Epinephrine-mediated stimulation of glucose uptake and lactate release by the perfused rat heart. Evidence for alpha- and beta-adrenergic mechanisms

Epinephrine treatment of the perfused rat heart led to an increase in the rate of glucose uptake and lactate release as well as increases in the rate of beating and the activity ratio of phosphofructokinase. The dose of epinephrine required for half maximal increases in the rate of beating, and glucose uptake and the activity ratio of phosphofructokinase was approx.10^?7M. Glucose uptake, lactate release and the activity ratio of phosphofructokinase were increased by the α-agonists methoxamine and phenylephrine, and the β agonist, isoproterenol. Propranolol and phenoxybenzamine each partially blocked the stimulatory effects of epinephrine on glucose uptake and lactate production. Phenoxybenzamine blocked the stimulatory effects of methoxamine but had no effect on those produced by isoproterenol which were blocked by propranolol. It is concluded that dual α and β adrenergic control of glycolysis occurs in cardiac muscle. It is proposed that the previously reported α-adrenergic control of phosphofructokinase plays a key role in the control of heart muscle glycolysis.     

Vasoconstrictor-mediated release of lactate from the perfused rat hindlimb.

The effects of different vasomodulators on lactate release by the constant-flow-perfused rat hindlimb were examined and compared with that by perfused mesenteric artery, incubated preparations of aortas, soleus and epitrochlearis muscles, and perifused soleus muscles. Infusion of vasopressin (0.5 nM), angiotensin II (5 nM), norepinephrine (50 nM), and methoxamine (10 microM) into the hindlimbs of 180- to 200-g rats increased the perfusion pressure by 112-167% from 30.4 +/- 0.8 mmHg, O2 consumption by 26-68% from 6.4 +/- 0.2 mumol.g-1 x h-1, and lactate efflux by 148-380% from 5.41 +/- 0.25 mumol.g-1 x h-1. Hindlimbs of 100- to 120-g rats responded similarly to angiotensin II. Isoproterenol (1 microM) had no effect on O2 uptake or perfusion pressure but increased lactate release by 118%. Nitroprusside (0.5 mM) markedly inhibited the vasoconstrictor-mediated increases in lactate release, perfusion pressure, and O2 consumption by the hindlimb but had no effect on isoproterenol-mediated lactate efflux. Serotonin (6.7 microM) increased lactate release from the perfused mesenteric artery by 120% from 5.48 mol.g-1 x h-1. Lactate release by incubated aorta was increased by angiotensin II (50 nM), isoproterenol (1 microM), and mechanical stretch. The increase mediated by angiotensin II was blocked by glycerol trinitrate (2.2 microM), which had no effect on lactate release by isoproterenol. Neither angiotensin II (5 nM) nor vasopressin (0.5 nM) increased lactate release from incubated soleus and epitrochlearis muscles; however, lactate release was increased by isoproterenol, and this increase was unaffected by glycerol trinitrate (2.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Myocardial uptake of thiopental enantiomers by the isolated perfused rat heart

Myocardial uptake of thiopental enantiomers by an isolated perfused rat heart preparation was examined after perfusion with protein-free perfusate. Outflow perfusate samples were collected at frequent intervals for 20 min during single-pass perfusion with 10 μg/ml racemic thiopental (washin phase) and for another 45 min during perfusion with drug-free perfusate (washout phase). (+)- and (−)-thiopental concentrations were assayed by chiral high-performance liquid chromatography. Heart rate, perfusion pressure, and electrocardiogram were also monitored. During the washin phase, there was no significant difference between the mean values of the equilibration rate constants of (+)- and (−)-thiopental enantiomers (0.44 ± 0.07 min⁻¹ and 0.43 ± 0.09 min⁻¹, respectively, P > 0.05). Mean volumes of distribution of (+)- and (−)-thiopental enantiomers were similar (6.34 ± 1.20 and 6.45 ± 1.29 ml/g for the washin phase and 7.22 ± 0.71 and 7.47 ± 0.81 ml/g for the washout phase, respectively, P > 0.05). This indicates that tissue accumulation of thiopental enantiomers in the isolated perfused rat heart was not stereoselective. Uptake of thiopental by the heart was perfusion flow rate-limited and independent of capillary permeability. These findings suggest that myocardial tissue concentration of racemic thiopental should be an accurate predictor of myocardial drug effect. © 1996 Wiley-Liss, Inc.     

Evidence for extracellular enzymic activity of the isolated perfused rat heart

1. The dissimilation of a number of externally added hexose phosphates and 5′-nucleotides by the perfused rat heart is described, and non-specific esterase and 5′-nucleotidase activity associated with the superficial cell membrane or vascular system has been demonstrated. 2. The rate of production of ¹⁴CO₂ from [U-¹⁴C]glucose 6-phosphate suggests that oxidation occurred after hydrolysis to glucose. The incorporation of isotope from [U-¹⁴C]glucose 6-phosphate into glycogen was small, and similar to that obtained with [U-¹⁴C]glucose as substrate. 3. Glucose 6-phosphate was also partially isomerized to fructose 6-phosphate. Similarly, fructose 6-phosphate was converted mainly into glucose 6-phosphate, but also into glucose and inorganic phosphate. When fructose 1,6-diphosphate was added to the perfusate, a mixture of glucose 6-phosphate, fructose 6-phosphate and triose phosphates accumulated in the medium approximately in the equilibrium proportions of the phosphohexose-isomerase and triose phosphate-isomerase reactions, together with inorganic phosphate and some glucose. Glucose 1-phosphate was hydrolysed to glucose, but was not converted into glucose 6-phosphate. Leakage of enzymes out into the perfusion fluid did not occur. 4. This demonstration that phosphohexose isomerase, triose phosphate isomerase and aldolase may react with extracellular substrates at an appreciable rate suggests that these enzymes are attached to the cell membrane.     

Intracellular accumulation of isoproterenol enhances the lactate production in the perfused rat heart

Effects of intracellular accumulation of isoproterenol (ISO) on lactate production were examined in perfused rat heart. The lactate production during ISO perfusion in rat heart was increased and subsequent addition of an inhibitor of catechol-O-methyl transferase (COMT) further enhanced the production, and the enhanced production was significantly reduced by uptake2 inhibitor. The perfusion with ISO free-medium in the heart with high intracellular accumulation of ISO produced lactate more than that in the low intracellular accumulation. The present experiments demonstrated that the enhanced lactate production is accompanied by intracellular accumulation of ISO in the perfused rat heart, and suggested that the accumulated ISO may activate intracellular beta-adrenoceptors in the rat heart.     

Stimulation by volume expansion of atrial natriuretic peptide release from perfused rat heart

The release of immunoreactive (ir-) rat atrial natriuretic peptide (rANP) with volume expansion in in situ retrograde perfused rat heart was examined. The volume expansion induced by the infusion of the perfusion medium into the right atrium increased the mean right atrial pressure and the ir-rANP release without changing the rate of the heart beat. There was a significant correlation between the peak values of ir-rANP release and those of mean atrial pressure. The bilateral cervical vagotomy did not effect the ir-rANP release induced by the volume expansion. Therefore, it is highly likely that the stimulatory effect of volume expansion on rANP release is due to, at least in part, the atrial distension accompanied by an increase in mean atrial pressure, not involving a vagal system.     

Control of oxygen uptake, microcirculation and glucose release by circulating noradrenaline in perfused rat liver

The effect of noradrenaline on oxygen uptake, on periportal and perivenous oxygen tension at surface acini, on microcirculation and on glucose output were studied in isolated rat livers perfused at constant flow with Krebs-Henseleit-hydrogen carbonate buffer containing 5mM glucose and 2mM lactate. Noradrenaline at 1 microM concentration caused a decrease in oxygen uptake, while at 0.1 microM it led to an increase. Both high and low doses of noradrenaline decreased the tissue surface oxygen tension in periportal and - after a transient rise - in perivenous areas. Noradrenaline at an overall constant flow caused an increase of portal pressure and an alteration of the intrahepatic distribution of the perfusate: at the surface of the liver and in cross sections infused trypan blue led to only a slightly heterogeneous staining after a low dose of noradrenaline but to a clearly heterogeneous staining after a high dose. Both high and low doses of noradrenaline stimulated glucose release. All effects could be inhibited by the alpha-blocking agent phentolamine. In conclusion, control of hepatic oxygen consumption by circulating noradrenaline is a complex result of opposing hemodynamic and metabolic components: the microcirculatory changes inhibit oxygen uptake; they dominate after high catecholamine doses. The metabolic effects include a stimulation of oxygen utilization; they prevail at low catecholamine levels. The noradrenergic control of glucose release is also very complex, involving direct, metabolic and indirect, hemodynamic components.     

Plasma ceruloplasmin Evidence for its presence in and uptake by heart and other organs of the rat

Evidence for the presence of the plasma protein, ceruloplasmin, in heart and other tissues of the rat was sought using various techniques. With p-phenylenediamine, ceruloplasmin-like oxidase activity was detected in heart postmitochondrial and 100 000 × g supernatants in amounts far exceeding those that could be accounted for by residual blood. Much lower levels were detected in kidney, brain and liver. Oxidase activity of heart purified on DEAE-cellulose in the same way as rat plasma ceruloplasmin and behaved identically also in disc gel electrophoresis. The presence of ceruloplasmin in heart extracts was confirmed immunologically by Ouchterlony diffusion, using rabbit antibody raised against pure rat ceruloplasmin. When pure [³H]leucin-labeled ceruloplasmin was infused intravenously into a copper-deficient rat, radioactivity was concentrated in the heart and brain within 2 h; radioactive counts per g attained 11 and 3 times those of plasma in the two organs, respectively. A lesser concentration occurred in the liver. The results suggest that circulating ceruloplasmin (made by the liver) finds its way into the cells of some organs, especially the heart, a phenomenon which may be related to the function of ceruloplasmin to provide copper to the cytochrome oxidase of various tissues.     

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

Glutathione peroxidase activity and release of glutathione from oxygen-deficient perfused rat heart.

The effect of cellular hypoxia on glutathione levels in rat hearts was determined. Hearts perfused with 95% N₂–5% CO₂ demonstrated a significant decrease in tissue reduced glutathione content when compared to control hearts perfused with 95% O₂–5% CO₂. The hypoxic perfusate contained reduced glutathione and its release was time dependent over a period of 60 minutes. The cellular depletion of oxidized glutathione and its release into coronary effluent were less evident with respect to reduced glutathione. Moreover during hypoxic perfusion we have observed a decrease of cytosol glutathione peroxidase activity. These results suggest that severe oxygen-deprivation causes in myocardial cells a significant perturbation of glutathione metabolism.     

Energy dependence of enzyme release from hypoxic isolated perfused rat heart tissue

There is a sudden release of intracellular constituents upon reoxygenation of isolated perfused hypoxic heart tissue (O2 paradox) or on perfusion with calcium-free medium after a period of hypoxia. Rat hearts were perfused by the method of Langendorff (Pfluegers Arch. 61: 291-332, 1895) with Krebs-Henseleit medium containing 10 mM glucose. Hearts were equilibrated for 30 min, followed by 90 min of hypoxia or 60 min of hypoxia and 30 min of reoxygenation. The massive enzyme release observed upon reoxygenation after 60 min of hypoxia was prevented by infusing 0.5 or 5 mM cyanide 5 min before reoxygenation. Lactate dehydrogenase (LDH) release commenced immediately upon withdrawal of cyanide. Hearts perfused with calcium-free medium throughout hypoxia did not release increased amounts of LDH at reoxygenation. Perfusing heart tissue with medium containing 0 or 25 microM calcium, but not 0.25 or 2.5 mM, after 50 min of hypoxia initiated a release of cardiac LDH, which was not further enhanced by reoxygenation. Enzyme release was significantly inhibited when the calcium-free perfusion medium included 10 mM 2-deoxyglucose (replacing glucose), 0.5 mM dinitrophenol, or 2.5 mM cyanide. Histologically, hearts perfused with calcium-free medium after 50 min of hypoxia showed areas of severe necrosis and contracture without any evidence of the contraction bands that were seen in hearts reoxygenated in the presence of calcium. Cardiac ATP and creatine phosphate (PCr) levels were significantly decreased after 50-60 min of hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

The uptake and release of calcium by heart mitochondria

The kinetics of uptake of Ca(2+) by rat heart mitochondria were studied by a spectrophotometric method with Arsenazo III indicator. The exponential rate coefficients measured with or without added phosphate increase with the amount of Ca(2+) added up to about 24mum. Evidence is given that the effect is attributable to a combination of formation of chelates at low concentrations to act as Ca(2+) buffers, with co-transport of substrate to provide more respiratory fuel. The inhibitory effect of Mg(2+) depends on the Ca(2+) concentration, so with a constant [Mg(2+)] the low concentrations of Ca(2+) are most inhibited, and the rate coefficients are still more Ca(2+)-dependent. Ca(2+) uptake is slowed by local anaesthetics such as butacaine and dibucaine, and also by propranolol and palmitoyl-CoA. After an uptake, the release of Ca(2+) was investigated. The spontaneous release involves an initially slow and small appearance of free Ca(2+) and is followed by an auto-accelerated phase. The release is accompanied by a gradual decrease in internal ATP; it is initiated by palmitoyl-CoA (reversed by carnitine), by lysophosphatidylcholine, by Na(+) salts (reversed by oligomycin) and by K(+) salts added to a K(+)-free medium containing valinomycin. The process is probably a response to an increased energy load imposed on the mitochondria by the various conditions, which include the spontaneous action of phospholipase activated by traces of Ca(2+). The problem of how much mitochondrial activity is participating in normal heart Ca(2+) turnover is discussed, and experiments showing only 7-14% exchange of the mitochondrial Ca(2+) occurring in vivo in 10 or 20min are reported.     

Regulation of production and release of lipoprotein by the perfused rat liver

The relationship between protein and triglyceride release into d < 1.007 lipoprotein was studied in the isolated perfused rat liver. Livers were perfused with a medium either high or low in linoleate content. Perfusion with the linoleate-rich medium resulted in a marked increase in the net release of both d < 1.007 lipoprotein triglyceride and lipoprotein protein, and caused a significant increase in amino acid incorporation into the protein moiety. Amino acid incorporation into d 1.008-1.21 protein was not affected by fatty acid concentration, while incorporation into whole perfusate and tissue proteins was depressed by a perfusate high in fatty acid content. Electron microscopic studies demonstrated that the livers with the higher rate of triglyceride release also produced a greater number of lipoprotein particles. The particles they released were also somewhat larger. These studies suggest that the intracellular concentration of newly esterified triglyceride and (or) some other lipid metabolite can specifically influence the release and perhaps the synthesis of d < 1.007 lipoprotein protein. They also suggest that the liver increases its rate of triglyceride release primarily by producing more lipoprotein particles.