Permeability of rat heart myocytes to cytochrome c.

Rat heart myocytes undergoing progressive damage demonstrate morphological changes of shortening and swelling followed by the formation of intracellular vacuoles and plasma membrane blebbing. The damaged myocytes displayed impaired N,N'-tetramethyl-p-phenyldiamine (TMPD) ascorbate-stimulated respiratory activity which was restored by the addition of reduced cytochrome c to the cell culture medium. To clarify the role played by cytochrome c in the impairment of cell respiration, polarographic, spectrophotometric and fluorescence as well as electron microscopy imaging experiments were performed. TMPD/ascorbate-stimulated respiratory activity returned to control levels, at approximately 20 microM cytochrome c, establishing the threshold below which the turnover rate by cytochrome c oxidase in the cell depends on cytochrome concentration. Mildly damaged cardiac myocytes, as indicated by cell shortening, retention of visible striations and free-fluorescein exclusion, together with the absence of lactate dehydrogenase leakage and exclusion of trypan blue, were able to oxidize exogenous cytochrome c and were permeable to fluorescein-conjugated cytochrome c. The results, while consistent with an early cytochrome c release observed at the beginning of cell death, elucidate the role played by cytochrome c in the kinetic control of mitochondrial electron transfer under pathological conditions, particularly those involving the terminal part of the respiratory chain. These data are the first to demonstrate that the sarcolemma of cardiac myocytes, damaged but still viable, is permeable to cytochrome c.

Identification of hepatocytes as the major locus of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1-related P450s, TCDDAA and TCDDAHH, in chick embryo liver.

It was recently shown that the pleiotropic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in chick embryo liver includes the induction of cytochrome P450-mediated arachidonic acid epoxygenation, as well as 7-ethoxyresorufin deethylation (EROD) and aryl hydrocarbon hydroxylation (AHH). The TCDD-induced arachidonic acid metabolism in avian liver microsomes is catalyzed by a 55 kDa P450, TCDDAA, whereas the TCDD-induced AHH and EROD are catalyzed by a different 54.5 kDa P450, TCDDAHH. In this study, we investigated the distribution and inducibility of TCDDAA and TCDDAHH in hepatocytes and nonparenchymal cells. Sonicates of freshly isolated hepatocytes from embryos treated with solvent alone (control) metabolized [14C]arachidonic acid principally to a single metabolite, omega-OH arachidonic acid. Treatment with TCDD increased total arachidonic acid metabolism 2.9-fold and epoxygenase products [epoxyeicosatrienoic acids (EETs) and EET-diols] 36-fold. After treatment, EETs and EET-diols constituted 59% of the total metabolites. EROD in hepatocyte sonicates was increased 32-fold by TCDD treatment. The same pattern of arachidonate metabolites and degree of increase in arachidonate metabolism and EROD by TCDD treatment was observed in the hepatocyte sonicates and liver microsomes. TCDD treatment increased arachidonic acid metabolism and EROD activity 3.6- and 50-fold, respectively, in the nonparenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

2,3,7,8-tetrachlorodibenzo-p-dioxin increases cardiac myocyte intracellular calcium and progressively impairs ventricular contractile responses to isoproterenol and to calcium in chick embryo hearts.

Binding by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the Ah receptor leads to transcriptional activation of several genes and a toxicity syndrome that includes tumor promotion, wasting, hormonal and immune system dysfunction, and death. Recent findings indicate that TCDD may also affect cardiac function. Here, we used the chick embryo, a TCDD-sensitive species, to further characterize the effects of TCDD on ventricular muscle contraction and on cardiac myocyte [Ca2+]i assessed with fura 2. The results show that TCDD causes an evolving sequence of contractile defects, independent of changes in diet, first impairing cAMP-modulated contraction (after 48 hr) and later (by seven days) decreasing responses to [Ca2+]o. Phenobarbital, even at high doses, failed to affect the inotropic response to isoproterenol, supporting the specificity of the ventricular contractile effects of TCDD. TCDD treatment also depressed inotropic responses to theophylline and forskolin, indicating that it has a post-beta-adrenergic receptor effect on cAMP action. In contrast to its depression of responses to beta-adrenergic stimuli and to [Ca2+]o, TCDD did not affect initial tensions of ventricular muscle stimulated at 1 Hz or the force-frequency response up to 1 Hz, indicating that TCDD-treated ventricles can respond normally at slow rates of stimulation. TCDD treatment depressed lusitropic (relaxation) responses to isoproterenol and to increasing [Ca2+]o indicating that it impairs the ability of the sarcoplasmic reticulum to sequester Ca2+. Fura 2-based measurements showed that [Ca2+]i was nearly doubled after TCDD treatment. The increase in [Ca2+]i is consistent with the decrease in the contractile response to [Ca2+]o, amelioration of the response to isoproterenol by subphysiologic concentrations of [Ca2+]o, and intermittent lack of response to electrical stimulation in high K+ observed in ventricles from TCDD-treated embryos. TCDD treatment also depressed the initial increase in [Ca2+]i by isoproterenol, consistent with the decreased contractile response to isoproterenol. The findings show that TCDD causes well defined, progressive impairment of avian ventricular responses to inotropic stimuli, providing new evidence that the heart is a target of TCDD action and that TCDD disturbs intracellular calcium processing.