Lactate metabolism in the perfused rat hindlimb.

A preparation of isolated rat hindleg was perfused with a medium consisting of bicarbonate buffer containing Ficoll and fluorocarbon, containing glucose and/or lactate. The leg was electrically prestimulated to deplete partially muscle glycogen. The glucose was labelled uniformly with 14C and with 3H in positions 2, 5 or 6, and lactate uniformly with 14C and with 3H in positions 2 or 3. Glucose carbon was predominantly recovered in glycogen, and to a lesser extent in lactate. The 3H/14C ration in glycogen from [5-3H,U-14C]- and [6-3H,U-14C]-glucose was the same as in glucose. Nearly all the utilized 3H from [2-3H]glucose was recovered as water. Insulin increased glucose uptake and glycogen synthesis 3-fold. When the muscle was perfused with a medium containing 10 mM-glucose and 2 mM-lactate, there was little change in lactate concentration. 14C from lactate was incorporated into glycogen. There was a marked exponential decrease in lactate specific radioactivity, much greater with [3H]- than with [14C]-lactate. The 'apparent turnover' of [U-14C]lactate was 0.28 mumol/min per g of muscle, and those of [2-3H]- and [3-3H]-lactate were both about 0.7 mumol/min per g. With 10 mM-lactate as sole substrate, there was a net uptake of lactate, at a rate of about 0.15 mumol/min per g, and the apparent turnover of [U-14C]lactate was 0.3 mumol/min per g. The apparent turnover of [3H]lactate was 3-5 times greater. When glycogen synthesis was low (no prestimulation, no insulin), the incorporation of lactate carbon into glycogen exceeded that from glucose, but at high rates of glycogen deposition the incorporation of lactate carbon was much less than that of glucose. Lactate incorporation into glycogen was similar in fast-twitch white and fast-twitch red muscle, but was very low in slow-twitch red fibres. We find that (a) pyruvate in muscle is incorporated into glycogen without randomization of carbon, and synthesis is not inhibited by mercaptopicolinate or cycloserine; (b) there is extensive lactate turnover in the absence of net lactate uptake, and there is a large dilution of 14C-labelled lactate from endogenous supply; (c) there is extensive detritiation of [2-3H]- and [3-3H]-lactate in excess of 14C utilization.     

Nutritive blood flow improves interstitial glucose and lactate exchange in perfused rat hindlimb

Microdialysis was used to assess the interstitial concentrations of glucose and lactate in the constant-flow-perfused rat hindlimb under varying levels of nutritive flow controlled by vasoconstrictors. Increased nutritive flow was achieved by norepinephrine (NE) or angiotensin II (ANG II) and decreased nutritive flow by serotonin (5-HT). NE and ANG II increased oxygen and glucose uptake as well as hindlimb lactate release by 50%. 5-HT decreased oxygen uptake by 15% but had no significant effect on glucose uptake or hindlimb lactate release. Microdialysis recovery of glucose and lactate was significantly elevated by NE and ANG II and decreased by 5-HT. The calculated interstitial concentration of glucose was increased by NE and ANG II but decreased by 5-HT. The interstitial concentration of lactate was decreased by NE and ANG II but increased by 5-HT. In all cases, nitroprusside reversed the effects of the vasoconstrictors. These data indicate that increased nutritive blood flow enhances the exchange of glucose and lactate by improving the supply of glucose to and the removal of lactate from the interstitium.     

Vasoconstrictor-mediated thermogenesis present in perfused skeletal muscle but absent from perfused heart

Resting muscle thermogenesis as controlled by vasocontrictors was compared in rat hindlimb and cardiac muscle. An α-adrenergic agonist combination of phenylephrine+atenolol increased oxygen uptake and perfusion pressure in the constant flow hindlimb and neither increase was blocked by 10 μM tetrodotoxin. The same adrenergic combination also increased oxygen uptake and perfusion pressure in the perfused heart but the former along with beating was completely blocked by tetrodotoxin. Vasoconstriction by phenylephrine occurs in the heart but is not linked to thermogenic increases as in hindlimb, implying that all metabolic energy in heart is conserved for contractile activity. The findings highlight a fundamental difference between skeletal and cardiac muscle.     

ATP stimulates the release of prostacyclin from perfused veins isolated from the hamster hindlimb

ATP-stimulated prostacyclin release from veins was investigated using epigastric veins isolated from hamsters. Veins were perfused with MOPS-buffered physiological salt solution (PSS). ATP was administered into the perfusate, and the bath solution (MOPS-PSS) was collected and assayed for the presence of the stable prostacyclin metabolite 6-keto-PGF1alpha. ATP (100 microM) resulted in reproducible increases in bath concentration from 73 +/- 22 to 279 +/- 50 pg/ml (P < 0.05, n = 5). This response was abolished by indomethacin (10 microM, P < 0.05). To ascertain whether the endothelium was the source of prostacyclin, endothelium was disrupted using air (n = 10) or deoxycholic acid (n = 6). Perfusion with air significantly reduced (P < 0.05) but did not completely abolish ATP-stimulated release of prostacyclin, while deoxycholic acid totally abolished the response (P < 0.05). The nonselective P2 receptor antagonist reactive blue 2 (100 microM) attenuated ATP-mediated release of prostacyclin but did not significantly alter ACh-stimulated release of prostacyclin. The nonselective adenosine receptor antagonist xanthine amine congener (1 microM) had no effect on ATP-stimulated release, and adenosine did not stimulate the release of prostacyclin. These results show that increases in intraluminal concentration of ATP stimulate abluminal release of prostacyclin from the venous endothelium. This effect is mediated by P2 receptors while adenosine and its receptors are not involved in this response.     

Somatostatin release from isolated perfused rat stomach.

Gastric somatostatin release from the isolated rat stomach was studied using a perfusion technique. Somatostatin released from the isolated perfused rat stomach was found to be identical in molecular size and immunoreactively with synthetic somatostatin. Infusion of glucagon (10^?7 M) caused biphasic increase of gastric somatostatin release. Gastric somatostatin release was also stimulated by infusion of theophylline (10^?3 M) and dibutyryl cyclic AMP (10^?3 M). These results indicate the possible involvement of adenylate cyclase-cyclic AMP system in the regulatory mechanism of gastric somatostatin release.     

Benzoate stimulates glutamate release from perfused rat liver.

In isolated perfused rat liver, benzoate addition to the influent perfusate led to a dose-dependent, rapid and reversible stimulation of glutamate output from the liver. This was accompanied by a decrease in glutamate and 2-oxoglutarate tissue levels and a net K+ release from the liver; withdrawal of benzoate was followed by re-uptake of K+. Benzoate-induced glutamate efflux from the liver was not dependent on the concentration (0-1 mM) of ammonia (NH3 + NH4+) in the influent perfusate, but was significantly increased after inhibition of glutamine synthetase by methionine sulphoximine or during the metabolism of added glutamine (5 mM). Maximal rates of benzoate-stimulated glutamate efflux were 0.8-0.9 mumol/min per g, and the effect of benzoate was half-maximal (K0.5) at 0.8 mM. Similar Vmax. values of glutamate efflux were obtained with 4-methyl-2-oxopentanoate, ketomethionine (4-methylthio-2-oxobutyrate) and phenylpyruvate; their respective K0.5 values were 1.2 mM, 3.0 mM and 3.8 mM. Benzoate decreased hepatic net ammonia uptake and synthesis of both urea and glutamine from added NH4Cl. Accordingly, the benzoate-induced shift of detoxication from urea and glutamine synthesis to glutamate formation and release was accompanied by a decreased hepatic ammonia uptake. The data show that benzoate exerts profound effects on hepatic glutamate and ammonia metabolism, providing a new insight into benzoate action in the treatment of hyperammonaemic syndromes.     

Evidence of separate pathways for lactate uptake and release by the perfused rat heart

The simultaneous release and uptake of lactate by the heart has been observed both in vivo and ex vivo; however, the pathways underlying these observations have not been satisfactorily explained. Consequently, the purpose of this study was to test the hypothesis that hearts release lactate from glycolysis while simultaneously taking up exogenous lactate. Therefore, we determined the effects of fatty acids and diabetes on the regulation of lactate uptake and release. Hearts from control and 1-wk diabetic animals were perfused with 5 mM glucose, 0.5 mM [3-(13)C]lactate, and 0, 0.1, 0.32, or 1.0 mM palmitate. Parameters measured include perfusate lactate concentrations, fractional enrichment, and coronary flow rates, which enabled the simultaneous, but independent, measurements of the rates of 1) uptake of exogenous [(13)C]lactate and 2) efflux of unlabeled lactate from metabolism of glucose. Although the rates of lactate uptake and efflux were both similarly inhibited by the addition of palmitate, (i.e., the ratio of lactate uptake to efflux remained constant), the ratio of lactate uptake to efflux was significantly higher in the controls compared with the diabetic group (1.00 +/- 0.14 vs. 0.50 +/- 0.07, P < 0.002). These data, combined with heterogeneous (13)C enrichment of tissue lactate, pyruvate, and alanine, suggest that glycolytically derived lactate production and oxidation of exogenous lactate operate as functionally separate metabolic pathways. These results are consistent with the concept of an intracellular lactate shuttle.     

Kinetic measurements of the release of prostaglandins from perfused rat lungs.

Prostaglandins released from isolated ventilated and perfused rat lungs were measured by a simple modification of the Vane technique using the rat stomach fundus as a continuous bioassay tissue. Exogenously supplied arachidonic acid was converted mainly to PGF2alpha which was determined by bioassay. A novel method for mixing a stream of inhibitors with the perfusate was used to determine PGF2alpha in the presence of substrate amounts of arachidonic acid. Using this system the apparent Km for PGF2alpha production with arachidonic acid as the substrate was found to be 1.90 X 10(-4)M, while the Ki for aspirin was found to 2.47 X 10(-4)M. These kinetic parameters are close to those reported for cell free systems and subcellular fractions suggesting that both substrate and inhibitor have ready access to the site of prostaglandin synthesis. The method appears to be generally useful to determine the effect of drugs and environment factors on the release of prostaglandins by the lung.     

Effects of glucagon on gluconeogenesis from lactate and propionate in the perfused rat liver.

Quinolinic acid (Q.A.) which inhibits gluconeogenesis at the site of phosphoenolpyruvate (PEP) synthesis, reduced the content of PEP while elevating that of aspartate and malate in rat livers perfused with a medium containing 10 mM L-lactate. Glucagon at 10(-9) M did not affect Q.A. inhibition of lactate gluconeogenesis nor the depression of PEP level, but further elevated malate and aspartate accumulation. Exogenous butyrate had the same effect as glucagon on these parameters. Butylmalonate (BM), an inhibitor of mitochondrial malate transport, inhibited lactate and propionate gluconeogenesis to similar extents. The addition of 10(-9) M glucagon had no effect on BM inhibition of lactate gluconeogenesis, but almost completely reversed BM inhibition of propionate gluconeogenesis. These results suggest that glucagon may act on at least two sites, resulting in elevated hepatic gluconeogenesis. First, it may stimulate dicarboxylic acid synthesis (malate and oxaloacetate, specifically) through activation of pyruvate carboxylation. Secondly, it may stimulate synthesis of other dicarboxylic acids (fumarate, for example) by activating certain steps of the tricarboxylic acid cycle. The stimulatory effect of glucagon on gluconeogenesis in the perfused rat liver is well documented (1, 2). Exton et al., who earlier located the site of stimulation between pyruvate and PEP synthesis (3), proposed that glucagon stimulated PEP synthesis in the perfused rat liver (4), while reports from Williamson et al. (5) suggested the pyruvate-carboxylase reaction as the site of glucagon action. Stimulation at sites above PEP formation and of portions of the tricarboxylic acid cycle (4) by glucagon have also been suggested (6). In the present experiments, we have used substrates entering at different parts of the gluconeogenic pathway, and specific inhibitors to further resolve the action of glucagon.     

Glutamine release from hindlimb and uptake by kidney in the acutely acidotic rat.

The arteriovenous difference (release) for glutamine across the hindlimb increases significantly during acute HCl-induced acidosis. This additional amount release by muscle tissue can account for the extra glutamine taken up by the kidney.     

Volume-regulatory potassium release from isolated perfused rat kidney

The present study has been performed to test for cell volume regulatory potassium release from the isolated perfused rat kidney exposed to hypotonic perfusate and for its sensitivity to potassium channel blocker barium and calcium channel blocker verapamil. Replacement of 25 mmol/l NaCl with 50 mmol/l mannitol has little effect on effluent potassium activity, whereas subsequent omission of mannitol from the perfusate leads to a transient increase of effluent potassium activity, reflecting volume regulatory potassium release. Barium (1 mmol/l) leads to a marked transient decrease of effluent potassium activity, pointing to net cellular uptake of potassium. Verapamil (1 mumol/l) leads to a slight decrease of effluent potassium activity. Both barium and verapamil virtually abolish the rapid, transient increase of effluent potassium activity upon exposure to hypotonic perfusates. Thus, the substances either block or markedly retard volume regulatory potassium release. The apparent renal vascular resistance is transiently increased by exposure to hypotonic perfusates and by barium, but is reduced by verapamil. Cell volume regulation of isolated perfused mouse straight proximal tubules is retarded but not abolished by verapamil (0.1 mmol/l). In conclusion, cellular potassium release from rat kidney can be determined by continuous measurement of effluent potassium activity. The volume regulatory potassium release and cell volume regulation are impaired by both barium and verapamil. The persisting cell volume regulation could be due either to slow potassium release and/or some mechanism independent of potassium.     

Insulin release from the perfused rat pancreas. Mode of action of tolbutamide

Dextran-linked tolbutamide causes first-phase insulin release when perfused through the isolated rat pancreas. The linked sulphonylurea is also able to stimulate the membranal adenylate cyclase present in mouse islets. These facts make it highly likely that sulphonylureas exert their pharmacological action by interaction with the plasma membrane. Their action may be mediated via the adenylate cyclase enzyme.     

Kinetic measurements of the release of prostaglandins from perfused rat lungs

Prostaglandins released from isolated, ventilated and perfused rat lungs were measured by a simple modification of the Vane technique using the rat stomach fundus as a continuous bioassay tissue. Exogeneously supplied arachidonic acid was converted mainly to PGF_2α which was determined by bioassay. A novel method for mixing a stream of inhibitors with the perfusate was used to determine PGF_2α in the presence of substrate amounts of arachidonic acid. Using this system the apparent K_m for PGF_2α production with arachidonic acid as the substrate was found to be 1.90 × 10⁻⁴M, while the K_i for aspirin was found to be 2.47 × 10⁻⁴M. These kinetic parameters are close to those reported for cell free systems and subcellular fractions suggesting that both substrate and inhibitor have ready access to the site of prostaglandin synthesis. The method appears to be generally useful to determine the effect of drugs and environmental factors on the release of prostaglandins by the lung.     

Kinetic measurements of the release of prostaglandins from perfused rat lungs

Prostaglandins released from isolated, ventilated and perfused rat lungs were measured by a simple modification of the Vane technique using the rat stomach fundus as a continuous bioassay tissue. Exogenously supplied arachidonic acid was converted mainly to PGF₂ which was determined by bioassay. A novel method for mixing a stream of inhibitors with the perfusate was used to determine PGF₂ in the presence of substrate amounts of arachidonic acid. Using this system the apparent K_m for PGF₂ production with arachidonic acid as the substrate was found to be 1.90 × 10⁻⁴M, while the K_i for aspirin was found to be 2.47 × 10⁻⁴M. These kinetic parameters are close to those reported for cell free systems and subcellular fractions suggesting that both substrate and inhibitor have ready access to the site of prostaglandin synthesis. The method appears to be generally useful to determine the effect of drugs and environmental factors on the release of prostaglandins by the lung.     

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.     

Regulation of gluconeogenesis from pyruvate and lactate in the isolated perfused rat liver

The effects of glucagon and the alpha-adrenergic agonist, phenylephrine, on the rate of 14CO2 production and gluconeogenesis from [1-14C]lactate and [1-14C]pyruvate were investigated in isolated perfused livers of 24-h-fasted rats. Both glucagon and phenylephrine stimulated the rate of 14CO2 production from [1-14C]lactate but not from [1-14C]pyruvate. Neither glucagon nor phenylephrine affected the activation state of the pyruvate dehydrogenase complex in perfused livers derived from 24-h-fasted rats. 3-Mercaptopicolinate, an inhibitor of the phosphoenolpyruvate carboxykinase reaction, inhibited the rates of 14CO2 production and glucose production from [1-14C]lactate by 50% and 100%, respectively. Furthermore, 3-mercaptopicolinate blocked the glucagon- and phenylephrine-stimulated 14CO2 production from [1-14C]lactate. Additionally, measurements of the specific radioactivity of glucose synthesized from [1-14C]lactate, [1-14C]pyruvate and [2-14C]pyruvate indicated that the 14C-labeled carboxyl groups of oxaloacetate synthesized from 1-14C-labeled precursors were completely randomized and pyruvate----oxaloacetate----pyruvate substrate cycle activity was minimal. The present study also demonstrates that glucagon and phenylephrine stimulation of the rate of 14CO2 production from [1-14C]lactate is a result of increased metabolic flux through the phosphoenolpyruvate carboxykinase reaction, and phenylephrine-stimulated gluconeogenesis from pyruvate is regulated at step(s) between phosphoenolpyruvate and glucose.     

Adiponectin opposes endothelin-1-mediated vasoconstriction in the perfused rat hindlimb

Recent studies have shown that adiponectin is able to increase nitric oxide (NO) production by the endothelium and relax preconstricted isolated aortic rings, suggesting that adiponectin may act as a vasodilator. Endothelin-1 (ET-1) is a potent vasoconstrictor, elevated levels of which are associated with obesity, type 2 diabetes, hypertension, and cardiovascular disease. We hypothesized that adiponectin has NO-dependent vascular actions opposing the vasoconstrictor actions of ET-1. We studied the vascular and metabolic effects of a physiological concentration of adiponectin (6.5 μg/ml) on hooded Wistar rats in the constant-flow pump-perfused rat hindlimb. Adiponectin alone had no observable vascular activity; however, adiponectin pretreatment and coinfusion inhibited the increase in perfusion pressure and associated metabolic stimulation caused by low-dose (1 nM) ET-1. Adiponectin was not able to oppose vasoconstriction when infusion was commenced after ET-1. This is in contrast to the NO donor sodium nitroprusside, which significantly reduced the pressure due to established ET-1 vasoconstriction, suggesting dissociation of the actions of adiponectin and NO. In addition, adiponectin had no effect on vasoconstriction caused by either high-dose (20 nM) ET-1 or low-dose (50 nM) norepinephrine. Our findings suggest that adiponectin has specific, apparently NO-independent, vascular activity to oppose the vasoconstrictor effects of ET-1. The hemodynamic actions of adiponectin may be an important aspect of its insulin-sensitizing ability by regulating access of insulin and glucose to myocytes. Imbalance in the relationship between adiponectin and ET-1 in obesity may contribute to the development of insulin resistance and cardiovascular disease.     

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.