Polyamines (putrescine, spermidine and spermine) are essential for cell growth and differentiation. Nitric oxideexhibits antihypertrophic functions and inhibits cardiac remodelling. However, the metabolism of polyamines and thepotential interactions with nitric oxide in cardiac hypertrophy remain unclear. We randomly divided Wistar rats into fourtreatment groups: controls, isoproterenol (ISO), ISO and -arginine, and -arginine. Isoproterenol (5mg/kg/day, subcutaneously)and/or?-arginine (800 mg/kg/day, intraperitoneally) was administered once daily for 7 days. The expression ofatrial natriuretic peptide mRNA was determined by reverse transcription-polymerase chain reaction, and fibrogenesis ofheart was assessed by Van Gieson staining. Polyamines were measured with high-performance liquid chromatography, andplasma nitric oxide content and lactate dehydrogenase (LDH) activity were determined with a spectrophotometer. Theexpression levels of ornithine decarboxylase, spermidine/spermine N1-acetyltransferase (SSAT), endothelial nitric oxidesynthase (eNOS) and inducible nitric oxide synthase (iNOS) were analysed by Western blot. Heart-to-body weight ratio,left ventricle-to-body weight ratio, atrial natriuretic peptide mRNA expression, collagen fibres and LDH activity wereelevated, both ornithine decarboxylase and SSAT proteins were up-regulated, and total polyamines were increased in thegroup treated with ISO. Additionally, the expression of iNOS was up-regulated, eNOS was down-regulated, and nitricoxide levels were low. Notably, cotreatment with -arginine reversed most of these changes except for SSAT expression,which was further up-regulated. We propose that increased polyamines and decreased nitric oxide are involved in cardiachypertrophy induced by ISO and suggest that -arginine pre-treatment can attenuate cardiac hypertrophy through theregulation of key enzymes of the polyamine and nitric oxide pathways.

Cardiac hypertrophy is a common pathological change accompanying cardiovascular disease. Recently, some evidence indicated thatcalcium-sensing receptor (CaSR) expressed in the cardiovascular tissue. However, the functional involvement of CaSR in cardiac hypertrophyremains unclear. Previous studies have shown that CaSR caused accumulation of inositol phosphate to increase the release ofintracellular calcium. Moreover, Ca2+-dependent phosphatase calcineurin (CaN) played a vital role in the development of cardiac hypertrophy.Therefore, we investigated the expression of CaSR in cardiac hypertrophy-induced by angiotensin II (AngII) and the effects ofCaSR activated by GdCl3 on the related signaling transduction pathways. The results showed that AngII induced cardiac hypertrophyand up-regulated the expression of CaSR, meanwhile increased the intracellular calcium concentration ([Ca2+]i) and activated CaNhypertrophic signaling pathway. Compared with AngII alone, the above changes were further obvious when adding GdCl3. But theeffects of GdCl3 on the cardiac hypertrophy were attenuated by CsA, a specific inhibitor of CaN. In conclusion, these results suggestthat CaSR is involved in cardiac hypertrophy-induced by AngII through CaN pathway in cultured neonatal rat cardiomyocytes.

Dopamine receptors exist in many tissues, including rat cardiac tissue. However, the physiological importance ofdopamine receptors in the homeostatic regulation of cardiac function is unclear. In this study, a model of ischaemia/reperfusion was established by culturing primary neonatal rat cardiomyocytes in ischaemia-mimetic solution for 2 hr, followedby incubation in normal culture medium for 24 hr. Lactate dehydrogenase activity, superoxide dismutase activity andmalondialdehyde content were determined colorimetrically with a spectrophotometer. Apoptotic cell death was assayed by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, terminal deoxynucleotidyl transferase-mediateddUTP-biotin nick end labelling staining and flow cytometry, and morphological alterations were observed with transmissionelectron microscopy. The intracellular free calcium concentration ([Ca2+]i) was measured by confocal laser scanningmicroscopy. Finally, the expression of dopamine receptor 1 (DR1), caspase-3, -8 and -9, Fas, Fas ligand and Bcl-2 and therelease of cytochromecwere analysed by Western blot. The results showed that DR1 expression was increased markedlyduring ischaemia/reperfusion. Treatment with 10M SKF-38393 (DR1 agonist) significantly increased lactate dehydrogenaseactivity, decreased superoxide dismutase activity and increased malondialdehyde content in the culture medium. The DR1agonist promoted the release of cytochromec, accumulation of [Ca2+]i, and apoptosis induced by ischaemia/reperfusion.Furthermore, SKF-38393 up-regulated the expression of caspase-3, -8 and -9, Fas and Fas ligand, and down-regulatedBcl-2 expression. In contrast, 10M SCH-23390 (DR1 antagonist) had no significant effects on the above indicators. Inconclusion, DR1 activation is involved in the apoptosis of cultured neonatal rat cardiomyocytes in simulated ischaemia/reperfusion through the mitochondrial and death receptor pathways.

The calcium-sensing receptor (CaR) is a G protein-coupled receptor. The CaR stimulation elicits phospholipaseC-mediated inositol triphosphate formation, leading to an elevation in the level of intracellular calcium released from endoplasmicreticulum (ER). Depletion of ER Ca2+ leads to ER stress, which is thought to induce apoptosis. Intracellular calciumoverload-induced apoptosis in cardiac myocytes during hypoxia–reoxygenation (H ?Re) has been demonstrated. However, thelinks between CaR, ER stress and apoptosis during H?Re are unclear. This study hypothesized that the CaR could induceapoptosis in neonatal rat cardiomyocytes during H?Re via the ER stress pathway. Neonatal rat cardiomyocytes were subjectedto 3 hr of hypoxia, followed by 6 hr of reoxygenation. CaR expression was elevated and the number of apoptotic cells wassignificantly increased, as shown by transferase-mediated dUTP nick end-labelling, with exposure to CaCl2, a CaR activator,during H?Re. The intracellular calcium concentration was significantly elevated and the Ca2+ concentration in the ER wasdramatically decreased during H?Re with CaCl2; both intracellular and ER calcium concentrations were detected by laserconfocal microscopy. Expression of GRP78 (glucose-regulated protein 78), the cleavage products of ATF6 (activating transcriptionfactor 6), phospho-PERK [pancreatic ER kinase (PKR)-like ER kinase], the activated fragments of caspase-12, andphospho-JNK (c-Jun NH2-terminal kinase) were increased following exposure to CaCl2 during H?Re. Our results confirmedthat the activated CaR can induce cardiomyocyte apoptosis via ER stress-associated apoptotic pathways during H? Re.

Polyamines (putrescine, spermidine, and spermine)play an essential role in cell growth, differentiation, andapoptosis. Protein kinase C (PKC) stimulates polyaminebiosynthesis through the induction of ornithine decarboxylase(ODC), a rate-limiting enzyme in polyaminebiosynthesis. Activation of PKC mediates ischemic preconditioningto reduce necrosis and apoptosis in intacthearts and in isolated culture cardiomyocytes. In this study,we examined whether the ODC/polyamine system isinvolved in the ischemic preconditioning signaling pathwayand whether this system interacts with PKC inpreconditioning-induced cardioprotection. Hearts werepreconditioned with three cycles of 5-min ischemia and5-min reflow, which caused an increase of ODC expressionand spermidine, spermine, and total polyamine pool levels.a-Difluoromethylornithine (DFMO) and ethylglyoxal bis(guanylhydrazone) (EGBG) inhibited the key enzymesinvolved in polyamine biosynthesis, and abolished thepreconditioning-induced reduction in infarct size andimprovement in postischemic heart contractility function.They also increased cell apoptosis extent and aggravatedmyocardium ultrastructure damage. Inhibition also attenuatedthe preconditioning-induced translocation and activationof the PKC-d,-e isoforms from the cytosol to theparticulate. Conversely, activation of PKC by phorbol 12-myristate 13-acetate (PMA) upregulated the ODC/polyaminesystem, whereas the PKC inhibitor chelerythrine(Che) downregulated the ODC/polyamine system. Thesefindings suggest that upregulation of the polyamine synthesismetabolism occurs in response to preconditioningand mediates preconditioning-induced cardioprotection.The ODC/polyamine system and PKC signals may "crosstalk" in preconditioned hearts such that inhibiting onepathway leads to a reduction in the activity of the otherpathway and vice versa.

1. Myocardial hypertrophy is a common pathologicalchange that accompanies cardiovascular disease. Dopamine D2receptors have been demonstrated in cardiovascular tissues.However, the pathophysiological involvement of D2 receptorsin myocardial hypertrophy is unclear. Therefore, the effects ofthe D2 receptor agonist bromocriptine and the D2 receptorantagonist haloperidol on angiotensin (Ang) II-or endothelin(ET)-1-induced hypertrophy of cultured neonatal rat ventricularmyocytes were investigated in the present study.2. Protein content and protein synthesis, determined byexamining [3H]-leucine uptake, were used as estimates of cardiomyocytehypertrophy. The expression of D2 receptor proteinin neonatal rat ventricular myocytes was determined usingwestern blotting. Changes in [Ca2?]i in cardiomyocytes wereobserved by laser scanning confocal microscopy.3. Angiotensin II and ET-1, both at 10 nmol/L, inducedmyocyte hypertrophy, as demonstrated by increased proteincontent and synthesis, [Ca2?]i levels, protein kinase C (PKC)activity and phosphorylation of extracellular signal-regulatedkinase, c-Jun N-terminal kinase and mitogen-activated proteinkinase (MAPK) p38 (p38). Concomitant treatment of cells with10 nmol/L AngII plus 10mol/L bromocriptine significantlyinhibited cardiomyocyte hypertrophy, MAPK phosphorylationand PKC activity in the membrane, as well as [Ca2?]i signallingpathways, compared with the effects of AngII alone. In addition, 10 mol/L bromocriptine significantly inhibited cardiomyocytehypertrophy induced by 10 nmol/L ET-1. However, pretreatmentwith haloperidol (10mol/L) had no significant effects oncardiomyocyte hypertrophy induced by either AngII or ET-1.4. In conclusion, D2 receptor stimulation inhibits AngIIinducedhypertrophy of cultured neonatal rat ventricularmyocytes via inhibition of MAPK, PKC and [Ca2?]i signallingpathways.

This study was focused on investigating the involvement of polyamine metabolism in the myocardial ischemia-reperfusion injury (MIRI)in an in vivo rat model. A branch of the descending left coronary artery was occluded for 30 min followed by 2 h, 6 h, 12 h, and 24 hreperfusion. Then the expression of spermidine/spermine N1-acetyltransferase (SSAT) and ornithine decarboxylase (ODC) and theconcentrations of polyamines were assessed. It was found that the expression of SSAT and ODC were upregulated after reperfusion and theconcentrations of spermidine and spermine were significantly decreased, while putrescine concentration was significantly increased. Theresults suggest that MIRI may cause disturbance of polyamine metabolism, and it may play a critical role in MIRI

Calcium sensing receptors (CaSR) is a member of super-family of G-protein coupling receptors.This review first introduced the concept, construction features, distribution, functions, decision methods, moderators, ge-netic locus of CaSR and its relationship with some diseases concisely. Then this article described the investigation progressof CaSR in cardiovascular system intensively, including the expression pattern, role and signal pathways of CaSR in rat my-ocardium in normal, ischemia-reperfusion injury, apoptosis and cardiac hypertrophy; the role and mechanism of CaSR incalcium homostasis regulation of rat myocardium, endoplasmic reticulum (ER) stress and cardiac ischemic preconditioningand postconditioning. The metabolism rule, physiological significance and pathological action of polyamine in cardiac cells; the increase of CaSR expression in cardiac tissue of artherosclerosic rat and its effect on sensitivity to acute myocardial in-farction are also discussed. In the end, the research perspective of CaSR in cardiovascular system was anticipated.

The intracellular Ca2? concentration ([Ca2?]i) isincreased during cardiac ischemia/reperfusion injury (IRI),leading to endo(sarco)plasmic reticulum (ER) stress. PersistentER stress, such as with the accumulation of [Ca2?]i,results in apoptosis. Ischemic post-conditioning (PC) canprotect cardiomyocytes from IRI by reducing the [Ca2?]i via protein kinase C (PKC). The calcium-sensing receptor(CaR), a G protein-coupled receptor, causes the productionof inositol phosphate (IP3) to increase therelease of intracellular Ca2? from the ER. This processcan be negatively regulated by PKC through the phosphorylationof Thr-888 of the CaR. This study testedthe hypothesis that PC prevents cardiomyocyte apoptosisby reducing the [Ca2?]i through an interaction of PKCwith CaR to alleviate [Ca2?]ER depletion and [Ca2?]melevation by the ER-mitochondrial associated membrane(MAM). Cardiomyocytes were post-conditioned after 3 hof ischemia by three cycles of 5 min of reperfusion and5 min of re-ischemia before 6 h of reperfusion. DuringPC, PKCe translocated to the cell membrane and interactedwith CaR. While PC led to a significant decreasein [Ca2?]i, the [Ca2?]ER was not reduced and [Ca2?]mwas not increased in the PC and GdCl3–PC groups.Furthermore, there was no evident Dwm collapse duringPC compared with ischemia/reperfusion (I/R) or PKCinhibitor groups, as evaluated by laser confocal scanningmicroscopy. The apoptotic rates detected by TUNEL andHoechst33342 were lower in PC and GdCl3–PC groupsthan those in I/R and PKC inhibitor groups. Apoptoticproteins, including m-calpain, BAP31, and caspase-12,were significantly increased in the I/R and PKC inhibitorgroups. These results suggested that PKCe interactingwith CaR protected post-conditioned cardiomyocytes fromprogrammed cell death by inhibiting disruption of themitochondria by the ER as well as preventing calciuminducedsignaling of the apoptotic pathway.

The expression and function of calcium-sensingreceptor (CaSR) in differentiated THP-1 (human acutemonocytic leukemia cell line) cells are unknown currently.This study investigated above-mentioned issues usingTRAP staining, immunofluorescence staining, Westernblotting, ELISA, and Laser Confocal Scanning Microscopytechniques. We found that CaSR protein was expressed, andmainly located in the membrane and cytoplasm in differentiatedTHP-1 cells. Elevated extracellular calcium orGdCl3 (an agonist of CaSR) raised intracellular calciumconcentration. And this increase was inhibited or abolishedby NPS2390 (an inhibitor of CaSR), U73122 (a specificinhibitor of phospholipase C, PLC) or thapsigargin (a Ca2?-ATPase inhibitor). The extracellular GdCl3 elevation stimulatedboth of IL-1b and TNFa release, and this effect ofGdCl3 was inhibited by NPS2390. In conclusion, CaSR isfunctionally expressed in differentiated THP-1 cells, and theactivated CaSR contributes to intracellular calcium incrementthrough Gq-PLC-inositol triphosphate (IP3) pathwayand commits to cytokine secretion. These results suggestthat CaSR might be involved in a variety of pathologicalprocesses mediated by activated monocyte-macrophages.