Signal transduction pathways involved in the mechanical responses to protease-activated receptors in rat colon

ABSTRACT Recording simultaneously in vitro the changes of endoluminal pressure (index of circular muscle activity) and isometric tension (index of longitudinal muscle activity), we examined the mechanisms responsible for the apamin-sensitive relaxant and contractile responses induced by protease-activated receptor (PAR)-1 and PAR-2 activating peptides, SFLLRN-NH 2 and SLIGRL-NH 2 , respectively, in rat colon. In the circular muscle, the inhibitory effects of SFLLRN-NH 2 and SLIGRL-NH 2 were significantly reduced by ryanodine, an inhibitor of Ca 2ϩ release from the sarcoplasmic reticulum, but unaffected by 1- , a protein kinase C (PKC) inhibitor, or genistein, a tyrosine kinase inhibitor. In the longitudinal muscle, the contractile responses to SFLLRN-NH 2 and SLIGRL-NH 2 were significantly reduced by nifedipine, an L-type calcium channel blocker, ryanodine, GF109203X, genistein, and abolished by U73122. The effects of genistein were additive with GF109203X but not with nifedipine. In the longitudinal muscle, the relaxant responses to the highest concentrations of SFLLRN-NH 2 and SLIGRL-NH 2 were abolished by nifedipine, reduced by genistein, and unaffected by ryanodine or GF109203X. In conclusion, influx of extracellular Ca 2ϩ through L-type voltage-dependent channels or release of Ca 2ϩ from intracellular stores are determining for the opening of the apamin-sensitive K ϩ channels responsible for longitudinal muscle relaxation or circular muscle inhibitory response, respectively, in rat colon. The longitudinal muscle contraction is mediated by activation of PLC; PKC and tyrosine kinase are involved in the cascade process, playing a parallel role. Indeed, tyrosine kinase and L-type Ca 2ϩ channels would act sequentially

Cardiac influence of the β3-adrenoceptor in the goldfish (Carassius auratus): a protective role under hypoxia?

The goldfish (Carassius auratus) exhibits a remarkable capacity to survive and remain active under prolonged and severe hypoxia, representing a well-suited model to study cardiac function when oxygen availability represents a limiting factor. Under hypoxia, the goldfish heart increases its performance, this representing a putative component of hypoxia tolerance; however, underlying mechanisms have not been yet elucidated. We aimed to investigate the role of β3-ARs in the mechanisms which modulate the goldfish heart performance, also in relation to oxygen levels. By western blotting analysis, we found that the goldfish heart expresses β3-ARs, and this expression increases under hypoxia. Effects of β3-ARs stimulation were analysed by using an ex vivo working heart preparation. Under normoxia, the β3-ARs selective agonist BRL37344 (10−12-10−7M) elicited a concentration-dependent increase of contractility abolished by the specific β3-AR antagonist (SR59230A; 10−8M), but not by α/β1/β2-ARs inhibitors (phentolamine, nadolol, and ICI118,551; 10−7M). Under acute hypoxia, BRL37344 did not affect the goldfish heart performance. However, SR59230A, but not phentolamine, nadolol, and ICI118,551, abolished the time-dependent enhancement of contractility which characterizes the hypoxic goldfish heart. Under both normoxia and hypoxia, adenylate cyclase and cAMP were found to be involved in the β3-ARs-dependent downstream transduction pathway. Our findings suggest the presence of functional β3-ARs in the goldfish heart, whose activation modulates the basal performance and contributes to the hypoxia-dependent increase of contractility

Crucial role of phospholamban phosphorylation and S-nitrosylation in the negative lusitropism induced by 17β-estradiol in the male rat heart.

Background/Aims: 17β-estradiol (17βE2) plays an important cardiovascular role by activating estrogen receptors (ER) α and ERβ. Previous studies demonstrated that the novel estrogen G protein-coupled receptor (GPR30/GPER) mediates estrogen action in different tissues. We have recently shown in the rat heart that 17βE2 elicits negative inotropism through ERα, ERβ and GPR30, by triggering activation of ERK1/2, phosphatidylinositol 3-kinase (PI3K), protein kinase A (PKA) and endothelial Nitric Oxide synthase (eNOS) signaling. Methods: In the present study, using the isolated and Langendorff-perfused rat heart as a model system we analyzed: i) whether and to which extent 17βE2 modifies mammalian ventricular myocardial relaxation (lusitropism); ii) the type of ERs and the signaling pathways involved in this effect. Results: We found that 17βE2 negatively modulated the ventricular lusitropic performance. This effect, which partially involved the vascular endothelium, recruited ERβ and occurred via PI3K, eNOS-NO-cGMP-protein kinase G (PKG) transduction cascade. Of note, 17βE2-mediated negative lusitropism associated with a modification of phospholamban (PLN) phosphorylation and S-nitrosylation (SNO) both in isolated Langendorff rat heart and in isolated cardiomyocytes. Conclusion: Taken together, our results allow including 17βE2 to the family of substances that control ventricular relaxation. This is of relevance in relation not only to the normal endocrine control of cardiac function, but also to physio-pathologic conditions characterized by an altered ventricular diastolic performance

Selenoprotein T as a new positive inotrope in the goldfish, Carassius auratus.

Selenoprotein T (SELENOT) is a thioredoxin-like protein, which mediates oxidoreductase functions via its redox active motif Cys-X-X-Sec. In mammals, SELENOT is expressed during ontogenesis and progressively decreases in adult tissues. In the heart, it is re-expressed after ischemia and induces cardioprotection against ischemia/reperfusion (I/R) injury. SELENOT is present in teleost fish, including the goldfish Carassius auratus. This study aimed to evaluate the cardiac expression of SELENOT, and the effects of exogenous PSELT (a 43-52 SELENOT derived-peptide) on the heart function of C. auratus, a hypoxia tolerance fish model. We found that SELENOT was expressed in cardiac extracts of juvenile and adult fish, located in the sarcoplasmic reticulum (SR) together with calsequestrin-2. Expression increased under acute hypoxia. On ex vivo isolated and perfused goldfish heart preparations, under normoxia, PSELT dose-dependently increased Stroke Volume (SV), Cardiac Output (Q̇), and Stroke Work (SW), by involving cAMP, PKA, L-type calcium channels, SERCA2a pumps, and pAkt. Under hypoxia, PSELT did not affect myocardial contractility. Only at higher concentrations (10−8 -10−7 M) an increase of SV and Q̇ was observed. It also reduced the cardiac expression of 3-NT, a tissue marker of nitrosative stress which increases under low oxygen availability. These data are the first to propose SELENOT 43-52, PSELT, as a cardiac modulator in fish, with a potential protective role under hypoxia

Quercetin derivatives as novel antihypertensive agents: Synthesis and physiological characterization

The antihypertensive flavonol quercetin (Q1) is endowedwith a cardioprotective effect againstmyocardial ischemic damage. Q1 inhibits angiotensin converting enzymeactivity, improves vascular relaxation, and decreases oxidative stress and gene expression. However, the clinical application of this flavonol is limited by its poor bioavailability and low stability in aqueous medium. In the aimto overcome these drawbacks and preserve the cardioprotective effects of quercetin, the present study reports on the preparation of five different Q1 analogs, in which all OH groups were replaced by hydrophobic functional moieties. Q1 derivatives have been synthesized by optimizing previously reported procedures and analyzed by spectroscopic analysis. The cardiovascular properties of the obtained compounds were also investigated in order to evaluate whether chemical modification affects their biological efficacy. The interaction with β-adrenergic receptors was evaluated by molecular docking and the cardiovascular efficacy was investigated on the ex vivo Langendorff perfused rat heart. Furthermore, the bioavailability and the antihypertensive properties of the most active derivative were evaluated by in vitro studies and in vivo administration (1month) on spontaneously hypertensive rats (SHRs), respectively. Among all studied Q1 derivatives, only the ethyl derivative reduced left ventricular pressure (at 10−8M÷10−6Mdoses) and improved relaxation and coronary dilation. NOSs inhibition by L-NAME abolished inotropism, lusitropism and coronary effects. Chronic administration of high doses of this compound on SHR reduced systolic and diastolic pressure. Differently, the acetyl derivative induced negative inotropism and lusitropism (at 10−10M and 10−8 ÷ 10−6 M doses), without affecting coronary pressure. Accordingly, docking studies suggested that these compounds bind both β1/β2-adrenergic receptors. Taking into consideration all the obtained results, the replacement of OHwith ethyl groups seems to improve Q1 bioavailability and stability; therefore, the ethyl derivative could represent a good candidate for clinical use in hypertension

HSP90 and eNOS partially co-localize and change cellular localization in relation to different ECM components in 2D and 3D cultures of adult rat cardiomyocytes.

Background information. Cultivation techniques promoting three-dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel-based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co-cultures to evaluate, on both two- and three-dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat-shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase). Results. Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co-culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel-cultured cardiomyocytes, areas of high co-localization between HSP90 and eNOS and co-localization coefficients, which indicated the highest correlation with respect to the other substrates. Conclusions. Our three-dimensional adult cardiomyocyte cultures are suitable for both analysing cell–ECM interactions at electron and confocal microscopy levels and monitoring micro-environment impact on cardiomyocyte phenotype

Catestatin Improves Post-Ischemic Left Ventricular Function and Decreases Ischemia/Reperfusion Injury in Heart

The Chromogranin A (CgA)-derived anti-hypertensive peptide catestatin (CST) antagonizes catecholamine secretion, and is a negative myocardial inotrope acting via a nitric oxide-dependent mechanism. It is not known whether CST contributes to ischemia/reperfusion injury or is a component of a cardioprotective response to limit injury. Here, we tested whether CST by virtue of its negative inotropic activity improves post-ischemic cardiac function and cardiomyocyte survival. Three groups of isolated perfused hearts from adult Wistar rats underwent 30-min ischemia and 120-min reperfusion (I/R, Group 1), or were post-conditioned by brief ischemic episodes (PostC, 5-cycles of 10-s I/R at the beginning of 120-min reperfusion, Group 2), or with exogenous CST (75 nM for 20 min, CST-Post, Group-3) at the onset of reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated with nitroblue-tetrazolium staining. The CST (5 nM) effects were also tested in simulated ischemia/reperfusion experiments on cardiomyocytes isolated from young-adult rats, evaluating cell survival with propidium iodide labeling. Infarct size was 61 ± 6% of risk area in hearts subjected to I/R only. PostC reduced infarct size to 34 ± 5%. Infarct size in CST-Post was 36 ± 3% of risk area (P < 0.05 respect to I/R). CST-Post reduced post-ischemic rise of diastolic LVP, an index of contracture, and significantly improved post-ischemic recovery of developed LVP. In isolated cardiomyocytes, CST increased the cell viability rate by about 65% after simulated ischemia/reperfusion. These results suggest a novel cardioprotective role for CST, which appears mainly due to a direct reduction of post-ischemic myocardial damages and dysfunction, rather than to an involvement of adrenergic terminals and/or endothelium

The Cholesterol Metabolite 25-Hydroxycholesterol Activates Estrogen Receptor α-Mediated Signaling in Cancer Cells and in Cardiomyocytes

The hydroxylated derivatives of cholesterol, such as the oxysterols, play important roles in lipid metabolism. In particular, 25-hydroxycholesterol (25 HC) has been implicated in a variety of metabolic events including cholesterol homeostasis and atherosclerosis. 25 HC is detectable in human plasma after ingestion of a meal rich in oxysterols and following a dietary cholesterol challenge. In addition, the levels of oxysterols, including 25 HC, have been found to be elevated in hypercholesterolemic serum.Here, we demonstrate that the estrogen receptor (ER) α mediates gene expression changes and growth responses induced by 25 HC in breast and ovarian cancer cells. Moreover, 25 HC exhibits the ERα-dependent ability like 17 β-estradiol (E2) to inhibit the up-regulation of HIF-1α and connective tissue growth factor by hypoxic conditions in cardiomyocytes and rat heart preparations and to prevent the hypoxia-induced apoptosis.The estrogen action exerted by 25 HC may be considered as an additional factor involved in the progression of breast and ovarian tumors. Moreover, the estrogen-like activity of 25 HC elicited in the cardiovascular system may play a role against hypoxic environments