Morris Karmazyn

Improved post-ischemic ventricular recovery in the absence of changes in energy metabolism in working rat hearts following heat-shock.

We have previously demonstrated that induction of the heat-shock response in rats results in improved recovery of isolated Langendorff-perfused rat hearts subjected to low-flow ischemia followed by reperfusion (Currie et al., 1988). The mechanisms underlying this protective effect of heat-shock are uncertain although the protection was associated with enhanced content of the antioxidant enzyme catalase but not superoxide dismutase or glutathione peroxidase (Currie et al., 1988). Various investigators have suggested the importance of improved energy metabolism in determining recovery following ischemia (Pasque and Wechsler, 1984; Haas et al., 1984; Devous and Lewandowski, 1987). We therefore examined, using a working rat heart model subjected to 10 or 15 min zero flow ischemia whether changes in energy metabolites could account for the protective effect of the heat-shock response. Hearts perfused 24 h after induction of heat-shock failed to demonstrate significant improvement of recovery following 10 min ischemia, however recovery was significantly enhanced in hearts reperfused after 15 min ischemia. Ischemia produced a depression in both ATP and creatine phosphate (CP) content whereas a moderate elevation in ADP and AMP and a marked increase in tissue lactate were evident. These changes were unaffected by prior heat-shock treatment. For both durations of ischemia tissue metabolites were determined during early (5 min) and late (30 min) reperfusion. Although partial recovery in high energy phosphates and a return of ADP, AMP and lactate to near-normal levels were evident, no differences in energy products were observed between hearts from normal or heat-shocked animals, in spite of significantly enhanced recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-ionophore stimulated release of leukotriene C4-like immunoreactive material from cardiac tissue

Administration of leukotrienes to cardiac tissue produces contractile depression and coronary artery constriction [5,8], thus making it possible that these substances mediate cardiac dysfunction under pathologic conditions. Up to now no studies have been performed to determine whether cardiac tissue has the inherent ability to produce leukotrienes. The present study was therefore carried out to ascertain whether isolated hearts perfused with saline buffer devoid of any blood constituents can produce leukotrienes under a variety of pharmacologic and pathologic situations. No leukotriene (LT) C4 was detected under control conditions or from hearts subjected to global ischemia and reperfusion or hypoxia and reoxygenation. A23187, a Ca2+ ionophore markedly stimulated LTC4 release. This effect was prevented by nordihydroguaiaretic acid, a selective lipoxygenase inhibitor. The addition of arachidonate as substrate had no effect on LTC4 release. In an attempt to divert arachidonate to LTC4 production, indomethacin, a cyclo-oxygenase inhibitor was added before arachidonate. No LTC4-like immunoreactivity was found in these experiments. These studies suggest that a lipoxygenase pathway for leukotriene production is present either in the coronary vasculature or myocardium. It was stimulated only by Ca2+ ionophore, probably indicating a requirement for high amounts of intracellular Ca2+.

Effect of D,L‐carnitine on the response of the isolated heart of the rat to ischaemia and reperfusion: relation to mitochondrial function

1 The effect of 100 μm (20 μg ml−1) of D,L‐carnitine was studied on the isolated heart of the rat subjected to 30 min of low flow ischaemia followed by reperfusion. 2 In untreated hearts (n = 30) ischaemia produced an almost total loss of contractility (P < 0.05 compared with non‐ischaemic time control) which was accompanied by an increase in resting tension of approximately 235% (P < 0.05). Ventricular arrhythmias developed during ischaemia in 100% (P < 0.05) of untreated hearts studied. Following reperfusion, untreated hearts recovered 16.3% of contractile function and demonstrated a 60% elevation in resting tension. The incidence of reperfusion‐associated ventricular fibrillation was 60%. 3 Carnitine treatment produced no effect on either the contractile depression or the elevation in resting tension during ischaemia but did significantly decrease the incidence of arrythmias at the termination of ischaemia to 63.3% (n = 30, P > 0.05). In the presence of carnitine, contractile recovery at the end of reperfusion was significantly increased to 30.2% (n = 10, P < 0.05) and the elevation in resting tension was decreased to 30% (n = 10, P > 0.05). The incidence of ventricular arrhythmias during reperfusion was significantly reduced by carnitine. 4 Two populations of mitochondria, subsarcolemmal (SLM) and interfibrillar (IFM) isolated at the end of the ischaemic period exhibited an overall increase in oxidative phosphorylation rates as well as uncoupled oxygen consumption; both phenomena were more pronounced with IFM. Carnitine generally potentiated this response. A 29% and 38% inhibition in atractyloside‐sensitive ADP uptake was observed in SLM and IFM, respectively, following ischaemia, which was partially prevented by carnitine. 5 After 10 min of reperfusion, adenosine diphosphate (ADP) uptake in SLM was further reduced to 55% of control whereas with IFM, uptake was not different from that seen at the end of ischaemia. Mitochondria isolated from hearts after 30 min of reperfusion revealed a significantly depressed oxidative phosphorylation as well as ADP/ATP translocase activity. These defects were partially reversed in hearts perfused with carnitine. 6 Our study demonstrates that D,L‐carnitine protects the rat isolated heart against injury associated with ischaemia and reperfusion through a mechanism associated with improved mitochondrial function.

Acute effects of hypoxia and phosphate on two populations of heart mitochondria

SummaryInitial Polytron treatment with subsequent exposure to the bacterial proteinase Nagarse has been shown to result in the isolation of two distinct populations of cardiac mitochondria, subsarcolemmal and interfibrillar mitochondria, respectively. Although these populations have been shown to possess distinct biochemical properties, few studies have been reported which document the potential differences in their response to pathological insult. We therefore examined the effect of acute hypoxia with or without reoxygenation as well as treatment with phosphate on oxidative phosphorylation on both groups of mitochondria. Freshly-isolated interfibrillar mitochondria (IFM) exhibited significantly higher respiratory values, with the exception of the ADP:O ratios, than subsarcolemmal mitochondria (SLM). With pyruvate-malate as respiratory substrate, 40 minutes hypoxia alone produced no effect on SLM whereas a stimulation in respiration was seen in IFM. A 40-minute reoxygenation period depressed the oxidative phosphorylation rate in SLM whereas it was stimulated in IFM. These treatments did not produce any effect in either population when succinate was the substrate of choice. Because of the latter observation, the possibility that increased lability of complex I of the electron transport chain accounted for the differences associated with NAD-linked substrates was studied by assessing NADH oxidation of sonicated mitochondria following the treatments. SLM exhibited enhanced permeability to exogenous NADH as well as increased sensitivity to sonication following either hypoxia or hypoxia/reoxygenation compared to IFM.Compared to hypoxia/reoxygenation, increasing concentrations of phosphate (5–15 mM) produced a marked depression in oxidative phosphorylation of SLM whereas IFM were relatively resistant. The toxic effects of phosphate were much more evident with pyruvate-malate as substrates; with succinate, oxidative phosphorylation of IFM was not depressed by phosphate whereas only a slight depression was observed with SLM. The latter population similarly exhibited reduced NADH oxidation following phosphate treatment whereas IFM were unaffected.Our studies show a differential sensitivity of two mitochondrial populations to hypoxia/reoxygenation, and, more markedly to phosphate. Since these effects were much less pronounced with succinate-linked respiration and since they were associated with defective NADH oxidation in SLM, it is suggested that the differences between the two populations may be accounted for by the increased lability of complex I of SLM due to hypoxia/reoxygenation or phosphate.

Calcium paradox-evoked release of prostacyclin and immunoreactive leukotriene C4 from rat and guinea-pig hearts. Evidence that endogenous prostaglandins inhibit leukotriene biosynthesis*

The purpose of this study was to assess the influence of the calcium paradox (5 min calcium-free perfusion followed by 15 min calcium repletion) on the release of immunoreactive leukotriene C4 and 6 Keto-prostaglandin F1 alpha from rat and guinea-pig hearts. Under control conditions or during the 5 min calcium-free perfusion period no immunoreactive leukotriene C4 was detectable in the coronary effluent. Following reperfusion with calcium-containing medium a large release of leukotriene C4 was observed although the amount was significantly greater in the rat heart. 6 keto-prostaglandin F1 alpha was detected during normal and calcium-free perfusion and the release was significantly stimulated during calcium repletion. Treatment with ibuprofen, a cyclo-oxygenase inhibitor, prevented the release of 6 keto-prostaglandin F1 alpha but increased the efflux of immunoreactive LTC4 during calcium repletion. Arachidonic acid, the substrate for prostaglandin and leukotriene synthesis increased the efflux of 6 keto-prostaglandin F1 alpha but decreased the release of leukotriene C4. The latter effect was reversed by perfusion with ibuprofen, and mimicked by prostacyclin, the primary cardiac prostaglandin. This study shows that the calcium paradox is a potent stimulus for eicosanoid release from rat and guinea-pig hearts, a phenomenon likely due to the activation of calcium-dependent enzymes. The study also suggests that endogenous prostaglandins inhibit leukotriene synthesis in cardiac tissue.

Role of prostaglandins in the arrhythmogenic effects of ouabain on isolated guinea pig hearts

We examined the hypothesis that endogenous prostaglandins participate in the arrhythmogenic influence of ouabain in guinea pig hearts. Addition of ouabain (10 ng/ml) resulted in a 5-fold increase in the release of 6-keto-prostaglandin F1 alpha in the coronary effluent. Ten of 13 hearts studied (77%) demonstrated arrhythmic activity with a mean time to the onset of arrhythmias of approximately 35 min. The nonsteroidal antiinflammatory drugs indomethacin and acetylsalicylic acid which significantly inhibited the efflux of 6-keto-prostaglandin F1 alpha also reduced the incidence of arrhythmias to 10 of 30 hearts studied. In those hearts in which arrhythmias occurred, the time to onset was significantly increased to approximately 50 and 55 min for acetylsalicylic acid and indomethacin, respectively. In contrast, exogenous prostaglandin F2 alpha (0.1 and 1 ng/ml) and prostacyclin (0.1 and 10 ng/ml) increased the incidence of arrhythmias to 100% (10 of 10 hearts studied) and decreased the time to onset to approximately 10 min. These prostaglandin pretreatments were also able to reverse the protective actions of both acetylsalicylic acid and indomethacin. Other concentrations (10 ng/ml prostaglandin F2 alpha and 1 ng/ml prostacyclin) had no influence either on the incidence of arrhythmias or their time to onset. Prostaglandin E2 (0.1 ng/ml) produced a modest but not significant decrease in the time to onset of arrhythmias although this concentration was significantly effective in reversing the nonsteroidal antiinflammatory drug effects. The inotropic, chronotropic and coronary constricting actions of ouabain were unaffected either by nonsteroidal antiinflammatory drug or prostaglandin pretreatment. These studies suggest that prostaglandins are involved, at least in part, in the arrhythmogenic actions of ouabain in the isolated guinea pig heart.

Activation of suppressor cells by low molecular weight factors secreted by spleen cells of tumor-bearing mice: Modulatory role of prostaglandins

We have shown previously that the spleens of mice bearing large M-1 fibrosarcomas contain inducer cells which secrete dialysable factors which activate suppressor T cells from unprimed, normal precursor spleen cells. Once activated, the suppressor cells inhibit the in vitro antibody synthesis of cocultured syngeneic splenocytes stimulated by T cell dependent antigens. In this paper we have examined the possibility that prostaglandins are involved in the activation process. Inducer and precursor cells were cultured in Marbrook vessels in chambers separated by dialysis membranes. Using this procedure, suppressor cells were activated following 12 h of culture but were not detectable after 6 h. The cyclooxygenase inhibitors, indomethacin, acetyl salicylic acid (ASA), and ibuprofen all prevented the activation of suppressor cells in a dose dependent manner. Prostaglandin (PG) E1, but not PGF2a or PGD2, restored the activation of suppressor cells in cultures containing the cyclooxygenase inhibitors. Restoration of suppressor cell activation was seen with 1 X 10(-7) M PGE1 but no activation of suppressor cells was seen in control cultures containing up to 1 X 10(-5) M PGE1. In addition, cultured spleen cells from tumor-bearing mice did not secrete higher quantities of PGE than did cells from age and sex matched normal mice. These data suggest that PGE has a modulatory rather than a direct role in the activation of suppressor cells by inducer factors from tumor-activated inducer cells.

A direct protective effect of sulphinpyrazone on ischaemic and reperfused rat hearts

1 Initiation of 60 min ischaemia to rat isolated hearts produced a depression in developed tension and heart rate. Subsequent reperfusion caused a greatly exacerbated creatine phosphokinase (CPK) efflux and limited functional recovery. 2 Sulphinpyrazone (100 ng ml−1 and 1 μg ml−1) significantly reduced CPK release, particularly after reperfusion, the lower concentration being more effective. 3 A reduction in the mechanical depression during ischaemia and enhanced recovery after reperfusion were seen only with 100 ng ml−1 sulphinpyrazone. Heart rate and coronary perfusion pressure were unaffected by drug treatment. 4 The reduction in reperfusion‐induced CPK efflux by 100 ng ml−1 sulphinpyrazone was maximal when the drug was present throughout the perfusion period although some protection was evident when sulphinpyrazone was present either during ischaemia or reperfusion only. An enhanced recovery in contractility was seen only when the drug was present throughout all phases of perfusion. 5 It is suggested that sulphinpyrazone exerts a direct protective effect on the heart particularly during reperfusion. The degree of protection is critically dependent on the concentration of sulphinpyrazone.

Effect of verapamil on phosphate-induced changes in oxidative phosphorylation and atractyloside-sensitive adenine nucleotide translocase activity in two populations of rat heart mitochondria

Phosphate (Pi)-induced depression in cardiac mitochondrial function was studied using mitochondria isolated by two different procedures which purportedly yield two distinct populations. Subsarcolemmal mitochondria (SLM) exhibited an enhanced sensitivity to 20 mM Pi with respect to oxidative phosphorylation. Thus, a significant depression in oxidative phosphorylation in this population was seen following only 1-min treatment, whereas interfibrillar mitochondria (IFM) were unaffected. Both populations showed a similar response to 5-min treatment with Pi. The Pi-induced depression in respiration was partially, although significantly, reversed by a 50 microM concentration of the calcium antagonist verapamil, an observation which suggests a contribution of calcium to the Pi-induced defect in respiration. Pi also produced a potent inhibition of ADP uptake in both mitochondrial populations, which was in close agreement to Pi-induced modification of low amplitude shrinkage-swelling responses following ADP addition. Both of these parameters were unaffected by verapamil. Our results show an enhanced sensitivity of SLM to a verapamil-sensitive Pi-induced depression in oxidative phosphorylation. However, the potent, verapamil-insensitive decrease in adenine nucleotide translocase activity by Pi demonstrates that calcium is likely only partially involved in Pi-induced depression in oxidative phosphorylation and that a further partial contribution arises from a decrease in adenine nucleotide translocase activity.

Reduction of phosphate-induced dysfunction in rat heart mitochondria by carnitine

The direct effects of varying concentrations (5-40 mM) of D,L-carnitine were studied in two populations, subsarcolemmal and interfibrillar, of cardiac mitochondria exposed to inorganic phosphate (Pi). After 5 min preincubation 20 mM Pi significantly depressed oxidative phosphorylation rate and ADP/ATP translocase activity, in both populations. Inclusion of D,L-carnitine during preincubation significantly prevented the Pi-induced depression in oxidative phosphorylation without affecting the ADP/ATP translocate system. The Pi-induced inhibition in mitochondrial oxygen consumption rate was seen with either pyruvate-malate, glutamate-malate or succinate as respiratory substrates and was also observed in uncoupled mitochondria treated with 2,4-dinitrophenol. Mitochondrial swelling and shrinkage studies revealed Pi-induced inner membrane instability, a phenomenon prevented by D,L-carnitine in a dose-dependent manner. The effect of Pi was also observed at a concentration of 5 mM which was also prevented by carnitine. Mepacrine, a phospholipase A2 inhibitor, failed to prevent any of the effects of Pi. The results therefore suggest that Pi can produce a depression in mitochondrial oxidative phosphorylation through a mechanism possibly associated with disturbed inner membrane structure and function but apparently unrelated to phospholipase A2 activation. The salutary actions of carnitine may partly explain its protective effects in the ischemic and reperfused heart, a phenomenon associated with enhanced intracellular Pi accumulation.