Louis G. Lange

Mechanism of cytokine inhibition of beta-adrenergic agonist stimulation of cyclic AMP in rat cardiac myocytes. Impairment of signal transduction.

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.

Immune Mechanisms of Cardiac Disease

It is evident that cellular infiltration can affect cardiac structure and function in a variety of disease states. Myocardial contractility can be impaired by cell-mediated injury or local release of cytokines. The study of immune cardiac disease has entered a period of rapid expansion that should be characterized by delineation of the mechanisms by which immune cells and factors localize in the myocardium, modulate myocyte function, and remodel myocardial architecture (Fig. 2). This new knowledge should result in the ability to target specifically both the pathways by which cardiac contractility is impaired by chronic inflammation and the sustained immune reactivity to cardiac antigens that underlies chronic myocardial inflammation. Nonspecific therapeutic interventions directed at congestive heart failure, currently the only acceptable approach to the treatment of immune myocarditis, should then serve a more ancillary function in the context of the use of rationally designed drugs. Such drugs could, for example, be specifically targeted to inhibiting the trafficking of leukocytes into the heart or the effects of their subsequent activation within the myocardium.

Cloning of the bovine pancreatic cholesterol esterase/lysophospholipase☆

A cDNA clone encoding for the bovine pancreatic cholesterol esterase has been sequenced. Pancreatic cholesterol esterases hydrolyze dietary cholesterol esters to cholesterol and free fatty acids, which are then absorbed from the gut. Northern blots reveal that the positive signal at 1.9 kilobases is much more intense in the cow than in calf pancreas, indicating that the induction of the enzyme is due to increased transcription or stability of mRNA. The primary structure of this enzyme is similar to that of the rat pancreatic lysophospholipase. We found that homogeneous human and bovine pancreatic cholesterol esterases have high levels of lysophospholipase activity, indicating that these two activities reside within the same protein. Therefore, the metabolism of dietary neutral lipids and polar lipids may be linked through a single enzyme.

Identification and quantitation of fatty acid ethyl esters in biological specimens.

Fatty acid ethyl esters, recently described as enzymatic products of nonoxidative ethanol metabolism in the heart, may represent a mediator or marker of ethanol-induced organ pathology such as alcoholic cardiomyopathy. This study was designed to develop a method for the extraction, quantitation, and definitive identification of fatty acid ethyl esters formed both in biological specimens and during enzymatic incubations. First, several potential sources of error were identified and characterized. Tissue extraction with alcohols led to the time, temperature, and concentration-dependent nonenzymatic formation of fatty acid alcohol esters. Contamination of both substrates, [14C]ethanol and 14C-fatty acid, used to measure enzymatically mediated fatty acid ethyl ester synthesis, could be removed by purification. Accurate quantitation of fatty acid ethyl esters in tissue was achieved using acetone as an extraction solvent, after which isolated lipids were thin-layer chromatographed on silica gel developed with an apolar solvent system (petroleum ether:diethyl ether:acetic acid, 75:5:1). Gas chromatography and mass spectroscopy identified individual fatty acid ethyl esters. The reproducibility of this assay was high, as assessed by quintuplicate determinations of fatty acid ethyl esters formed in liver and heart homogenates, a method with standard deviations 4 to 11% of the mean.

Immune cytokines and cardiac disease.

In conditions such as idiopathic dilated congestive cardiomyopathy associated with lymphocytic myocarditis and cardiac allograft rejection, the immune system can reversibly impair cardiac function. Cytokines interleukin-1 and tumor necrosis factor disrupt β-adrenergic signal transduction and agonist stimulation of contractility. Identification of this reversible effect potentially offers a novel pathophysiologic mechanism for producing cardiac injury.

Purification to homogeneity and characterization of major fatty acid ethyl ester synthase from human myocardium

Non‐oxidative metabolism of ethanol via fatty acid ethyl ester synthase is present in those extrahepatic organs most commonly damaged by alcohol abuse. DEAE‐cellulose chromatography of human myocardial cytosol at pH 8.0 separated synthase I, minor and major activities, eluting at conductivities of 5,7 and 11 mS, respectively. The major synthase was purified 8900‐fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti‐human albumin affinity‐chromatographies with an overall yield of 2596. SDS‐PAOE showed a single polypeptide with a molecular mass of 26 kDa and gel permeation chromatography under nondenaturing conditions indicated a molecular mass of 54 kDa for the active enzyme. The purified enzyme catalyzed ethyl ester synthesis at the highest rates with unsaturated octadecanoic fatty acid substrates (V max = 100 and 65 nmol/mg/h for oleate and linoleate, respectively). K m values for oleate, linoleate, arachidonate, palmitate and stéarate were 0.22 mM, 0.20 mM, 0.13 mM, 0.18 mM and 0.12 mM, respectively. Thus, human heart fatty acid ethyl ester synthase (major form) is a soluble dimeric enzyme comprised of two identical, or nearly identical, subunits (M r = 26000).

Immune cytokine inhibition of beta-adrenergic agonist stimulated cyclic AMP generation in cardiac myocytes

We hypothesize that reversible depression of cardiac function in cardiac allograft rejection and lymphocytic myocarditis reflects down modulation of the beta-adrenergic receptor system by a soluble product of activated immune cells. Thus, exposure of cultured cardiac myocytes to mixed lymphocyte culture or activated splenocyte supernatants produces 70% inhibition of isoproterenol-stimulated cAMP concentrations (Ki = 5% supernatant) in the absence of gross cellular injury or control media effects. This cAMP suppressive factor is not dialyzable and is ammonium sulfate precipitable. Beta-adrenergic receptor density, binding constant and affinity states are unaffected. These results demonstrate the existence of a cytokine inhibitor of cAMP accumulation that may mediate, in part, depression of cardiac contractility observed when immune cells invade the myocardium.

Nonoxidative ethanol metabolism in human leukocytes: Detection of fatty acid ethyl ester synthase activity

Oxidative pathways of alcohol metabolism such as alcohol dehydrogenase usually are not present in human blood and therefore clinical studies correlating ethanol metabolism with alcohol abuse syndromes have not been performed. To assess the activity of nonoxidative ethanol metabolism in blood, we assayed for the activity of fatty acid ethyl ester synthase, a pathway recently described as abundant in the human organs most commonly damaged by alcohol. Indeed, peripheral human leukocytes contain detectable fatty acid ethyl ester synthase activity: 1.2 X 10(6) leukocytes from 10 ml blood catalyze the synthesis of ethyl oleate at 1.4 nmol/4 hr. The reaction is linear with respect to cell number and expended time; Km oleate = 600 microM, Km ethanol = 600 mM. DEAE cellulose chromatography partially purifies synthase activity into a minor and major form (activity ratio = 10/1). Thus, gene products exist in human blood that recognize ethanol and whose biological activity is conveniently assayable for clinical investigations of alcohol metabolism and abuse.

Nonoxidative ethanol metabolism: expression of fatty acid ethyl ester synthase-III in cultured neural cells.

Alcohol metabolism in the human brain has been characterized as essentially nonoxidative in nature, with the esterification of ethanol with fatty acids via fatty acid ethyl ester synthase. This pathway of ethanol metabolism is related to end organ damage in the brain but the neural cell type expressing FAEES has not been identified. In this study human and rodent neuroblastoma and glioma cell lines are assayed for fatty acid ethyl ester synthase activity. Cells with neuronal properties demonstrated higher activity than glioma cell lines. We confirmed the presence of the mRNA for one type of synthase, fatty acid ethyl ester synthase-III in three neuronal cell lines--N1E115 cells, PC12 cells, and SK-N-MC cells. These results support the hypothesis that FAEES activity is expressed chiefly in cells with neuronal properties and suggest that non-oxidative ethanol metabolism is potentially related to the toxic effect of ethanol on the human brain.

RELATIONSHIP OF HUMAN PANCREATIC CHOLESTEROL ESTERASE GENE STRUCTURE WITH LIPID PHENOTYPES

Pancreatic cholesterol esterase is one of the enzymes that plays a pivotal role in cholesterol absorption. Differences in the genotype of this enzyme could affect the susceptibility of individuals to dyslipidemia and/or cardiovascular disease. We undertook this study to investigate if any correlation exists between restriction fragment length polymorphism in the human pancreatic cholesterol esterase gene and serum lipid levels. DNA from 96 healthy adults was restricted with Stu I, Southern blotted, and probed with cDNA of human pancreatic cholesterol esterase. Results revealed six distinct patterns which were classified as A, B, C, D, E, and F which had a population frequency of 1%, 34.5%, 49%, 12.5%, 1% and 2% respectively. Correlation of the distribution of lipid and lipoprotein levels by pattern and sex revealed a significant interaction between pattern type and HDL (p=0.03) in the most common group (group C) for males. Male patients of pattern C tended to have a lower LDL cholesterol than non-pattern C males (p=0.07); in addition, 80% of all males in the study population with LDL cholesterol under 100 mg/dl were found in pattern C. Thus, the most common Stu I RFLP genotype is associated with a favorable lipid phenotype. This report shows an association between the human pancreatic cholesterol esterase genotype and serum lipid levels. Further analysis of a larger study group with Stu I and alternative polymorphic restriction enzymes is warranted, to confirm this biologically plausible result.