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.     

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.     

Differential control of glycogenolysis and flow by arterial and portal acetylcholine in perfused rat liver.

The effects of acetylcholine on glucose and lactate balance and on perfusion flow were studied in isolated rat livers perfused simultaneously via the hepatic artery (100 mmHg, 25-35% of flow) and the portal vein (10 mmHg, 75-65% of flow) with a Krebs-Henseleit bicarbonate buffer containing 5 mM-glucose, 2 mM-lactate and 0.2 mM-pyruvate. Arterial acetylcholine (10 microM sinusoidal concentration) caused an increase in glucose and lactate output and a slight decrease in arterial and portal flow. These effects were accompanied by an output of noradrenaline and adrenaline into the hepatic vein. Portal acetylcholine elicited only minor increases in glucose and lactate output, a slight decrease in portal flow and a small increase in arterial flow, and no noradrenaline and adrenaline release. The metabolic and haemodynamic effects of arterial acetylcholine and the output of noradrenaline and adrenaline were strongly inhibited by the muscarinic antagonist atropine (10 microM). The acetylcholine-dependent alterations of metabolism and the output of noradrenaline were not influenced by the alpha 1-blocker prazosin (5 microM), whereas the output of adrenaline was increased. The acetylcholine-dependent metabolic alterations were not inhibited by the beta 2-antagonist butoxamine (10 microM), although the overflow of noradrenaline was nearly completely blocked and the output of adrenaline was slightly decreased. These results allow the conclusion that arterial, but not portal, acetylcholine caused sympathomimetic metabolic effects, without noradrenaline or adrenaline being involved in signal transduction.     

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.     

Eicosanoid-mediated increase in glucose and lactate output as well as decrease and redistribution of flow by complement-activated rat serum in perfused rat liver

Rat serum, in which the complement system had been activated by incubation with zymosan, increased the glucose and lactate output, and reduced and redistributed the flow in isolated perfused rat liver clearly more than the control serum. Heat inactivation of the rat serum prior to zymosan incubation abolished this difference. Metabolic and hemodynamic alterations caused by the activated serum were dose dependent. They were almost completely inhibited by the cyclooxygenase inhibitor indomethacin and by the thromboxane antagonist 4-[2-(4-chlorobenzesulfonamide)-ethyl]-benzene-acetic acid (BM 13505), but clearly less efficiently by the 5'-lipoxygenase inhibitor nordihydroguaiaretic acid and the leukotriene antagonist N-(3-[3-(4-acetyl-3-hydroxy-2-propyl-phenoxy)-propoxy]-4-chlorine-6-meth yl- phenyl)-1H-tetrazole-5-carboxamide sodium salt (CGP 35949 B). Control serum and to a much larger extent complement-activated serum, caused an overflow of thromboxane B2 and prostaglandin F2 alpha into the hepatic vein. It is concluded that the activated complement system of rat serum can influence liver metabolism and hemodynamics via release from nonparenchymal liver cells of thromboxane and prostaglandins, the latter of which can in turn act on the parenchymal cells.     

Different accessibility from the artery and the portal vein of alpha- and beta-receptors involved in the sympathetic nerve action on glycogenolysis and hemodynamics in perfused rat liver

In perfused rat liver perivascular nerve stimulation (7.5 Hz, 20 V, 2 ms, 5 min) at the liver hilus caused an increase in glucose and lactate output and a decrease in flow. The influence of the alpha 1-receptor blocker prazosine and the beta-blocker propranolol on these nerve effects was studied in the isolated rat liver perfused classically via the portal vein only and, as developed recently, via both the hepatic artery and the portal vein. 1) In livers perfused via the portal vein only the nerve stimulation-dependent metabolic alterations were nearly completely inhibited by prazosine (5 microM), but not influenced by propranolol (10 microM). The hemodynamic changes were lowered to only 33% by prazosine and not altered by propranolol either. 2) In livers perfused via the hepatic artery (100 mm Hg, 20-40% of flow) and the portal vein (10 mm Hg, 80-60% of flow)--similar to portal perfusions--the nerve stimulation--dependent metabolic alterations were almost completely blocked by arterial, portal or simultaneously applied arterial and portal prazosine. However--in contrast to portal perfusions--the metabolic alterations were reduced to about 20% (glucose) and 50% (lactate) also by propranolol independently of its site of application. The decrease in flow was reduced by prazosine to about 60%, 50% and 30% when applied via the artery, the portal vein or via both vessels, respectively. The hemodynamic alterations were not influenced by propranolol. These results allow the following conclusions: A subpopulation of beta-receptors can play a permissive role in the alpha 1-receptor-mediated sympathetic nerve action on glucose and lactate metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Increase in glucose and lactate output and perfusion resistance by stimulation of hepatic nerves in isolated perfused rat liver: role of alpha 1-, alpha 2-, beta 1- and beta 2-receptors.

Rat liver was perfused in situ via the portal vein without recirculation: 1) Electrical stimulation of the nerve bundles around hepatic artery and portal vein increased glucose and lactate output, reduced flow and caused an overflow of noradrenaline into the hepatic vein. The alpha-agonist phenylephrine also augmented glucose and lactate output and lowered flow with an ED50 of about 1 microM, while the beta-agonist isoproterenol increased glucose output but reduced lactate output with an ED50 of about 0.2 microM and left flow unaltered. 2) The alpha 1-receptor antagonist prazosin (KI at alpha 1-sites approximately 1 nM, at alpha 2-sites approximately 100 nM) inhibited the nerve stimulation-dependent increase in glucose and lactate output and reduction of flow with an ID50 of about 1 nM, while the alpha 2-receptor antagonist yohimbine (KI at alpha 2-sites approximately 10 nM, at alpha 1-sites approximately 1500 nM) was inhibitory only with an ID50 of about 400 nM. 10 nM prazosin clearly reduced the nerve actions, completely blocked the effects of 1 microM phenylephrine and left the effects of 0.2 microM isoproterenol unaltered. 10 nM yohimbine did not affect the nerve actions nor the effects of phenylephrine or isoproterenol. 3) The beta 1-receptor antagonist metoprolol (KI at beta 1-sites approximately 100 nM, at beta 2-sites approximately 1.2 microM) at 10 microM concentrations did not interfere with the nerve stimulation-dependent increase in glucose and lactate output or the decrease in flow. It did not have any specific alpha-antagonistic influence either on the changes brought about by 1 microM phenylephrine; however, it blocked the beta 2-mediated increase in glucose output by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leukotrienes increase glucose and lactate output and decrease flow in perfused rat liver

In isolated perfused rat liver leukotriene C4 and D4 but not B4 and E4 enhanced glucose and lactate output and lowered perfusion flow similar to the thromboxane A2 analogue U46619, extracellular ATP and prostaglandin F2 alpha. The kinetics of the metabolic changes caused by leukotriene C4 and D4 resembled those effected by U46619 and ATP but not those elicited by prostaglandin F2 alpha; the kinetics of the hemodynamic changes were similar only to those caused by U46619. The results show that leukotrienes could be important modulators of hepatic metabolism and hemodynamics and point to a complex intra-organ cell-cell communication between non-parenchymal and parenchymal cells.     

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.     

Receptor subtype mediating the action of circulating endothelin on glucose metabolism and hemodynamics in perfused rat liver.

The subtype of endothelin receptor that mediates metabolic and hemodynamic effects of circulating endothelin was explored using perfused rat liver. Infusion of endothelin (ET)-1 or ET-3 into the portal vein at a concentration of 0.3 nM increased glucose and lactate output and decreased perfusion flow, although ET-3 was less effective than ET-1. The metabolic effects of ET-1 were observed even under costant-flow perfusion. Infusion of either sarafotoxin S6b or S6c, an ET(A)- or ET(B)-receptor agonist, mimicked the actions of ET-1 to an equal extent. The flow reduction and glucose production induced by ET-1 were partly attenuated by the ET(A)-receptor antagonist BQ485. By contrast, ET(B)-receptor antagonist BQ788 enhanced glucose production caused by ET-1 and ET-3 without affecting the hemodynamic change. The effects of ET-1 and ET-3 were almost totally inhibited by the combination of BQ485 and BQ788. These results suggest that both ET(A) and ET(B) receptors are involved in the metabolic and hemodynamic effects of circulating endothelin in rat liver, while the ET(A)-receptor-mediated action appears to be dominant.     

Potential role for prostaglandin F2 alpha, D2, E2 and thromboxane A2 in mediating the metabolic and hemodynamic actions of sympathetic nerves in perfused rat liver

In isolated rat liver perfused at constant pressure perivascular nerve stimulation caused an increase of glucose and lactate output and a reduction of perfusion flow. The metabolic and hemodynamic nerve effects could be inhibited by inhibitors of prostanoid synthesis, which led to the suggestion that the effects of nerve stimulation were, at least partially, mediated by prostanoids [Iwai, M. & Jungermann, K. (1987) FEBS Lett. 221, 155-160]. This suggestion is corroborated by the present study. 1. Prostaglandin D2, E2 and F2 alpha as well as the thromboxane A2 analogue U46619 enhanced glucose and lactate release and lowered perfusion flow similar to nerve stimulation. 2. The extents, the kinetics and the concentration dependencies of the metabolic and hemodynamic actions of the various prostanoids were different. Prostaglandin F2 alpha and D2 caused relatively stronger changes of metabolism, while prostaglandin E2 and U46619 had stronger effects on hemodynamics. Prostaglandin F2 alpha elicited greater maximal alterations than D2 with similar half-maximally effective concentrations. Prostaglandin F2 alpha mimicked the nerve actions on both metabolism and hemodynamics best with respect to the relative extents and the kinetics of the alterations. 3. The hemodynamic effects of prostaglandin F2 alpha could be prevented completely by the calcium antagonist nifedipine without impairing the metabolic actions of the prostanoid. Apparently, prostaglandin F2 alpha influenced metabolism directly rather than indirectly via hemodynamic changes. The present results, together with the previously described effects of prostanoid synthesis inhibitors, suggest that prostanoids, probably prostaglandin F2 alpha and/or D2, could be involved in the actions of sympathetic hepatic nerves on liver carbohydrate metabolism. Since prostanoids are synthesized only in non-parenchymal cells, nervous control of metabolism appears to depend on complex intra-organ cell-cell interactions between the nerve, non-parenchymal and parenchymal cells.     

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)     

Activation of glycogenolysis by stimulation of the hepatic nerves in perfused livers of guinea pig and tree shrew as compared to rat: differences in the mode of action

A study on the metabolic and hemodynamic actions of hepatic nerve stimulation in the perfused liver of guinea pig and tree shrew as compared to rat was performed, since the density of liver innervation was reported to be different. 1) Nerve stimulation resulted in an increase in glucose release and decrease in lactate uptake or in a shift to output as well as a decrease in portal flow in all three species. The change in glucose output was very similar, that in lactate balance and flow was smaller in tree shrew than in guinea pig and rat. Apparently, the metabolic and hemodynamic changes did not reflect the different densities of liver innervation. 2) The overflow of the neurotransmitter noradrenaline into the hepatic vein differed very clearly in the three animals. In the guinea pig and tree shrew the maximal increase in noradrenaline concentration measured in the effluent was about 6-7-fold higher than in the rat. 3) The content of noradrenaline in the liver in vivo was about five-fold higher in the guinea pig and again another four-fold higher in the tree shrew than in the rat. The contents of adrenaline and dopamine were very low in comparison to those of noradrenaline. The different hepatic noradrenaline contents of the three species investigated are in line with the anatomical findings on the different innervation density. 4) Inhibitors of eicosanoid synthesis reduced the nerve stimulation-dependent metabolic and hemodynamic alterations in guinea pig liver as in rat liver indicating a similar mechanism in these species. Apparently, prostaglandins might be involved as mediators or modulators of nerve actions also in the more densely innervated guinea pig liver and not only in the less densely innervated rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Signal propagation via gap junctions, a key step in the regulation of liver metabolism by the sympathetic hepatic nerves.

Cell-to-cell communication via gap junctions has been proposed to be involved in the metabolic actions of sympathetic liver nerves in the rat. The effects of hepatic nerve stimulation and noradrenaline-, PGF2 alpha- and glucagon infusion on glucose metabolism and perfusion flow were studied in perfused rat liver in the absence and presence of the gap junctional inhibitors, heptanol, carbenoxolone and (4 beta)phorbol 12-myristate 13-acetate (4 beta PMA). (i) Stimulation of the hepatic nerve plexus increased glucose output, decreased flow and caused an overflow of noradrenaline into the hepatic vein. (ii) Heptanol completely inhibited not only the nerve stimulation-dependent metabolic and hemodynamic alterations but also the noradrenaline overflow. Thus the heptanol-dependent inhibitions were caused primarily by a strong impairment of transmitter release. (iii) Carbenoxolone inhibited the effects of neurostimulation on glucose metabolism partially by about 50%, whereas it left perfusion flow and noradrenaline overflow essentially unaltered. (iv) 4 beta PMA reduced the nerve stimulation-dependent enhancement of glucose release by about 80% but the noradrenaline-dependent increase in glucose output only by about 30%; the increase in glucose release by PGF2 alpha and by glucagon remained essentially unaltered. 4 beta PMA reduced the nerve stimulation-dependent decrease in portal flow by about 35% but did not affect the noradrenaline-and PGF2 alpha-elicited alterations, nor did it alter noradrenaline overflow. The results allow the conclusion that gap junctional communication plays a major role in the regulation of hepatic carbohydrate metabolism by sympathetic liver nerves, but not by circulating noradrenaline, PGF2 alpha or glucagon.     

Control of glycogenolysis and hemodynamics in perfused rat liver by the sympathetic innervation. Dependence on stimulation frequency and duration

Perivascular stimulation of the hepatic nerves in the in situ perfused rat liver with a constant frequency of 20 Hz over a constant period of 5 min had previously been shown to cause an increase of glucose output, a shift from lactate uptake to release, a reduction in perfusion flow (Hartmann et al. (1982) Eur. J. Biochem. 123, 521-526) and an overflow of noradrenaline into the hepatic vein (Beckh et al. (1982) FEBS Lett. 149, 261-265). In the present study the dependence of the metabolic and hemodynamic effects on the frequency between 1 and 30 Hz and duration of stimulation between 0.5 and 5 min was investigated. Over a constant stimulation period of 5 min the alteration in glucose exchange was maximal with a frequency of 10 Hz and half-maximal with 4 Hz. The corresponding values for the exchange of lactate were 5 Hz and 2 Hz, respectively, and for the perfusion flow 2.5 Hz and 1.5 Hz, respectively. An increase of noradrenaline overflow was not observed with the lower frequencies of 1 and 2.5 Hz; it was maximal at 10 Hz and half-maximal at 6.5 Hz. At a constant frequency of 20 Hz the increase in glucose release was maximal with a total stimulation period of 1 min and half-maximal with a period of 0.4 min. An essentially maximal alteration of lactate exchange and perfusion flow as well as of noradrenaline overflow was also effected by a stimulation period of 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of inositol phosphate formation by circulating noradrenaline but not by sympathetic nerve stimulation with a similar increase of glucose release in perfused rat liver

In the isolated rat liver perfused in situ, stimulation of the nerve bundles around the hepatic artery and portal vein caused an increase of glucose and lactate output and a reduction of perfusion flow. These changes could be inhibited completely by alpha-receptor blockers. The possible involvement of inositol phosphates in the intracellular signal transmission was studied. 1. In cell-suspension experiments, which were performed as a positive control, noradrenaline caused an increase in glucose output and, in the presence of 10 mM LiCl, a dose-dependent and time-dependent increase of inositol mono, bis and trisphosphate. 2. In the perfused rat liver 1 microM noradrenaline caused an increase of glucose and lactate output and in the presence of 10 mM LiCl a time-dependent increase of inositol mono, bis and trisphosphate that was comparable to that observed in cell suspensions. 3. In the perfused rat liver stimulation of the nerve bundles around the portal vein and hepatic artery caused a similar increase in glucose and lactate output to that produced by noradrenaline, but in the presence of 10 mM LiCl there was a smaller increase of inositol monophosphate and no increase of inositol bis and trisphosphate. These findings are in line with the proposal that circulating noradrenaline reaches every hepatocyte, causing a clear overall increase of inositol phosphate formation and thus calcium release from the endoplasmic reticulum, while the hepatic nerves reach only a few cells causing there a small local change of inositol phosphate metabolism and thence a propagation of the signal via gap junctions.     

Direct control of glycogen metabolism in the perfused rat liver by the sympathetic innervation

The mode of action of hepatic nerves on the metabolism of carbohydrates was studied in the rat liver perfused in situ. 1. Electrical stimulation of the nerve bundles around the hepatic artery and the portal vein resulted in an increase of glucose and lactate output, an enhancement of phosphorylase a activity and a decrease of portal flow. 2. Sodium nitroprusside prevented the hemodynamic changes after nerve stimulation without affecting the metabolic alterations. 3. Phentolamine or an extracellular calcium level below 300 mumol x 1(-1) abolished both hemodynamic and metabolic changes after nerve stimulation, while propranolol or atropine were without effect. 4. Norepinephrine infusion mimicked nerve stimulation only at the highly unphysiological concentration of 0.1 microM; it was not effective at a concentration of 0.01 microM, which might be reached in the sinusoidal blood due to an overflow from intrahepatic synapses. The present results suggest that, in rat liver, glycogen breakdown is regulated by alpha-sympathetic nerves directly rather than indirectly via hemodynamic changes or via norepinephrine overflow.     

Profiling substrate fluxes in the isolated working mouse heart using 13C-labeled substrates: focusing on the origin and fate of pyruvate and citrate carbons

The availability of genetically modified mice requires the development of methods to assess heart function and metabolism in the intact beating organ. With the use of radioactive substrates and ex vivo perfusion of the mouse heart in the working mode, previous studies have documented glucose and fatty acid oxidation pathways. This study was aimed at characterizing the metabolism of other potentially important exogenous carbohydrate sources, namely, lactate and pyruvate. This was achieved by using (13)C-labeling methods. The mouse heart perfusion setup and buffer composition were optimized to reproduce conditions close to the in vivo milieu in terms of workload, cardiac functions, and substrate-hormone supply to the heart (11 mM glucose, 0.8 nM insulin, 50 microM carnitine, 1.5 mM lactate, 0.2 mM pyruvate, 5 nM epinephrine, 0.7 mM oleate, and 3% albumin). The use of three differentially (13)C-labeled carbohydrates and a (13)C-labeled long-chain fatty acid allowed the quantitative assessment of the metabolic origin and fate of tissue pyruvate as well as the relative contribution of substrates feeding acetyl-CoA (pyruvate and fatty acids) and oxaloacetate (pyruvate) for mitochondrial citrate synthesis. Beyond concurring with the notion that the mouse heart preferentially uses fatty acids for energy production (63.5 +/- 3.9%) and regulates its fuel selection according to the Randle cycle, our study reports for the first time in the mouse heart the following findings. First, exogenous lactate is the major carbohydrate contributing to pyruvate formation (42.0 +/- 2.3%). Second, lactate and pyruvate are constantly being taken up and released by the heart, supporting the concept of compartmentation of lactate and glucose metabolism. Finally, mitochondrial anaplerotic pyruvate carboxylation and citrate efflux represent 4.9 +/- 1.8 and 0.8 +/- 0.1%, respectively, of the citric acid cycle flux and are modulated by substrate supply. The described (13)C-labeling strategy combined with an experimental setup that enables continuous monitoring of physiological parameters offers a unique model to clarify the link between metabolic alterations, cardiac dysfunction, and disease development.     

Chronotropic response to (+/-)-CGP12177 in right atria of stressed rats

Foot-shock stress changes the sensitivity of the rat right atria to beta1- and beta2-adrenoceptor (AR) agonists. We investigated whether the same stress protocol also changes the atrial sensitivity to the non conventional agonist, (+/-)-CGP12177. Concentration-response curves to (+/-)-CGP12177, a beta1- and beta2-adrenoceptor antagonist with agonist properties at the putative beta4-adrenoceptors, were obtained in the absence and presence of propranolol (200 nM or 2 microM), CGP20712A 10 nM plus ICI118,551 50 nM, or CGP20712A (1 microM or 3 microM), in right atria from rats submitted to three daily foot-shock sessions (120 mA pulses of 1.0 s duration applied at random intervals of 5-25 s over 30 min) and killed after the third session. The pD2 for (+/-)-CGP12177 was not influenced by foot-shock stress. The stimulant effect of (+/-)-CGP12177 was resistant to blockade by 200 nM and 2 microM (+/-)-propranolol, and to combined blockade by CGP20712A and IC1118,551. However, in right atria from stressed rats given 200 nM propranolol, the concentration-response curve to the agonist was shifted 2.0-fold to the right. CGP20712A shifted the concentration-response curve to (+/-)-CGP12177 to the right by 4.6- (1 microM) and 19-fold (3 microM) in atria of control rats, and by 2.2- (1 microM) and 43-fold (3 microM) in atria of stressed rats. Maximum response to CGP12177 was not affected by propranolol or CGP20712A in concentrations ranging from 0.1 nM to 10 microM. These results show that the chronotropic effect of (+/-)-CGP12177 is mediated by atypical beta4-adrenoceptors. In constrast with to beta1-and (or) beta2-AR, this receptor is resistant to the effects of foot-shock stress, suggesting that the putative beta4-AR is a different receptor from a low affinity state of beta1-adrenoceptor, as previously proposed, unless both proposed isoforms of beta1-adrenoceptor show independent stress-induced behavior.