Stimulation by alpha-adrenergic agonists of Ca2+ fluxes, mitochondrial oxidation and gluconeogenesis in perfused rat liver.

Glucose output from perfused livers of 48 h-starved rats was stimulated by phenylephrine (2 microM) when lactate, pyruvate, alanine, glycerol, sorbitol, dihydroxyacetone or fructose were used as gluconeogenic precursors. Phenylephrine-induced increases in glucose output were immediately preceded by a transient efflux of Ca2+ and a sustained increase in oxygen uptake. Phenylephrine decreased the perfusate [lactate]/[pyruvate] ratio when sorbitol or glycerol was present, but increased the ratio when alanine, dihydroxyacetone or fructose was present. Phenylephrine induced a rapid increase in the perfusate [beta-hydroxybutyrate]/[acetoacetate] ratio and increased total ketone-body output by 40-50% with all substrates. The oxidation of [1-14C]octanoate or 2-oxo[1-14C]glutarate to 14CO2 was increased by up to 200% by phenylephrine. All responses to phenylephrine infusion were diminished after depletion of the hepatic alpha-agonist-sensitive pool of Ca2+ and returned toward maximal responses after Ca2+ re-addition. Phenylephrine-induced increases in glucose output from lactate, sorbitol and glycerol were inhibited by the transaminase inhibitor amino-oxyacetate by 95%, 75% and 66% respectively. Data presented suggest that the mobilization of an intracellular pool of Ca2+ is involved in the activation of gluconeogenesis by alpha-adrenergic agonists in perfused rat liver. alpha-Adrenergic activation of gluconeogenesis is apparently accompanied by increases in fatty acid oxidation and tricarboxylic acid-cycle flux. An enhanced transfer of reducing equivalents from the cytoplasmic to the mitochondrial compartment may also be involved in the stimulation of glucose output from the relatively reduced substrates glycerol and sorbitol and may arise principally from an increased flux through the malate-aspartate shuttle.     

The Ca2+-dependent actions of the alpha-adrenergic agonist phenylephrine on hepatic glycogenolysis differ from those of vasopressin and angiotensin

The stimulation of hepatic glycogenolysis by the Ca2+-dependent hormones phenylephrine, vasopressin and angiotensin II was studied as a function of intracellular and extracellular Ca2+. In the isolated perfused rat liver the decline in glucose formation was monophasic ('half-life' approximately equal to 3 min) with vasopressin (1 nM) or angiotensin II (0.05 microM), but biphasic (half-life of 4.8 min and 17.6 min) in the presence of the alpha-agonist phenylephrine (0.01 mM), indicating either a different mode of mobilization or the mobilization of additional intracellular calcium stores. Under comparable conditions an elevated [Ca2+] level was maintained in the cytosol of hepatocytes for at least 10 min in the presence of phenylephrine, but not vasopressin. Titration experiments performed in the isolated perfused liver to restore cellular calcium revealed differences in the hormone-mediated uptake of Ca2+. The onset in glucose formation above that seen in the absence of exogenous calcium occurred at approximately 30 microM or 70-80 microM Ca2+ in the presence of phenylephrine or vasopressin respectively. The shape of the response curve was sigmoidal for vasopressin and angiotensin II, but showed a distinct plateau between 0.09 mM and 0.18 mM in the presence of phenylephrine. The plateau was also observed at phenylephrine concentrations as low as 0.5 microM. The formation of plateaus observed after treatment of the liver with A 23187, but not after EGTA, is taken as an indication that intracellular calcium stores are replenished. A participation of the mitochondrial compartment could be excluded by pretreatment of the liver with the uncoupler 2,4-dinitrophenol. Differences in the Ca2+ dependence of the glycogenolytic effects of these hormones were also revealed by kinetic analysis. It is concluded that phenylephrine differs from vasopressin and angiotensin II in that, in addition to a more common, non-mitochondrial pool, which is also responsive to the vasoactive peptides, the agonist mobilizes Ca2+ from a second, non-mitochondrial pool. The results are consistent with the proposal that Ca2+ transport across subcellular membranes may be subject to different hormonal control.     

Thyroid hormone and dehydroepiandrosterone permit gluconeogenic hormone responses in hepatocytes

The importance of the sn-glycerol- 3-phosphate (G-3-P) electron transfer shuttle in hormonal regulation of gluconeogenesis was examined in hepatocytes from rats with decreased mitochondrial G-3-P dehydrogenase activity (thyroidectomized) or increased G-3-P dehydrogenase activity [triiodothyronine (T(3)) or dehydroepiandrosterone (DHEA) treated]. Rates of glucose formation from 10 mM lactate, 10 mM pyruvate, or 2.5 mM dihydroxyacetone were somewhat less in hypothyroid cells than in cells from normal rats but gluconeogenic responses to calcium addition and to norepinephrine (NE), glucagon (G), or vasopressin (VP) were similar to the responses observed in cells from normal rats. However, with 2. 5 mM glycerol or 2.5 mM sorbitol, substrates that must be oxidized in the cytosol before conversion to glucose, basal gluconeogenesis was not appreciably altered by hypothyroidism but responses to calcium and to the calcium-mobilizing hormones were abolished. Injecting thyroidectomized rats with T(3) 2 days before preparing the hepatocytes greatly enhanced gluconeogenesis from glyc erol and restored the response to Ca(2+) and gluconeogenic hormones. Feeding dehydroepiandrosterone for 6 days depressed gluconeogenesis from lactate or pyruvate but substantially increased glucose production from glycerol in euthyroid cells and restored responses to Ca(2+) in hypothyroid cells metabolizing glycerol. Euthyroid cells metabolizing glycerol or sorbitol use the G-3-P and malate/aspartate shuttles to oxidize excess NADH generated in the cytosol. The transaminase inhibitor aminooxyacetate (AOA) decreased gluconeogenesis from glycerol 40%, but had little effect on responses to Ca(2+) and NE. However, in hypothyroid cells, with minimal G-3-P dehydrogenase, AOA decreased gluconeogenesis from glycerol more than 90%. Thus, the basal rate of gluconeogenesis from glycerol in the euthyroid cells is only partly dependent on electron transport from cytosol to mitochondria via the malate/aspartate shuttle and almost completely dependent in the hypothyroid state, and the hormone enhancement of the rate in euthyroid cells involves primarily the G-3-P cycle. These data are consistent with Ca(2+) being mobilized by gluconeogenic hormones and G-3-P dehydrogenase being activated by Ca(2+) so as to permit it to transfer reducing equivalents from the cytosol to the mitochondria.     

Regulation of gluconeogenesis by norepinephrine, vasopressin, and angiotensin II: a comparative study in the absence and presence of extracellular Ca2+1

In hepatocytes isolated from fasted rats, vasopressin and angiotensin II stimulate the rate of gluconeogenesis from lactate or pyruvate in a Ca2+-dependent manner similar to that previously reported for norepinephrine. Actions of the peptide hormones on gluconeogenesis from glycerol or sorbitol, reduced substrates that require oxidation before they enter the gluconeogenic pathway at triosephosphate, also resemble those of norepinephrine. Stimulation of glucose production from these substrates is observed only in the presence of extracellular Ca2+. Actions of the peptide hormones on gluconeogenesis from dihydroxyacetone or fructose, the oxidized counterparts of glycerol and sorbitol, respectively, do not resemble those of norepinephrine. While norepinephrine enhances rates of glucose production from dihydroxyacetone or fructose in the absence of extracellular Ca2+, vasopressin and angiotensin II are ineffective either in the absence or presence of extracellular Ca2+. When the oxidation-reduction state in hepatocytes metabolizing dihydroxyacetone is altered by adding an equimolar concentration of ethanol (to provide cytosolic reducing equivalents), the results are similar to those obtained when cells are incubated with the reduced counterpart of dihydroxyacetone, glycerol, i.e., the peptide hormones cause an apparent increase in the rate of glucose production in a Ca2+-dependent manner. If, on the other hand, hepatocytes are incubated with glycerol or sorbitol and an equimolar concentration of pyruvate (to provide a cytosolic hydrogen acceptor), the peptide hormones, unlike norepinephrine, are ineffective in stimulating gluconeogenesis in the absence of extracellular Ca2+. These results indicate that whereas many of the actions of vasopressin and angiotensin II are similar to those of alpha 1-adrenergic agents, there are major differences in the manner in which the hormones act at various sites to regulate gluconeogenesis.     

Suppression of the mitochondrial oxidation of (-)-palmitylcarnitine by the malate-aspartate and alpha-glycerophosphate shuttles.

Palmitylcarnitine oxidation by isolated liver mitochondria has been used to investigate the interaction of fatty acid oxidation with malate, glutamate, succinate, and the malate-aspartate shuttle. Mitochondria preincubated with fluorocitrate were added to a medium containing 2mM ATP and ATPase. This system, characterized by a high energy change, allowed titration of respiration to any desired rate between States 4 and 3 (Chance, B., and Williams, G. R. (1956) Adv. Enzymol. Relat. Areas Mol. Biol. 17, 65-134). When respiration (reference, with palmitylcarnitine and malate as substrates) was set at 75% of State 3, the oxidation of palmitylcarnitine was limited by acetoacetate formation. The addition of malate or glutamate approximately doubled the rate of beta oxidation. Malate circumvented this limitation by citrate formation, but the effect of glutamate apparently was due to enhancement of the capacity for ketogenesis. The rate of beta oxidation was curtailed when malate and glutamate were both present. This curtailment was more pronounced when the malate-aspartate shuttle was fully reconstituted. Among the oxidizable substrates examined, succinate was most effective in inhibiting palmitylcarnitine oxidation. Mitochondrial NADH/NAD+ ratios were correlated positively with suppression of beta oxidation. The degree of suppression of beta oxidation by the malate-aspartate shuttle (NADH oxidation) or by succinate oxidation was dependent on the respiratory state. Both substrates extensively reduced mitochondrial NAD+ and markedly suppressed beta oxidation as respiration approached State 4. Calculations of the rates of flux of hydrogen equivalents through beta oxidation show that the suppression of beta oxidation by glutamate or by the malate-aspartate shuttle is accounted for by increased flux of reducing equivalents through mitochondrial malic dehydrogenase. This increased Flux is accompanied by an increase in the steady state NADH/NAD+ ratio and a marked decrease in the synthesis of citrate. The alpha-glycerophosphate shuttle was reconstituted with mitochondria isolated from rats treated with L-thyroxine. This shuttle was about equal to the reconstructed malate-aspartate shuttle in supression of palmitylcarnitine oxidation. This interaction could not be demonstrated in euthyroid animals owing to the low activity of the mitochondrial alpha-glycerol phosphate dehydrogenase. It is concluded that beta oxidation can be regulated by the NADH/NAD+ ratio. The observed stimulation of flux through malate dehydrogenase both by glutamate and by the malate-aspartate shuttle results in an increased steady state NADH/NAD+ ratio, and is linked to a stoichiometric outward transport of aspartate. We suggest, therefore, that some of the reducing pressure exerted by the malate-aspartate shuttle and by glutamate plus malate is provided through the energy-linked, electrogenic transport of aspartate out of the mitochondria. These results are discussed with respect to the mechanism of the genesis of ethanol-induced fatty liver.     

Vasopressin decreases total free fatty acids but enhances release of radioactivity from isolated hepatocytes labelled with [3H]arachidonic acid

The short-term effects of vasopressin on free fatty acids and lysophospholipids were investigated in hepatocytes isolated from fed rats. Over the time period 0.25 to 10 min vasopressin decreased the steady-state concentrations of palmitic, stearic and oleic acids measured by gas liquid chromatography in extracts of cells incubated at 0.1 mM extracellular Ca2+. The concentrations of arachidonic and linoleic acids did not change. In hepatocytes labelled with [3H]arachidonic acid and incubated at 1.3 mM extracellular Ca2+ vasopressin or the Ca2+-selective ionophore A23187 increased the rate of accumulation of radioactivity in the incubation medium by 40%. The action of A23187 was dependent on extracellular Ca2+. When hepatocytes labelled with 32Pi were treated with vasopressin, no change in the amounts of [32P]lysophosphatidylethanolamine or [32P]lysophosphatidylcholine was observed. It is concluded that the action of vasopressin on hepatocytes is associated with the release of arachidonic acid or metabolites of arachidonic acid but is not accompanied by a general increase in the steady-state concentrations of free fatty acids and lysophospholipids.     

Phosphate and calcium uptake by mitochondria and by perfused rat liver induced by the synergistic action of glucagon and vasopressin.

Co-administration of glucagon and vasopressin to rat liver perfused with buffer containing 1.3 mM-Ca2+ induces a 4-fold increase in Pi in the subsequently isolated mitochondria (from approx. 9 to approx. 40 nmol/mg of mitochondrial protein). This increase is not attributable to PPi hydrolysis, and is not observed if the perfusate Ca2+ is lowered from 1.3 mM to 50 microM. The increase in mitochondrial Pi closely parallels that of mitochondrial Ca2+; when the increase in Pi and Ca2+ accumulation is maximal, the molar ratio is close to that in Ca3(PO4)2. Measurement of changes in the perfusate Pi revealed that, whereas administration of glucagon or vasopressin alone brought about a rapid decline in perfusate Pi, the largest decrease (reflecting net retention of Pi by the liver) was observed when the hormone was co-administered in the presence of 1.3 mM-Ca2+. The synergistic action of glucagon plus vasopressin was nullified by lowering the perfusate Ca2+ to 50 microM. The data provide evidence that, whereas glucagon may be able to alter Pi fluxes directly in intact liver, any alterations induced by vasopressin are indirect and result only from its action of mobilizing Ca2+.     

Acute and chronic ethanol treatment in vivo increases malate-aspartate shuttle capacity in perfused rat liver

The effects of acute and chronic treatment with ethanol on transport of reducing equivalents into mitochondria via the malate-aspartate shuttle were studied in perfused rat liver. The shuttle capacity was estimated from the decrease in rates of glucose production from the reduced substrate sorbitol caused by an increase in the NADH/NAD+ ratio in the cytosol due to metabolism of ethanol. The greater the capacity of the malate-aspartate shuttle, the smaller the inhibition of glucose synthesis by ethanol. Glucose synthesis was decreased about 2-fold less in livers from fasted rats treated acutely 2.5 h earlier with ethanol than in untreated controls. Chronic treatment with ethanol for 3-5 weeks prevented completely the decrease in glucose synthesis from sorbitol due to ethanol oxidation. Rates of ethanol uptake were elevated significantly from 69 +/- 7 mumols/g/h in livers from control rats up to 92 +/- 7 mumols/g/h in livers from SIAM rats. Similarly, rates of ethanol uptake were stimulated by chronic ethanol treatment from 71 +/- 6 to 222 +/- 15 mumols/g/h; this increase was largely sensitive to aminooxyacetate. Taken together, these data indicate that flux of reducing equivalents over the malate-aspartate shuttle is increased by both acute and chronic treatment with ethanol and that movement of reducing equivalents from the cytosol into the mitochondria via the malate-aspartate shuttle is an important rate determinant in hepatic ethanol oxidation.     

Stimulation of glycine catabolism in isolated perfused rat liver by calcium mobilizing hormones and in isolated rat liver mitochondria by submicromolar concentrations of calcium.

Glucagon stimulates flux through the glycine cleavage system (GCS) in isolated rat hepatocytes (Jois, M., Hall, B., Fewer, K., and Brosnan, J. T. (1989) J. Biol. Chem. 264, 3347-3351. In the present study, flux through GCS was measured in isolated rat liver perfused with 100 nM glucagon, 1 microM epinephrine, 1 microM norepinephrine, 10 microM phenylephrine, or 100 nM vasopressin. These hormones increased flux through GCS in perfused rat liver by 100-200% above the basal rate. The possibility that the stimulation of flux by adrenergic agonists and vasopressin is mediated by increases in cytoplasmic Ca2+ which in turn could regulate mitochondrial glycine catabolism was examined by measuring flux through GCS in isolated mitochondria in the presence of 0.04-2.88 microM free Ca2+. Flux through GCS in isolated mitochondria was exquisitely sensitive to free Ca2+ in the medium; half-maximal stimulation occurred at about 0.4 microM free Ca2+ and maximal stimulation (7-fold) was reached when the free Ca2+ in the medium was 1 microM. The Vmax (nanomoles/mg protein/min) and Km (millimolar) values for the flux through GCS in intact mitochondria were 0.67 +/- 0.16 and 20.66 +/- 4.82 in the presence of 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and 3.28 +/- 0.76 and 10.98 +/- 1.91 in presence of 0.5 microM free Ca2+, respectively. The results show that the flux through GCS is sensitive to concentrations of calcium which would be achieved in the cytoplasm of hepatocytes stimulated by calcium-mobilizing hormones.     

Stimulation of CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by calcium in rat hepatocytes

The effects of Ca2+, ionophore A23187, and vasopressin on CTP:phosphocholine cytidylyltransferase were investigated. Cytidylyltransferase is present in the cytosol and in a membrane-bound form on the microsomes. Digitonin treatment caused release of the cytosolic form rapidly. Addition of 7 mM Ca2+ to hepatocyte medium resulted in a 3-fold decrease in cytidylyltransferase released by digitonin treatment (1.7 +/- 0.1 nmol/min per mg compared to 5.1 +/- 0.2 nmol/min per mg in the control). Verapamil, a calcium channel blocker, partially overcame this effect of Ca2+. Ionophore A23187 and vasopressin both mimicked the effect of Ca2+ and resulted in a decrease in cytidylyltransferase release (2.4 +/- 0.1 nmol/min per mg and 2.5 +/- 0.2 nmol/min per mg, respectively) compared to control (3.4 +/- 0.1 nmol/min per mg). In agreement with the digitonin experiments, incubation with 7 mM Ca2+ resulted in a decrease in cytidylyltransferase in the cytosol (from 4.0 to 1.2 mol/min per mg) and a corresponding increase in the microsomes (from 0.6 to 2.4 nmol/min per mg). Verapamil partially blocked this translocation caused by Ca2+. Ionophore A23187 and vasopressin also caused translocation of the cytidylyltransferase from the cytosol to the microsomes. The addition of Ca2+ also resulted in an increase in PC synthesis. With 7 mM Ca2+ in the medium, the label associated with PC increased to 3.8 +/- 0.1.10(6) dpm/dish from 2.7 +/- 0.1.10(6) dpm/dish after 10 min. PC degradation was also affected, since 7 mM Ca2+ in the medium resulted in an increase in LPC formation both in the cell and the medium. We conclude that high concentrations of calcium in the hepatocyte medium can cause a stimulation of CTP:phosphocholine cytidylyltransferase and PC synthesis in cultured hepatocytes.     

Effects of vasopressin and La3+ on plasma-membrane Ca2+ inflow and Ca2+ disposition in isolated hepatocytes. Evidence that vasopressin inhibits Ca2+ disposition.

Vasopressin caused a 40% inhibition of 45Ca uptake after the addition of 0.1 mM-45Ca2+ to Ca2+-deprived hepatocytes. At 1.3 mM-45Ca2+, vasopressin and ionophore A23187 each caused a 10% inhibition of 45Ca2+ uptake, whereas La3+ increased the rate of 45Ca2+ uptake by Ca2+-deprived cells. Under steady-state conditions at 1.3 mM extracellular Ca2+ (Ca2+o), vasopressin and La3+ each increased the rate of 45Ca2+ exchange. The concentrations of vasopressin that gave half-maximal stimulation of 45Ca2+ exchange and glycogen phosphorylase activity were similar. At 0.1 mM-Ca2+o, La3+ increased, but vasopressin did not alter, the rate of 45Ca2+ exchange. The results of experiments performed with EGTA or A23187 or by subcellular fractionation indicate that the Ca2+ taken up by hepatocytes in the presence of La3+ is located within the cell. The addition of 1.3 mM-Ca2+o to Ca2+-deprived cells caused increases of approx. 50% in the concentration of free Ca2+ in the cytoplasm [( Ca2+]i) and in glycogen phosphorylase activity. Much larger increases in these parameters were observed in the presence of vasopressin or ionophore A23187. In contrast with vasopressin, La3+ did not cause a detectable increase in glycogen phosphorylase activity or in [Ca2+]i. It is concluded that an increase in plasma membrane Ca2+ inflow does not by itself increase [Ca2+]i, and hence that the ability of vasopressin to maintain increased [Ca2+]i over a period of time is dependent on inhibition of the intracellular removal of Ca2+.     

Thiophosphorylation causes Ca2+-independent norepinephrine secretion from permeabilized PC12 cells

Adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) was used to examine the role of phosphorylation in the regulation of norepinephrine secretion by digitonin-permeabilized PC12 cells. While most kinases will use ATP gamma S to thiophosphorylate proteins, thiophosphorylated proteins are relatively resistant to dethiophosphorylation by protein phosphatases. Norepinephrine secretion by permeabilized PC12 cells was ATP- and Ca2+-dependent but resistant to calmodulin antagonists. Half-maximum secretion was obtained in 0.75 microM Ca2+. Permeabilized PC12 cells were incubated with ATP gamma S in the absence of Ca2+, the ATP gamma S was removed, and norepinephrine secretion was determined. Preincubation with ATP gamma S increased the amount of norepinephrine secreted in the absence of Ca2+, but it had no effect on the amount released in the presence of Ca2+. After a 15-min preincubation in 1 mM ATP gamma S, there was almost as much secretion in the absence of Ca2+ as in its presence. Inclusion of ATP in the preincubation inhibited the effect of ATP gamma S. Ca2+ stimulated the rate of modification by ATP gamma S as brief preincubations with ATP gamma S in the presence of Ca2+ resulted in higher levels of Ca2+-independent secretion than did preincubations with ATP gamma S in the absence of Ca2+. Similarly, brief preincubations of permeabilized cells with ATP in the presence of Ca2+ resulted in elevated levels of Ca2+-independent secretion. Secretion of norepinephrine from ATP gamma S-treated cells was ATP-dependent. These results suggest that norepinephrine secretion by PC12 cells is regulated by a Ca2+-dependent phosphorylation. Once this phosphorylation has occurred, secretion is still ATP-dependent, but it no longer requires Ca2+.     

On the source of the vasopressin-induced increases in diacylglycerol in hepatocytes

In hepatocytes pre-labelled with [3H]glycerol, vasopressin increased by 20% the amount of radioactivity present in diacylglycerols. The effect of vasopressin was partially dependent on Ca2+. The magnitude of the increase in [3H]diacylglycerol was 5-times the sum of the radioactivity present in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. No stimulation by vasopressin of the initial rate of incorporation of radioactivity into diacylglycerols was observed in cells incubated in the presence of 10 mM [3H]glycerol. Treatment of hepatocytes labelled with either [3H]ethanolamine or [3H]choline with vasopressin, ionophore A23187 or phospholipase C increased the amount of radioactivity present in trichloroacetic acid extracts of the cells. The effect of vasopressin was dependent on extracellular Ca2+. It is concluded that in hepatocytes vasopressin increases diacylglycerols by a process which does not principally involve the conversion of phosphoinositides to diacylglycerol or the de novo synthesis of diacylglycerol from glycerol 3-phosphate, but does involve the Ca2+-dependent conversion of phosphatidylethanolamine and phosphatidylcholine to diacylglycerol.     

Glutamine metabolism of isolated rat hepatocytes. Evidence for catecholamine activation of alpha-ketoglutarate dehydrogenase

Effects of norepinephrine on gluconeogenesis and ureogenesis from glutamine by hepatocytes from fasted rats were assessed. Comparisons were made to asparagine metabolism and to the effects of NH4Cl and dibutyryl cyclic AMP. With asparagine as substrate, aspartate content was very high but norepinephrine, dibutyryl cyclic AMP, or NH4Cl had little effect on gluconeogenesis or ureogenesis. Metabolism of asparagine could be greatly enhanced by the combination of oleate, ornithine, and NH4Cl. However, even under these conditions, asparatate content remained high, and norepinephrine and dibutyryl cyclic AMP had little influence on glucose or urea synthesis. With glutamine as substrate, aspartate content was much lower, but was greatly elevated by norepinephrine, dibutyryl cyclic AMP, or NH4Cl. Each of these effectors strongly stimulated glucose and urea formation from glutamine. NH4Cl stimulation was accompanied by an increased glutamate and decreased alpha-ketoglutarate content. This suggests the mechanism for NH4Cl stimulation is a near-equilibrium adjustment to ammonia by glutamate dehydrogenase and aspartate aminotransferase rather than a principal involvement of glutaminase. Although both norepinephrine and dibutyryl cyclic AMP lowered alpha-ketoglutarate to the same extent, norepinephrine more rapidly increased aspartate content and led to a smaller accumulation of glutamate than did dibutyryl cyclic AMP. Moreover, only norepinephrine led to a rapid increase in succinyl-CoA concentration. The catecholamine effect could not be explained by specific changes in cytosolic or mitochondrial redox states. The results suggest that alpha-ketoglutarate dehydrogenase is a site of catecholamine action in rat liver. Since purified alpha-ketoglutarate dehydrogenase is known to be Ca2+ stimulated and Ca2+ flux is involved in catecholamine action, these findings also suggest that mitochondrial Ca2+ is elevated by catecholamines.     

The effect of ionophore A23187 on calcium ion fluxes and alpha-adrenergic-agonist action in perfused rat liver.

The effect of ionophore A23187 on cellular Ca2+ fluxes, glycogenolysis and respiration was examined in perfused liver. At low extracellular Ca2+ concentrations (less than 4 microM), A23187 induced the mobilization of intracellular Ca2+ and stimulated the rate of glycogenolysis and respiration. As the extracellular Ca2+ concentration was elevated, biphasic cellular Ca2+ fluxes were observed, with Ca2+ uptake preceding Ca2+ efflux. Under these conditions, both the glycogenolytic response and the respiratory response also became biphasic, allowing the differentiation between the effects of extracellular and intracellular Ca2+. Under all conditions examined the rate of Ca2+ efflux induced by A23187 was much slower than the rate of phenylephrine-induced Ca2+ efflux, although the net amounts of Ca2+ effluxed were similar for both agents. The effect of A23187 on phenylephrine-induced Ca2+ fluxes, glycogenolysis and respiration is dependent on the extracellular Ca2+ concentration. At concentrations of less than 50 microM-Ca2+, A23187 only partially inhibited alpha-agonist action, whereas at 1.3 mM-Ca2+ almost total inhibition was observed. The action of A23187 at the cellular level is complex, dependent on the experimental conditions used, and shows both differences from and similarities to the hepatic action of alpha-adrenergic agonists.     

Decreased flux through pyruvate dehydrogenase during calcium ion movements induced by vasopressin, alpha-adrenergic agonists and the ionophore A23187 in perfused rat liver.

Vasopressin or alpha-adrenergic agents such as phenylephrine or adrenaline, but not glucagon, elicited an initial decrease in flux through pyruvate dehydrogenase assayed by 14CO2 production from [1-14C]pyruvate in perfused rat liver. This rapid decrease in 14CO2 production was maximal within 1-2 min of exposure, concomitant with a rise in effluent pyruvate concentration: a subsequent return towards initial values in both parameters was completed well before 5 min. This time course was superposed with Ca2+ efflux from perfused liver, maximal (at 116 nmol/min per g wet wt. of liver) at 1-2 min of exposure. The percentage of the active (dephospho) form of pyruvate dehydrogenase was not decreased at 2 min of exposure. The effect on flux through pyruvate dehydrogenase by phenylephrine was abolished by prazosine, phentolamine or phenoxybenzamine. Ionophore A23187 also caused a depression in 14CO2 production from [1-14C]pyruvate and a rise in effluent pyruvate concentration, but this effect was stable for longer times, and it was delayed when Ca2+ was omitted from the perfusion medium. Responses of phenylephrine and A23187 were not additive. The results demonstrate that under the experimental conditions employed in intact perfused liver, the mitochondrial multienzyme system of pyruvate dehydrogenase is sensitive to vasopressin, alpha-adrenergic agents and A23187. The similar time course in Ca2+ efflux may be indicative of the involvement of Ca2+ in mediating this effect.     

Calcium ion fluxes induced by the action of alpha-adrenergic agonists in perfused rat liver.

Phenylephrine (2.0 microM) induces an alpha 1-receptor-mediated net efflux of Ca2+ from livers of fed rats perfused with medium containing physiological concentrations (1.3 mM) of Ca2+. The onset of efflux (7.1 +/- 0.5 s; n = 16) immediately precedes a stimulation of mitochondrial respiration and glycogenolysis. Maximal rates of efflux are observed between 35 s and 45 s after alpha-agonist administration; thereafter the rate decreases, to be no longer detectable after 3 min. Within seconds of terminating phenylephrine infusion, a net transient uptake of Ca2+ by the liver is observed. Similar effects were observed with vasopressin (1 m-unit/ml) and angiotensin (6 nM). Reducing the perfusate [Ca2+] from 1.3 mM to 10 microM had little effect on alpha-agonist-induced Ca2+ efflux, but abolished the subsequent Ca2+ re-uptake, and hence led to a net loss of 80-120 nmol of Ca2+/g of liver from the tissue. The administration at 5 min intervals of short pulses (90 s) of phenylephrine under these conditions resulted in diminishing amounts of Ca2+ efflux being detected, and these could be correlated with decreased rates of alpha-agonist-induced mitochondrial respiration and glucose output. An examination of the Ca2+ pool mobilized by alpha-adrenergic agonists revealed that a loss of Ca2+ from mitochondria and from a fraction enriched in microsomes accounts for all the Ca2+ efflux detected. It is proposed that the alpha-adrenergic agonists, vasopressin and angiotensin mobilize Ca2+ from the same readily depleted intracellular pool consisting predominantly of mitochondria and the endoplasmic reticulum, and that the hormone-induced enhanced rate of mitochondrial respiration and glycogenolysis is directly dependent on this mobilization.     

Insulin blockade of norepinephrine tachyphylaxis in the perfused kidney

Tachyphylaxis to norepinephrine (NOR) was determined in the rabbit kidney perfused with Krebs-Henseleit solution by using different calcium concentrations (2.5 mM; 5 mM; 12.5 mM) in the perfusate. The addition of insulin to the perfusion fluid causes a reversion of the tachyphylaxis which is seen at those Ca2+ concentrations. This effect is demonstrated mainly at 5 mM Ca2+. When kidneys were perfused with 12.5 mM calcium there was disappearance of NOR-mediated tachyphylaxis, both in the absence and in the presence of insulin. In this calcium concentration, insulin decreases vascular reactivity to NOR. These results suggest that insulin blockade of alpha adrenergic tachyphylaxis is a calcium-mediated effect which is thought to be due to an enhancement of calcium pumping inside the cells.     

Influence of the Malate-Aspartate Shuttle on Oxidative Metabolism in Synaptosomes

beta-Methyleneaspartate, a specific inhibitor of aspartate aminotransferase (EC 2.6.1.1.), was used to investigate the role of the malate-aspartate shuttle in rat brain synaptosomes. Incubation of rat brain cytosol, "free" mitochondria, synaptosol, and synaptic mitochondria, with 2 mM beta-methyleneaspartate resulted in inhibition of aspartate aminotransferase by 69%, 67%, 49%, and 76%, respectively. The reconstituted malate-aspartate shuttle of "free" brain mitochondria was inhibited by a similar degree (53%). As a consequence of the inhibition of the aspartate aminotransferase, and hence the malate-aspartate shuttle, the following changes were observed in synaptosomes: decreased glucose oxidation via the pyruvate dehydrogenase reaction and the tricarboxylic acid cycle; decreased acetylcholine synthesis; and an increase in the cytosolic redox state, as measured by the lactate/pyruvate ratio. The main reason for these changes can be attributed to decreased carbon flow through the tricarboxylic acid cycle (i.e., decreased formation of oxaloacetate), rather than as a direct consequence of changes in the NAD+/NADH ratio. Malate/glutamate oxidation in "free" mitochondria was also decreased in the presence of 2 mM beta-methyleneaspartate. This is probably a result of decreased glutamate transport into mitochondria as a result of low levels of aspartate, which are needed for the exchange with glutamate by the energy-dependent glutamate-aspartate translocator.     

Ca2+-dependent activation of oxoglutarate dehydrogenase by vasopressin in isolated hepatocytes.

Vasopressin stimulated gluconeogenesis from proline in hepatocytes from starved rats; this was attributed to an activation of oxoglutarate dehydrogenase (EC 1.2.4.2) [Staddon & McGivan (1984) Biochem. J. 217, 477-483]. The role of Ca2+ in the activation mechanism was investigated. (1) In the absence of extracellular Ca2+, vasopressin caused a stimulation of gluconeogenesis and a decrease in cell oxoglutarate content that were markedly transient when compared with the effects in the presence of Ca2+. (2) Ca2+ added to cells stimulated for 2 min by vasopressin in the absence of extracellular Ca2+ sustained the initial effects of vasopressin. Ca2+ added 15 min after vasopressin, a time at which both the rate of gluconeogenesis and the cell oxoglutarate content were close to the control values, caused a stimulation of gluconeogenesis and a decrease in cell oxoglutarate content. (3) Under conditions of cell-Ca2+ depletion, vasopressin had no effect on gluconeogenesis or cell oxoglutarate content. (4) Ionophore A23187 stimulated gluconeogenesis and caused a decrease in cell oxoglutarate content, but the phorbol ester 4 beta-phorbol 12-myristate 13-acetate had no effects. (5) These data suggest that the initial activation of oxoglutarate dehydrogenase by vasopressin is dependent on an intracellular Ca2+ pool and independent of extracellular Ca2+. For activation of a greater duration, a requirement for extracellular Ca2+ occurs. The activation of oxoglutarate dehydrogenase by A23187 is consistent with a mechanism involving Ca2+, but the lack of effect of 4 beta-phorbol 12-myristate 13-acetate indicates that protein kinase C is not involved in the mechanism of activation by vasopressin.