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Intermedin (IMD), a paracrine/autocrine peptide, protects against cardiac fibrosis. However, the underlying mechanism remains poorly understood. Previous study reports that activation of nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cardiac fibrosis. In this study, we aimed to investigate whether IMD mitigated cardiac fibrosis by inhibiting NLRP3. Cardiac fibrosis was induced by angiotensin II (Ang II) infusion for 2 weeks in rats. Western blot, real-time PCR, histological staining, immunofluorescence assay, RNA sequencing, echocardiography, and hemodynamics were used to detect the role and the mechanism of IMD in cardiac fibrosis. Ang II infusion resulted in rat cardiac fibrosis, shown as over-deposition of myocardial interstitial collagen and cardiac dysfunction. Importantly, NLRP3 activation and endoplasmic reticulum stress (ERS) were found in Ang II-treated rat myocardium. Ang II infusion decreased the expression of IMD and increased the expression of the receptor system of IMD in the fibrotic rat myocardium. IMD treatment attenuated the cardiac fibrosis and improved cardiac function. In addition, IMD inhibited the upregulation of NLRP3 markers and ERS markers induced by Ang II. In vitro, IMD knockdown by small interfering RNA significantly promoted the Ang II-induced cardiac fibroblast and NLRP3 activation. Moreover, silencing of inositol requiring enzyme 1 α (IRE1α) blocked the effects of IMD inhibiting fibroblast and NLRP3 activation. Pre-incubation with PKA pathway inhibitor H89 blocked the effects of IMD on the anti-ERS, anti-NLRP3, and anti-fibrotic response. In conclusion, IMD alleviated cardiac fibrosis by inhibiting NLRP3 inflammasome activation through suppressing IRE1α via the cAMP/PKA pathway.

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.

Aim: Endoplasmic reticulum stress (ERS) and inflammation participate in cardiac fibrosis. Importantly, a novel paracrine/autocrine peptide intermedin1–53 (IMD1–53) in the heart inhibits myocardial fibrosis in rats. However, the mechanisms are yet to be fully elucidated. Methods: Myocardial fibrosis in apolipoprotein E-deficient (ApoE -/-) mice and neonatal rat cardiac fibroblasts (CFs) were induced using homocysteine (Hcy). Results: IMD1–53 inhibited myocardial fibrosis in vivo and in vitro. Picrosirius red staining showed that IMD1–53 reduced myocardial interstitial collagen deposition in ApoE-/- mice treated with Hcy and decreased the expression of myocardial collagen I and III, which was further verified in rat CFs. IMD1–53 attenuated myocardial hypertrophy, as shown by cardiomyocyte cross-sectional area, ratio of heart weight to body weight, and mRNA levels of atrial natriuretic peptide and brain natriuretic peptide. IMD1–53 inhibited the upregulation of ERS hallmarkers such as glucose-regulated protein 78 (GRP78), GRP94, activating transcription factor 6 (ATF6), ATF4, inositol-requiring enzyme 1α, spliced-X-box-binding protein-1, protein kinase receptor-like ER kinase, and eukaryotic translation initiation factor 2α in mouse myocardium and rat CFs treated with Hcy. In addition, IMD1–53 decreased the production of inflammatory factors such as tumor necrosis factor-α, monocyte chemotactic protein-1, interleukin-6 (IL-6), and IL-1β in the mouse myocardium and rat CFs treated with Hcy. Concurrently, IMD1–53 ameliorated the expression of nuclear factor-κB, transforming growth factor-β1, and c-Jun N-terminal kinase in the mouse myocardium and rat CFs treated with Hcy. Conclusions: IMD potentially protects against myocardial fibrosis induced by Hcy in ApoE-/- mice, possibly via attenuating myocardial ERS and inflammation.

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.

This study compared angiotensin II (Ang II) and angiotensin III (Ang III) for their effects on rat neonatal cardiomyocytes and cardiac fibroblasts in vitro and discussed the possible role of Ang III in the pathogenesis of cardiac remodeling. To do so, protein synthesis, cardiac fibroblast proliferation, collagen synthesis, and secretion in response to treatment with Ang III and Ang II were investigated. Protein synthesis rate was assessed by 3H-Leucine (3H-Leu) incorporation; the content of DNA was defined by 3H-thymidine (3H-TdR) incorporation; and collagen synthesis and secretion were assessed by 3H-proline (3H-Pro) incorporation. In neonatal cardiomyocytes, Ang III stimulated protein synthesis in a concentration-dependent manner, whereas in neonatal cardiac fibroblasts, DNA synthesis as well as collagen synthesis and secretion were increased in a concentration-dependent manner. Treatment with captopril, selective aminopeptidase A (APA) inhibitor (EC33), or selective aminopeptidase N inhibitor (PC18) had no effect on these outcomes. Treatment with losartan significantly decreased the effects of Ang III, except for cardiomyocyte protein synthesis. Compared with Ang II, Ang III could stimulate cardiomyocyte protein synthesis, cardiac fibroblast proliferation, and collagen synthesis and secretion. Furthermore, 10-7 mol/L Ang II but not Ang III significantly increased APA activity in both cardiomyocytes and fibroblasts. All these results show the bioactive effects of Ang III on myocardial cells and suggest that Ang III could be an important independent factor besides Ang II in the regulation of cardiac function and may affect the pathogenesis of cardiac remodeling.

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. We aimed to determine whether the endogenous apelin/APJ system is an intrinsic protective pathway in ischemic/reperfusion injury. A Langendorff model of perfused isolated rat hearts and primary cultured myocardial cells from neonatal rats were used. Cardiac function was monitored and apelin/APJ expression was determined by real-time PCR and Western blot analysis. In rats under I/R, cardiac function was significantly decreased as compared with controls, and APJ was over-expressed at both the mRNA and protein levels (by 7-fold and 35%, respectively, both p

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.

Atrogin-1/MAFbx is an ubiquitin E3 ligase that regulates myocardial structure and function through the ubiquitin-dependent protein modification. However, little is known about the effect of atrogin-1 activation on the gene expression changes in cardiomyocytes. Neonatal rat cardiomyocytes were infected with adenovirus atrogin-1 (Ad-atrogin-1) or GFP control (Ad-GFP) for 24 hours. The gene expression profiles were compared with microarray analysis. 314 genes were identified as differentially expressed by overexpression of atrogin-1, of which 222 were up-regulated and 92 were down-regulated. Atrogin-1 overexpression significantly modulated the expression of genes in 30 main functional categories, most genes clustered around the regulation of cell death, proliferation, inflammation, metabolism and cardiomyoctye structure and function. Moreover, overexpression of atrogin-1 significantly inhibited cardiomyocyte survival, hypertrophy and inflammation under basal condition or in response to lipopolysaccharide (LPS). In contrast, knockdown of atrogin-1 by siRNA had opposite effects. The mechanisms underlying these effects were associated with inhibition of MAPK (ERK1/2, JNK1/2 and p38) and NF-κB signaling pathways. In conclusion, the present microarray analysis reveals previously unappreciated atrogin-1 regulation of genes that could contribute to the effects of atrogin-1 on cardiomyocyte survival, hypertrophy and inflammation in response to endotoxin, and may provide novel insight into how atrogin-1 modulates the programming of cardiac muscle gene expression.

Proliferation and migration of vascular smooth-muscle cells (VSMCs) are essential events in neointimal hyperplasia. Recent findings that active oxygen species induce pro-oncogene expression, and stimulate VSMC DNA synthesis and cell division, suggest that active oxygen species may play an important role in intimal proliferation after arterial injury. To determine how the redox state of the artery was altered by injury, the levels of thiobarbituric-acid-reactive substances (TBARs, markers of lipid oxidation) and glutathione peroxidase (GSH-PX, the enzyme responsible for the production of glutathione, a major intracellular antioxidant) were measured in the rat aorta after balloon injury. There was an inverse relationship between the level of TBARs, which increased significantly to a maximum 17% greater than normal at 10 days after injury (p0.05, n=7), and the activity of GSH-PX, which decreased significantly to a minimum 36.5% less than normal at 10 days after injury (p0.05, n=7). To determine whether maintaining a more reduced vessel environment would inhibit intimal proliferation, L-cysteine was administered by intraperitoneal injection from 3 days before to 14 days after balloon injury. At 14 days after the arterial injury, the aorta was harvested for histological and morphometric measurements of TBARs and GSH-PX. At 7 and 14 days after balloon injury, the aorta was harvested for [3H]thymidine incorporation studies. Efficacy of therapy was demonstrated by a significant decrease in the level of TBARs and an increase in the activity of GSH-PX (p0.05 vs. the control group, n=7). In the L-cysteine group, all parameters of intimal proliferation were significantly reduced compared to the controls, including the intima:media ratio (0.091 vs. 0.253, p0.05); the cross-sectional area of the neointima (0.0563 compared to 0.140 mm2, p0.05), coverage of the internal elastic lamina by the neointima (23.44 compared to 43.27%, p0.05), and [3H]thymidine incorporation (at 7 days 240.58 vs 350.68 cpm/mg tissue, p0.05; at 14 days 181.71 vs. 275.30 cpm/mg tissue, p0.05). These results demonstrate dynamic alterations in the vessel redox state after arterial injury, and suggest that maintaining a more reduced environment (e.g. administration of L-cysteine) will reduce intimal proliferation after arterial injury.

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.

To explore the impact of sulfur dioxide (SO(2)) on hydrogen sulfide (H(2)S)/cystathionine-γ-lyase (CSE) and H(2)S/mercaptopyruvate sulfurtransferase (MPST) pathways in the pathogenesis of hypoxic pulmonary hypertension.Thirty-two male Wistar rats were randomly divided into four groups: control group (n = 8), hypoxic group (n = 8), hypoxic + SO(2) group (n = 8) and hypoxic + hydroxamate (HDX) group (n = 8). After 21 days of experiment, the concentration and production of H(2)S in lung tissues were measured respectively for each rat. The protein expression of CSE and MPST in intima and media of small pulmonary arteries in rats was detected with immunohistochemical method.Compared with control group, the mean pulmonary artery pressure (mPAP) in rats of hypoxic group was increased significantly [(33.38 ± 6.32) mm Hg vs. (16.74 ± 3.81) mm Hg, P0.01]. Compared with hypoxic group, the mPAP in rats of hypoxic + SO(2) group was decreased significantly [(29.65 ± 2.53) mm Hg vs. (33.38 ± 6.32) mm Hg, P0.01]. However, compared with hypoxic group, the mPAP in rats of hypoxic + HDX group was increased significantly [(39.44 ± 6.26) mm Hg vs. (33.38 ± 6.32) mm Hg, P0.01]. Compared with control group, the concentration [(2.02 ± 0.43) µmol/g vs. (3.11 ± 0.42) µmol/g, P0.01] and production [(19.64 ± 3.48) nmol/(g·min)vs. (28.20 ± 5.95) nmol/(g·min), P0.05] of H(2)S were decreased significantly in rats of hypoxic group, respectively. When treated with SO(2), hypoxic rats showed an increased concentration [(2.73 ± 0.20) µmol/g vs. (2.02 ± 0.43) µmol/g, P0.01] and production [(26.24 ± 1.92) nmol/(g·min) vs. (19.64 ± 3.48) nmol/(g·min), P0.01] of H(2)S in lung tissue compared with those without receiving SO(2) treatment. When treated with HDX, hypoxic rats showed a significant decrease in concentration [(1.64 ± 0.23) µmol/g vs. (2.02 ± 0.43) µmol/g, P0.05] and production [(13.94 ± 3.63) nmol/(g·min) vs. (19.64 ± 3.48) nmol/(g·min), P0.05] of H(2)S in lung tissue compared with those without receiving HDX treatment. As for the expression of CSE in small pulmonary arteries (SPAs), compared with control group, the expression of CSE in intima [(0.31 ± 0.02) vs. (0.36 ± 0.01), P0.01] and media [(0.27 ± 0.01) vs. (0.30 ± 0.01), P0.01] in rats of hypoxic group was decreased significantly. While compared with hypoxic group, the expression of CSE in intima [(0.35 ± 0.02) vs. (0.31 ± 0.02), P0.01] in SPAs of hypoxic + SO(2) group was increased significantly. With HDX treatment, the expression of CSE in intima [(0.26 ± 0.01) vs. (0.31 ± 0.02), P0.01] in SPAs of hypoxic group was lower than that without HDX treatment. As for the expression of MPST in SPAs, compared with hypoxic group, the expression of MPST in media [(0.32 ± 0.02) vs. (0.29 ± 0.01), P0.01] in SPAs of hypoxic + SO(2) group was increased significantly.SO(2) might upregulate H(2)S/CSE and H(2)S/MPST pathways in pulmonary arteries of hypoxic rats.

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.

Sulfur dioxide was considered to be toxic and detrimental to human health. However, this review highlights recent advances that suggest sulfur dioxide might be a novel endogenous gaseous signaling molecule involved in the regulation of cardiovascular functions.The data used in this review were mainly from the studies reported in Medline and PubMed published from 1986 to 2010.Original articles and critical reviews selected were relevant to exogenous and endogenous sulfur dioxide.The sulfur dioxide/aspartate amino transferase pathway is endogenously generated in the cardiovascular system, and sulfur dioxide shows broad bioactive effects, such as antihypertension, vasodilation, and amelioration of vascular remodeling. A disturbed sulfur dioxide/aspartate amino transferase pathway is known to be involved in the pathogenesis of many cardiovascular diseases, such as ischemia-reperfusion injury, monocrotaline-induced pulmonary hypertension, athrosclerosis, spontaneous hypertension and hypoxic pulmonary hypertension. Furthermore, in experimental studies the prognosis of these cardiovascular diseases can be improved by targeting endogenous sulfur dioxide.The findings suggest that sulfur dioxide is a novel endogenous gaseous signaling molecule involved in the regulation of cardiovascular functions.

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 investigate the impact of human urotensin II (hUII) on pulmonary arterial smooth muscle cell (PASMCs) cycle in vitro.PASMCs dissected from Wistar rats were cultured in vitro, and incubated with series of concentrations of hUII (10(-7) mol/L, 10(-8) mol/L, 10(-9) mol/L) for 12 hours under normoxia or hypoxia condition, in order to analyze cell cycle progression and sub-G1 of PASMCs by using flow cytometric analysis stain of propidium iodide, which represented the proliferative and apoptotic changes in PASMCs.The study showed a dose-dependent effect of hUII on PASMCs proliferation, which reflected the increase both in percentage of S phase of cell cycle and proliferative index (PI). The response of PASMCs to hUII was different under normoxic and hypoxic conditions. Compared with the control group, the treatment of 10(-7) mol/L, 10(-8) mol/L and 10(-9) mol/L hUII produced an increase of 175%, 136% and 118% under normoxia, respectively, and 135%, 118% and 103% under hypoxia, respectively. The concentration 10(-7) mol/L hUII played a significant role in PASMCs proliferation both under hypoxia and normoxia (P0.01). The results of cell cycle did not show sub-G1 of PASMCs at various concentrations of hUII.hUII may stimulate DNA synthesis in S phase cell cycle of PASMCs and the proliferation of PASMCs under normoxia and hypoxia conditions, which promote cell growth in a dose-dependent manner.

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 role of mitogen-activated protein kinase phosphatase-1 (MKP-1) in the regulation of cells proliferation, the expression of MKP-1 and extracellular signal-regulated kinase-1 (ERK-1) in heart and aorta of spontaneous hypertensive rat (SHR) and WKY were studied. We also investigated the effect of MKP-1 genes,which were transfected into vascular smooth muscle cells (VSMC) using the classical calcium phosphate coprecipitation technique, on the incorporation of 3H-TdR in VSMC stimulated by angiotensin II (Ang II).(1) Compared with that of WKY, MKP-1 expression in heart and aorta were significantly decreased by 53% (P0.01) and 45% (P0.01) in SHR, respectively. While the expression of ERK-1 in heart and aorta of SHR were higher than that of WKY (P0.01). The ratio of ERK-1/MKP-1 in heart and aorta of SHR were significantly higher than that of WKY. (2) 3H-TdR incorporation in VSMC stimulated by Ang II (10(-7) mol/L) was increased by 207% (P0.01), compared with control group. In the transfected cells with wild MKP-1 gene, Ang II-induced incorporation of 3H-TdR lowered 63%, compared with untransfected cells (P0.05). There were no marked inhibitive role between mutant MKP-1-transfected cells and blank vector-transfected cells in response to Ang II, compared with Ang II group (P0.05).These results showed that the expression of ERK-1 in heart and aorta isolated from SHR, which stimulated proliferation and hypertrophy of cells, is higher than that of MKP-1 which dephosphorylates and inactivated ERK-1. In addition, MKP-1 significantly inhibits Ang II-stimulated proliferation of VSMC.

To observe the effect of myocardial mitochondrial L-arginine (L-Arg)/nitric oxide (NO) system on mitochondrial Ca2+ transport by using purified rat mitochondria and incubation of them in vitro.Compared with control group, incubation of mitochondria with L-Arg (10(-4) mol/L, NO substrate) or sodium nitroprusside (5 x 10(-7) mol/L, the donor of exogenous NO, SNP) increased significantly mitochondrial NO2- (66% and 89%, P0.01), respectively, and decreased the Ca2+ content (40% and 54%, P0.01). After L-Arg or SNP treatment, mitochondrial Ca2+ uptake were decreased by 67% and 85%, respectively (P0.01), vs control. The rate of mitochondrial Ca2+ release decreased by 11% and 8%, respectively (P0.01). When L-NAME (NO synthase inhibitor) was incubated with mitochondria and the L-Arg together, it inhibited the effects of L-Arg, NO2 on the mitochondrial NO2 formation, Ca2+ content descending, and decrease of Ca2+ uptake and release.The data suggest that myocardial mitochondrial L-Arg /NO systems take part in the regulation of cardiomyocytes Ca2+ transportation.

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 coordinated role and its mechanism of the high protein and hypercholesterol intake on inducing rat myocardial fibrosis.The tissue level of the collagen in left ventricule, the concentrations of the plasma and the cardiac tissue angiotensin II (Ang II) and Aldosterone (Ald), the serum concentration of nitrite (NO2-), in the Wistar rats on diet which adding 20% protein or/and 100 mg/d cholesterin in the rat standard foods for 8 weeks, were measured by the colorimetric analysis of the hydroxyproline, by the radioimmunoassay, and by the assay of Griess, respectively.1.69 times left ventricular collagen contents, 0.7 times plasma concentrations of total cholesterin, 1.5 times levels of the plasma Ang II and 1 time myocardial ald contents were higher, and the serum NO2- concentration was significant lower, in the rats of the high protein and hypercholesterol intake than in the rats of the high protein intake. That 0.48 times left ventricular collagen contents, 0.23 times plasma Ang II in the high protein and hypercholesterol intake rats were higher than in the high cholesterin intake rats.The excessive protein and cholesterin intake can induce the coordinated effect on developing the myocardial fibrosis of rats. And the mechanism of the fibrosis in rat left ventricule maybe result with the activation of RAAS and the endothelial injury.

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.

Adventitia plays an important role in vascular injury diseases. Nitric oxide (NO) from inducible nitric oxide synthase (iNOS) is involved in many cardiovascular diseases. iNOS/NO pathway is activated in aorta of spontaneously hypertensive rats (SHRs). The role of aldosterone in L-arginine (L-Arg)/iNOS/NO pathway of aortic adventitia is uncertain. We investigated the effect of aldosterone antagonist spironolactone on adventitial L-Arg/iNOS/NO pathway in SHRs.Twenty male SHR (10 weeks old, 220-280 g) were randomly divided into 2 groups (10 in each): untreated or treated with the aldosterone receptor antagonist, spironolactone (20 mg/kg per day) for 6 weeks. Age-matched Wistar-Kyoto rats (WKY) were used as control. Systolic blood pressure (tail-cuff method) was measured weekly. Six weeks later, the rats were sacrificed. The whole aorta was collected and aortic adventitia was isolated. iNOS activity, [(3)H]-L-Arg transport assay of aortic adventitia were carried out by isotope-labeled L-arginine conversion rate measurement, and measurement of nitrate/nitrite (NOx), an index of NO production of aortic adventitia was assayed with the Griess reaction.iNOS activity, [(3)H]-L-Arg transport and NO production were greatly increased in aortic adventitia of SHR [(12.55 +/- 2.27) pmol x mg(-1) x min(-1), (0.88 +/- 0.19) pmol x mg(-1) x min(-1) and (2.07 +/- 0.39) micromol/L)] compare versus WKY [(5.96 +/- 1.87) pmol x mg(-1) x min(-1), (0.51 +/- 0.15) pmolxmg(-1) x min(-1) and (1.55 +/- 0.32) micromol/L, P0.01], and decreased significantly by spironolactone treatment [(8.32 +/- 1.84) pmol x mg(-1) x min(-1), (0.61 +/- 0.15) pmol x mg(-1) x min(-1) and (1.64 +/- 0.27) micromol/L, P0.01].L-Arg/iNOS/NO is activated in aortic adventitia of SHR. Spironolactone can inhibit the activation of L-Arg/iNOS/NO pathway. Aldosterone may play an important role in some cardiovascular diseases such as atherosclerosis through iNOS/NO pathway.

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.

Hydrogen sulfide (H(2)S) dilates blood vessels in vivo and in vitro probably by opening vascular smooth muscle K(+)-ATP channels. The study was designed to observe the role of mitochondria membrane K(ATP) channel blocker (5-HD) in the regulation of cardiac function isolated perfused heart of rat with H(2)S.The isolated rat heart was perfused in a Langendorff apparatus. After 20 minutes of stabilization, physiological concentration of NaHS (H(2)S donor, 100 micromol/L) was continuously perfused for 20 min in group A (n = 6), isolated hearts in group B (n = 6) and C (n = 7) were pretreated with nonspecific K(ATP) channel blocker glibenclamide (100 micromol/L) or 5-HD (100 micromol/L) for 5 minutes then perfused with NaHS (100 micromol/L) for 10 minutes. Heart rate (HR), left ventricular developed pressure (DeltaLVP), dp/dt(max) and dp/dt(min) and coronary perfusion flow (CPF) were measured.Post continuous perfusion of NaHS at physiological concentration for 20 minutes, DeltaLVP, dp/dt(max) and dp/dt(min) all significantly decreased while HR and CPF remained unchanged compared to baseline levels (all P0.05). The negative inotropic effect of H(2)S could partly be blocked by nonspecific K(ATP) channel blocker glibenclamide and mitochondria membrane K(ATP) channel blocker 5-HD.Present findings suggested that H(2)S at physiological concentration could produce negative inotropic effect in isolated hearts and this effect was mediated by K(ATP) channel and mitochondria membrane K(ATP) channel.

1. The sulphur-containing gases hydrogen sulphide and sulphur dioxide can be generated endogenously in mammalian tissues and exert significant biological effects in the cardiovascular system. Hydrogen sulphide is considered to be the third novel gasotransmitter in addition to nitric oxide and carbon monoxide. The present review describes the effects of hydrogen sulphide on the cardiovascular system and its possible mechanisms under physiological conditions. We also discuss the pathophysiological effects of hydrogen sulphide on cardiovascular diseases. The therapeutic potential of hydrogen sulphide is summarized. 2. We recently discovered that sulphur dioxide, another endogenous sulphur-containing gas, has important physiological and pathophysiological roles in the cardiovascular system. To some extent, the effect of sulphur dioxide is similar to that of the other gasotransmitters nitric oxide, carbon monoxide and hydrogen sulphide. Sulphur dioxide may also be a novel gas mediator in the cardiovascular system.

To study the modulatory effect of sulfur dioxide (SO(2)) on the accumulation of collagen type I and type III in the wall of aorta during spontaneously hypertensive rat (SHR) vascular remodeling.Eight male Wistar Kyoto rats at the age of 4 weeks with normal blood pressure were used as a control group. And sixteen male SHRs at the age of 4 weeks were randomly divided into an SHR control group and SHR + Na(2)SO(3)/NaHSO(3) (SO(2) donor) group. Na(2)SO(3)/NaHSO(3) solution was injected intraperitoneally everyday to the rats in the SHR + Na(2)SO(3)/NaHSO(3) group. After 5 weeks, the systemic blood pressure was measured. The weight ratio of left ventricle to the whole heart was also measured. The rat aorta was dyed with Hart's method. The morphometric parameters including outer radius, lumen radius and the wall thickness were calculated by Leica workstation. The plasma level of SO(2) was determined by HPLC method. The expressions of collagen type I and type III in aorta were detected by immunohistochemistry.(1) Compared with the WKY rat control group, the systolic blood pressure increased by 53%, the weight ratio of left ventricle to the whole heart increased by 6% but the plasma level of SO(2) decreased by 44% for rats in the SHR group (P0.01 or P0.05). Compared with the SHR control group, the systolic blood pressure decreased by 26%, but the plasma level of SO(2) increased by 28% (all P0.01) for rats in the SHR + Na(2)SO(3)/NaHSO(3) group. (2) Compared with the WKY rats, the ratio of media to lumen radius increased by 28% for SHR. Compared with the SHR group, the ratio of media to lumen radius decreased by 10% (P0.01) in rats of the SHR + Na(2)SO(3)/NaHSO(3) group. (3) Compared with rats in the WKY control group, collagen type I expression increased by 10% for rats in the SHR group (P0.01). Compared with the SHR group, however, the expression decreased by 58% for rats in the SHR + Na(2)SO(3)/NaHSO(3) group (P0.01). (4) Compared with rats in the WKY control group, the expression increased by 13% for rats in the hypoxia SHR group (P0.01); however, compared with rats in the SHR group, the expression decreased by 8% in the rats of the SHR +Na(2)SO(3)/NaHSO(3) group (P0.01).In the process of SHR vascular collagen remodeling in the rats, SO(2) could inhibit the abnormal accumulation of collagen type I and type III in the wall of aorta. This effect may be one of the mechanisms by which SO(2) ameliorates SHR vascular remodeling.

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 and blood pressure in spontaneously hypertensive rats (SHR).Four weeks old male SHR rats were treated with saline (control, n = 7), sodium hydrosulfide (NaHS, a H2S donor, n = 7) and propargylglycine (PPG, endogenous H2S production inhibitor, n = 6) for 5 weeks. Age-natched male Wistar Kyoto (WKY) rats served as normotensive controls (n = 8). Five weeks later, systolic blood pressure (SBP) was measured in conscious and quiet rats by means of the standard tail-cuff method. The protein expressions of intercellular adhesive molecule-1 (ICAM-1), nuclear transcriptional factor-kappaB p65 (NF-kappaB p65) and inhibitor of nuclear transcriptional factor-kappaB (IkappaB-alpha) in thoracic aorta of rats were detected by immunohistochemical assay, while the expression of ICAM-1 mRNA in thoracic aorta of rats were investigated by in situ hybridization.SBP of control SHR rats was significantly higher than that of WKY rats (P0.05) accompanied by significantly upregulated expressions of ICAM-1 mRNA, ICAM-1 protein, NF-kappaB p65 protein in aortic endothelial cells (all P0.01), while the expression of IkappaB-alpha protein in aortic endothelial cells in SHR control group was significantly lower than that of WKY control group (P0.01). NaHS treated SHR rats showed significantly reduced SBP and downregulated expressions of ICAM-1 mRNA, ICAM-1, NF-kappaB p65 in aortic endothelial cells and upregulated expression of IkappaB-alpha protein in aortic endothelial cells compared to untreated control SHR rats (all P0.05). In SHR rats treated with PPG, the expressions of ICAM-1 mRNA, ICAM-1 protein, NF-kappaB p65 protein in aortic endothelial cells were further increased while the expression of IkappaB-alpha protein further decreased compared with control SHR rats (all P0.05).H2S might attenuate the development of hypertension through by attenuating vascular inflammation reactions.

Hypoxic pulmonary hypertension is an important pathophysiologic process of various cardiovascular diseases. Sulfur dioxide (SO2) was considered as a kind of toxic gas previously, but recent studies suggested that SO2 could act as a key bioactive molecule in the pathogenesis of cardiovascular diseases. Therefore, this study was designed to examine the effect of sulfur dioxide on pulmonary vascular structure of hypoxic pulmonary hypertensive rats treated with SO2 donor substances.The rats were randomly divided into 3 groups: control group(n = 8), hypoxic group(n = 8) and hypoxic + SO2 group (n = 10, treated with SO2 donor Na2SO3/NaHSO3). The rats of hypoxic group and hypoxic + SO2 group were under a hypoxic condition for 21 days, while the rats of control group were exposed to room air. The mean pulmonary artery pressure was tested by means of right cardiac catheterization and the content of SO2 in plasma was investigated by high performance liquid chromatography (HPLC). The change in relative medial thickness (RMT) of pulmonary arteries was examined under optical microscope. The ultra-structural changes were observed under a transmission electron microscope. The data were analyzed through one-way analysis of variance (ANOVA) by SPSS 13.0 software.Compared with control group [(2.25 +/- 0.50) kPa], the mean pulmonary artery pressure of hypoxic group [(5.12 +/- 0.51) kPa] raised significantly (t = 5.091, P0.01) and RMT of hypoxic group (9.66 +/- 1.27) compared with control group (6.83 +/- 1.57) significantly raised (t = 3.392, P0.01). Ultrastructural observation showed the proliferation and degeneration of endothelial cells in small pulmonary arteries in rats with pulmonary hypertension. The internal elastic lamina was irregular. The proliferation of medial smooth muscle cells of arteries was shown at the level of respiratory bronchioles. The collagens also increased. Meanwhile, compared with control group [(33.36 +/- 5.62) micromol/L], the content of SO2 in plasma of hypoxic group [(27.01 +/- 4.17) micromol/L] declined (t = 2.067, P0.05). Whereas compared with that of hypoxic group [(5.12 +/- 0.51) kPa], the mean pulmonary artery pressure of hypoxic + SO2 group [(3.94 +/- 0.33) kPa] declined (t = 2.712, P0.01) and RMT of hypoxic + SO2 group (6.97 +/- 1.83) decreased compared with hypoxic group (9.66 +/- 1.27) (t = 3.009, P0.01). Compared with those of hypoxic group, the pulmonary artery ultrastructural changes in hypoxic group ameliorated obviously after using exogenous sulfur dioxide donor. The endothelial cells became flat and the smooth muscle cells of arteries slightly enlarged and arranged regularly. At the same time, compared with hypoxic group [(27.01 +/- 4.17) micromol/L], the content of SO2 in plasma of hypoxic + SO2 group [(29.89 +/- 4.52) micromol/L] raised (t = 1.263, P0.05).Sulfur dioxide plays an important role in the regulation of small pulmonary artery structural changes in hypoxic pulmonary hypertensive rats. The hypoxic pulmonary hypertensive damages can be ameliorated significantly after using exogenous SO2 donor.

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.

To explore the effects of sulfur dioxide (SO2) on the proliferation and apoptosis of aorta smooth muscle cells in hypertension rats and possible mechanism thereof.Sixteen 4-week-old male spontaneously hypertensive rats (SHRs) were randomly divided into 2 equal groups: control and Na2SO3/NaHSO3 (a SO2 donor)-treated group. Eight 4-week-old male WKY (Wistar Kyoto) rats were assigned for normal control group. Five weeks later, the pressure was measured. The rat aortas were dyed with Hart's method. The morphometric parameters were calculated by Leica workstation. The plasma level of SO2 was determined by HPLC method. VSMC apoptosis was measured by TUNEL technique. The expression levels of proliferating cell nuclear antigen (PCNA), Bcl-2, Fas and caspase-3 were detected by immunohistochemical assay.(1) Compared with those of the WKY rats, the blood pressure, ratio of media to lumen radius, and proliferation index (PI) of the SHRs were increased [(172 +/- 10) mm Hg vs (112 +/- 9) mm Hg, 0.073 +/- 0.004 vs 0.057 +/- 0.004, 0.32 +/- 0.06 vs 0.05 +/- 0.03, respectively], but the plasma level of SO2 and the apoptosis index (AI) were decreased in the SHRs [(6.4 +/- 1.5) micromol/L vs (11.3 +/- 1.0) micromol/L, 0.16 +/- 0.07 vs 0.30 +/- 0.19, respectively]. The expression of Bcl-2 was increased (0.209 +/- 0.007 vs 0.202 +/- 0.006), and the expression levels of Fas and caspase-3 of SHRs were both lower than those of the WKY rats (0.205 +/- 0.006 vs 0.211 +/- 0.005, 0.229 +/- 0.005 vs 0.244 +/- 0.010, respectively). (2) Compared with the SHR control group, the systolic blood and the ratio of media to lumen radius were decreased [(128 +/- 7) mm Hg, 0.066 +/- 0.002, respectively], but the plasma level of SO2 was increased [(8.3 +/- 1) micromol/L] for the SHR + Na2SO3/NaHSO3 group. PI was lower (0.14 +/- 0.03) and AI was higher (0.40 +/- 0.11) in SHR + Na2SO3/NaHSO3 group than those in SHR control group. The expression of Bcl-2 of VSMCs was down-regulated (0.199 +/- 0.006), but the levels of Fas and caspase-3 were up-regulated (0.218 +/- 0.003 and 0.251 +/- 0.011 respectively) in the SHR + Na2SO3/NaHSO3 group.SO2 may attenuate the structural remodeling through reducing the proliferation and enhancing the apoptosis of smooth muscle cells in SHRs. SO2 may modulate the process of apoptosis possibly through the downward regulation of the level of Bcl-2 and enhance the expression of Fas and caspase-3.

The present study aimed to explore the protective effect of endogenous sulfur dioxide (SO2) in the development of monocrotaline (MCT)-induced pulmonary hypertension (PH) in rats.Forty Wistar rats were randomly divided into the MCT group receiving MCT treatment, the MCT+L-aspartate-beta- hydroxamate (HDX) group receiving MCT plus HDX treatment, the MCT+SO2 group receiving MCT plus SO2 donor treatment, and the control group. Mean pulmonary artery pressure (mPAP) and structural changes in pulmonary arteries were evaluated. SO2 content, aspartate aminotransferase activity, and gene expression were measured. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), reduced glutathione (GSH), oxidized glutathione, and malondialdehyde (MDA) levels were assayed.In the MCT-treated rats, mPAP and right ventricle/(left ventricle+septum) increased significantly (P0.01), pulmonary vascular structural remodeling developed, and SOD, GSHPx, CAT, GSH, and MDA levels of lung homogenates significantly increased (P0.01) in association with the elevated SO2 content, aspartate aminotransferase activity, and gene expression, compared with the control rats. In the MCT+HDXtreated rats, lung tissues and plasma SO2 content and aspartate aminotransferase activities decreased significantly, whereas the mPAP and pulmonary vascular structural remodeling were markedly aggravated with the decreased SOD, CAT, and GSH levels of lung tissue homogenates compared with the MCT-treated rats (P0.01). In contrast, with the use of a SO2 donor, the pulmonary vascular structural remodeling was obviously lessened with elevated lung tissue SOD, GSH-Px, and MDA content, and plasma SOD, GSH-Px, and CAT levels.Endogenous SO2 might play a protective role in the pathogenesis of MCT-induced PH and promote endogenous antioxidative capacities.

To observe the effects of sulfur dioxide (SO2) on cardiac function of isolated perfused heart of rat and to explore the physiological regulation of endogenous SO2 on myocardial action.The hearts of 64 Wistar rats were isolated, perfused with Krebs-Henseleit (KH) solution through Langendorff apparatus, and randomly divided into 8 equal groups: Four groups underwent perfusion of SO2 of the concentrations 1, 10, 100, 1000 micromol/L respectively for 5 min and then perfused with KH solution for 15 min. Eight hearts underwent perfusion of SO2 of the physiological concentration (10 micromol/L) for 20 min. The control group underwent perfusion of KH solution for 20 min. Eight hearts of the nicardipine group underwent perfusion of nicardipine, a L-type calcium channel blocker, 2.5 micromol/L for 5 min, SO2 10 micromol/L for 5 min, and then KH solution for 10 min. The heart in the hydroxamate (HDX) group underwent perfusion of HDX, an inhibitor of SO2 endogenous generating enzymes, for 5 min, and then perfused by KH solution for 15 min. The heart rate (HR), difference of left ventricular pressure (LVP), left ventricular peak rate of contraction (+ dp/dtmax), peak rate of relaxation (- dp/dtmax), and coronary flow (CF) were measured. Then transmission electron microscopy was conducted.SO2 concentration-dependently inhibited the left ventricular +/- dp/dtmax, LVP, HR, and CF (all P0.01). The left ventricular +/- dp/dtmax, LVP, and HR were inhibited (P0.05) by the physiological concentration (10 micromol/L) SO2 donor continuous perfusion for 20 min. During perfusion 20 min, the LVP, + dp/dtmax, - dp/dtmax, and HR after perfusion for 20 min of the physiological concentration (10 micromol/L) SO2 donor continuous perfusion group were (15 +/- 3) mm Hg, (485 +/- 74) mm Hg/s, (339 +/- 64) mm Hg/s, and (114 +/- 26)/min respectively, all significantly lower than those 5 min after perfusion [(23 +/- 7) mm Hg, (595 +/- 93)mm Hg/s, (436 +/- 83) mm Hg/s, and (159 +/- 31)/min, all P0.05]. The LVP, + dp/ dtmax, -dp/dtmax, and HR of the nicardipine group were(37 +/- l0)mm Hg, (1025 +/- 287)mm Hg/s, (570 +/- 181)mm Hg/s, and (139 +/- 48)/min respectively, all not significantly different from those of the control group. The LVP, + dp/dtmax, - dp/dtmax, and CF after perfusion of the HDX group were (50 +/- 11)mm Hg, (1167 +/- 270) mm Hg/s, (889 +/- 72) mm Hg/s, and (6.3 +/- 1.9) ml/min respectively, all significantly lower than those before perfusion [(69 +/- 16) mm Hg, (1579 +/- 315) mm Hg/s, (1186 +/- 263) mm Hg/s, and (9.5 +/- 1.3) ml/min, all P0.05].Exogenous SO2 has negative inotropic effect on myocardium. by the mechanism related to voltage-gated calcium channel. Nicardipine blocks the inhibitory effect of SO2 at physiological concentration.

The present study was designed to explore the endogenous production and localization of the sulfur dioxide (SO2)/aspartate aminotransferase pathway in vascular tissues of rats and to examine its vasorelaxant effect on isolated aortic rings,as well as the possible mechanisms.The content of SO2 in the samples was determined by using high performance liquid chromatography with fluorescence detection. Aspartate aminotransferase activity and its gene expression were measured by an enzymatic method and quantitative RT-PCR, respectively. Aspartate aminotransferase mRNA location in aorta was detected by in situ hybridization. The vasorelaxant effect of SO2 on isolated aortic rings of the rats was investigated in vitro. L-type calcium channel blocker, nicardipine, and L-type calcium channel agonist, Bay K8644, were used to explore the mechanisms by which SO2 relaxed the aortic rings.Aorta had the highest SO2 content among the vascular tissues tested (P0.01). The aortic aspartate aminotransferase mRNA located in endothelia and vascular smooth muscle cells beneath the endothelial layer.Furthermore, a physiological dose of the SO2 derivatives (Na2SO3/NaHSO3) relaxed isolated artery rings slightly, whereas higher doses (1-12 mmol/L) relaxed rings in a concentration-dependent manner. Pretreatment with nicardipine eliminated the vasorelaxant response of the norepinephrine-contracted rings to SO2 completely. Incubation with nicardipine or SO2 derivatives successfully prevented vasoconstriction induced by Bay K8644.Endogenous SO2 and its derivatives have a vasorelaxant function, the mechanisms of which might involve the inhibition of the L-type calcium channel.

To investigate the effects of 11, 12-epoxyeicosatrienoic acid (11, 12-EET) preconditioning and postconditioning on Ca(2+)-handling proteins in myocardial ischemia/reperfusion (IR) injury in rats and reveal the effects and mechanism of 11, 12-EET on cardioprotection. METHODS The IR injury model was built by stopping perfusion for 40 minutes followed by reperfusion for 30 minutes. The isolated Langendorff-perfused rat hearts were divided into 4 groups: control group, IR group, EET preconditioning (Pre-EET) group and EET postconditioning (Post-EET) group. The computer-based electrophysiological recorder system was used to measure the changes of the maximal rate of pressure increased in the contraction phase (+dp/dt(max)), the maximal rate of pressure decreased in the diastole phase (-dp/dt(max)), the left ventricular end diastolic pressure (LVEDP) and the difference of left ventricular pressure (delta LVP). The activity of Ca(2+)-ATPase in sarcoplasmic reticulum was measured with colorimetric method. Reverse transcription-polymerase chain reaction was used to assess the gene expression of C(a2+)-handling protein [sarcoplasic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), ryanodine receptor type 2 (RyR,), and 1, 4, 5-trisphosphate inositol receptor type 2 (IP3 R2) ] mRNAs level.Compared with IR group, the myocardial functions, the value of Ca(2+)-ATPase, and the expressions of IP3 R2 mRNA were significantly increased and the expression of PLB mRNA was significantly decreased in both Pre-EET group and Post-EET group (P0.05, P0.01). And the expression of SERCA mRNA was significantly increased in Pre-EET group (P0. 05). However, no significant differences were detected between Pre-EET and Post-EET groups. Moreover, the expression of RyR2 mRNA was not significantly different among all groups.11, 12-EET preconditioning and post-conditioning can protect myocardium from IR injury by elevating the activity of Ca(2+)-ATPase in sarcoplasmic reticulum, up-regulating the expression of IP3 R2 mRNA, and down-regulating the expression of PLB mRNA. Moreover, up-regulating the expression of SERCA mRNA maybe one of mechanisms of 11, 12-EET preconditioning on cardio protection against IR injury.

To explore the effects of 11,12-epoxyeicosatrienoic acid (11,12-EET) preconditioning and postconditioning on myocardial ischemia/reperfusion (IR) injury in rats, the IR injury model was built by stopping perfusion for 40 min followed by reperfusion for 30 min, and the changes of mitochondrial functions, myocardial metabolism and function were measured. Langendorff-perfused isolated rat hearts were divided into 4 groups: control group, persistently perfused with Krebs-Henseleit (K-H) fluid for 100 min; IR group, stopped perfusion for 40 min followed by reperfusion for 30 min; Pre-EET group, preconditioned with 6.24×10(-9) mol/L 11,12-EET for 5 min twice before subjected to ischemia; Post-EET group, postconditioned with 6.24×10(-9) mol/L 11,12-EET for 30 s twice before reperfusion. The computer-based electrophysiological recording system was used to measure the changes of maximal rate of the pressure increase in contract phase (+dp/dt(max)), maximal rate of the pressure decrease in diastole phase of heart (-dp/dt(max)), left ventricular end-diastolic pressure (LVEDP) and difference of left ventricular pressure (DLVP). The activities of lactate dehydrogenase (LDH) in effluent, Ca(2+)-ATPase, Na(+)-K(+)-ATPase and succinate dehydrogenase (SDH) in mitochondria were measured with colorimetry method; superoxide dismutase (SOD) activity was measured with hydroxylamine method and malondialdehyde (MDA) content in myocardial tissues was measured with TBA method. The results showed that: (1) Compared with that in the control group, the myocardial functions, the values of SOD, SDH and Na(+)-K(+)-ATPase were decreased in IR group (P0.05); the values of LDH, MDA and Ca(2+)-ATPase were increased (P0.05) in IR group. (2) Compared with that in IR group, the values of SDH and Na(+)-K(+)-ATPase were increased (P0.05) and the value of Ca(2+)-ATPase was decreased (P0.05) in both Pre-EET and Post-EET groups. But no significant differences were detected between Pre-EET and Post-EET groups. (3) Compared with IR treatment, both 11,12-EET preconditioning and postconditioning caused significant decreases in MDA content and leakage of LDH, amendment of heart functions and increases in SOD activity (P0.05). But there were no significant differences between 11,12-EET preconditioning and postconditioning. These results indicate that 11,12-EET preconditioning and postconditioning can protect myocardium from IR injury by improving mitochondrial functions, up-regulating the activities of Na(+)-K(+)-ATPase and SDH, and down-regulating the activity of Ca(2+)-ATPase in mitochondria. Moreover, 11,12-EET preconditioning and postconditioning also elevate the activity of SOD and reduce the content of MDA, suggesting that 11,12-EET can depress the oxidative stress in IR rat heart.

To study the modulatory effect of hydrogen sulfide (H(2)S) on oxidative stress in the development of pulmonary hypertension induced by hypoxia.Twenty male Wistar rats were randomly divided into control group (n=6), hypoxic group (n=6) and hypoxia+NaHS group (n=8). Hypoxic challenge was performed everyday for 21 days. NaHS solution was injected intra-peritoneally everyday before hypoxic challenge for rats in the hypoxia+NaHS group. After 21 days of hypoxia, the mean pulmonary artery pressure(mPAP) was measured by cardiac catheterization. The weight ratio of right ventricle to left ventricle+septum [RV/(LV+SP)] was also measured. The lung homogenates were assayed for total antioxidant capacity(T-AOC), superoxide dismutase (SOD), oxidized glutathione (GSSG), reduced glutathione (GSH), malondialdehyde(MDA) and hydroxy radical(*OH), and the SOD mRNA levels were assayed by real time polymerase chain reaction.After three weeks of hypoxic disposure, hypoxic hypertension and vascular remodeling developed. Compared with the control group, the mPAP[(23.7+/-2.2) mm Hg vs (16.3+/-3.7) mm Hg,P0.01] and the weight ratio of RV/(LV+SP) increased (P0.01), but lung tissue T-AOC was decreased by 21.4% (P0.01).But GSSG was increased by 68.5% (P0.01) as compared with those of the control rats. However, compared with those of the hypoxic group, the mPAP in rats of hypoxia+NaHS group was decreased [(16.3+/-2.8) mm Hg vs (23.7 +/-2.2) mm Hg]. Administration of NaHS increased T-AOC by 18.8% (P0.05) but eliminated GSSG by 23.2% (P0.05) in rats of hypoxia+NaHS group as compared with the hypoxic group. There were no significant changes in lung tissue SOD mRNA level and its capacity among the three groups.Hydrogen sulfide acted as antioxidant during the oxidative stress of hypoxic pulmonary hypertension, and the mechanism was partly through attenuating the content of GSSG.