P3491Mechano-chemo-transduction through nitric oxide pathway enhances calcium cycling in cardiomyocytes (original) (raw)
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Endothelial cell modulation of cardiomyocyte gene expression
Experimental Cell Research, 2019
WB Western blot WGA Wheat germ agglutinin β-MHC -Myosin heavy chain 2013, Fredriksen et al., 2001). Kidney disease is commonly found in heart failure (HF) patients (Grande et al., 2017). It was found that GFR is associated with the degree of cardiac diastolic dysfunction and adverse clinical outcomes (Jain et al., 2017). A recent nationwide population-based cohort study showed that heart failure patients have a higher risk of developing dementia (Adelborg et al., 2017). Studies also revealed post-mortem that 73.6% ± 7.0% of all cardiomyocytes were mono-nucleated, 25.5% ± 7.4% were bi-nucleated, and 1.0% ± 1.2% were tri-nucleated in the heart of individuals aged one month to 73 years. In agreement with previous studies (Mollova et al., 2013, Olivetti et al., 1996), they established that already 1 month after birth; the final number of cardiomyocytes was reached (3.2 ± 0.75 × 10 9 cells) and remained constant throughout life. By stereological measuring the average volume of cardiomyocytes at different ages, it was found that the adaptive about 3-fold greater (endothelium-derived contracting factors) (Flammer et al., 2012). Furchgott and Zawadzki (Furchgott and Zawadzki, 1980) first described the phenomenon of endothelium-dependent relaxation in 1980. Subsequently, the identification of nitric oxide (NO) as the crucial endothelium-derived mediator of vascular relaxation led to substantial progress in cardiovascular research (Flammer et al., 2012). In addition to with arterial hypertension and echocardiographically documented left ventricular hypertrophy, who received either the AT 1 receptor blocker losartan or the β 1 receptor blocker atenolol, were much less likely to experience such a major cardiovascular event (Kjeldsen et al., 2002). Also whereby NT-proBNP levels are more sensitive and specific to distinguish HF from non-HF subjects (Fonseca et al., 2004). NT-proBNP levels were also suggested to be highly predictive of incident atrial fibrillation, the most common cardiac rhythm abnormality, after adjustment for an extensive number of covariates (Patton et al., 2009). Although ANP and BNP have been recommended to provide prognostic information
Regulation ofPulmonary Endothelial CellShape byTRP- Mediated CalciumEntry*
2017
dent on thelevel ofshear andtheduration. Todetermine therelative roles ofhemodynamics and chronic hypoxia inregulating eNOS andpreproET-1 expression inthepulmonary circulation, we studied adult maleratsafter leftpulmonary arteiy (LPA) banding followed by4weeksofnormoxia or 1weekofnormoxia and3weeks ofhypoxia (FIo2=0.12). Methods Hemodynamic measurements were madeon catheterized animals. Western blotanalysis ofeNOSprotein was performed on crudelung homogenates. We also carried outNorthern blotanalysis forpreproET-1 messenger RNA(mRNA) on total RNA extracted fromthelungs of therats. Results Innormoxic rats, LPAstenosis reduced blood flow tothe left lung from9.8±0.9 (sham surgeiy controls) to0.8±0.4 mL/100mg/min (p<0.05); there was nota significant in¬ creaseinright lung blood flow. eNOSprotein content inthe left lung ofnormoxic ratswith LPAstenosis wasdecreased by 32±7%(p<0.05) incomparison with the content intheright lung, andright lung eNOS content was unchanged in comparison ...
A vascular bed–specific pathway regulates cardiac expression of endothelial nitric oxide synthase
Journal of Clinical Investigation, 1999
The endothelial nitric oxide synthase (eNOS) gene is induced by a variety of extracellular signals under both in vitro and in vivo conditions. To gain insight into the mechanisms underlying environmental regulation of eNos expression, transgenic mice were generated with the 1,600-bp 5′ flanking region of the human eNos promoter coupled to the coding region of the LacZ gene. In multiple independent lines of mice, transgene expression was detected within the endothelium of the brain, heart, skeletal muscle, and aorta. β-galactosidase activity was consistently absent in the vascular beds of the liver, kidney, and spleen. In stable transfection assays of murine endothelial progenitor cells, the 1,600-bp promoter region was selectively induced by conditioned media from cardiac myocytes, skeletal myocytes, and brain astrocytes. Cardiac myocyte-mediated induction was partly abrogated by neutralizing anti-plateletderived growth factor (PDGF) antibodies. In addition, promoter activity was upregulated by PDGF-AB. Analysis of promoter deletions revealed that a PDGF response element lies between-744 and-1,600 relative to the start site of transcription, whereas a PDGF-independent cardiac myocyte response element is present within the first 166 bp of the 5′ flanking region. Taken together, these results suggest that the eNos gene is regulated in the cardiac endothelium by both a PDGF-dependent and PDGF-independent microvascular bed-specific signaling pathway.
Hypertension, 2002
NO acting through soluble guanylyl cyclase and cGMP formation is a negative regulator of cardiomyocyte hypertrophy. Downstream targets mediating the inhibitory effects of NO/cGMP on cardiomyocyte hypertrophy have not been elucidated. In addition to its antihypertrophic effects, NO promotes apoptosis in cardiomyocytes, presumably through cGMP-independent pathways. We investigated the role of cGMP-dependent protein kinase (PKG) in the antihypertrophic and proapoptotic effects of NO. Incubation of neonatal rat cardiomyocytes with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (250 mol/L) or the PKG-selective cGMP analog 8-pCPT-cGMP (500 mol/L) activated endogenous PKG type I, as shown by the site-specific phosphorylation of vasodilator-stimulated phosphoprotein, a well-characterized PKG substrate. SNAP (250 mol/L) and 8-pCPT-cGMP (500 mol/L) modestly attenuated the hypertrophic response to ␣ 1 -adrenergic stimulation with phenylephrine. Although a high concentration of SNAP (1000 mol/L) promoted apoptosis in cardiomyocytes, as evidenced by the formation of histone-associated DNA fragments, antihypertrophic concentrations of SNAP (250 mol/L) and 8-pCPT-cGMP (500 mol/L) did not promote cell death. Because chronic activation downregulated endogenous PKG I, we explored whether gene transfer of PKG I would enhance the sensitivity of cardiomyocytes to the antihypertrophic effects of NO/cGMP. Indeed, after adenoviral overexpression of PKG I, SNAP (250 mol/L) and 8-pCPT-cGMP (500 mol/L) completely suppressed the hypertrophic response to ␣ 1 -adrenergic stimulation. As observed in noninfected cells, SNAP (250 mol/L) and 8-pCPT-cGMP (500 mol/L) did not promote apoptosis in cardiomyocytes overexpressing PKG I. Moreover, overexpression of PKG I did not enhance the proapoptotic effects of 1000 mol/L SNAP, implying PKG-independent effects of NO on apoptosis. Endogenous PKG I mediates antihypertrophic but not proapoptotic effects of NO in a cell culture model of cardiomyocyte hypertrophy. Adenoviral gene transfer of PKG I selectively enhances the antihypertrophic effects of NO without increasing the susceptibility to apoptosis. (Hypertension. 2002;39:87-92.)
Biological Procedures Online, 2002
We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This method makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed.
Journal of Biological Chemistry, 2000
Endothelial nitric-oxide synthase (NOS-III) is defined as being strictly dependent on Ca 2؉ /calmodulin (CaM) for activity, although NO release from endothelial cells has been reported to also occur at intracellular free Ca 2؉ levels that are substimulatory for the purified enzyme. We demonstrate here that NOS-III, but neither NOS-I nor-II, is rapidly and strongly activated and phosphorylated on both Ser and Thr in the presence of cGMP-dependent protein kinase II (cGK II) and the catalytic subunit of cAMP-dependent protein kinase (cAK) in vitro. Phosphopeptide analysis by mass spectrometry identified Ser 1177 , as well as Ser 633 which is situated in a recently defined CaM autoinhibitory domain within the flavin-binding region of human NOS-III. Phosphoamino acid analysis identified a putative phosphorylation site at Thr 495 in the CaM-binding domain. Importantly, both cAK and cGK phosphorylation of NOS-III in vitro caused a highly reproducible partial (10-20%) NOS-III activation which was independent of Ca 2؉ /CaM, and as much as a 4-fold increase in V max in the presence of Ca 2؉ /CaM. cAK stimulation in intact endothelial cells also increased both Ca 2؉/ CaM-independent and-dependent activation of NOS-III. These data collectively provide new evidence for cAK and cGK stimulation of both Ca 2؉ / CaM-independent and-dependent NOS-III activity, and suggest possible cross-talk between the NO and prostaglandin I 2 pathways and a positive feedback mechanism for NO/cGMP signaling. The family of homodimeric nitric-oxide synthases (NOS) 1 catalyze the formation of nitric oxide (NO) and L-citrulline from the precursor amino acid, L-arginine. To date, three genetically
The nitric oxide pathway in the cardiovascular system
Journal of Physiology and Biochemistry, 2002
The present review analyzes the role nitric oxide (NO) plays in the homeostasis of the cardiovascular system. By regulating vascular smooth muscle cell and myocyte contractility, myocardial oxygen consumption and renal tubular transport, this simple molecule plays a central role in the control of vascular tone, cardiac contractility and short and long tern regulation of arterial pressure. Fifteen years ago, all we knew about NO is that it had very similar properties as those of endothelium-derived relaxing factor and that its action was probably mediated by cGMP. An enormous amount of knowledge has since been amassed on the biochemical pathways that NO follows from the moment it is synthesized from L-arginine until the physiological or pathological actions take place in the effector cells. This review intends to organize this knowledge in a fashion that is easy to understand. We will dissect the NO pathway in different steps, focusing on the physiological and pathophysiological actions of the isoenzymes which synthesize NO, the molecules involved in this synthesis such as caveolins, protein kinases and cofactors, the situations in which endogenous inhibitors of NO synthase are formed from L-arginine instead of NO, the way in which NO exerts its physiological action through cGMP-dependent protein kinases and finally, the pathological routes NO may follow when the oxidative status of the cell is high. Esta revisión aborda el importante papel que juega el óxido nítrico (NO) en la homeostasis del sistema cardiovascular. Esta simple molécula regula la contractilidad de las células del músculo liso vascular y de los miocitos, el consumo de oxígeno del miocardio y el transporte tubular renal. Así pues, juega un papel clave en el control del tono vascular, en la contractilidad cardiaca y en la regulación a corto y largo plazo de la presión arterial. Hace quince años lo único que se sabía del NO era que poseía propiedades muy similares a las del factor relajante derivado del endotelio y que su acción, probablemente, estaba mediada por el GMP cíclico. A lo largo de los últimos años, se ha ido recopilando mucha información relativa a las vías metabólicas que sigue el NO, desde que es sintetizado a partir de la L-arginina, hasta que tienen lugar sus acciones fisiológicas o patológicas en las células efectoras del sistema cardiovascular. Esta revisión pretende organizar esta información de una manera fácilmente comprensible. La vía del NO se divide en diferentes pasos, centrándose en las acciones fisiológicas y patofisiológicas de las isoenzimas que sintetizan NO, en las moléculas involucradas en esta síntesis, tales como las caveolinas, las proteín cinasas y los cofactores de la NO sintasa (NOS). También se analizan las situaciones en las que, a partir de la L-arginina, se forman inhibidores endógenos de la NOS, los mecanismos por los que el NO ejerce sus acciones fisiológicas a través de proteín cinasas dependientes de GMPc y, finalmente, se estudian las rutas patológicas que puede seguir el NO cuando el estado oxidativo de la célula es elevado.
Molecular Pharmacology, 2005
Cardiac hypertrophy is a compensatory mechanism in response to a variety of cardiovascular diseases. Recently, reactive oxygen species and nitric oxide (NO) have been demonstrated to be involved in the pathogenesis of atherosclerosis; however, the role of these free radicals in the development of cardiac hypertrophy remains unclear. Here, we investigate NO modulation of cellular signaling in endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy in culture. ET-1 treatment of cardiomyocytes increased constitutive NO synthase activity and induced NO production via the stimulation of ET-receptor subtype ET B. Using Northern blot analysis and chloramphenicol acetyltransferase assay, we found that NO suppressed the ET-1-induced increase in c-fos mRNA level and promoter activity. In contrast, ET-1 stimulation of c-fos expression was augmented by depletion of endogenous NO generation with the addition of NO scavenger PTIO into cardiomyocytes. Cells cotransfected with the dominant negative and positive mutants of signaling molecules revealed that the Ras/Raf/extracellular-signal regulated kinase (ERK) signaling pathway is involved in ET-induced c-fos gene expression. Furthermore, NO directly inhibited ET-1-induced ERK phosphorylation and activation in a cGMP-dependent manner, indicating that NO modulates ET-1-induced c-fos expression via its inhibitory effect on ERK signaling pathway. The ET-1-stimulated activator protein-1 (AP-1) DNA binding activity and AP-1-mediated reporter activity were attenuated by NO. In addition, NO also significantly inhibited ET-1-stimulated promoter activity of hypertrophic marker gene β-myosin heavy chain and the enhanced protein synthesis. Taken together, our findings provide the molecular basis of NO as a negative regulator in ET-1-induced cardiac hypertrophy.
Vascular smooth muscle and nitric oxide synthase
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2002
The concept of endothelium-derived relaxing factor (EDRF) put forward in 1980 by Furchgott and Zawadzki implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium diffuses to the underlying vascular smooth muscle, where it modulates vascular tone as well as vascular smooth muscle cell (VSMC) proliferation by increasing cGMP formation with subsequent activation of cGMP-dependent protein kinase. According to this concept, VSMC do not express NOS by themselves. This attractive, simple scheme is now under considerable debate. To address this issue, we designed this study with the use of a novel supersensitive immunocytochemical technique of signal amplification with tyramide and electron microscopic immunogold labeling complemented with Western blotting, as in our recent studies demonstrating NOS in the myocardial and skeletal muscles. We provide the first evidence that, in contrast to the currently accepted view, VSMC in various blood vessels express all three NOS ...