry. The mRNA expressions of ADM, ADM2/IMD and their receptors C (RLR, RAMP1, RAMP2 and RAMP3 in the lung tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR).(1) The rat model of chronic pulmonary hypertension was confirmed by the increased mean pulmonary arterial pressure (mPAP) and weight ratio of right ventricle to left ventricle plus septum [RV/(LV + S)] in 4H group compared to NC group. (2) The concentrations of ADM in the plasma and lung homogenate of 4H group were 2.3 and 3.2 folds of NC group, respectively (all P0.01). The levels of ADM2/IMD were higher 89.6% and 45.0% in the plasma and lung homogenate of 4H group than those of NC group (respectively, P0.01, P0.05). (3) The mRNA expressions of ADM2/IMD and ADM in the lung of 4H group were up-regulated (respectively, P0.01, P0.05 vs. NC group). The expressions of CRLR and RAMP1 mRNAs were down-regulated (all P0.01 vs. NC group), while the levels of RAMP2 and RAMP3 mRNAs were no significant difference between the two groups. (4) The strong ADM2/IMD immunostaining was detected in the endothelial and adventitial cells of the rat pulmonary arteriole.ADM2/IMD, like its paralog ADM, might be closely related to the chronic hypoxic pulmonary hypertension in rats. The disorders of the gene expression and/or the synthesis and metabolism of ADM2/IMD and its receptor CRLR/RAMP1 possibly take part in the pathogenesis of chronic hypoxic pulmonary hypertension in rats.

The characteristics of ryanodine receptor in rat cardiac sarcoplasmic reticulum (SR) and nuclear envelope (NE) were studied.Velocity and isopyknic gradient centrifugation was employed to fractionate rat SR and NE. Ryanodine receptor was assayed with [3H] ryanodine saturate binding to the preparations.The maximal binding (Bmax) and dissociating constant (Kd) of ryanodine receptor in rat cardiac NE were, 1.7% and 60% of those in SR respectively. Phosphorylation in vitro by PKA and PKC increased Bmax of the receptors in SR by 372% and 121%, and augmented those in NE by 221% and 306%, without any effects on Kd.Ryanodine receptors were present in rat myocardial NE, with lower density and higher affinity than those located in SR, which can be activated by PKA and PKC.

To explore the time-dependent changes of endogenous hydrogen sulfide system at the early stage of pulmonary hypertension induced by high pulmonary flow in rats.Eighty male SD rats, whose weight ranged 140 - 160 g, were randomly divided into control group (n = 40) and shunt group (n = 40). Rats in shunt group were subjected to an abdominal aorta-inferior vena cava shunt to create an animal model of high pulmonary flow. After 1 d, 3 d, 1 week, 4 week and 8 weeks of experiment, systolic pulmonary artery pressure (SPAP) of each rat, the H2S of rat lung tissue and CSEmRNA of rat lung tissue were evaluated, respectively.SPAP increased significantly as compared with those in control group in 1 week and 8 weeks of experiment. In contrast to control group, the H2S of rat lung tissue increased significantly on 3 d and in 4 weeks, respectively. Meanwhile, in contrast to control group, relative amount of CSE mRNA of lung tissues elevated significantly on 3 d and in 4 weeks, respectively. Moreover, SPAP and the H2S of rat lung tissue, the CSE mRNA of rat lung tissue correlated negatively in 1 week, 4 weeks and 8 weeks of experiment.Animal model of rats with high pulmonary blood flow exhibited pulmonary hypertension. Lung tissue H2S and CSE mRNA of rats exhibited double peaks within 8 weeks. These results revealed that endogenous H2S system might be relevant with the development of pulmonary hypertension induced by high pulmonary blood flow, and probably, it played a protective role in the regulation of pulmonary hypertension, especially, at its early stage.

To investigate the effects of human urotensin II (hUII) on in vivo pia mater microcirculation in rats.Adult SD rats were randomly assigned to the following groups: control, sodium chloride injection (NS), UII(10(-6) mol/L), noradrenaline (NA, 10(-6) mol/L), and UII (10(-6) mol/L) + NA (10(-6) mol/L) groups. For recording of microcirculation images in pia mater, skull windows were performed and mounted on the stage of an intravital microscope equipped with a TV camera. Video images of microcirculation were stored by a video cassette recorder. Temporal changes in internal diameter and microcirculatory velocity of microvessels were measured by computer using the Image Pro software. The blood flow in cerebral tissues were measured with PIMII laser Doppler perfusion Imager (Lisca, Sweden).The internal diameters of arterioles and venules in control group were (35.4 +/- 3.6) microm and (40.6 +/- 8.5) microm, respectively. In UII group, the arterioles and venules contracted immediately after treated with UII and up to the peak at 1 min, the internal diameters of arterioles and venules were (25.6 +/- 3.4) microm and (23.4 +/- 3.3) microm, respectively (P0.05). Both microcirculatory velocity in arterioles and venules had no significant changes in UII group (P0.05). The blood flow in meninges increased 1 min after treated with UII and up to high peak at 5 min (3.5 +/- 0.4 perfusion unit vs. control 2.3 +/- 0.6, P0.05).hUII can contract microvessels in pia mater of rats and increase microcirculatory blood perfusion to cerebral tissue involved.

To observe the effect of angiotensin-converting enzyme inhibitors (ACEI) and aldosterone receptor blockers on cardiac function to explore the mechanism of cardiac function descending and myocardial injury in calcium-overload rats.Calcium-overload in rat was induced by administration of Vitamin D3 plus nicotine. To Estimate the extent of calcium-overload by calcium content. Angiotension II and aldosterone levels in the myocardia were measured by radioimmunoassay. Cardiac function (+/- LVdp/dt, LVESP and LVEDP) were measured by Powerlab. The malondialdehyde (MDA) content, activities of lactate dehydrogenase (LDH) and creatine kinase (CPK) were measured by biochemistry.Calcium content increased by 3.2-, 5.8 -fold in myocardial and artery, compared with controls. VDN-treated survivors showed lower + LVdp/dt(max) and -LVdp/dt(max) values, by 27% and 34%, respectively (both P0.01). Higher LVESP, and LVEDP by 42 % and 32% (P0.01); heart rate and mean arterial pressure were not significantly altered (P0.05). The lipid peroxidation products MDA and conjugated diene in myocardia were increased 22% (P0.01), 68% (P0.05) (P0.05), respectively. The plasma activity of CPK and LDH was greatly increased by 4.5-and 3.1-fold (P0.01), respectively. ACEI and spironolactone obviously relieved degree of calcium-overload and improved cardiac function and myocardial injury(P0.01). Calcium content in myocardia and artery was lower 44%, 39% and 57%, 34%. Lower MDA by 20%, 30%, lower conjugated diene by 44%, 35% than calcium-overload group. The plasma activity of CPK and LDH were obviously decreased 28%, 34% and 20%, 27%, compared with calcium-overload group.Calcium-overload could lead to cardiac function descending and myocardial injury in calcium-overload rats by VDN. ACEI and spironolactone could reduce calcium-overload in myocardial and ameliorate cardiac function and decrease myocardial injury.

To explore the effect of hyperhomocysteinemia on vascular calcification and the underlying mechanism of it.Arterial calcification of Sprague-Dawley rats was induced by vitamin D3 plus nicotine. Hyperhomocysteinemia was established by feeding high methionine diet for six weeks and was assessed b y plasma total homocysteine (tHcy) level detected by HPLC method. Calcification was confirmed by von Kossa staining, measurement of calcium content, alkaline phosphatases (ALP) activity and osteocalcin (OC) concentration of vascular tissue. Lipid conjugated dienes formation were determined to reflecting the production of lipid peroxide.The results showed that there were mass black granules deposited in aortic wall of the calcified rats by von Kossa staining. Calcium content, ALP activity, OC concentration in calcified rats increased by 8.09-fold, 45.57% and 2.81-fold compared with those of the control group (P0.01). Calcium content in calcified rats with high methionine diet increased by 34.29% compared with that of the calcified rats, while ALP activity and OC content decreased by 29.13% and 74.69% compared with that of the calcified rats. Lipid conjugated dienes formation in plasma of the rat with high methionine diet and of calcified rats with high methionine diet increased by 1.93 and 2.89-fold compared with those of the control group, respectively (P0.01), and in calcified rats with high methionine diet group was increased by 32.90% compared with that of high methionine diet group (P0.01).Hyperhomocysteinemia could promote vascular calcification, which might be mediated through the production of lipid peroxide.

Pulmonary hypertension is a common complication of congenital heart disease with a left-to right shunt. The mechanism of pulmonary hypertension induced by high pulmonary blood flow is still not fully understood. Recent studies showed that hydrogen sulfide (H2S) could relax vascular smooth muscle cells. But the change of the system of H2S in pulmonary hypertension induced by high pulmonary blood flow was not reported. We studied the influence on expression of CSE mRNA and production of hydrogen sulfide in rat lung tissues by L-Arginine, in order to demonstrate a regulating role of nitric oxide (NO) in the regulation of cystathionine-gamma-lyase/hydrogen sulfide system (CSE/H2S).Thirty male SD rats were randomly divided into shunting group, shunting with L-Arginine group, and control group. Abdominal aorta and inferior vena cava shunting was produced in rats of the later group. Pulmonary artery mean pressure (mPAP) and the hypertrophy of right ventricle of each rat were analyzed. The expression of lung tissue CSE mRNA was measured using quantitative reverse transcription-polymerase chain reaction and in situ hybridization. The activity of CSE in lung tissue was measured according to chemical analysis.mPAP was significantly increased in shunted rats compared with normal control (P0.01), the expression of lung tissue CSE mRNA and the activity of CSE in lung tissue were decreased in shunt group (P0.01). However, L-arginine significantly attenuated pulmonary artery pressure, but augmented the expression of lung tissue CSE mRNA as well as the activity of CSE in lung tissue.L-Arginine reverses the down-regulation of CSE/H2S system in high pulmonary blood flow-induced pulmonary hypertension.

The present study was designed to determined the cardiovascular effects of IMD1-53 in rats and its possible mechanism.Isolated rat hearts were perfused by Iangendorff mode, and ventricular function was measured after IMD1-53 perfusion. Meanwhere, we investigated the effects of IMDI) on arterial pressure after intravenous administration of IMD. And cAMP content was detected in rat ventricular and aortic tissues.The results showed that perfusion with IMD significantly enhanced cardiac function and resulted in higher LVSP, +dp/dt(max) and -dp/dt(max) by 45%, 51% and 37%, respectively, compared with control and increased coronary infusion flow. The effects of IMD1-53 on cardiac function were antagonized by H-89, an inhibitor of PKA. The content of cAMP in the ventricular tissues after IMD perfusion was 131% higher than control. In addition, intravenous administration of IMD induced a potent decrease in arterial pressureand heart rate, and in aortic tissues, IMD incubation resulted in a 236% increase in cAMP content compared with control group.The study reveals that IMD can increase cardiac function and decrease arterial pressure in rat and the effects may be related to cAMP pathway.

To explore the possible impact of endogenous hydrogen sulfide (H2S) on the content and metabolism of collagen in rats with high pulmonary blood flow.Thirty-two male SD rats, weighing 120-140 g, were randomly divided into 4 groups (n = 8), shunt group, shunt + PPG (propargylglycine, an antagonist of endogenous H2S producing enzyme) group, sham group and sham + PPG group. Rats in shunt group and shunt + PPG group were subjected to an abdominal aorta-inferior vena cava shunt to create an animal model of high pulmonary flow. In the sham group and sham + PPG group, rats experienced the same experimental processes except the shunting procedure. After 4 weeks of experiment, lung tissue H2S content of rat was determined by a modified sulfide electrode method. Pulmonary artery collagen I, collagen III, MMP-13 and TIMP-1 protein expressions of rat were investigated by immunohistochemistry.After 4 weeks of experiment, lung tissue H2S content increased significantly in rats of shunt group as compared with that of sham group (P0.05). Pulmonary artery collagen I and collagen III protein expression increased obviously in rats of shunt group as compared with that of sham group (P0.01). After administration of PPG for 4 weeks, lung tissue H2S content decreased significantly in rats of shunt + PPG group as compared with that of shunt group (P0.05). In contrast to rats in shunt group, collagen I and collagen III protein expression in pulmonary arteries of shunt + PPG group increased significantly, respectively (P0.05). Compared with rats of shunt group, pulmonary artery MMP-13, TIMP-1 and the ratio of MMP-13/TIMP-1 in shunt + PPG group down-regulated significantly (P0.05).Endogenous H2S might play a protective regulatory role in the development of pulmonary hypertension and pulmonary vascular structural remodelling in rats by decreasing the content of pulmonary artery collagen resulting from catabolism of collagen.

Abnormal apoptosis of pulmonary artery smooth muscle cells (PASMCs) is an important pathophysiological process in the pulmonary artery structural remodeling and pulmonary hypertension. We investigated possible effect of endogenous hydrogen sulfide (H2S) on apoptosis of PASMCs during the development of pulmonary hypertension induced by high pulmonary blood flow.Thirty-nine male Sprague-Dawley rats were randomly assigned to 4-week control, 4-week shunt, 4-week shunt + propargylglycine (PPG), 11-week control, 11-week shunt and 11-week shunt + sodium hydrosulfide (NaHS) groups. Rats in 4-week shunt, 4-week shunt + PPG, 11-week shunt and 11-week shunt + NaHS groups underwent an abdominal aorta-inferior vena cava shunt. Rats in 4-week shunt + PPG group were intraperitoneally injected with PPG, an inhibitor of endogenous H2S production, for 4 weeks. Rats in 11-week shunt + NaHS group were intraperitoneally injected with NaHS, a H2S donor, for 11 weeks. Lung tissue H2S was evaluated by sulfide-sensitive electrode. Apoptosis of PASMCs were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). Expressions of Fas, bcl-2 and caspase-3 in the PASMCs were analyzed with immunochemical staining.Four weeks after the shunting operation, the apoptosis of PASMCs and expression of Fas and caspase-3 were significantly decreased (P0.01), but expression of bcl-2 increased significantly (P0.01). PPG administration further inhibited the apoptosis of PASMCs, downregulated the expression of Fas and caspase-3 (P0.01), but increased the expression of bcl-2 (P0.01). After 11 weeks of shunting operation, the apoptosis of PASMCs and expression of Fas and caspase-3 were significantly decreased (P0.01), but expression of bcl-2 increased obviously (P0.01). NaHS administration significantly increased the apoptosis of PASMCs, upregulated the expression of Fas and caspase-3, but inhibited the expression of bcl-2.H2S induces the apoptosis of PASMCs in the development of high pulmonary blood flow-induced pulmonary hypertension by activating the Fas pathway and inhibiting the bcl-2 pathway.

To investigate the effects of carvedilol and metoprolol on cardiac fibrosis in rats with experimental myocardial infarction (MI).MI was induced in male Sprague-Dawley rats by ligating the left coronary artery. Rats randomly received saline, carvedilol (10 mg.kg(-1).d(-1)) or metoprolol (20 mg.kg(-1).d(-1)) beginning at 4 weeks post MI for 8 weeks per gavage. Sham-operated rats serve as control. Collagen perivascular circumferential collagen area (PCVA), peri-coronary circumferential collagen area (VLCA) and interstitial collagen volume fraction (ICVF) as well as myocardial hydroxyproline content were determined after hemodynamic measurements at the study end.LVEDP were significantly lower and +/- dp/dt significantly higher in carvedilol and metoprolol treated MI rats than that in saline treated MI rats. Myocardial hydroxyproline, PCVA/VLCA ratio and ICVF were significantly reduced in metoprolol, more significantly reduced in carvedilol treated MI rats compared to saline treated MI rats (all P0.05).Metoprolol and carvedilol could decrease the concentration of hydroxyproline and ICVF in MI rats.

To investigate the effects of hydrogen sulfide (H2S) on vascular inflammation in pulmonary hypertension induced by high pulmonary blood flow.Forty-four male SD rats were randomly divided into 8 groups: 4-week control group (n = 7), 4-week shunt group (n = 7), 4-week shunt + propargylglycine (PPG, an endogenous H2S release inhibitor) intraperitoneal injection group (n = 8), 11-week control group (n = 7), 11-week shunt group (n = 7), and 11-week shunt + sodium hydrosulfide (NaHS, a H2S donor) intraperitoneal injection group (n = 8). Right ventricular catheterization was used to measure the mean pulmonary arterial pressure (mPAP). Immunohistochemistry was used to detect the expression of inflammatory related factor intercellular adhesion molecule-1 (ICAM-1), and the key molecules of nuclear factor-kappaB (NF-kappaB) signal transduction pathway, including NF-kappaB p65 and inhibitor of NF-kappaB (IkappaBalpha), in the pulmonary artery, and ELISA was used to detect the concentrations of the inflammatory related factors, including ICAM-1, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) in blood plasma and lung tissues so as to reflect the corresponding inflammatory responsiveness.The plasma and lung tissue ICAM-1, IL-8 and MCP-1 contents of the 4-week shunt group were all significantly higher than those of the 4-week control group (P0.05 or P0.01). The mPAP of the 4 week shunt + PPG group was (20.3 +/- 1.7) mm Hg, significantly higher than that of the 4-week shunt group [(16.2 +/- 1.5) mm Hg, P0.01]. The expression levels of ICAM-1 and NF-kappaB p65 in the small and median pulmonary artery endothelin cells of the 4-week shunt + PPG group were both significantly stronger than those of the 4-week shunt group (P0.05 or P0.01), whereas the expression of IkappaBalpha was weaker than that of the 4-week shunt group (P0.05). The plasma IL-8 content of the 4-week shunt + PPG group was (148 +/- 29) micromol/L, significantly higher than that of the 4 week-shunt group [(118 +/- 23) micromol/L, P0.05], and the lung tissue ICAM-1 and MCP-1 levels of the 4-week shunt + PPG group were (27.3 +/- 5.0) micromol/g and (12.9 +/- 1.1) micromol/g respectively, both significantly higher than those of the 4-week shunt group [(21.9 +/- 2.1) and (10.2 +/- 1.4) micromol/g respectively, both P0.05]. The mPAP and expression levels of ICAM-1 and NF-kappaB p65 of the large, median, and small pulmonary artery endothelia cells of the 11-week shunt group were all higher than those of the 11-week control group (P0.05 or P0.01), whereas the expression levels of IkappaBalpha were all less obvious (P0.05 or P0.01). The plasma and lung tissue ICAM-1, IL-8, and MCP-1 levels of the 11-week shunt group were all significantly higher than those of the 11-week control group (all P0.01). The mPAP of the 11 week shunt + NaHS group was (23.2 +/- 3.0) mm Hg, significantly lower than that of the 11-week shunt group [(27.5 +/- 1.9) mm Hg, P0.05]. The ICAM-1 and NF-kappaB p65 expression levels of large, median, and small pulmonary artery endothelia cells of the 11-week shunt + NaHS group were all significantly weaker than those of the 11-week shunt group (P0.05 or P0.01), whereas the protein expression levels of IkappaBalpha in small and median pulmonary artery endothelia cells of the 11-week shunt + NaHS group were significantly higher than those of the 11-week shunt group (both P0.05). The plasma and lung tissue ICAM-1 contents of the 11-week shunt + NaHS group were (124 +/- 11) micromol/L and (19.9 +/- 2.5) micromol/g, both significantly lower than those of the 11-week shunt group [(154 +/- 20) micromol/L and (23.9 +/- 3.6) micromol/g respectively, both P0.01]. The plasma and lung tissue IL-8 contents of the 11-week shunt + NaHS group were (92 +/- 11) micromol/L and (15.0 +/- 1.7) micromol/g, both significantly lower than those of the 11-week shunt group [(121 +/- 17) micromol/L and (19.0 +/- 3.9) micromol/g respectively, both P0.01]. The lung tissue MCP-1 content of the 11-week shunt + NaHS group was (10.8 +/- 1.6) micromol/g, significantly lower than that of the 11-week shunt group [(13.5 +/- 1.4) micromol/g, P0.01].H2S attenuates the development of pulmonary hypertension induced by high pulmonary blood flow through ameliorating pulmonary vascular inflammation. The inhibitory effect of H2S on the pulmonary vascular inflammation involves elevating IkappaBalpha expression, down-regulating NF-kappaB p65 expression and then inhibiting the expression of inflammatory related factors.

The mechanism of high pulmonary blood flow-induced pulmonary hypertension remains unclear. The aim of this study was to explore the effect of proadrenomedullin N-terminal 20-peptide (PAMP) on pulmonary hypertension, through examining the alterations of pulmonary PAMP expression and plasma PAMP concentration in rats with pulmonary hypertension induced by high pulmonary blood flow.Sixteen male Sprague-Dawley rats were randomly divided into control (n=8) and shunt groups (n=8). Aortocaval shunting was produced in the shunt group. After 11 weeks of shunting, systolic pulmonary artery pressure (sPAP), diastolic pulmonary artery pressure (dPAP) and mean pulmonary artery pressure (mPAP) were evaluated by using a right cardiac catheterization procedure. The ultrastructural changes in intra-acinar pulmonary arteries were observed. The concentration of plasma PAMP was measured by radioimmunoassay. The expression of PAMP in pulmonary arteries was detected by immunohistochemical assay.sPAP, dPAP and mPAP were significantly increased in shunt rats compared with controls (P0.01). Ultrastructural changes, such as hyperplasia and swelling of endothelial cells, irregularity of internal elastic laminar, and hypertrophy and increased number of synthetic phenotype of smooth muscle cells, were found in intra-acinar pulmonary muscularized arteries in the shunt group. Plasma PAMP concentration (616 +/- 195 pg /mL vs 427 +/- 90 pg /mL) and PAMP expression in endothelial cells (0.62 +/- 0.09 vs 0.38 +/- 0.12) and in smooth muscle cells (0.24 +/- 0.07 vs 0.14 +/- 0.05) of pulmonary arteries increased significantly in the shut group compared with controls.The up-regulation of pulmonary and plasm PAMP expression might be involved in the development of high pulmonary blood flow-induced pulmonary hypertension.

To study the changes of endogenous hydrogen sulfide (H(2)S) and the effect of exogenously applied H(2)S on ovalbumin-induced acute asthma.Twenty-four Male SD rats were randomly divided into a control group (n = 8), an asthma group (n = 8) and a NaHS group (n = 8). Pulmonary function was measured, and the pulmonary pathology changes, the content of H(2)S in lung tissue and plasma and the activity of H(2)S generating enzymes in lung tissue were detected at the 28 th day after ovalbumin administration. Western blotting was used to detect the endothelial cystathionine-gamma-lyase CSE protein in the lung tissues.In the asthma group, the peak expiratory flow (PEF) was (2.90 +/- 0.70) L/s, the contents of H(2)S in the plasma was (10 +/- 3) micromol/L, in the lung tissue was (4.9 +/- 1.3) micromol/L. The H(2)S generating enzyme activity in the lung tissue of the asthma group was (1.00 +/- 0.10) nmolxmin(-1)xmg(-1) pro, and the lung CSE content of the asthma group was significantly lower than that of the control group (6.50 +/- 0.10) L/s, (54 +/- 10), (24.1 +/- 8.0) micromol/L, (1.80 +/- 0.10) nmolxmin(-1)xmg(-1), F = 112.13, 110.10, 27.34, 79.39, 12.28, all P0.05). The pulmonary pathology score of the asthma group was 3 (2 - 4), significantly higher than that of the control group [1 (0 - 1), H = 16.93, P0.01]. In the NaHS group, the PEF was (5.70 +/- 0.50) L/s, the content of H(2)S in the plasma was (17 +/- 4) micromol/L, in the lung tissue was (15.3 +/- 4.0) micromol/L, the H(2)S generating enzyme activity in the lung tissue was (1.60 +/- 0.20) nmolxmin(-1)xmg(-1) pro, the lung CSE content of the NaHS group was significantly higher than that of the asthma group (F = 112.13, 110.10, 27.34, 79.39, 12.28, all P0.05). The pulmonary pathology score of the NaHS group was 1 (1 - 2), significantly lower than that of the asthma group [3 (2 - 4) score, H = 16.93, P0.01]. There was a significantly positive correlation between the content of the content of H(2)S in lung tissue and PEF (r = 0.74, P0.01). There was a significantly negative correlation between the content of H(2)S in lung tissue and the pulmonary pathology score (r = -0.64, P0.01).Endogenous H(2)S is involved in the pathogenesis of asthma in this animal model. Exogenously applied H(2)S can attenuate inflammation of asthma and exert protective effect from asthma.

To construct a detection method of endogenous hydrogen sulfide (H2S) from erythrocytes.Prepared rat erythrocyte (10(6) cell) was added in 4 mL 2-amino-2-methyl-1,3-propanediol(225 mmol/L, pH=9.55) and ultrasonically lysed for 3 times. The erythrocyte lysis was transferred into a 25 mL Erlenmeyer flask and beta-mercaptopyruvate was added for final concentration of 2 mmol/L. Cryovial test tubes (2 mL) were used as the centre wells each contained 0.5 mL of 1% zinc acetate as trapping solution and a filter paper of 2.0x2.5 cm2 to increase the air/liquid contacting surface. The flasks containing reaction mixture and centre wells were flushed with N2 before being sealed with a double layer of Parafilm. Reaction was initiated by transferring the flasks from ice to a 37 degrees C shaking water bath. After incubation at 37 degrees C for 60 min, 0.5 mL of 50% trichloroacetic acid was added into the reaction mixture to stop the reaction. The flasks were sealed again and incubated at 37 degrees C for another 60 min to ensure a complete trapping of the H2S released from the mixture. Released H2S is absorbed by zinc acetate and generation zinc sulfide. The zinc sulfide formed is dissolved in a hydrochloric acid solution of amino-dimethylaniline (N,N-dimethyl-p-phenylenediamine), and methylene blue is formed within 10 min at room temperature in the presence of ferric chloride. The blue color of methylene blue is measured at 670 or 650 nm on spectrophotometer.We first demonstrated the key enzymes of endogenous H2S generation-3-mercaptopyruvate sulfurtransferase (MPST) gene expression by RT-PCR. Endogenous H2S production from rat erythrocytes was 22.76+/-1.53 micromol/min/10(8) cells, about 4-folds as compared with to the liver and kidney tissues. However, the endogenous H2S production from erythrocyte by L-cysteine pathway was little detected.Endogenous H2S released from erythrocytes depended mainly on MPST pathway. Our method is effective to detect the erythrocytic endogenous H2S.

To evaluate the effect and related mechanisms of aldosterone (ALD) on inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) production in aortic adventitia.Aortic adventitias from SD rats were incubated for 6 hours with various protocols: buffer alone (control), ALD (10(-8) mol/L - 10(-6) mol/L), ALD + spironolactone (10(-5) mol/L, ALD + SP), ALD + RU486 (10(-5) mol/L), LPS 10 ng/ml (LPS), ALD + LPS (10 ng/ml), ALD + LPS + SP (10(-5) mol/L), and ALD + LPS + RU486. Nitrate/nitrite (NOx), an index of NO production, was measured by Greiss Reaction. iNOS activity was determined by isotope-labeled L-arginine convertion rate.(1) NOx production and iNOS activity were similar between ALD and control groups (P0.05). NOx production was significantly reduced while iNOS activity remained unchanged in the ALD (10(-6) mol/L) + SP group compared to ALD (10(-6) mol/L) group. NOx production by 10(-7) mol/L and 10(-6) mol/L ALD increased by 50.0% and 58.7% respectively (P0.01) and iNOS activity was also significantly increased (P0.01) in ALD + RU486 group than that in ALD group. (2) LPS significantly increased the NOx production and iNOS activity (P0.01) and these effects were not augmented by adding ALD to LPS (P0.05) and SP significantly blocked and RU486 significantly enhanced the effects by LSP and ALD on NOx production and iNOS activity (P0.05).Aldosterone has a dual effect on iNOS/NO through mineralocorticoid receptor and glucocorticoid receptor pathway.

The purpose of the present study was to explore the expression changes of intermedin/adrenomedullin 2 (IMD/ADM2), a novel small molecular bioactive peptide, and its receptors, calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMP1, RAMP2, RAMP3) in the right ventricle of rats with chronic hypoxia-induced pulmonary hypertension. Twenty male Sprague-Dawley rats were randomly divided into 4-week hypoxia group and normal control group (each n=10). The rats in hypoxia group were placed in an isobaric hypoxic chamber, in which O(2) content was maintained at 9%-11% by delivering N(2), and CO(2) content was maintained at3% for 4 weeks (8 h/d, 6 d/week). The rats in the control group were housed in room air. The protein levels of IMD/ADM2 and adrenomedullin (ADM) in blood plasma and right ventricular tissue were measured by radioimmunoassay. The mRNA expressions of IMD/ADM2, ADM and their receptors CRLR, RAMP1, RAMP2, RAMP3 in right ventricular tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the ratio of right ventricle weight to left ventricle plus septum weight [RV/(LV+S)] and mean pulmonary arterial pressure (mPAP) were higher in hypoxia group than those in the control group (all P0.01), suggesting that the rat model of pulmonary hypertension was successfully established. However, the mean carotid arterial pressure (mCAP) between the two groups had no significant difference. Compared with that in the control group, ADM contents in plasma and right ventricular tissue in hypoxia group increased by 1.26 and 1.68 folds (all P0.01), respectively. Likewise, IMD/ADM2 contents in blood plasma and right ventricular tissue in hypoxia group increased by 0.90 and 1.19 folds (P0.01), respectively, compared with that in the control group. The data of RT-PCR showed that mRNA levels of ADM, IMD/ADM2 and RAMP2 in hypoxia group increased by 155.1% (P0.01), 80.9% (P0.01) and 52.9% (P0.05), respectively, compared with those in the control group. There were no significant differences in mRNA expressions of CRLR, RAMP1 and RAMP3 between the two groups (all P0.05). Taken together, the results show that the level of IMD/ADM2 increases in the rats with chronic hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) is a common complication of congenital heart defects with a left-to-right shunt characterized by high pulmonary blood flow. Pulmonary vascular structural remodeling (PVSR) is the pathological basis of PH. However, the pathophysiologic features and mechanisms responsible for PH and PVSR induced by increased pulmonary blood flow have not been fully understood. The present study was designed to explore the possible effect and mechanism of hydrogen sulfide (H(2)S) on the regulation of PVSR induced by high pulmonary flow in rats.Thirty-two male SD rats, weighing 120 - 140 g, were randomly divided into shunt group (n = 8), shunt + NaHS group (n = 8), control group (n = 8) and control + NaHS group (n = 8). Rats in shunt group and shunt + NaHS group were subjected to an abdominal aorta-inferior vena cava shunt to create an animal model of high pulmonary flow. Rats in the control and control + NaHS groups underwent the same experimental protocol as mentioned above except for the shunt procedure. Rats in the shunt + NaHS and control + NaHS groups were intraperitoneally injected with NaHS at 56 micromol/(kgxd), and rats in the shunt and control groups were injected with the same volume of physiological saline. After 11 weeks of experiment, rats were sacrificed and lung tissues were obtained. The percentage of muscularized artery (MA) was calculated. The changes in relative medial thickness (RMT) in small pulmonary arteries and median pulmonary arteries were examined. Proliferative cell nuclear antigen (PCNA), extracellular signal-regulated kinase (ERK1) and phosphorylation extracellular signal-regulated kinase (P-ERK1) protein expression were examined by Western blot, and at the same time, PCNA protein expression by pulmonary artery smooth muscle cells was observed by immunohistochemistry.After 11 weeks of shunt, compared with control group, the percentage of MA increased significantly (25.12 +/- 2.26 vs 14.42 +/- 3.41, P0.05), and RMT in small pulmonary arteries and median pulmonary arteries increased significantly in rats of shunt group (23.6 +/- 3.5 vs 12.6 +/- 2.1, 24.8 +/- 1.9 vs 13.5 +/- 2.2, P0.05 for all). PCNA protein expression in small and median pulmonary arteries increased significantly (0.49 +/- 0.04 vs 0.39 +/- 0.07, 0.46 +/- 0.08 vs 0.36 +/- 0.05, P0.01 for all), and the ratio of PERK/ERK1 protein expression of pulmonary arteries increased significantly (P0.01) in rats of shunt group compared with those of control group. After the administration of exogenous H(2)S donor, NaHS, for 11 weeks, in contrast to rats in shunt group, the percentage of MA decreased significantly (21.5 +/- 2.0 vs 25.1 +/- 2.3, P0.05), and RMT in small and median pulmonary arteries decreased significantly (20.2 +/- 2.8 vs 23.6 +/- 3.5, 20.8 +/- 3.1 vs 20.8 +/- 3.1, P0.05 for all) in rats of shunt + NaHS group. PCNA protein expression in small and median pulmonary artery smooth muscle cells decreased significantly (0.32 +/- 0.06 vs 0.49 +/- 0.04, 0.29 +/- 0.07 vs 0.46 +/- 0.08, P0.01 for all), and the ratio of PERK/ERK1 protein expression of pulmonary arteries decreased significantly (P0.01) in rats of shunt + NaHS group compared with that of shunt group.H(2)S may play a regulatory role in pulmonary vascular structural remodeling induced by high pulmonary blood flow via mitogen-activated protein kinase (MAPK)/ERK signal transduction pathway.

Endothelin-1(1-31) (ET-1(1-31)) is a novel member of the endothelin family, which comprises 31 amino acids and derived from the selective hydrolysis of big ET-1 by chymase. Although ET-1(1-31) has been reported to be involved in biological effects via direct or indirect (converting to ET-1(1-21)) mechanisms, the cardiovascular effects of central ET-1(1-31) are not fully identified. The present study was designed to comparatively investigate the cardiovascular effects of intracerebroventricular (icv) application of ET-1(1-31) or ET-1(1-21) in anesthetized rats. Injection (icv) of ET-1(1-31) (500 pmol) produced a biphasic blood pressure response: an initial increase (from 118+/-8 to 138+/-14 mmHg, P

To explore the possible role of endogenous hydrogen sulfide (H(2)S), a novel gasotransmitter, in the pathogenesis of pulmonary vascular structural remodeling (PVSR) induced by high pulmonary blood flow.Thirty-two Sprague-Dawley male rats were randomly divided into sham, shunt, sham+NaHS (a H(2)S donor) and shunt+NaHS groups. Rats in shunt and shunt+NaHS groups underwent an abdominal aorta-inferior vena cava shunt, and rats in shunt+NaHS and sham+NaHS groups were intraperitoneally injected with NaHS. PVSR was investigated using optical microscope and transmission electron microscope. Lung tissue H(2)S was evaluated by sulfide-sensitive electrodes. Nitric oxide synthase (NOS), heme oxygenase (HO-1), proliferative cell nuclear antigen (PCNA) and extracellular signal-regulated kinase (ERK) activation were analyzed by Western blotting.After 11 weeks of shunting, PVSR developed with a decrease in lung tissue H(2)S production and an increase in nitric oxide (NO). However, lung tissue carbon monoxide (CO) did not change. After the treatment with NaHS for 11 weeks, H(2)S donor ameliorated PVSR and downregulated PCNA expression and ERK activation with an increase in lung tissue CO production and HO-1 protein expression but a decrease in NO production, NOS activity and eNOS protein expression in shunted rats.H(2)S exerted a regulatory effect on PVSR induced by high pulmonary blood flow. Meanwhile, H(2)S down-regulated the ERK/MAPK signal pathway, inhibited the NO/NOS pathway and enhanced the CO/HO pathway in rats with high pulmonary blood flow.

To explore the effects of proadrenomedullin N-terminal 20 peptide (PAMP) on nitric oxide (NO) production in rat cardiac fibroblasts (CFs) induced by angiotensin II (AngII) stimulation.Neonatal SD rat CFs isolated by trypsin digestion were cultured and stimulated with PAMP, AngII or their combination, and NO production in the CFs in response to the treatments was measured by nitric acid reductase method.NO levels in the cell culture treated with 1x10(-9), 1x10(-8), 1x10(-7), and 1x10(-6) micromol/L AngII were 73.88-/+2.23, 64.34-/+3.02, 54.12-/+2.82, and 40.21-/+1.45 micromol/L, respectively, showing significant differences between the groups (P0.01), whereas treatment of the cells with 1x10(-9), 1x10(-8), 1x10(-7), and 1x10(-6) micromol/L PAMP did not result in significant variation in NO production (74.40-/+3.42, 74.91-/+2.66, 75.77-/+3.31, and 74.23-/+2.43 micromol/L, respectively) in comparison with that of the blank control group (74.57-/+2.49 micromol/L, P0.05). Combined treatments with 1x10(-7) micromol/L AngII and PAMP at 1x10(-9), 1x10(-8), 1x10(-7), and 1x10(-6) micromol/L PAMP caused significant increment of NO production (66.15-/+2.95, 80.58-/+3.77, 88.67-/+1.46, and 96.22-/+2.96 micromol/L, respectively, P0.01) in a PAMP dose-dependent manner, suggesting the abolishment of AngII-induced enhancement of NO production in the CFs by PAMP.PAMP increases NO production in the CFs in the presence of AngII but it does not induce significant changes in NO production when used alone.

To explore the impact of endogenous hydrogen sulfide (H(2)S), a novel gaseous signal molecule, on the nitric oxide (NO)/nitric oxide synthase (NOS) pathway in left-to-right shunt.Thirty-two male SD rats were randomly divided into 4 equal groups: shunt group undergoing abdominal aorta-inferior vena cava puncture so as to establish model of left-to-right shunt; shunt + PPG group undergoing abdominal aorta-inferior vena cava puncture so as to establish model of left-to-right shunt and then intraperitoneal injection of propargylglycine (PPG), an inhibitor of cystathionine-gamma-lyase: sham group undergoing sham operation; and sham + PPG group undergoing sham operation and then intraperitoneal injection of PPG. Four weeks later, right cardiac catheterization was conducted to measure the mean pulmonary artery pressure (MPAP). Then the rats were killed and their lung tissues and samples of plasma were collected. The contents of H(2)S, nitric oxide (NO), and nitrogen oxide synthase (NOS) activity, and the content of plasma NO were calculated. Western blotting was used to detect the endothelial nitric oxide synthase (eNOS) protein in the lung tissues. The correlation of MPAP with lung H(2)S and NO was analyzed.The MPAP of the shunt group was not significantly different from that of the sham group, and the MPAP of the shunt + PPG group was significantly higher then those of the shunt group and sham group by 15.82% and 20.55% respectively (both P0.05). The content of lung tissue H(2)S of the shunt group was 37.56 +/- 2.13 micromol/mg, significantly higher than that of the shunt group (14.35 +/- 1.76, P0.05), the content of lung tissue H(2)S of the shunt + PPG group was 28.76 +/- 2.24 micromol/mg, significantly lower than that of the shunt group (P0.05). The lung tissue NO content of the shunt group was 38.48 micro +/- 6.53 micromol/microg, significantly higher than that of the sham group (31.78 +/- 6.51 micromol/microg). The NOS activity of the shunt group was 15.12 +/- 2.44 U/mg protein, significantly higher than that of the sham group (12.00 +/- 1.40 U/mg protein, P0.05). The lung eNOS content of the shunt group was significantly higher than that of the sham group (P0.05). The plasma NO content of the shunt group was 23.18 +/- 3.56 micromol/L, significantly higher than that of the sham group (17.94 +/- 3.39 micromol/L, P0.05). The lung tissues NO and NOS activity, and plasma NO of the shunt + PPG group were 46.04 +/- 5.95 micromol/microg, 20.89 +/- 3.94 U/mg protein, and 27.79 +/- 4.82 micromol/L respectively, all significantly higher than those of the shunt group (38.48 +/- 6.53 micromol/microg, 15.12 +/- 2.44 U/mg protein, and 23.18 +/- 3.56 micromol/L, all P0.05). The eNOS content of the shunt + PPG group was significantly higher than that of the shunt group (P0.05). The lung H(2)S content was negatively correlated with the MPAP and lung NO content (r = -0.705, P = 0.005; and r = -0.645, P = 0.013).Endogenous H(2)S may play a regulatory role in the pulmonary artery pressure of left-to-right shunt through inhibiting NO/NOS pathway.

To investigate alteration and cross link of the aortic intima and platelet endogenous L-Arg/NOS/NO pathway induced by water immersion restraint (WIR) stress.After 7 h of WIR stress, the aortic intima was isolated and prepared the platelet, then NO2- production released from aortic intima and platelet was measured with Greiss regent, NOS activity and L-arginine transport activity were detected by isotope tracer method.After 7 h of WIR stress, the levels of NO2- from platelet and aortic intima obviously decreased by 57% and 46% respectively as compared with the control rats (P0.01). The inhibitory effects of NO2- showed a positive correlation between platelet and aortic intima by person regression analysis (r2=0.9835, P0.01). The kinetics analysis of NOS activity of platelet showed that the enzymatic catalytic maximum velocity (Vmax) decreased by 44%, Km values increase by 18% and the enzymatic catalytic efficiency was obviously reduced in the WIR stress rats (P0.01). The aortic intima NOS activity was inhibited by 50% after WIR stress (P0.01). The person analysis showed the obvious correlation between them (r2=0.9726, P0.01). In the WIR stress platelet, the L-Arg transport Vmax obviously reduced by 32%, Km parameter increased 37%, and the transport efficiency was inhibited by 50% (P0.01), which exhibited obvious positive correlation with the inhibition of L-Arg transport activity in aortic intima (27%) of WIR stress rats (r2=0.9887, P0.01).WIR stress down-regulated endogenous L-Arg/NOS/NO pathway of aortic intima and platelet, whose alteration showed obvious positive correlation. Detection of the alteration of endogenous L-Arg/NOS/NO pathway in platelet might act as an indirect method to assess the endothelial dysfunction involving the pathogensis of stress.

Endothelin (ET)-1(1-21) is known to play an important role in the pathogenesis of acute ischemic arrhythmia. In the present study, we attempted to determine whether administration of ET-1(1-31) would result in arrhythmia in perfused isolated rat hearts. Forty-eight Sprague-Dawley rats weighing approximately 250-350 g were randomized into 6 groups. Heart was isolated and perfused in a Langendorff mode. The effects of ET-1(1-31) on arrhythmia, heart rate, coronary flow, and heart function were analyzed. Perfusion with 1 nM ET-1(1-31) resulted in frequent ventricular ectopic beats (VEBs) and ventricular tachycardia (VT). Overall VEB was 128.0 (approximately 66.0-1015.0), and the arrhythmia score (AS) was 2.18 +/- 0.87; both were significantly higher than those of the control group (P0.01). Pretreatment with perfusion of 10 nM of the ETA-receptor antagonist BQ(123) markedly attenuated the occurrence of VEB and VT induced by ET-1(1-31). AS in 10 nM BQ123 group was significantly lower than that in 1 nM ET-1(1-31) group (P0.01). The arrhythmia induced by 1 nM ET-1(1-31) was partially but significantly reduced by phosphoramidon (1 microM), a neutral endopeptidase/ET-converting enzyme inhibitor. ET-1(1-31) per se caused arrhythmia in perfused isolated rat hearts. This arrhythmogenic action is in part mediated by ET(A) receptor and may be attributed mainly to the conversion of ET-1(1-31) to ET-1(1-21.).

To explore the impact of hydrogen sulfide (H2S) donor, sodium hydrosulfide (NaHS), on pulmonary vascular structure and vasoactive peptides in rats with pulmonary hypertension induced by high pulmonary blood flow.Thirty-two male Wistar rats, weighing 120-140 g, were randomly divided into shunt group (n=8), shunt + NaHS group (n=8), sham group (n=8), and sham + NaHS group (n=8). Rats in shunt group and shunt + NaHS group were subjected to an abdominal aorta-inferior vena cava shunt to create an animal model of high pulmonary flow. In the sham group and sham + NaHS group, rats experienced the same experimental processes except the shunting procedure. Rats in shunt + NaHS group and sham + NaHS group were intraperitoneally injected with an exogenous H2S donor--NaHS, at a dose of 56 micromol/(kg x d). Meanwhile, rats in shunt group and sham group were injected with the same volume of physiological saline. After 11 weeks of experiment, systolic pulmonary artery pressure (SPAP) of each rat was evaluated by using a right cardiac catheterization procedure. Heart tissues were separated as right ventricular (RV) and left ventricular plus septum (LV + SP), and the ratio of RV to LV + SP [RV/(LV + SP)] was calculated. The morphologic changes including micro-and ultra-structural changes of pulmonary arteries of rats were observed under optical microscope and electro-microscope, respectively. The percentage of muscular artery (MA) in small pulmonary arteries was calculated. The change of relative medial thickness (RMT) of pulmonary arteries was examined. H2S concentration in plasma was evaluated by modified sulfide electrode method. Endothelin-1 (ET-1), atrial natriuretic peptide (ANP), calcitonin gene related peptide (CGRP), and proadrenomedullin peptide (PAMP) were calculated by radioimmunoassay kit.After 11 weeks of shunt, compared with sham group, SPAP increased by 48.63% (P0.01 ) and RV/ (LV + SP) increased by 21.95% (P0.01). Plasma H2S decreased significantly (P0.01). The percentage of MA increased significantly (P0.01); RMT increased significantly (P0.01). The changes of ultra-structure of pulmonary arteries showed that endothelial cells became swollen and desquamation, internal elastic lamina became irregular, and smooth muscular cells increased in size, showing synthetic phenotype. After the rats with shunt was administered with NaHS for 11 weeks, plasma H2S increased significantly (P0.01). SPAP decreased by 19.82% and RV/(LV + SP) decreased by 7.31% (P0.01). The percentage of MA decreased significantly and RMT decreased significantly (P0.01). The changes of ultra-structure of the pulmonary arteries showed lighten significantly. Plasma ET-1, ANP, and CGRP decreased significantly (all P0.01), whereas PAMP increased significantly than that of shunt group (P0.01).The reduced production of endogenous H2S is one of mechanism of pulmonary hypertension and pulmonary vascular structure remodeling in rats with high pulmonary blood flow. H2S plays an important regulatory effect on vasoactive peptide ET-1,+ ANP, CGRP and PAMP.

To determine the cardioprotective action of ghrelin and des-octanoyl ghrelin in rats with isoproterenol-induced myocardial injury.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 microg/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 [3H]ghrelin were determined by receptor binding assays.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% (P0.01). ISO-treated rats had 95% (P0.01) higher GHSR mRNA levels in the myocardia. The maximal binding capacity of [3H]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 (P0.01), with ghrelin administration as compared with that in controls, but was unaltered in des-octanoyl ghrelin group.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 explore the possible impact of hydrogen sulfide donor-NaHS (sodium hydrosulfide) on experimental pulmonary artery structural remodeling induced by high pulmonary flow and endogenous carbon monoxide/heme oxygenase pathway.Thirty-two male SD rats were randomly divided into shunt group (n=8), shunt+NaHS group (n=8), sham group (n=8) and sham+NaHS group (n=8). Rats in shunt group and shunt+NaHS group were subjected to an abdominal aorta-inferior vena cava shunt to create an animal model of high pulmonary flow. In the sham group and sham+NaHS group, rats experienced the same experimental processes except the shunting procedure. After 11 weeks of operation, systolic pulmonary artery pressure (SPAP) was detected using a right cardiac catheterization procedure. The percentage of muscularized artery (MA), partly muscularized artery (PMA) and non-muscularized artery (NMA) was calculated. Relative medial thickness (RMT) and relative medial area (RMA) of MA were observed under optical-microscope, respectively. The ultra-structure of pulmonary arteries was observed under electro-microscope. Lung tissue carbon monoxide (CO) was measured. Western-blot was used to analyze lung tissue heme oxygenase (HO-1) protein expression.After 11 weeks of shunt, compared with sham group, SPAP in shunt group increased by 48.6%.The percentages of MA and PMA increased by 74.2% and 90.9%, but the percentage of NMA decreased 32.2%, respectively, in rats of shunt group as compared with that of sham group. In contrast to those in sham group, RMT and RMA in median MA and small MA of rats in shunt group increased by 83.6%, 86.9%, and 74.4%, 39.9%, respectively. Ultra-structural changes in rats of shunt group showed that endothelial cells were swollen and denatured, inner elastic membrane became irregular, and smooth muscle cells became synthetic phenotype. The sham and shunt groups did not differ significantly in CO and HO-1 protein expression of lung tissues. In contrast to that of shunt group, SPAP in rats of shunt+NaHS group decreased by 19.8%. The percentages of MA and PMA decreased by 14.4% and 12.2%, but the percentage of NMA increased 13.9%, respectively, in rats of shunt+NaHS group as compared with that of shunt group. In contrast to those of shunt group, RMT and RMA in median MA and small MA in rats of shunt+NaHS group decreased by 16.2%, 14.3%, and 26.9%, 14.3%, respectively. For rats in shunt+NaHS group ultra-structural changes showed that flat endothelial cells, inner elastic membrane became regular, and smooth muscle cells were contractile phenotype. The content of CO increased by 25.5% and the HO-1 protein expression increased by 114.3% in shunt+NaHS group as compared with those of shunt group.NaHS might prevent pulmonary hypertension and pulmonary artery structural remodeling induced by high pulmonary flow, and its mechanism might be associated with the changes in endogenous CO/HO pathway.

To investigate the role of the endogenous cystathionine gamma-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 (P0.01). 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 (P0.01), 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 (P0.01). 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.

To investigate the mechanism of platelet function caused by Lysophosphatidic acid (LPA), by observing the change of the L-arginine/nitric oxide synthase/nitric oxide (L-Arg/NOS/NO) pathway of platelet in rats.LPA (10(-6), 10(-5) and 5x10(-5) mol/L) was administrated in rats and incubated for 30 and 60 minutes. The nitrite production was measured by Greiss assay; NOS activities and L-arginine transportation were detected by isotope tracer method and intracellular [Ca(2+)]i changes by fluorescent probe.LPA increased NO release by 35% and 56%, after incubating for 30 and 60 minutes, respectively. LPA (10(-6), 10(-5)aand 5x10(-5) mol/L) enhanced the NO productions of platelets in a concentration-dependent manner (P0.01). EC(50) was 17.8 micromol/L, and 95% CI was 13.3-24.2 micromol/L, involved in the physiological concentration of LPA in plasma (P0.01). Simultaneously, different doses of LPA increased NOS activities and L-arginine uptake in a dose-dependent manner (P0.01). In this study, LPA (50 micromol/L) increased the intracellular free calcium ion concentration ([Ca(2+)]i, P0.01), after incubating for 30 and 60 minutes. Pre-treated with NOS inhibitor-L-NAME for 20 minutes, LPA obviously enhanced the effects by 20% and 32% respectively (P0.01). On the contrary, pre-treated with L-arginine (200 micromol/L) for the same times obviously reduced the effects by 14% and 18% respectively (P0.01).LPA increased NO release by enhancing L-arginine uptake and NOS activities, up-regulating L-arginine/NOS/NO pathway in platelets of rats.

Endothelin-1 1-31 (ET-1 1-31), a novel member of the endothelin family comprising 31 amino acids and derived from the selective hydrolysis of big ET-1 by chymase, directly activates endothelin receptors or converts to ET-1 1-21 by ET converting enzyme (ECE). The cardiovascular effects of central ET-1 1-31 are not identified. The present study was designed to investigate the cardiovascular actions of ET-1 1-31 within the rostral ventrolateral medulla (RVLM) in anesthetized rats. Bilateral injection of ET-1 1-31 (0.5, 1, and 2 pmol for each side) into the rostral ventrolateral medulla produced an initial pressor and/or a long-lasting hypotensive action but did not affect HR. Unilateral microinjection of 2 and 4 pmol of ET-1 1-31 into the rostral ventrolateral medulla only produced a significant (P0.05) transient increase in blood pressure by an average of 13 and 12 mm Hg, respectively, whereas unilateral microinjection of 8 pmol of ET-1 1-31 produced a sustained fall in blood pressure (from 92 +/- 6 to 69 +/- 8 mm Hg, P0.05). The transient pressor effect of unilaterally injecting ET-1 1-31 (4 pmol) into the rostral ventrolateral medulla was completely abolished by pretreatment with either ETA receptor antagonist BQ123 (83 +/- 2 versus 84 +/- 5 mm Hg, P0.05) or ET converting enzyme inhibitor phosphoramidon (99 +/- 5 versus 99 +/- 7 mm Hg, P0.05) but not ETB receptor antagonist IRL1038 (89 +/- 6 versus 96 +/- 7 mm Hg, P0.05). In addition, prior injection of phosphoramidon also completely abolished the long-lasting hypotension of intra-RVLM ET-1 1-31 (8 pmol) but did not modify the depressor action of intra-RVLM ET-1 1-21 (from 100 +/- 6 to 76 +/- 8 mm Hg, P0.05). In conclusion, the current results suggest that the cardiovascular effects of intra-RVLM ET-1 1-31 might be the result of conversion of ET-1 1-31 to ET-1 1-21 through activation of ETA receptors.

To construct a method of measurement microamount hydrogen sulfide (H(2)S) using sensitive sulphur electrode.According to the physical and chemical characters of H(2)S, H(2)S, which in the fluids by mean of physical dissolve and chemical shape, is turned to sulphur ion (S(2-)) by chemical responses. After the microamount of S(2-) was measured by sensitive sulphur electrode, and the concentration of H(2)S was converted, a method was constructed to measure the H(2)S. It was used to analyze the concentrations of H(2)S of plasma in rats and humans, the endogenous concentration of H(2)S of cardiovascular tissue in rats, and CSE activity of cardiovascular tissues and cells.The exponential regression of S(2-) in the extent including 1 to 80 micromol/L was found using sensitive sulphur electrode. The H(2)S levels of plasma in male and female rats were 40+/-4 and 41+/-5 micromol/L, respectively, and significant difference was not found; those in venous blood plasma of men and women were 33+/-4 micromol/L and 35+/-5 micromol/L respectively, without significant difference. There were not significant differences in the aortic endogenous levels of H(2)S (24+/-6 and 25+/-5 nmol/mg pro) and myocardial levels (19+/-4 and 19+/-6 nmol/mg protein) between female and male rats. There were no different results of CSE activity in aortal tissue using sensitive sulphur electrode or traditional methods, however, the CSE activity of vascular smooth muscle cells could be accurately measured using sensitive sulphur electrode, which was difficult in using traditional method.The sensitive sulphur electrode assay was fit for the analysis of CSE/H(2)S pathway in cardiovascular research.

To explore the changes of time-dependent pulmonary artery structural remodeling in pulmonary hypertension induced by high pulmonary flow in rats.Eighty male SD rats were randomly divided into control group (n =40) and shunt group (n = 40). Rats in shunt group were subjected to an abdominal aorta-inferior vena cava shunt to create an animal model of high pulmonary flow. In the control group, rats experienced the same experimental processes except the shunting procedure. After 1 day, 3 days, 1 week, 4 weeks, and 8 weeks of experiment, systolic pulmonary artery pressure (SPAP) and mean pulmonary artery pressure (MPAP) of each rat were evaluated by using a right cardiac catheterization procedure. Heart tissues were separated as right ventricle (RV) and left ventricle plus septum (LV+SP), and the ratio of RV to LV+SP [RV/ (LV+ SP)] was calculated. The morphologic changes including micro- and ultra-structural changes of pulmonary arteries of rats were observed under optical microscope and electro-microscope, respectively. The percentages of muscularized artery (MA), partial muscularized artery (PMA) and non-muscularized artery (NMA) in small pulmonary arteries and median pulmonary arteries were calculated. The changes of relative medial thickness (RMT) and relative medial area (RMA) of pulmonary arteries were examined.Compared with control group, SPAP and MPAP did not change on day 1, day 3, and week 4. However, in week 1 and week 8 of experiment, SPAP and MPAP increased significantly (P0.01). Meanwhile, in week 8 of experiment, RV/ (LV+SP) increased significantly (P0.05). In contrast to control group, the percentages of MA, PMA, and NMA did not change in day 1, day 3 and week 1. But in week 4 and week 8, the percentages of MA and PMA increased significantly (P0.01) but that of NMA decreased significantly (P0.01). RMT and RMA did not change in day 1, day 3, week 1 and even week 4 in shunt group as compared with those of control group, but they increased significantly in week 8 (P0.05). The changes of ultra-structure of pulmonary arteries included that endothelial cells became swollen and large in size on day 3, smooth muscular cells increased in size besides the change of endothelial cells in week 1, and they changed from contractile phenotype to synthetic phenotype in week 4. Collagen deposited in pulmonary arteries markedly in week 8.Pulmonary artery structural remodeling develops in a time-dependent manner. Endothelial cells of pulmonary arteries become swollen firstly, followed by the proliferation of smooth muscular cells and finally by remodeling of extra cellular matrix.

To investigate the cardiac protective effects of ghrelin in rat with myocardial injury induced by isoproterenol (ISO).Rats were subcutaneously injected ISO 40 mg/kg/d with or without ghrelin 1 or 10 nmol/kg/d for 2 d. Hemodynamic parameters including mean arterial blood pressure and left ventricular pressure were measured at 12 h after the last injection with ISO and/or ghrelin. Plasma lactate dehydrogenase (LDH) activity, plasma and myocardial contents of malondialdehyde (MDA), and conjugated diene were measured. Plasma ghrelin and endothelin-1 levels were assayed using radioimmunoassay methods. Endothelin-1 and ghrelin mRNA were determined using RT-PCR.About 45 % (5/11) of rats after treatment with ISO alone died during experimental periods. However, no rats died after administration with ghrelin 10 nmol/kg/d (0/11, P0.05). Ghrelin also obviously ameliorated the hemodynamic disturbance in rats induced by ISO. The plasma LDH activity, contents of myocardial and plasma MDA, and conjugated diene level in plasma in ISO+G10 nmol/kg/d group were decreased by 28 %, 34 %, 73 %, and 38 % compared with those of ISO group (all P0.01) respectively. ISO-induced endothelin-1 mRNA over-expression was inhibited and endothelin-1 level in plasma were inhibited by ghrelin 1 and 10 nmol/kg/d. The ghrelin levels in plasma and ghrelin mRNA in myocardium were increased in the rats after injection of ISO. The plasma ghrelin level was further increased after ghrelin administration.Ghrelin has a protective effect against ISO-induced myocardial injury.

To explore the production of ADM, changes and significance of adrenomedullin (ADM) mRNA and ADM receptor system-calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) mRNA in calcified myocardium and aorta of rats.Contents of ADM in plasma, myocardium and vessel were measured by radioimmunoassay (RIA). The amount of ADM, CRLR and RAMPs mRNA was determined by semi-quantitative RT-PCR.The contents of calcium in calcified myocardium and aorta were increased by 342% and 606% (all P0.01), respectively, and alkaline phosphatase activities in calcified myocardium and vessel were increased by 66.5% and 82.7% (all P0.01), respectively, compared with the control. Contents of ADM in plasma, myocardium and aorta were increased by 58 % (P0.01), 14.3%(P0.01) and 27.8%(P0.05). Furthermore, it was found that the levels of ADM, CRLR and RAMP2 mRNA in calcified myocardium were elevated by 90.6% 157.5% and 119.6%(all P0.01), respectively, but RAMP3 mRNA was decreased by 14.1 %(P0.01). The levels of ADM, CRLR, RAMP2 and RAMP3 mRNA in calcified aorta were elevated by 37.7%(P0.01), 41.4%(P0.01),60.1%(P0.05) and 13%(P0.01) respectively, compared with the control. The elevated levels of CRLR, RAMP2 or RAMP3 mRNA were positively correlated with up regulations of ADM mRNA in calcified myocardium and aorta.Calcified myocardium and aorta may generate an increased amount of ADM, different alterations of gene expressions of ADM and its receptor system. These RESULTS suggest that ADM and its receptor system might participate in the regulation of calcification in heart and vessel.

It is known that the caudal ventrolateral medulla (CVLM) plays an important role in controlling blood pressure and mediating the cardiovascular effects of centrally acting antihypertensive drugs such as clonidine. Recently, the effect of clonidine was believed to be related to the functional states of N-methyl-D-aspartate (NMDA) receptors. The present work was designed to observe the interactions between clonidine and NMDA receptor in the CVLM. Unilaterally injected clonidine (6 nmol) into the CVLM not only produced a pressor action, but also effectively (P0.01, n=8) antagonized the decreases in both mean arterial pressure (MAP) (-22.3+/-5.0 to -7.9+/-2.3 mmHg) and heart rate (HR) (-31.9+/-5.9 to -10.3+/-2.7 beats/min) evoked by L-glutamate in the CVLM. Unilaterally injected NMDA receptor antagonist MK801 (200 pmol) into the CVLM significantly increased MAP by 26.5+/-3.7 mmHg and HR by 37.1+/-7.6 beats/min, and completely (P0.01, n=10) abolished the pressor effect (16.1+/-6.6 to 1.5+/-2.8 mmHg) of clonidine in the CVLM. In conclusion, these findings show that NMDA receptors within the CVLM contribute to clonidine-induced action, and suggest that the CVLM plays an important role in the interaction between clonidine and NMDA receptors.

To observe protective effects of taurine on reactive oxygen species generation induced by homocysteine in rat vascular smooth muscle cells (VSMC).Rat VSMC was incubated with various concentrations of homocysteine and taurine. The lactate dehydrogenase (LDH) activity which released into culture medium was elevated as an indicator for VSMC injury. The reactive oxygen species (ROS)--hydrogen peroxide (H2O2) and superoxide anion (O2(-*)) were measured with luminol or lucigenin chemiluminescence method, and the mitochondria Mn-superoxide dismutase (Mn-SOD) and catalase (CAT) were also measured in treated VSMC.LDH leakage from cultured VSMC treated with homocysteine, was increased (P0.01 vs control), and it was markedly inhibited when co-incubated with taurine (P0.01). Homocysteine induced H2O2 generation from VSMC in a concentration dependent manner (P0.01 vs control). However, taurine (5, 10, and 20 mmol/L) significantly antagonized 0.5 mmol/L homocysteine-induced H2O2 generation in VSMC in a concentration dependent manner (P0.01 vs homocysteine alone group), although taurine itself did not alter the H2O2 generation in VSMC (P0.05 vs control). In this study, the superoxide anion in VSMC was not detectable by chemiluminescence method. In addition, treatment of VSMC with taurine increased mitochondria Mn-SOD and CAT activity in a concentration dependent manner (P0.05), but homocysteine decreased mitochondria Mn-SOD and CAT activity (P0.01 vs control). In addition, co-administration of taurine markedly ameliorated homocysteine-induced inhibition of Mn-SOD and CAT activity in VSMC (P0.01 vs homocysteine alone group).Taurine antagonized the effects of homocysteine on ROS generation and anti-oxidant enzyme activities in rat VSMC in vitro.