Mechanism of action of cysteinyl leukotrienes on glucose and lactate balance and on flow in perfused rat liver. Comparison with the effects of sympathetic nerve stimulation and noradrenaline

Rat livers were perfused at constant pressure via the portal vein with media containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate. 1. Leukotrienes C4 and D4 enhanced glucose and lactate output and reduced perfusion flow to the same extent and with essentially identical kinetics. They both caused half-maximal alterations (area under the curve) of carbohydrate metabolism at a concentration of about 1 nM and of flow at about 5 nM. The leukotriene-C4/D4 antagonist CGP 35949 B inhibited the metabolic and hemodynamic effects of 5 nM leukotrienes C4 and D4 with the same efficiency, causing 50% inhibition at about 0.1 microM. 2. Leukotriene C4 elicited the same metabolic and hemodynamic alterations with the same kinetics as leukotriene D4 in livers from rats pretreated with the gamma-glutamyltransferase inhibitor, acivicin. 3. The calcium antagonist, nifedipine, at a concentration of 50 microM did not affect the metabolic and hemodynamic changes caused by 5 nM leukotriene D4. The smooth-muscle relaxant, nitroprussiate, at a concentration of 10 microM reduced flow changes, without significantly affecting the metabolic alterations. 4. Leukotriene D4 not only reduced flow; it also caused an intrahepatic redistribution of flow, restricting some areas from perfusion. Thus, leukotrienes increased glucose and lactate output directly in the accessible parenchyma and, in addition, indirectly by washout from restricted areas during their reopening upon termination of application. 5. The phospholipase A2 inhibitor, bromophenacyl bromide, but not the cyclooxygenase inhibitor, indomethacin, at a concentration of 20 microM reduced the metabolic and hemodynamic effects of 5 mM leukotriene D4. 6. Stimulation of the sympathetic hepatic nerves with 2-ms rectangular pulses at 20 Hz and infusion of 1 microM noradrenaline increased glucose and lactate output and decreased flow, similar to 10 nM leukotrienes C4 and D4. The kinetics of the metabolic and hemodynamic changes caused by the leukotrienes differed, however, from those due to nerve stimulation and noradrenaline. 7. The leukotriene-C4/D4 antagonist, CGP 35949 B, even at very high concentrations (20 microM) inhibited the metabolic and hemodynamic alterations caused by nerve stimulation or noradrenaline infusion only slightly and unspecifically.(ABSTRACT TRUNCATED AT 400 WORDS)     

Possible involvement of eicosanoids in the actions of sympathetic hepatic nerves on carbohydrate metabolism and hemodynamics in perfused rat liver

In isolated rat liver perfused at constant pressure with Krebs-Henseleit buffer containing 5 mM glucose, 2 mM lactate, 0.2 mM pyruvate and 0.1% bovine serum albumin, perivascular nerve stimulation (20 V, 20 Hz, 2 ms) and infusion of ATP (100 microM), noradrenaline (1 microM) or arachidonic acid (100 microM) caused an increase in glucose and lactate output and a reduction of perfusion flow. The metabolic effects of nerve stimulation but not those of ATP and noradrenaline were inhibited strongly by the phospholipase A2 inhibitor bromophenacyl bromide (BPB, 20 microM) and the cyclooxygenase inhibitor indomethacin (Indo, 20 microM) and only slightly by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 20 microM). In contrast, the hemodynamic effects not only of nerve stimulation but also of ATP and noradrenaline were inhibited strongly by BPB and Indo and slightly by NDGA. The metabolic and hemodynamic actions of arachidonate were inhibited specifically by Indo. These results suggest that the effects of nerve stimulation were at least partially mediated or modulated by eicosanoids, especially by prostanoids.     

Role of extracellular calcium in the metabolic and hemodynamic actions of sympathetic nerve stimulation, noradrenaline and prostaglandin F2 alpha in perfused rat liver. Differential inhibition by nifedipine and verapamil

In perfused rat liver hepatic nerve stimulation (10 Hz, 2 ms) caused an increase in glucose and lactate output, a decrease in flow and an overflow of noradrenaline into the hepatic vein. Noradrenaline (1 microM) (NA) and prostaglandin F2 alpha (5 microM) (PGF2 alpha), which are implicated as mediators of nerve action, elicited similar effects. 1) All actions of nerve stimulation and the hemodynamic but not the metabolic effects of noradrenaline and PGF2 alpha were largely dependent on extracellular calcium. 2) The dihydropyridine type calcium antagonist nifedipine (5 microM) inhibited the hemodynamic but not the metabolic actions of nerve stimulation, NA and PGF2 alpha, while the phenylalkylamine type calcium antagonist verapamil (5 microM) had no effect. These findings allow the following conclusions: Calcium influx into I nerve endings, necessary for the release of neurotransmitter, II parenchymal cells, for the display of metabolic effects induced by nerve stimulation, and III the actions of NA and PGF2 alpha, do not appear to be mediated by the normal affinity nifedipine- or the verapamil-sensitive channels. Calcium influx into vascular smooth muscle and/or endothelial cells for the display of hemodynamic action induced by nerve stimulation and the NA and PGF2 alpha effects, appear to occur through nifedipine-sensitive but verapamil-insensitive channels.     

Control of ketogenesis in the perfused rat liver by the sympathetic innervation

The regulation of ketogenesis by the hepatic nerves was investigated in the rat liver perfused in situ. Electrical stimulation of the hepatic nerves around the portal vein and the hepatic artery caused a reduction of basal ketogenesis owing to a decrease in acetoacetate release to 30% with essentially no change in 3-hydroxybutyrate release. At the same time, as observed before [Hartmann et al. (1982) Eur. J. Biochem. 123, 521-526], nerve stimulation increased glucose output, shifted lactate uptake to output and decreased perfusion flow. Ketogenesis from oleate, which enters the mitochondria via the carnitine system, was also lowered after nerve stimulation owing to a decrease of acetoacetate release to 30% with no alteration in 3-hydroxybutyrate release. Ketogenesis from octanoate, which enters the mitochondria independently of the carnitine system, was decreased after nerve stimulation as a result of a drastic decrease of acetoacetate output to 15% and a less pronounced decrease of 3-hydroxybutyrate release to 65%. Noradrenaline mimicked the metabolic nerve effects on ketogenesis only at the highly unphysiological concentration of 0.1 microM under basal conditions and in the presence of oleate as well as partly in the presence of octanoate. It was essentially not effective at a concentration of 0.01 microM, which might be reached in the sinusoids owing to overflow from the hepatic vasculature. Sodium nitroprusside prevented the hemodynamic changes after nerve stimulation; it did not affect the nerve-dependent reduction of ketogenesis under basal conditions and in the presence of oleate, yet it diminished the nerve effect on octanoate-dependent ketogenesis. Phentolamine clearly reduced the metabolic and hemodynamic nerve effects, while propranolol was without effect. The present data suggest that hepatic ketogenesis was inhibited by stimulation of alpha-sympathetic liver nerves directly rather than indirectly via hemodynamic changes or noradrenaline overflow from the vessels and that the site of regulation should be mainly intramitochondrial.     

Rate of oxygen consumption by isolated articular chondrocytes is sensitive to medium glucose concentration

Cartilage tissue engineering typically involves the culture of isolated chondrocytes within a scaffold material. The oxygen tension within the engineered tissue is known to be an essential parameter for implant success. This will be sensitive to the oxygen consumption behavior of the embedded chondrocytes, which remains to be characterized. We report that the oxygen consumption of bovine articular chondrocytes is sensitive to glucose deprivation below 2.7 mM, increasing from a basal level of 9.6x10(-16) to <18.4x10(-16) mol/cell.h in 1.3 mM glucose. Further studies examined the influence of selecting high (18.4 mM) or low (5.1 mM) glucose medium on the oxygen tension in 2 mm thick cellular agarose constructs. A relative upregulation of oxygen consumption was observed in constructs cultured in low glucose medium. This resulted in the near-anoxic oxygen concentration of 5 microM oxygen in constructs seeded with 40x10(6) cells/ml, compared to 57 microM in the corresponding high glucose culture. The upregulation of oxygen consumption generally corresponded to the inhibition of glycolysis, which is consistent with the Crabtree phenomenon. Medium osmolarity (316-600 mOsm) had minimal effects on chondrocyte oxygen consumption rate. In conclusion, glucose availability is a critical parameter that regulates the oxygen tension within tissue engineered constructs.     

Hepatocyte heterogeneity in response to extracellular ATP

1. The metabolic and hemodynamic effects of extracellular ATP in perfused rat liver were compared during physiologically antegrade (portal to hepatic vein) and retrograde (hepatic to portal vein) perfusion. ATP in concentrations up to 100 microM was completely hydrolyzed during a single liver passage regardless of the perfusion direction. 2. The ATP(20 microM)-induced increases of glucose output, perfusion pressure and ammonium ion release seen during antegrade perfusions were diminished by 85-95% when the perfusion was in the retrograde direction, whereas the amount of Ca2+ mobilized from the liver was decreased by only 60%. The maximal rate of initial K+ uptake following ATP was dependent on the amount of Ca2+ mobilized regardless of the direction of perfusion. In the presence of UMP (1 mM), an inhibitor of ATP hydrolysis by membrane-bound nucleotide pyrophosphatase, the effect of the direction of perfusion on the glycogenolytic response to ATP (20 microM) was largely diminished. 3. For a maximal response of glucose output, Ca2+ release and perfusion pressure to extracellular ATP, concentrations of about 20 microM, 50 microM and 100 microM were required during antegrade perfusion, respectively. These maximal responses could also be obtained during retrograde perfusion, but higher ATP concentrations were required (120 microM, 80 microM, above 200 microM, respectively). 4. 14CO2 production from [1-14C]glutamate which occurs predominantly in the perivenous hepatocytes capable of glutamine synthesis was stimulated by extracellular ATP (20 microM); it was only slightly affected by the direction of perfusion. In antegrade perfusions, ATP (20 microM) increased 14CO2 production from 88 to 162 nmol g-1 min-1, compared to an increase from 91 to 148 nmol g-1 min-1 in retrograde perfusion. 5. The data are interpreted to suggest that (a) extracellular ATP is predominantly hydrolyzed by a small hepatocyte population located at the perivenous outflow of the acinus; (b) glycogenolysis to glucose is predominantly localized in the periportal area; (c) contractile elements (sphincters) exist near the inflow of the sinusoidal bed; (d) a considerable portion of the Ca2+ mobilized by ATP is derived from liver cells that do not contribute to hepatic glucose output.     

Inhibition of glucose production during hepatic nerve stimulation in regenerating rat liver perfused in situ. Possible involvement of gap junctions in the action of sympathetic nerves.

To explore the possible role of gap junctions in neural regulation of hepatic glucose metabolism, the effects of hepatic nerve stimulation on metabolic and hemodynamic changes were examined in normal and regenerating rat liver which was perfused in situ at constant pressure via the portal vein with a medium containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate. 1. The content of connexin 32, a major component of gap junctions in rat liver, decreased transiently to about 25% of the control level in regenerating liver 48-72 h after partial hepatectomy and recovered to normal by the 11th day after the operation. 2. In normal liver, electrical stimulation of the hepatic nerves (10 Hz, 20 V, 2 ms) and infusion of noradrenaline (1 microM) both increased glucose and lactate output and reduced perfusion flow. 3. In early stage of regenerating liver 48 h and 72 h after partial hepatectomy, the increase in glucose output in response to nerve stimulation was almost completely inhibited, whereas the change in lactate balance was partially suppressed and the reduction of flow rate was retained. The response of glucose output to nerve stimulation recovered by the 11th day after partial hepatectomy. In contrast, exogenous application of noradrenaline increased glucose output even in the early stage of regenerating liver. 4. The increase in noradrenaline overflow during hepatic nerve stimulation in the early stage of regenerating liver was approximately the same as in normal liver. Liver glycogen was sufficiently preserved in the early stage of regenerating liver. However, noradrenaline infusion could no more increase glucose output both in normal and in regenerating livers after 24 h of fasting that depleted liver glycogen. These results suggest that the impaired effects of sympathetic nerve stimulation on glucose metabolism observed in regenerating liver are derived neither from reduced release of noradrenaline nor from depletion of liver glycogen, but rather from transient reduction of gap junctions which assist signal propagation of the nerve action through intercellular communication in rat liver.     

The effects of endogenous opioids on tension development of isolated, electrically stimulated rat atria

The possible inotropic effects of all three classes of endogenous opioids were tested alone or in combination with noradrenaline, adrenaline, or carbachol on electrically stimulated atria isolated from male Sprague-Dawley rats. Noradrenaline (6.0 and 12 microM) and adrenaline (4.0 and 8.0 microM) injections caused marked but transient (5 min) dose-related increases in atrial tension compared with preinjection control values, whereas carbachol (0.14 and 1.4 microM) caused a more potent and prolonged (over 15 min) dose-related decrease in atrial tension development. Adrenal enkephalins (0.3-4.0 microM) of methionine enkephalin, leucine enkephalin, Met-enkephalin-Arg6-Phe7, and Met-enkephalin-Arg6-Gly7-Leu8, beta-endorphin (0.2-2.0 microM), or dynorphin A(1-13) (0.2-2.0 microM) did not change atrial tension for a 15-min postadministration test period. In addition, these opioids did not affect the positive inotropic effects of noradrenaline (12 microM) or adrenaline (8.0 microM) or the negative inotropic actions of carbachol (1.4 microM) when the same doses of noradrenaline, adrenaline, or carbachol were given alone. These data indicate that endogenous opioids given in micromolar concentrations tested did not affect atrial tension development of electrically stimulated rat atria. Comparing these data with those of past literature, it is suggested that circulating endogenous opioids probably do not have any direct effects on the rat myocardium to affect myocardial contractility.     

Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans.

Noradrenaline (norepinephrine) was shown to be a potent inhibitor of glucose-induced insulin release from rat pancreatic islets, with half-maximal inhibition of the secretory response to 20 mM-glucose occurring at approx. 0.3 microM, and complete suppression of the response occurring at 4 microM-noradrenaline. Inhibition of insulin secretion by noradrenaline was antagonized by the alpha 2-adrenergic antagonist yohimbine (half maximally effective dose approximately 1 microM), but was largely unaffected by the alpha 1-adrenergic antagonist prazosin at concentrations up to 50 microM, suggesting that the response was mediated by alpha 2-adrenergic receptors. Noradrenaline significantly reduced the extent of 45Ca2+ accumulation in glucose-stimulated islets, although as much as 5 microM-noradrenaline was required for 50% inhibition of this response. The ability of noradrenaline to inhibit islet-cell 45Ca2+ uptake was totally abolished in media containing 1 mM-dibutyryl cyclic AMP, suggesting that the response may have been secondary to lowering of islet cyclic AMP. Under these conditions, however, noradrenaline was still able to inhibit insulin secretion maximally. The data suggest that the site(s) at which noradrenaline acts to mediate inhibition of insulin secretion in rat islets lies distal to both islet-cell cyclic AMP accumulation and Ca2+ uptake.     

Rapid modulation of adipocyte phosphofructokinase activity by noradrenaline and insulin.

1. Alterations in phosphofructokinase properties can be reproducibly seen in tissue extracts prepared and rapidly assayed after exposure of rat adipocytes to hormones. 2. Noradrenaline, corticotropin or isoprenaline (isoproterenol; beta-adrenergic agonist) decreased the activity measured with high fructose 6-phosphate concentrations (3--6 mM), but increased activity measured with lower concentrations of this substrate (0.3--0.9 mM). Noradrenaline decreased the Vmax. and the concentration of fructose 6-phosphate that gave half the Vmax.. 3. Insulin opposed the actions of noradrenaline and itself increased phosphofructokinase activity. 4. The effect of noradrenaline appeared to be exerted through a beta- rather than an alpha-type of adrenoceptor. 5. The effects of noradrenaline to decrease phosphofructokinase activity at high [fructose 6-phosphate] and to increase activity at low [fructose 6-phosphate] could be rapidly reversed in cells by addition of the beta-blocker propranolol. 6. The effect of noradrenaline seen at low [fructose 6-phosphate] could be abolished by homogenization of cells in buffer containing albumin or reversed by brief incubation of tissue extracts with albumin, suggesting that this effect of the hormone is due to the association of some ligand with the enzyme.     

Early circulatory and metabolic events in island skin flaps of the pig

Circulatory and metabolic skin-flap events were studied prior to and up to 6 hours after elevation of buttock island flaps in pigs. During the elevation, significant reductions in superficial skin blood flow, measured by laser Doppler flowmetry (LDF) and dermal flap temperature, were seen. Significant correlations were found between blood flow and temperature. Total flap blood flow, measured as venous outflow, also showed an initial transient decrease, but 2 hours after flap construction, venous outflow had returned to preoperative values. A significant increase in lactate release, together with increased oxygen consumption and glucose uptake, was seen 4 hours after the surgical intervention. Hypoxanthine release, indicating ischemia, was seen only during the first hour after flap elevation. Noradrenaline outflow was noted after 4 and 6 hours, but there was no parallel reduction in flap blood flow. A great deal of the flow reduction in acutely elevated island flaps may thus be due to primary hypothermia rather than to the degenerative release of noradrenaline, which seems to have no early effect on skin flap blood flow. On the other hand, the noradrenaline release may be linked to an increased metabolic activity in the skin flaps.     

Zonation of the metabolic action of vasopressin in the bivascularly perfused rat liver

Predominance of the vasopressin binding capacity in the hepatic perivenous area leads to the hypothesis that the metabolic effects of the hormone should also be more pronounced in this area. Until now this question has been approached solely by experiments with isolated hepatocytes where an apparent absence of metabolic zonation was found. We have reexamined this question using the bivascularly perfused liver. In this system periportal cells can be reached in a selective manner with substrates and effectors via the hepatic artery when retrograde perfusion (hepatic vein --> portal vein) is done. The action of vasopressin (1-10 nM) on glycogenolysis, initial calcium efflux, glycolysis and oxygen uptake were measured. The results revealed that the action of vasopressin in the liver is heterogeneously distributed. Glycogenolysis stimulation and initial calcium efflux were predominant in the perivenous area, irrespective of the vasopressin concentration. Oxygen uptake was stimulated in the perivenous area; in the periportal area it ranged from inhibition at low vasopressin concentrations to stimulation at high ones. Lactate production was generally greater in the perivenous zone, whereas the opposite occurred with pyruvate production. Analysis of these and other results suggests that at least three factors are contributing to the heterogenic response of the liver parenchyma to vasopressin: a) receptor density, which tends to favour the perivenous zone; b) cell-to-cell interactions, which tend to favour situations where the perivenous zone is amply supplied with vasopressin; and c) the different response capacities of perivenous and periportal cells.     

Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species

The effect of enzymatically generated reduced oxygen metabolites on the activity of hepatic microsomal glutathione S-transferase activity was studied to explore possible physiological regulatory mechanisms of the enzyme. Noradrenaline and the microsomal cytochrome P-450-dependent monooxygenase system were used to generate reduced oxygen species. When noradrenaline (greater than 0.1 mM) was incubated with rat liver microsomes in phosphate buffer (pH 7.4), an increase in microsomal glutathione S-transferase activity was observed, and this activation was potentiated in the presence of a NADPH-generating system; the glutathione S-transferase activity was increased to 180% of the control with 1 mM noradrenaline and to 400% with both noradrenaline and NADPH. Superoxide dismutase and catalase inhibited partially the noradrenaline-dependent activation of the enzyme. In the presence of dithiothreitol and glutathione, the activation of the glutathione S-transferase by noradrenaline, with or without NADPH, was not observed. In addition, the activation of glutathione S-transferase activity by noradrenaline and glutathione disulfide was not additive when both compounds were incubated together. These results indicate that the microsomal glutathione S-transferase is activated by reduced oxygen species, such as superoxide anion and hydrogen peroxide. Thus, metabolic processes that generate high concentrations of reduced oxygen species may activate the microsomal glutathione S-transferase, presumably by the oxidation of the sulfhydryl group of the enzyme, and this increased catalytic activity may help protect cells from oxidant-induced damage.     

Unspecific inhibition by the calmodulin antagonist calmidazolium and the intracellular calcium antagonist TMB-8 of the actions of sympathetic hepatic nerves and noradrenaline on glucose balance and flow in perfused rat liver

In perfused rat liver hepatic nerve stimulation (10 Hz, 2 ms) (NS) increased glucose and lactate output, decreased flow and was accompanied by an overflow of noradrenaline into the hepatic vein. These effects were dependent on extracellular and partly on intracellular calcium. Infusion of noradrenaline (1 microM) (NA) elicited similar effects. 1) Calmidazolium at 1, 2 and 5 microM caused an increase in basal glucose output and a decrease and intrahepatic redistribution of flow after a lag of 30, 20 and 5 min, respectively. 2) After 5 min of 1 microM calmidazolium, i.e. before it altered basal metabolism and flow, the actions of NS and NA remained unaltered. 3) After 40 min of 1 microM calmidazolium, i.e. after it had just begun to alter basal metabolism and flow, NS caused a decrease in glucose and lactate output rather than an increase and the metabolic effects of NA were strongly reduced whereas the hemodynamic changes of both stimuli were not altered. 4) TMB-8 at 25, 50 and 100 microM caused a transient increase in lactate output and a decrease and intrahepatic redistribution of flow after a lag of 5 min only at 100 microM concentrations. 5) The effects of NS were inhibited already by 25 microM TMB-8 which reduced NA release whereas the effects of NA were not influenced. Thus, calmidazolium and TMB-8 did not act as a calmodulin and intracellular calcium antagonist, respectively, but had unspecific "side effects" in the complex system of the perfused liver. The antagonists cannot be used to study the role of intracellular calcium in intact organs.     

Oxygen and temperature dependence of stimulated insulin secretion in isolated rat islets of Langerhans

The effects of lowered O2 tension on insulin secretion and changes in cellular energy parameters were investigated in isolated rat pancreatic islets perifused with buffers equilibrated with 21, 9, 5, and 1% oxygen and containing 5 mM glucose. Decreasing the external [O2] reduced the amount of insulin released in response to 16 mM glucose, 20 mM alpha-ketoisocaproic acid, and 40 mM KCl. Secretion elicited by high glucose or KCl had declined significantly at 9% oxygen, whereas that caused by alpha-ketoisocaproic acid became inhibited below 5% O2. Lowering the oxygen tension also decreased the ability of islets to respond with a rise in [ATP]/[ADP] upon stimulation with metabolic secretagogues. This reduction in the evoked increase in the nucleotide ratios paralleled the inhibition of stimulated insulin secretion. Addition of 2 mM amytal markedly decreased the islet energy level and eliminated the secretory response to 16 mM glucose. The results suggest that enhancement of B-cell energy production and a consequent rise in [ATP] (or [ATP]/[ADP]) are a necessary event for the hormone release elicited by high glucose and alpha-ketoisocaproic acid. A decrease in temperature inhibited insulin secretion with all three secretagogues tested. The energies of activation were similar for high glucose and KCl-induced secretion, about 20 kcal/mol, but were higher for alpha-ketoisocaproic acid, about 35 kcal/mol. At 28 degrees C, the [ATP]/[ADP] was larger than that at 38 degrees C (8 versus 5) and was not increased further upon addition of 16 mM glucose. It is suggested that a decrease in the rate of energy production at lowered temperatures may contribute to the inhibition of insulin release caused by metabolic secretagogues.     

The metabolism and binding of catecholamines by the hepatic microsomal mixed-function oxidase of the rat.

Noradrenaline and adrenaline were metabolized by an NADPH- and oxygen-dependent process located within the hepatic microsomal fraction of the rat. Metabolism was inhibited by CO and compound SKF 525A, but not by pargyline, an inhibitor of monoamine oxidase, or by 3,4-dimethoxy-5-hydroxybenzoic acid, an inhibitor of catechol O-methyltransferase. It is concluded that the enzyme system responsible for the metabolism of the catecholamines was the microsomal mixed-function oxidase. The Km for noradrenaline was 2.4 mM and for adrenaline 1.0 mM, and V 15.6 and 3.6 nmol/min per mg of microsomal protein respectively. Both catecholamines bound to the microsomal fraction, producing a type II spectral change, with a Ks for noradrenaline of 0.9 mM and for adrenaline of 1.0 mM, and showed other characteristics of type II compounds by inhibited the reduction of cytochrome P-450 by NADPH and exhibiting an enhanced metabolism in the presence of acetone. The major product of catecholamine metabolism was an as yet unidentified alkali-labile compound, which did not correspond to any of the recognized catecholamine metabolites.     

Stimulation of inositol phospholipid breakdown in pig brain miniprisms by carbachol and monoamines: effect of K+

1. The effects of carbachol, monoamines and K+ upon the rate of inositol phospholipid breakdown in pig brain miniprisms have been investigated. 2. In the striatum, carbachol (EC50 approx. 1 microM) and noradrenaline (EC50 approx. 25 microM) stimulated inositol phospholipid breakdown, whereas 5-hydroxytryptamine (1-1000 microM) was without effect. 3. The rate of inositol phospholipid breakdown was increased by raising the assay [K+] to greater than or equal to 40 mM. In the hippocampus and hypothalamus, a synergistic effect between K+ and carbachol was noted, whereas in the striatum, the effects were additive. 4. In striatal and hippocampal miniprisms, dopamine also increased inositol phospholipid breakdown, albeit only at high (greater than or equal to 1 mM) concentrations. Dopamine (1 mM) reduced the stimulation produced by noradrenaline (1 mM), suggesting that the effect of dopamine is due to a weak noradrenergic action of this catecholamine.     

Mechanism of action of sympathetic hepatic nerves on carbohydrate metabolism in perfused rat liver

In the perfused rat liver stimulation of the hepatic nerves around the portal vein and the hepatic artery was previously shown to increase glucose output, to shift lactate uptake to output, to decrease and re-distribute intrahepatic perfusion flow and to cause an overflow of noradrenaline into the hepatic vein. The metabolic effects could be caused directly via nerve hepatocyte contacts or indirectly by the hemodynamic changes and/or by noradrenaline overflow from the afferent vasculature into the sinusoids. Evidence against the indirect modes of nerve action is presented. Reduction of perfusion flow by lowering the perfusion pressure from 2 to 1 ml X min-1 X g-1--as after nerve stimulation--or to 0.35 ml X min-1 X g-1--far beyond the nerve stimulation-dependent effect--did not change glucose output and lowered lactate uptake only slightly. Only re-increase of flow to 2 ml X min-1 X g-1 enhanced glucose and lactate release transiently due to washout of glucose and lactate accumulated in parenchymal areas not perfused during low perfusion flow. In chemically sympathectomized livers nerve stimulation decreased perfusion flow almost normally but without changing the intrahepatic microcirculation; yet it enhanced glucose and lactate output only insignificantly and caused noradrenaline overflow of less than 10% of normal. Conversely, in the presence of nitroprussiate (III) nerve stimulation reduced overall flow only slightly without intrahepatic redistribution but still increased glucose and lactate output strongly and caused normal noradrenaline overflow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatocyte heterogeneity in glutamate metabolism and bidirectional transport in perfused rat liver

1. The metabolic fate of infused [1-14C]glutamate was studied in perfused rat liver. The 14C label taken up by the liver was recovered to 85 +/- 2% as 14CO2 and [14C]glutamine. Whereas 14CO2 production accounted for about 70% of the [1-14C]glutamate taken up under conditions of low endogenous rates of glutamine synthesis, stepwise stimulation of glutamine synthesis by NH4Cl increased 14C incorporation into glutamine at the expense of 14CO2 production. Extrapolation to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of vascular glutamate taken up by the liver for glutamine synthesis. This was observed in both, antegrade and retrograde perfusions and suggests an almost exclusive uptake of glutamate into perivenous glutamine-synthetase-containing hepatocytes. 2. Glutamate was simultaneously taken up and released from perfused rat liver. At a near-physiological influent glutamate concentration (0.1 mM), the rates of unidirectional glutamate influx and efflux were similar (about 100 and 120 nmol g-1 min-1, respectively). 3. During infusion of [1-14C]oxoglutarate (50 microM), addition of glutamate (2 mM) did not affect hepatic uptake of [1-14C]oxoglutarate. However, it increased labeled glutamate release from the liver about 10-fold (from 9 +/- 2 to 86 +/- 20 nmol g-1 min-1; n = 4), whereas 14CO2 production from labeled oxoglutarate decreased by about 40%. This suggests not only different mechanisms of oxoglutarate and glutamate transport across the plasma membrane, but also points to a glutamate/glutamate exchange. 4. Oxoglutarate was recently shown to be taken up almost exclusively by perivenous glutamine-synthetase-containing hepatocytes [Stoll, B & H?ussinger, D. (1989) Eur. J. Biochem. 181, 709-716] and [1-14C]oxoglutarate (9 microM) was used to label selectively the intracellular glutamate pool in this perivenous cell population. The specific radioactivity of this intracellular (perivenous) glutamate pool was assessed by measuring the specific radioactivity of newly synthesized glutamine which is continuously released from these cells into the perfusate. Comparison of the specific radioactivities of glutamine and glutamate released from perivenous cells indicates that about 60% of total glutamate release from the liver is derived from the perivenous glutamine-synthetase-containing cell population. Following addition of unlabeled glutamate (0.1 mM), unidirectional glutamate efflux from perivenous cells increased from about 30 to 80 nmol g-1 min-1, whereas glutamate efflux from non-perivenous (presumably periportal) hepatocytes remained largely unaltered (i.e. 20-30 nmol g-1 min-1). 5. It is concluded that, in the intact liver, vascular glutamate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase.     

Atrial supersensitivity to noradrenaline in stressed female rats

Stress can change the responses to catecholamines in many tissues. The aim of this study was to investigate the influence of the estrous cycle on the sensitivity of right atria to noradrenaline in female rats subjected to acute swimming stress. Female Wistar rats in proestrus, estrus, metestrus or diestrus were submitted to a 50 min-swimming session. Immediately after the exercise, the rats were killed and their right atria were mounted for isometric recording of the spontaneous beating rate. Concentration-effect curves to noradrenaline were obtained before and after the inhibition of neuronal uptake with phenoxybenzamine (10 microM) and of extraneuronal uptake with estradiol (5 microM). Acute swimming stress did not change the right atrial sensitivity to noradrenaline in rats in estrus, metestrus and diestrus. However, swimming stress produced supersensitivity to noradrenaline in proestrus (pD(2) control: 7.14 +/- 0.03 vs. pD(2) swimming: 7.55 +/- 0.04; p<0.05). This supersensitivity was still observed after uptake inhibition. When catecholamine uptake was inhibited, the concentration-effect curve to noradrenaline was shifted to the left 2.5-fold in the proestrus control group and 1.7-fold in the proestrus stress group (p<0.05). In conclusion, the estrous cycle influenced the acute stress-induced atrial supersensitivity to noradrenaline.