Previous studies have demonstrated the existence of a circulating myocardial depressant substance during human septic shock. We have recently identified this substance as a synergistic combination of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). This study utilized an in vitro cardiac myocyte assay to evaluate the potential mechanistic role of nitric oxide (NO) and cGMP in depression of myocyte contractility induced by TNF-α, IL-1β, TNF-α + IL-1β (at low concentrations), and human septic shock serum (HSS). TNF-α, IL-1β, TNF-α + IL-1β, and each of 5 sera from patients with acute septic shock caused depression of both maximum extent and peak velocity of cardiac myocyte shortening and an increase in intracellular cGMP concentration during 30 min of exposure (minimum P < 0.01). NO synthetase (NOS) and guanylate cyclase inhibitors such as N-methyl-l-arginine (l-NMA) and methylene blue prevented these effects; an excess ofl-arginine withl-NMA restored them (minimum P < 0.01). In contrast,d-arginine failed to reestablish cytokine-induced myocyte depression and cGMP accumulation prevented byl-NMA. Exposure of myocytes to TNF-α, IL-1β, or TNF-α + IL-1β produced a concentration-dependent increase in intracellular cGMP that paralleled the depression of cardiac myocyte contractility (minimum P < 0.001). In addition, TNF-α, IL-1β, TNF-α + IL-1β, or HSS application to cardiac myocytes resulted in increased NO gas generation, which was inhibited byl-NMA (minimum P < 0.01). Furthermore, unstimulated cardiac myocytes were shown to harbor constitutive but not inducible NOS activity. These data suggest that the sequential generation of NO by a constitutive NOS and cGMP by guanylate cyclase represents an important mechanism of cardiac myocyte depression by TNF-α, IL-1β, TNF-α + IL-1β, and the myocardial depressant substance(s) of septic shock.

. Enhanced NO and superoxide generation in dysfunctional hearts from endotoxemic rats. Am J Physiol Heart Circ Physiol 283: H1108-H1115, 2002. First published May 16, 2002 10.1152/ajpheart.00549. 2001.-Free radicals have been implicated in the etiology of cardiac dysfunction during sepsis, but the actual species responsible remains unclear. We studied the alterations in myocardial nitric oxide (NO), superoxide, and peroxynitrite generation along with cardiac mechanical function and efficiency in hearts from lipopolysaccharide (LPS)-treated rats. Six hours after LPS (4 mg/kg ip) or saline (control) treatment, hearts were isolated and perfused for 1 h with recirculating Krebs-Henseleit buffer and paced at 300 beats/min. Cardiac work, O2 consumption, and cardiac efficiency were markedly depressed in LPS hearts compared with controls. Plasma nitrate/nitrite level was elevated in LPS rats, and ventricular NO production was enhanced as measured by electron spin resonance spectroscopy, Ca 2ϩ -independent NO synthase (NOS) activity, and inducible NOS immunohistochemistry. Ventricular superoxide production was also enhanced in LPS-treated hearts as seen by lucigenin chemiluminescence and xanthine oxidase activity. Increased nitrotyrosine staining (immunohistochemistry) and higher lipid hydroperoxides levels were also detected in LPS-treated hearts, indicating oxygen radical-induced stress. Enhanced generation of both NO and superoxide, and thus peroxynitrite, occur in dysfunctional hearts from endotoxemic rats. sepsis; cardiac dysfunction; nitric oxide; superoxide and peroxynitrite SEPTIC SHOCK is characterized by severe hypotension with profound vasodilatation and multiple organ failure resulting from systemic release of inflammatory cytokines in response to an infective organism (35). Depression of myocardial contractility is a well-documented feature of septic shock (15, 34) despite the fact that assessment of intrinsic cardiac function is complicated by a marked increase in heart rate and decreased preload and afterload. Data from both clinical (32) and experimental (29) studies indicate the presence of genuine myocardial dysfunction when assessed independently of changes in hemodynamics. However, the etiological mechanism(s) of cardiac dysfunction in sepsis is not well understood, but various circulating and/or locally produced mediators have been implicated (for review see Ref. 19).Evidence from our laboratory (39, 40) and from others (2, 3) suggests that exposure of animal hearts or isolated cardiac myocytes to bacterial endotoxin (lipopolysaccharides, LPS) or proinflammatory cytokines enhanced nitric oxide (NO) generation via induction of NO synthase (iNOS). The production of large amounts of NO by this enzyme may have detrimental effects on the myocardium (2, 9, 39). On the other hand, NO may be cardioprotective (37) and may also act as an antioxidant molecule (45).Recent studies have indicated that many of the deleterious effects of NO are mediated by peroxynitrite; this powerful oxidant is generated from a f...

Proinflammatory cytokines (interleukin-1β, tumor necrosis factor-α, and interferon-γ; Cytomix) depress myocardial contractile work partially by stimulating expression of inducible nitric oxide (NO) synthase (iNOS). Because NO and peroxynitrite inhibit myocardial O2 consumption (MV˙o 2), we examined whether this mechanism contributes to reduced cardiac work. In control isolated working rat hearts, cardiac work was stable for 60 min, followed by a decline from 60 to 120 min, without change in MV˙o 2. Cardiac efficiency (work/MV˙o 2) was therefore reduced from 60 to 120 min. Cytomix shortened the onset (within 20–40 min) and enhanced the depression in cardiac work and efficiency and inhibited MV˙o 2 after 80 min. Mercaptoethylguanidine (MEG), an iNOS inhibitor and peroxynitrite scavenger, or the glucocorticoid dexamethasone (Dex) abolished the effects of Cytomix. iNOS expression was increased 10-fold by Cytomix and abolished by Dex but not MEG. That cytokine-induced depression in cardiac work precedes the reduction in MV˙o 2 suggests, at least in the early response, that NO and/or peroxynitrite may not impair heart function by inhibiting mitochondrial respiration but reduce the heart’s ability to utilize ATP for contractile work.

Exercise enhances cardiac output and blood flow to working skeletal muscles but decreases visceral perfusion. The alterations in nitric oxide synthase (NOS) activity and/or expression of the cardiopulmonary, skeletal muscle, and visceral organs induced by swim training are unknown. In sedentary and swim-trained rats (60 min twice/day for 3-4 wk), we studied the alterations in NOS in different tissues along with hindquarter vasoreactivity in vivo during rest and mesenteric vascular bed reactivity in vitro. Hindquarter blood flow and conductance were reduced by norepinephrine in both groups to a similar degree, whereas N(G)-nitro-L-arginine methyl ester reduced both indexes to a greater extent in swim-trained rats. Vasodilator responses to ACh, but not bradykinin or S-nitroso-N-acetyl-penicillamine, were increased in swim-trained rats. Ca(2+)-dependent NOS activity was enhanced in the hindquarter skeletal muscle, lung, aorta, and atria of swim-trained rats together with increased expression of neuronal NOS in the hindquarter skeletal muscle and endothelial NOS in the cardiopulmonary organs. Mesenteric arterial bed vasoreactivity was unaltered by swim training. Physiological adaptations to swim training are characterized by enhanced hindquarter ACh-induced vasodilation with upregulation of neuronal NOS in skeletal muscle and endothelial NOS in the lung, atria, and aorta.

1 We have studied the e ects of a novel agonist, solid-phase von Willebrand Factor (sVWF), on tumour cell-induced platelet aggregation (TCIPA). 2 Washed platelet suspensions were obtained from human blood and the e ects of HT-1080 human ®brosarcoma cells and sVWF on platelets were studied using aggregometry, phase-contrast microscopy, and¯ow cytometry. 3 Incubation of platelets with sVWF (1.2 mg ml 71 ) and HT-1080 cells (5610 3 ml 71 ) resulted in a two-phased reaction characterized ®rst by the adhesion of platelets to sVWF, then by aggregation. 4 TCIPA in the presence of sVWF was inhibited by S-nitroso-glutathione (GSNO, 100 mM) and prostacyclin (PGI 2 , 30 nM). 5 Platelet activation in the presence of tumour cells and sVWF resulted in the decreased surface expression of platelet glycoprotein (GP)Ib and up-regulation of GPIIb/IIIa receptors. 6 Pre-incubation of platelets with PGI 2 (30 nM) resulted in inhibition of sVWF-tumour cellstimulated platelet surface expression of GPIIb/IIIa as measured by¯ow cytometry using antibodies directed against both non-activated and activated receptor. In contrast, GSNO (100 mM) did not a ect sVWF-tumour cell-stimulated platelet surface expression of GPIIb/IIIa. 7 Flow cytometry performed with PAC-1 antibodies that bind only to the activated GPIIb/IIIa revealed that GSNO (100 mM) caused inhibition of activation of GPIIb/IIIa. 8 The inhibitors exerted no signi®cant e ects on TCIPA-mediated changes in GPIb. 9 Thus, sVWF potentiates the platelet-aggregatory activity of HT-1080 cells and these e ects appear to be mediated via up-regulation of platelet GPIIb/IIIa. 10 Prostacyclin and NO inhibit TCIPA-sVWF-mediated platelet aggregation. The mechanisms of inhibition of this aggregation by PGI 2 di er from those of NO.