Acetaldehyde, the end product of oxidative ethanol metabolism, contributes to alcohol-induced disease in the liver, but cannot account for damage in organs such as the pancreas, heart, or brain, where oxidative metabolism is minimal or absent; nor can it account for the varied patterns of organ damage found in chronic alcoholics. Thus other biochemical mediators may be important in the pathogenesis of alcohol-induced organ damage. Many human organs were found to metabolize ethanol through a recently described nonoxidative pathway to form fatty acid ethyl esters. Organs lacking oxidative alcohol metabolism yet frequently damaged by ethanol abuse have high fatty acid ethyl ester synthetic activities and show substantial transient accumulations of fatty acid ethyl esters. Thus nonoxidative ethanol metabolism in addition to the oxidative pathway may be important in the pathophysiology of ethanol-induced disease in humans.

Reversible congestive heart failure can accompany cardiac allograft rejection and inflammatory myocarditis, conditions associated with an immune cell infiltrate of the myocardium. To determine whether immune cell secretory products alter cardiac muscle metabolism without cytotoxicity, we cultured cardiac myocytes in the presence of culture supernatants from activated immune cells. We observed that these culture supernatants inhibit -adrenergic agonist-mediated increases in cultured cardiac myocyte contractility and intracellular cAMP accumulation. The myocyte contrai response to increased extracellular Ca2+ concentration is unaltered by prior exposure to these culture supernatants, as is the increase in myocyte intracellular cAMP concentration in response to stimulation with forskolin, a direct adenyl cyclase activator.Inhibition occurs in the absence of alteration in P-adrenergic receptor density or ligand binding affinity. Suppressive activity is attributable to the macrophage-derived cytokines interleukin 1 and tumor necrosis factor. Thus, these observations describe a role for defined cytokines in regulating the hormonal respon- Profound suppression of cardiac contractile function can accompany inflammatory myocarditis associated with idiopathic dilated congestive cardiomyopathy (1) and cardiac allograft rejection (2), despite minimal necrosis of cardiac muscle (3). Contractile performance can be restored in a subset of patients after immunosuppressive therapy (4). The cellular basis of this reversible pathophysiology has not been established. These Immune Cell Cultures. Rat bidirectional primary mixed lymphocyte cultures (MLCs) were established using splenocytes harvested from Lewis strain and outbred SpragueDawley adult rats. Cellular activation was confirmed by documentation of accelerated proliferation in MLCs compared to syngeneic splenocyte cultures by using [3H]thymidine incorporation rates. Culture supernatants were harvested after 96 hr, clarified by centrifugation (2000 X g, 10 min), sterile-filtered, and stored in aliquots at -20TC. Activated splenocyte culture supernatants were obtained from 24-hr cultures of adult rat splenocytes suspended in medium containing Con A (5 ,ug/ml). Lectin was removed by batch adsorption to Sephadex G-25 prior to storage and use. Immune cell cultures were established in the same culture medium used for myocytes, except for the chromatographic fractionations when serum-free lectin-stimulated splenocyte culture supernatants were used. Myocyte culture medium served as control medium for experiments using MLC supernatants. Culture medium adsorbed with Sephadex G-25, in a manner identical to that used for lectin removal, served as control medium for experiments using activated splenocyte culture supernatants. All cell culture reagents used contained lipopolysaccharide at less than 10 pg/ml, as measured in the amebocyte lysate assay (Sigma) tTo whom reprint requests should be addressed. 6753The publication costs of this article were defrayed in part by page charg...

A B S T R A C T Mechanisms responsible for alcohol-induced heart muscle disease have been difficult to elucidate partly because of previously obscure, demonstrable cardiac metabolism of ethanol. Recently, fatty acid ethyl esters were identified in our laboratory and found to be myocardial metabolites of ethanol. In the present study, they have been shown to induce mitochondrial dysfunction. Incubation of isolated myocardial mitochondria with fatty acid ethyl esters led to a concentration-dependent reduction of the respiratory control ratio index of coupling of oxidative phosphorylation and decrement of maximal rate of oxygen consumption. Furthermore, fatty acid ethyl esters were demonstrated to bind to mitochondria in vitro, and, importantly, 72% of intracellularly synthesized ethyl esters were found to bind to mitochondria isolated from intact tissue incubated with ethanol. Protein binding of fatty acid ethyl esters was markedly less than that of fatty acids. Because uncoupling of mitochondrial oxidative phosphorylation correlated with the cleavage of fatty acid ethyl ester shown to be initially bound to mitochondria, with resultant generation of fatty acid, a potent uncoupler, in a locus in or near the mitochondrial membrane, fatty acid ethyl esters may contribute to a potentially toxic shuttle for fatty acid with transport from physiological intracellular binding sites to the mitochondrial membrane; direct effects of fatty acid ethyl esters may also be deleterious. Operation of this shuttle as a result of ethanol ingestion and subsequent accumulation of fatty acid ethyl esters may account for the impaired mitochondrial function and inefficient energy production associated with toxic effects of ethanol on the heart.

Studies conducted in our laboratory have demonstrated that activated immune cells produce a soluble inhibitor(s) of cardiac myocyte contractile and cyclic AMP (cAMP) responses to beta-adrenergic stimulation. To examine the mechanism of this effect, metabolic assays were conducted on cultured rat cardiac myocytes incubated in the presence and absence of supernatants harvested from rat activated splenocyte cultures. Intracellular cAMP accumulation in response to isoproterenol was inhibited by up to 74% in a dose-dependent fashion by conditioned media containing soluble cytokines from activated immune cells. By use of myocyte cultures in which contaminating nonmyocyte proliferation was inhibited by nonlethal irradiation, this phenomenon was shown to be independent of mitogenic effects. Isobutylmethylxanthine, a phosphodiesterase inhibitor, did not ablate cytokine-induced inhibition of cAMP accumulation. Parameters of beta-adrenergic receptor binding and affinity were also unaffected. cAMP suppression was maintained after cholera toxin stimulation of cAMP production via stimulatory G protein ADP-ribosylation. cAMP inhibition was not apparent when cells were stimulated with forskolin, a direct adenylate cyclase activator. Importantly, pertussis toxin treatment significantly ablated cytokine-induced cAMP inhibition. Thus, interference with agonist-occupied beta-adrenergic receptor coupling to adenylate cyclase to produce cAMP and subsequent contractile responses is induced by a factor(s) elaborated by activated immune cells. This interference occurs at the level of signal transduction across the membrane, can be overridden by pertussis toxin, and may involve changes in the coupling of the stimulatory/inhibitory G proteins to adenylate cyclase. These results demonstrate a novel mechanism of cytokine-induced myocyte dysfunction and may have important pathophysiological ramifications in immune-mediated myocardial diseases.