Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the last few years, the importance of steady state NO concentrations have emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose 5 basic distinct concentration levels of NO activity; cGMP mediated processes ([NO] <1-30 nM; Akt phosphorylation ([NO] = 30-100 nM); stabilization of HIF-1α ([NO] = 100-300 nM); phosphorylation of p53 ([NO] > 400 nM) and nitrosative stress (1 µM). In general, lower NO concentrations promote cell survival and proliferation, while higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species (ROS) generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell cycle arrest profile to a cell survival one. The resulting reactive nitrogen species (RNS) that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability which are referred to as Kinetic Determinants for Molecular Target Interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.

A, Stanley WC, Recchia FA. Impaired myocardial metabolic reserve and substrate selection flexibility during stress in patients with idiopathic dilated cardiomyopathy. Am J Physiol Heart Circ Physiol 293: H3270-H3278, 2007. First published October 5, 2007; doi:10.1152/ajpheart.00887.2007.-Under resting conditions, the failing heart shifts fuel use toward greater glucose and lower free fatty acid (FFA) oxidation. We hypothesized that chronic metabolic abnormalities in patients with dilated cardiomyopathy (DCM) are associated with the absence of the normal increase in myocardial glucose uptake and maintenance of cardiac mechanical efficiency in response to pacing stress. In 10 DCM patients and 6 control subjects, we measured coronary flow by intravascular ultrasonometry and sampled arterial and coronary sinus blood. Myocardial metabolism was determined at baseline, during atrial pacing at 130 beats/min, and at 15 min of recovery by infusion of [ 3 H]oleate and [ 13 C]lactate and measurement of transmyocardial arteriovenous differences of oxygen and metabolites. At baseline, DCM patients showed depressed coronary flow, reduced uptake and oxidation of FFA, and preferential utilization of carbohydrates. During pacing, glucose uptake increased by 106% in control subjects but did not change from baseline in DCM patients. Lactate release increased by 122% in DCM patients but not in control subjects. Cardiac mechanical efficiency in DCM patients was not different compared with control subjects at baseline but was 34% lower during stress. Fatty acid uptake and oxidation did not change with pacing in either group. Our results show that in DCM there is preferential utilization of carbohydrates, which is associated with reduced flow and oxygen consumption at rest and an impaired ability to increase glucose uptake during stress. These metabolic abnormalities might contribute to progressive cardiac deterioration and represent a target for therapeutic strategies aimed at modulating cardiac substrate utilization. microcirculation FATTY ACIDS provide 60 -90% of the energy necessary to sustain contractile function in the resting fasting state, with the remainder coming from glucose and lactate oxidation. During acute cardiac stress, such as pacing or exercise, the healthy human heart rapidly increases glucose and lactate uptake, with a relative decline in free fatty acid (FFA) uptake (1,3,4,9). Clinical and animal studies have suggested that this response is advantageous: it increases myocardial metabolic efficiency, since carbohydrates are a more efficient substrate than lipids, generating more contractile power for any given rate of myocardial oxygen consumption (MV O 2 ) (20,29,31).Dilated cardiomyopathy (DCM) is often characterized by reduced FFA uptake and oxidation and accelerated glycolysis and glucose oxidation under resting conditions (7,21,31). This metabolic shift may act to optimize the limited oxidative capacity of cardiomyopathic hearts resulting from impairment of mitochondrial function (28) and/or ATP transfer from ...

Transient reduction in coronary perfusion pressure in the isolated mouse heart increases microvascular resistance (paradoxical vasoconstriction) by an endothelium-mediated mechanism. To assess the presence and extent of paradoxical vasoconstriction in hearts from normal and diabetic rats and to determine whether increased heme oxygenase (HO)-1 expression and HO activity, using cobalt protoporphyrin (CoPP), attenuates coronary microvascular response, male Wistar rats were rendered diabetic with nicotinamide/streptozotocin for 2 wk and either CoPP or vehicle was administered by intraperitoneal injection weekly for 3 wk (0.5 mg/100 g body wt). The isolated beating nonworking heart was submitted to transient low perfusion pressure (20 mmHg), and coronary resistance (CR) was measured. During low perfusion pressure, CR increased and was associated with increased lactate release. In diabetic rats, CR was higher, HO-1 expression and endothelial nitric oxide synthase were downregulated, and inducible nitric oxide synthase and O(2)(-) were upregulated. After 3 wk of CoPP treatment, HO activity was significantly increased in the heart. Upregulation of HO-1 expression and HO activity by CoPP resulted in the abolition of paradoxical vasoconstriction and a reduction in oxidative ischemic damage. In addition, there was a marked increase in serum adiponectin. Elevated HO-1 expression was associated with increased expression of cardiac endothelial nitric oxide synthase, B-cell leukemia/lymphoma extra long, and phospho activator protein kinase levels and decreased levels of inducible nitric oxide synthase and malondialdehyde. These results suggest a critical role for HO-1 in microvascular tone control and myocardial protection during ischemia in both normal and mildly diabetic rats through the modulation of constitutive and inducible nitric oxide synthase expression and activity, and an increase in serum adiponectin.

In addition to O-phosphorylation, O-linked modifications of serine and threonine by ␤-N-acetyl-D-glucosamine (GlcNAc) may regulate muscle contractile function. This study assessed the potential role of O-GlcNAcylation in cardiac muscle contractile activation. To identify specific sites of O-GlcNAcylation in cardiac myofilament proteins, a recently developed methodology based on GalNAz-biotin labeling followed by dithiothreitol replacement and light chromatography/tandem mass spectrometry site mapping was adopted. Thirty-two O-GlcNAcylated peptides from cardiac myofilaments were identified on cardiac myosin heavy chain, actin, myosin light chains, and troponin I. To assess the potential physiological role of the GlcNAc, force- [Ca 2؉ ] relationships were studied in skinned rat trabeculae. Exposure to GlcNAc significantly decreased calcium sensitivity (pCa50), whereas maximal force (F max ) and Hill coefficient (n) were not modified. Using a pan-specific O-GlcNAc antibody, it was determined that acute exposure of myofilaments to GlcNAc induced a significant increase in actin O-GlcNAcylation. This study provides the first identification of O-GlcNAcylation sites in cardiac myofilament proteins and demonstrates their potential role in regulating myocardial contractile function. D iabetes mellitus is a risk factor for the development of heart failure, 1 and abnormal glucose metabolism may contribute directly to depressed cardiac function. Studies in humans and animal models of diabetes mellitus have demonstrated abnormal myofilament function 2 and impaired excitation-contraction coupling, 3,4 which may depress myocardial function. Posttranslational modifications of myofilament proteins regulate cardiac function and phosphorylation of myofilament proteins may result in functional abnormalities in heart failure. [5][6][7] In addition to O-linked phosphorylation of serine (Ser) and threonine (Thr) residues of proteins, dynamic Materials and Methods Mass Spectrometric Identification of O-GlcNAc-Modified ProteinsTo label the specific sites (further details are in the online data supplement, available at http://circres.ahajournals.org), GlcNAcmodified peptides were labeled with GalNAz-biotin and enriched by avidin chromatography, and then dithiothreitol (DTT) was used to replace the GlcNAc-GalNAz-biotin by ␤-elimination and Michael addition (BEMAD) as previously described. 14 Isolated Skinned Fiber StudiesFor skinned fiber studies, cardiac trabeculae were isolated and mounted as previously described. 5 ImmunoblotsMyofilament proteins were isolated as previously described, 15 with minor modifications. To determine the global GlcNAc modifications of myofilament proteins, a pan-GlcNAc antibody (CTD 110.6, Covance) was used as previously described. 16 To assess cardiac troponin I (cTnI) phosphorylation, a phospho-TnI (Ser23/Ser24) antibody (Cell Signaling, Danvers, Mass) was used as previously described. 5 Results and Discussion Myofilament Proteins Are Modified by O-GlcNAcWith the enrichment and BEMAD experiments describ...

BackgroundThe purpose of our study was to investigate the potential contribution of germline mutations in NOTCH1, GATA5 and TGFBR1 and TGFBR2 genes in a cohort of Italian patients with familial Bicuspid Aortic Valve (BAV).MethodsAll the coding exons including adjacent intronic as well as 5′ and 3′ untranslated (UTR) sequences of NOTCH1, GATA5, TGFBR1 and TGFBR2 genes were screened by direct gene sequencing in 11 index patients (8 males; age = 42 ± 19 years) with familial BAV defined as two or more affected members.ResultsTwo novel mutations, a missense and a nonsense mutation (Exon 5, p.P284L; Exon 26, p.Y1619X), were found in the NOTCH1 gene in two unrelated families. The mutations segregated with the disease in these families, and they were not found on 200 unrelated chromosomes from ethnically matched controls. No pathogenetic mutation was identified in GATA5, TGFBR1 and TGFBR2 genes.ConclusionsTwo novel NOTCH1 mutations were identified in two Italian families with BAV, highlighting the role of a NOTCH1 signaling pathway in BAV and its aortic complications. These findings are of relevance for genetic counseling and clinical care of families presenting with BAV. Future studies are needed in order to unravel the still largely unknown genetics of BAV.

Congenital heart diseases (CHDs) are recognized as the most common type of birth malformations. Although recent advances in pre- and neonatal diagnosis as well as in surgical procedures have reduced the morbidity and mortality for many CHD, the etiology for CHD remains undefined. In non-syndromic and isolated (without a familial history or a Mendelian inheritance) forms of CHDs, a multifactorial pathogenesis with interplay between inherited and non-inherited causes is recognized. In this paper, we discuss the current knowledge of the potential molecular mechanisms, mediating abnormal cardiac development in non-syndromic and isolated CHD, including mutations in cardiac transcription factors, the role of somatic mutations and epigenetic alterations as well as the influence of gene-environment interactions. In the near future, the advent of high-throughput genomic technologies with the integration of system biology will expand our understanding of isolated, non-syndromic CHDs for their prevention, early diagnosis and therapy.

Plasmid-mediated 3-lactamases from strains of Escherichia coli and Pseudomonas aeruginosa were separated by isoelectric focusing on a 0.8-mm thin-layer agarose gel with a pH gradient of 3.5 to 9.5. Their banding patterns and isoelectric points were compared with those obtained with a 2.0-mm polyacrylamide gel as the support medium. The agarose method produced banding patterns and isoelectric points which corresponded to the polyacrylamide gel data for most samples. Differences were observed for HMS-1 and PSE-1 ,B-lactamases. The HMS-1 sample produced two highly resolvable enzyme bands in agarose gels rather than the single faint enzyme band observed on polyacrylamide gels. The PSE-1 sample showed an isoelectric point shift of 0.2 pH unit between polyacrylamide and agarose gel (pl 5.7 and 5.5, respectively). The short focusing time, lack of toxic hazard, and ease of formulation make agarose a practical medium for the characterization of P-lactamases.Analytical isoelectric focusing (IEF) for the detection of P-lactamase enzymes from gramnegative bacteria has been done since the mid1970s (7). This method allows for the characterization and differentiation of many types of Plactamases by visual comparison of the banding patterns and by the isoelectric points of the constituent proteins. Currently, ,-lactamases are characterized by IEF in polyacrylamide gel, a procedure which is a modification of the original work of Matthew et al. in 1975 (7). This experimental procedure requires 18 h of IEF to achieve adequate identification of most ,B-lactamase types. The resulting banding patterns and isoelectric points are consistent, although the origins and chemical composition of the patterns have not been clearly delineated.One alternative to polyacrylamide is agarose, a purified galactan hydrocolloid which was recently introduced as a medium for analytical IEF. Agarose media offer a macroporous matrix of considerable strength at concentrations 51% (4). The macroporous matrix and the low ionic character of agarose minimize the problems of electroendosmosis and molecular sieving associated with polyacrylamide. Agarose is also simpler in gel formulation and is nontoxic, in contrast to the reported toxicity of unpolymerized t Reprint requests should be directed to Antone A. Medeiros, Miriam Hospital, 176 Summit Avenue, Providence, RI 02906.polyacrylamide (10). These factors led us to explore the use of agarose gel IEF for the identification of ,-lactamase enzymes and to compare these results with those established by polyacrylamide gel IEF. MATERIALS AND METHODSBacterial strains. The Escherichia coli and Pseudomonas aeruginosa strains containing plasmid-mediated P-lactamases used in this study were kindly supplied by Margaret Matthew, R. W. Hedges, and George A. Jacoby. E. coli K-12 strains included R6K(TEM-1) (2, 5); RP1(TEM-2) (5); RGN238(OXA-1) (1, 5); R46(OXA-2) (1, 5); R57b(OXA-3) (1, 5); R997(HMS-1) (8); and R1O1O(SHV-1) (8). P. aeruginosa PU21 strains contained plasmids RPL11(PSE-1) (9); R151(PSE-2) (6); Rmsl49(PSE-3) (1...

S-nitrosoglutathione (GSNO) is a nitric oxide (NO) donor compound which has been postulated to be involved in transport of NO in vivo. It is known that gamma-glutamyl transpeptidase (GGT) is one of the enzymes involved in the enzyme-mediated decomposition of GSNO, but no kinetics studies of the reaction GSNO-GGT are reported in literature. In this study we directly investigated the kinetics of GGT with respect to GSNO as a substrate and glycyl-glycine (GG) as acceptor co-substrate by spectrophotometry at 334 nm. GGT hydrolyses the gamma-glutamyl moiety of GSNO to give S-nitroso-cysteinylglycine (CGNO) and gamma-glutamyl-GG. However, as both the substrate GSNO and the first product CGNO absorb at 334 nm, we optimized an ancillary reaction coupled to the enzymatic reaction, based on the copper-mediated decomposition of CGNO yielding oxidized cysteinyl-glycine and NO. The ancillary reaction allowed us to study directly the GSNO/GGT kinetics by following the decrease of the characteristic absorbance of nitrosothiols at 334 nm. A K(m) of GGT for GSNO of 0.398+/-31 mM was thus found, comparable with K(m) values reported for other gamma-glutamyl substrates of GGT.