Abstract-The aim of the present study was to determine whether cardiac nitric oxide (NO) production changes during the progression of pacing-induced heart failure and whether this occurs in association with alterations in myocardial metabolism. Dogs (nϭ8) were instrumented and the heart paced until left ventricular end-diastolic pressure reached 25 mm Hg and clinical signs of severe failure were evident. Every week, hemodynamic measurements were recorded and blood samples were withdrawn from the aorta and the coronary sinus for measurement of NO metabolites, O 2 content, free fatty acids (FFAs), and lactate and glucose concentrations. ne of the proposed mechanisms of cardiac dysfunction in heart failure attributes a major role to an excessive production of nitric oxide (NO) in the heart and, specifically, in myocytes.1,2 Circulating proinflammatory cytokines, found in high concentrations in plasma of patients with heart failure, 3 would stimulate the expression of inducible NO synthase (iNOS) with consequent overproduction of NO. NO has, among other functions, negative inotropic effects at high concentrations, as demonstrated in vitro.4-6 A similar mechanism of cardiac depression has been recognized previously in septic shock, although the levels of circulating cytokines and NO catabolites in this syndrome are much higher than those found during heart failure. 3,[7][8][9] However, to date, evidence of an increase in cardiac production of NO, sufficient to cause a negative inotropic action during heart failure, has not been provided. The finding that iNOS is expressed in tissue from failing hearts does not necessarily imply that cardiomyocytes are exposed to toxic concentrations of NO. Indeed, it is the amount of NO and not the enzyme isotype generating it that determines the degree to which cardiac function would be depressed. Numerous studies suggest that NO release is rather low in heart failure. In clinical and animal studies, 2,10 pharmacological blockade of NO synthases (NOS) did not alter baseline hemodynamics, indicating that, rather than being characterized by overproduction, systemic NO synthesis could already be minimal. Several investigations, including our own, in patients and in animal models of heart failure report an impairment of endothelial release of NO in large arteries and in coronary microvessels.11-14 The reduced vascular NO

Cytokines can initiate and perpetuate human diseases, and are among the best-validated of therapeutic targets. Cytokines can be blocked by the use of soluble receptors; however, the use of this approach for cytokines such as interleukin (IL)-1, IL-4, IL-6 and IL-13 that use multi-component receptor systems is limited because monomeric soluble receptors generally exhibit low affinity or function as agonists. We describe here a generally applicable method to create very high-affinity blockers called 'cytokine traps' consisting of fusions between the constant region of IgG and the extracellular domains of two distinct cytokine receptor components involved in binding the cytokine. Traps potently block cytokines in vitro and in vivo and represent a substantial advance in creating novel therapeutic candidates for cytokine-driven diseases.

The role of nitric oxide (NO) in the regulation of 02 consumption was studied in chronically instrumented conscious dogs. A specific NO synthesis inhibitor, nitro-L-arginine (NLA, 30 mg/kg IV), significantly increased mean arterial pressure from 100±4 to 134±5 mm Hg (mean±SEM) and total peripheral resistance by 157±16% and reduced cardiac output by 47±3% and heart rate by 34±6% after 120 minutes. Changes in arterial blood gases were not observed. There were significant changes in Po2 (-14±2 mm Hg), 02 saturation (-21±2%), the percentage of hemoglobin as oxyhemoglobin (-21±2%), and 02 content (-3.0±0.9 vol%) and a significant increase in percent reduced hemoglobin (21±1%) in mixed venous blood, associated with an increase in 02 extraction (5.1±0.2 vol%) (all P<.01). 02 consumption was increased from 124± 6 to 155 ±9 mL/min (P<.05). Methoxamine, titrated to have hemodynamic effects similar to those of NLA (eg, itric oxide (NO) formed during the metabolism N of L-arginine1-3 has been identified as an important endothelium-derived relaxing factor (EDRF), which is released from the endothelium and results in vascular smooth muscle relaxation through a cGMP-dependent mechanism.3-5 NO is also found in the central nervous system, where it may mediate some forms of interneuronal communication.6 In addition, NO has been found in activated macrophages, where it is responsible for cytotoxicity and its production is induced after cytokine stimulation.7-9 The mechanism for cytotoxicity of activated macrophages is dependent on NO production, which has multiple actions including inhibition of mitochondrial respiration in target cells. Three mitochondrial enzymes are affected by NO: (1) aconitase in the tricarboxylic acid cycle and (2) NADH+-ubiquinone oxidoreductase and (3) succinateubiquinone oxidoreductase, which are part of complex I and complex II of the mitochondrial electron transport chain.9-11It was recently reported that in cultured rat hepatocytes and rat aortic smooth muscle cells, NO production induced by cytokines results in significant inhibition of aerobic energy metabolism through the inhibition of the mitochondrial enzymes.12-14 Because all observations of the effects of NO on respiratory function previous to these findings were examined in vitro and involved the induction of NO synthase, which is associated with the production of relatively large local concentrations of Received January 28, 1994; accepted August 24, 1994 (Circ Res. 1994;75:1086-1095 Key Words * constitutive nitric oxide synthase * cardiac output * nitro-L-arginine * mitochondrial function . oxygen extraction * barbiturates NO and with pathophysiological conditions, we wondered whether the constitutive pathways of NO production could regulate tissue metabolism under normal physiological conditions. Therefore, the present study addressed the question whether tissue 02 consumption is modulated physiologically in vivo by NO produced by the constitutive NO synthase.In the present study, nitro-L-arginine (NLA), a specific NO synthesis i...

The one-disulfide intermediates formed during the oxidative refolding of ribonuclease A (RNase A) have been characterized. This information is important for understanding the folding pathways of RNase A. The one-disulfide intermediates were blocked with 2-aminoethyl methanethiosulfonate, fractionated using ion-exchange chromatography, and digested with trypsin and chymotrypsin. The resulting peptide fragments were fractionated using reversed phase high-performance liquid chromatography, and identified using mass spectrometry. The relative population of each one-disulfide intermediate was determined from its disulfide bond concentration using a postcolumn disulfide detection system. A total of 24 out of 28 possible one-disulfide intermediates were found to be populated (greater than 0.3%) in the one-disulfide mixture. The population of one-disulfide intermediates displays a nonrandom distribution. All four native disulfide pairings have populations greater than those predicted by loop entropy calculations, suggesting the presence of enthalpic contributions stabilizing these species. The one-disulfide intermediate [65, 72], containing the disulfide bond between cysteines 65 and 72, comprises 40% of the entire one-disulfide population. The interactions that stabilize this intermediate may play an important role in the regeneration pathways of RNase A.

Endothelium-dependent responses are depressed in coronary and peripheral blood vessels after the onset of pacing-induced heart failure in dogs and heart failure of various etiologies in humans. The present study was designed to examine whether these responses were due to decreases in the expression of endothelial cell NO synthase (ecNOS) and cyclooxygenase-1 (COX-1). After 1 month of left ventricular pacing, 8 mongrel dogs were monitored for heart failure as defined by clinical signs and left ventricular end diastolic pressures > 25 mm Hg. Total RNA and protein were isolated from endothelial cells scraped from the thoracic aorta and analyzed by Northern and Western blotting, respectively. Blots probed with 32P-labeled cDNAs for ecNOS and COX-1 were quantified densitometrically, and results were normalized against GAPDH or von Willebrand factor (vWF). In arbitrary units, the ratios of ecNOS to GAPDH were 2.66 +/- 0.77 (mean +/- SEM, n = 17) and 1.12 +/- 0.37 (n = 6 and the ratios of COX-1 to GAPDH were 1.52 +/- 0.52 and 0.56 +/- 0.15 before and after heart failure, respectively. These represent 56% to 64% (P < .05) reductions in ecNOS and COX-1 gene expression. There was no change in the ratios of either COX-1 or ecNOS to vWF. There was also a marked reduction in ecNOS protein after heart failure, estimated at 70%. A marked reduction in nitrite production, a measure of enzyme activity, from thoracic aortas in response to stimulation by either acetylcholine or bradykinin also occurred. To determine whether ecNOS and COX-1 could be independently regulated, an orally active NO-releasing agent, CAS 936, was given to 7 normal dogs for 7 days, and aortic ecNOS and COX-1 mRNAs were analyzed. The ratio of ecNOS to GAPDH was depressed by 52% (P < .05) in aortas from these dogs, whereas the ratio of COX-1 to GAPDH was unchanged. Similar results were found when data were normalized to vWF. These results suggest that at least two endothelial vasodilator gene products are reduced in heart failure, as opposed to a selective defect in NO synthase gene expression.