Hormones and triacylglycerol metabolism under normoxic and ischemic conditions

Summary Fatty acids, the preferred substrate in normoxic myocardium, are derived from either exogenous or endogenous triacylglycerols. The supply of exogenous fatty acids is dependent of the rate of lipolysis in adipose tissue and of the lipoprotein lipase activity at the coronary vascular endothelium. A large part of the liberated fatty acids is reesterified with glycerol-3-phosphate and converted to triacylglycerols. Endogenous lipolysis and lipogenesis are intracellular compartmentalized multienzyme processes of which individual hormone-sensitive steps have been demonstrated in adipose tissue. The triacylglycerol lipase is the rate-limiting enzyme of lipolysis and glycerol-3-phosphate acyltransferase and possibly phosphatidate phosphohydrolase are the rate-limiting enzymes of lipogenesis. The hormonal regulation of both processes in heart is still a matter of dispute. Triacylglycerol lipase activity in myocardial tissue has two intracellular sources: 1, the endoplasmic reticular and soluble neutral lipase, and 2. the lysosomal acid lipase. Studies in our laboratory have indicated that whereas lipolysis is enhanced during global ischemia and anoxia, overall lipolytic enzyme activities in heart homogenates were not altered. In addition we were unable to demonstrate alterations in tissue triacylglycerol content and glycerol-3-phosphate acyltransferase activity under these conditions. Lipolysis, is subject to feedback inhibition by product fatty acids. Therefore all processes leading to an increased removal of fatty acids from the catalytic site of the lipase will stimulate lipolysis. These studies will be reviewed. In addition, studies from our department have demonstrated the capacity of myocardial lysosomes to take up and degrade added triacylglycerol-particles in vitro. Such a process, stimulated by Ca²⁺ and stimulated by acidosis, offers another physiological target for hormone actions.     

Glycine metabolism in rat kidney cortex slices.

We have previously described a method for measuring the rotational diffusion of membrane proteins by using fluorescent triplet probes [Johnson & Garland (1981) FEBS Lett. 135, 252-256]. We now describe the criteria by which the suitability of such probes may be judged. In general, the greatest sensitivity is achievable with probes where the ratio of the quantum yields for prompt fluorescene (phi f) and triplet formation (phi t) are high, as with Rhodamine (phi f/phi t congruent to 10(3)). However, considerations of heat generation at the sample membrane, of time resolution of fast rotations and of irreversible bleaching of the fluorescent probe also apply. The immediate environment of a probe molecule at a membrane protein must also be important in determining the performance of a given probe. Nevertheless, we describe guidelines for evaluating the likely usefulness of fluorescent triplet probes in measurements of membrane protein rotation.     

Effect of exogenous CDP-choline on choline metabolism in isolated adult rat ventricular myocytes under normoxic and hypoxic conditions

The purpose of this study was to examine the effect of exogenous CDP-choline on choline metabolism and phosphatidylcholine biosynthesis in adult rat ventricular myocytes. Choline uptake and metabolism were examined, using [methyl³ H] choline. CDP-choline in the medium produced a concentration dependent reduction in the amount of radio-label in phosphocholine and phospholipid but it did not alter choline uptake into the myocytes. CDP-choline also did not antagonize the effect of hypoxia on phosphatidylcholine synthesis; rather it accentuated the hypoxia-induced reductions in cellular phosphocholine and phosphatidylcholine biosynthesis. These results indicate that the exogenous administration of CDP-choline alters choline metabolism in the heart by reducing the formation of phosphocholine and phosphatidylcholine without altering choline uptake and suggest an effect of a CDP-choline metabolite on choline metabolism which is not effective in opposing the effect of hypoxia on phosphatidylcholine biosynthesis.     

Role of P2 purinergic receptors in synaptic transmission under normoxic and ischaemic conditions in the CA1 region of rat hippocampal slices

The role of ATP and its stable analogue ATPγS [adenosine-5′-o-(3-thio)triphosphate] was studied in rat hippocampal neurotransmission under normoxic conditions and during oxygen and glucose deprivation (OGD). Field excitatory postsynaptic potentials (fEPSPs) from the dendritic layer or population spikes (PSs) from the soma were extracellularly recorded in the CA1 area of the rat hippocampus. Exogenous application of ATP or ATPγS reduced fEPSP and PS amplitudes. In both cases the inhibitory effect was blocked by the selective A₁ adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and was potentiated by different ecto-ATPase inhibitors: ARL 67156 (6-N,N-diethyl-D-β,γ-dibromomethylene), BGO 136 (1-hydroxynaphthalene-3,6-disulfonate) and PV4 [hexapotassium dihydrogen monotitanoundecatungstocobaltate(II) tridecahydrate, K₆H₂[TiW₁₁CoO₄₀]·13H₂O]. ATPγS-mediated inhibition was reduced by the P2 antagonist suramin [8-(3-benzamido-4-methylbenzamido)naphthalene-1,3,5-trisulfonate] at the somatic level and by other P2 blockers, PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate) and MRS 2179 (2′-deoxy-N ⁶-methyladenosine 3′,5′-bisphosphate), at the dendritic level. After removal of both P2 agonists, a persistent increase in evoked synaptic responses was recorded both at the dendritic and somatic levels. This effect was prevented in the presence of different P2 antagonists. A 7-min OGD induced tissue anoxic depolarization and was invariably followed by irreversible loss of fEPSP. PPADS, suramin, MRS2179 or BBG (brilliant blue G) significantly prevented the irreversible failure of neurotransmission induced by 7-min OGD. Furthermore, in the presence of these P2 antagonists, the development of anoxic depolarization was blocked or significantly delayed. Our results indicate that P2 receptors modulate CA1 synaptic transmission under normoxic conditions by eliciting both inhibitory and excitatory effects. In the same brain region, P2 receptor stimulation plays a deleterious role during a severe OGD insult.     

A behavioural study of neuroglobin-overexpressing mice under normoxic and hypoxic conditions

Neuroglobin (Ngb), a neuron-specific heme-binding protein that binds O₂, CO and NO reversibly, and promotes in vivo and in vitro cell survival after hypoxic and ischaemic insult. Although the mechanisms of this neuroprotection remain unknown, Ngb might play an important role in counteracting the adverse effects of ischaemic stroke and cerebral hypoxia. Several Ngb overexpressing mouse models have confirmed this hypothesis; however, these models were not yet exposed to in-depth behavioural characterisations. To investigate the potential changes in behaviour due to Ngb overexpression, heterozygous mice and wild type (WT) littermates were subjected to a series of cognitive and behavioural tests (i.e., the SHIRPA primary screening, the hidden-platform Morris water maze, passive avoidance learning, 47 h cage activity, open field exploration, a dark–light transition box, an accelerating rotarod, a stationary beam, a wire suspension task and a gait test) under normoxic and hypoxic conditions. No significant behavioural differences were found between WT and Ngb-overexpressing mice at three months old. However, one-year-old Ngb-overexpressing mice travelled more distance on the stationary beam compared with WT littermates. This result shows that the constitutive overexpression of Ngb might counteract the endogenous decrease of Ngb in crucial brain regions such as the cerebellum, thereby counteracting age-induced neuromotor dysfunction. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Adenosine 3':5'-monophosphate metabolism and turnover in dog thyroid slices.

The metabolism and turnover of adenosine 3':5'-monophosphate (cyclic AMP) have been studied in intact thyroid cells incubated in vitro. Thyroid slices have been stimulated by 1 mU thyrotropin/ml, then washed with buffer, or with buffer containing thyrotropin antibody, or trypsin so as to cut off the stimulation. The decline of cyclic AMP levels has been followed and the time required to decrease this level to half of the initial value estimated. Computer simulation taking into account the penetration of trypsin in the slices, the kinetics of thyrotropin inactivation and the relation between thyrotropin concentration and cyclic AMP concentration at the steady state has made it possible to estimate the true cellular half-life of cyclic AMP in the stimulated cell to 1 min 50 s. The method provides an experimental approach to the demonstration in intact cells of effective on cyclic AMP disappearance. The methodology of the calculation of half-life and turnover from such data is discussed.     

Effects of the ionophore grisorixin on myocardial function and metabolism in isolated perfused working rat heart under normoxic and hypoxic conditions

The effects of grisorixin, a monocarboxylic ionophore, were studied on isolated working rat hearts perfused with a suspension of washed pig erythrocytes (10% hematocrit). Grisorixin (2.5 microM) induced a transient stimulation of heart work, maximal at 5 min, expressed by an increase in heart rate (+21%) and aortic flow (+17%) and by an increase in coronary flow, maximal at 10 min (+47%). Concomitantly, myocardial Vo2 was slightly enhanced and the myocardial creatine phosphate level dropped (2 min). The lactate production increased by 82% (5 min) then dropped to the control value (10 min) and increased again till the 45th min (+211%), indicating a cardiac metabolic drift towards anaerobic glycolysis due to partial inhibition of the oxidative metabolism. Owing to its properties as an ionophore, grisorixin also induced a strong and rapid increase of potassium concentration in the perfusate and a decrease of sodium. Grisorixin was tested on hearts submitted to 20 min of hypoxic conditions. The hypoxia was rather mild and induced only very slight modifications of the ultrastructure. In the control series, heart rate and aortic flow decreased regularly while coronary flow and lactate production increased. Upon reoxygenation, the heart performances were rapidly restored. Grisorixin was administered according to four different protocols. When injected at the onset of hypoxia or 5 min later, it was able to maintain the aortic flow during the first minutes and induce a higher coronary dilation. These beneficial effects were short-lasting and no deleterious effects were found on the ultrastructure of hearts subjected to grisorixin whether after hypoxia or after reoxygenation.     

Contribution of endothelin to pulmonary vascular tone under normoxic and hypoxic conditions

The contribution of endothelin to resting pulmonary vascular tone and hypoxic pulmonary vasoconstriction in humans is unknown. We studied the hemodynamic effects of BQ-123, an endothelin type A receptor antagonist, on healthy volunteers exposed to normoxia and hypoxia. Hemodynamics were measured at room air and after 15 min of exposure to hypoxia (arterial PO(2) 99.8 +/- 1.8 and 49.4 +/- 0.4 mmHg, respectively). Measurements were then repeated in the presence of BQ-123. BQ-123 decreased pulmonary vascular resistance (PVR) 26% and systemic vascular resistance (SVR) 21%, whereas it increased cardiac output (CO) 22% (all P < 0.05). Hypoxia raised CO 28% and PVR 95%, whereas it reduced SVR 23% (all P < 0.01). During BQ-123 infusion, hypoxia increased CO 29% and PVR 97% and decreased SVR 22% (all P < 0.01). The pulmonary vasoconstrictive response to hypoxia was similar in the absence and presence of BQ-123 [P = not significant (NS)]. In vehicle-treated control subjects, hypoxic pulmonary vasoconstriction did not change with repeated exposure to hypoxia (P = NS). Endothelin contributes to basal pulmonary and systemic vascular tone during normoxia, but does not mediate the additional pulmonary vasoconstriction induced by acute hypoxia.     

Metabolic effects induced by epinephrine in Rana balcanica erythrocytes under normoxic and hypoxic conditions

The metabolic effects of epinephrine on Rana balacanica erythrocyte suspension were studied under normoxia and hypoxia. After epinephrine treatment, a 1.2-fold increase of lactate formation and a 20 per cent decrease of ATP concentration was found under normoxic conditions. These effects were rapid and specific to beta, alpha(1) and alpha(2) antagonists. Glycolysis was stimulated to almost the same extent by both epinephrine and forskolin as normoxic conditions. The stimulation of glycolysis was probably due to stimulation of phosphofructokinase (PFK) as well as to activation of Na(+), K(+)-ATPase. The decrease of ATP was a contributing factor to PFK activation. Despite the high levels of c-AMP at hypoxia, glycolysis was not further induced by epinephrine.     

Effect of isoproterenol on lipid metabolism and prostaglandin production in cultures of newborn rat heart cells, under normoxic and hypoxic conditions

Catecholamines are known to exert deleterious effects on heart cells and to provoke biochemical alterations similar to those observed during myocardial infarction. In order to investigate the mechanisms of these effects, we have studied in cultures of muscle (M) and fibroblast-like (F) cells derived from newborn rat hearts, the action of isoproterenol on membrane lipid metabolism and on prostaglandin production. We showed in F cells that beta-agonist stimulation produced a striking loss of membrane phospholipids and a moderate hydrolysis of cell triglycerides. In addition, isoproterenol treatment induced a significant stimulation of the secretion of prostacyclin but not of prostaglandin E2 by F cells. None of these effects were potentiated by oxygen deprivation. In contrast, M cells, which are sensitive to ischemia, failed to respond to isoproterenol treatment. These results suggest that catecholamines and hypoxia may exert combined deleterious effects on heart tissue by acting separately on the different target cells in vivo.     

Taurine release in mouse brain stem slices under cell-damaging conditions

Summary. Taurine has been thought to be essential for the development and survival of neural cells and to protect them under cell-damaging conditions. In the brain stem taurine regulates many vital functions, including cardiovascular control and arterial blood pressure. We have recently characterized the release of taurine in the adult and developing brain stem under normal conditions. Now we studied the properties of preloaded [³H]taurine release under various cell-damaging conditions (hypoxia, hypoglycemia, ischemia, the presence of metabolic poisons and free radicals) in slices prepared from the mouse brain stem from developing (7-day-old) and young adult (3-month-old) mice, using a superfusion system. Taurine release was greatly enhanced under these cell-damaging conditions, the only exception being the presence of free radicals in both age groups. The ischemia-induced release was characterized to consist of both Ca²⁺-dependent and -independent components. Moreover, the release was mediated by Na⁺-, Cl⁻-dependent transporters operating outwards, particularly in the immature brain stem. Cl⁻ channel antagonists reduced the release at both ages, indicating that a part of the release occurs through ion channels, and protein kinase C appeared to be involved. The release was also modulated by cyclic GMP second messenger systems, since inhibitors of soluble guanylyl cyclase and phosphodiesterases suppressed ischemic taurine release. The inhibition of phospholipases also reduced taurine release at both ages. This ischemia-induced taurine release could constitute an important mechanism against excitotoxicity, protecting the brain stem under cell-damaging conditions.     

Activities of some enzymes of phospholipid metabolism in cultured rat ventricular myocytes in normoxic and hypoxic conditions

Phospholipid catabolism is thought to be one of the critical events in membrane injury during heart ischemia. In this work, the enzymes involved in phospholipid metabolism were studied in purified cultured ventricular myocytes in normoxic and hypoxic conditions. Purified ventricular myocytes exhibited an alkaline phospholipase A activity which had sn-2 specificity and which was calcium dependent, and an acid phospholipase A activity with sn-1 specificity. These cells also exhibited lysophospholipase and acyl-CoA/lysophosphatidylcholine acyltransferase activities. Oxygen deprivation of the myocardial cells for 4 h resulted in a sharp reduction of both phospholipase A2 and A1 activities. The activities of the other lipolytic enzymes were unaffected by hypoxia. Although hypoxia resulted in a marked increase of lactate dehydrogenase leakage in the bathing fluid, no additional release of the lipolytic enzymes and mitochondrial enzyme was observed. However, we noted an important alkaline phospholipase A2 leakage during normoxia. It is suggested that ventricular myocytes, under hypoxia, tend to prevent phospholipid degradation by reducing their phospholipase A activities.     

Adenosine deamination increases the survival under acidic conditions in Escherichia coli

Aims: Resistance to acidic stress contributes to bacterial persistence in the host and is thought to promote their passage through the human gastric barrier. The aim of this study was to examine whether nucleosides have a role in the survival under acidic conditions in Escherichia coli. Methods and Results: We found that adenosine has a function to survive against extremely acidic stress. The deletion of add encoding adenosine deaminase that converts adenosine into inosine and NH₃ attenuated the survival in the presence of adenosine. The addition of adenosine increased intracellular pH of E. coli cells in pH 2·5 medium. Addition of inosine or adenine did not increase the resistance to acidic conditions. Conclusions: Our present results imply that adenosine was used to survive under extremely acidic conditions via the production of NH₃. Significance and Impact of the Study: It has been proposed that amino acid decarboxylation is the major system for the resistance of E. coli to acidic stress. In this study, the adenosine deamination was shown to induce the survival under acidic conditions, demonstrating that bacteria have alternative strategies to survive under acidic conditions besides amino acid decarboxylation.