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.