Hepatic zonation of carbon and nitrogen fluxes derived from glutamine and ammonia transformations.

BACKGROUND: Glutaminase predominates in periportal hepatocytes and it has been proposed that it determines the glutamine-derived nitrogen flow through the urea cycle. Glutamine-derived urea production should, thus, be considerably faster in periportal hepatocytes. This postulate, based on indirect observations, has not yet been unequivocally demonstrated, making a direct investigation of ureogenesis from glutamine highly desirable. METHODS: Zonation of glutamine metabolism was investigated in the bivascularly perfused rat liver with [U-14C]glutamine infusion (0.6 mM) into the portal vein (antegrade perfusion) or into the hepatic vein (retrograde perfusion). RESULTS: Ammonia infusion into the hepatic artery in retrograde and antegrade perfusion allowed to promote glutamine metabolism in the periportal region and in the whole liver parenchyma, respectively. The results revealed that the space-normalized glutamine uptake, indicated by (14)CO(2) production, gluconeogenesis, lactate production and the associated oxygen uptake, predominates in the periportal region. Periportal predominance was especially pronounced for gluconeogenesis. Ureogenesis, however, tended to be uniformly distributed over the whole liver parenchyma at low ammonia concentrations (up to 1.0 mM); periportal predominance was found only at ammonia concentrations above 1 mM. The proportions between the carbon and nitrogen fluxes in periportal cells are not the same along the liver acinus. CONCLUSIONS: In conclusion, the results of the present work indicate that the glutaminase activity in periportal hepatocytes is not the rate-controlling step of the glutamine-derived nitrogen flow through the urea cycle. The findings corroborate recent work indicating that ureogenesis is also an important ammonia-detoxifying mechanism in cells situated downstream to the periportal region.

Zonation of the action of ethanol on gluconeogenesis and ketogenesis studied in the bivascularly perfused rat liver.

Zonation of the actions of ethanol on gluconeogenesis and ketogenesis from lactate were investigated in the bivascularly perfused rat liver. Livers from fasted rats were perfused bivascularly in the antegrade and retrograde modes. Ethanol and lactate were infused into the hepatic artery (antegrade and retrograde) and portal vein. A previously described quantitative analysis that takes into account the microcirculatory characteristics of the rat liver was extended to the analysis of zone-specific effects of inhibitors. Confirming previous reports, gluconeogenesis and the corresponding oxygen uptake increment due to saturable lactate infusions were more pronounced in the periportal region. Arterially infused ethanol inhibited gluconeogenesis more strongly in the periportal region (inhibition constant=3.99+/-0.22mM) when compared to downstream localized regions (inhibition constant=8.64+/-2.73mM). The decrease in oxygen uptake caused by ethanol was also more pronounced in the periportal zone. Lactate decreased ketogenesis dependent on endogenous substrates in both regions, periportal and perivenous, but more strongly in the former. Ethanol further inhibited ketogenesis, but only in the periportal zone. Stimulation was found for the perivenous zone. The predominance of most ethanol effects in the periportal region of the liver is probably related to the fact that its transformation is also clearly predominant in this region, as demonstrated in a previous study. The differential effect on ketogenesis, on the other hand, suggest that the net effects of ethanol are the consequence of a summation of several partial effects with different intensities along the hepatic acini.

Glycogen levels and energy status of the liver of fasting rats with diabetes types 1 and 2

Glycogen levels and the energy status of livers from fasting rats with diabetes types 1 and 2 were measured. After a 24 h fast, the hepatic glycogen levels of rats with diabetes1 and diabetes2 were, 18.7 and 2.6 times higher, respectively, than those of livers from the normal rats. In diabetes1 rats, the glycogen levels decreased when the fasting period was extended to 48 and 72 h. The opposite occurred with the control and diabetes2 rats. Consistently, glucose release by the perfused livers from diabetes1 rats was considerably higher during at least 60 minutes after initiating perfusion. The hepatic ATP content of diabetes1 rats was similar to that of the control rats; in diabetes2 rats, the hepatic ATP content was increased. It could be concluded that regulation of glycogen deposition and degradation in rats with diabetes1 differed markedly from that of rats with diabetes2 which, in turn, behaved similarly to normal healthy rats.

Teores de glicogênio e os estados energéticos de fígados de ratos com diabete dos tipos 1 e 2 foram medidos. Após um jejum de 24 horas os teores de glicogênio de ratos com diabete1 e diabete2 foram, respectivamente 18,7 e 2,6 vezes superiores àqueles de fígados de animais controle. Em ratos com diabete1 o conteúdo de glicogênio diminuiu quando o período de jejum foi prolongado para 48 e 72 horas. O oposto ocorreu em ratos controle e ratos com diabete2. Consistentemente, a liberação de glicose por fígados em perfusão isolada obtidos de ratos com diabete1 foi consideravelmente maior durante ao menos 60 minutos após o início da perfusão. O conteúdo hepático de ATP de ratos com diabete1 foi similar àquele de ratos controle; em ratos com diabete2 o conteúdo hepático de ATP foi maior. Pode-se concluir que a regulação da deposição e degradação do glicogênio em ratos com diabete1 difere marcadamente daquela de ratos com diabete2, os quais, por seu turno, comportam-se similarmente a ratos normais e saudáveis.

Flexibility of the hepatic zonation of carbon and nitrogen fluxes linked to lactate and pyruvate transformations in the presence of ammonia.

It has been proposed that key enzymes of ureagenesis and the alanine aminotransferase activity predominate in periportal hepatocytes. However, ureagenesis from alanine, when measured in the perfused liver, did not show periportal predominance and even the release of the direct products of alanine transformation, lactate and pyruvate, was higher in perivenous cells. An alternative way of analyzing the functional distributions of alanine aminotransferase and the urea cycle along the hepatic acini would be to measure alanine and urea production from precursors such as lactate or pyruvate plus ammonia. In the present work these aspects were investigated in the bivascularly perfused rat liver. The results of the present study confirm that gluconeogenesis and the associated oxygen uptake tend to predominate in the periportal region. Alanine synthesis from lactate and pyruvate plus ammonia, however, predominated in the perivenous region. Furthermore, no predominance of ureagenesis in the periportal region was found, except for conditions of high ammonia concentrations plus oxidizing conditions induced by pyruvate. These observations corroborate the view that data on enzyme activity or expression alone cannot be extrapolated unconditionally to the living cell. The current view of the hepatic ammonia-detoxifying system proposes that the small perivenous fraction of glutamine synthesizing perivenous cells removes a minor fraction of ammonia that escapes from ureagenesis in periportal cells. However, since urea synthesis occurs at high rates in all hepatocytes with the possible exclusion of those cells not possessing carbamoyl-phosphate synthase, it is probable that ureagenesis is equally important as an ammonia-detoxifying mechanism in the perivenous region.

The action of glibenclamide on glycogen catabolism and related parameters in the isolated perfused rat liver.

Inhibitory effects on glycogenolysis have been reported for glibenclamide in the presence of insulin after stimulation of glycogenolysis by glucagon. Inhibition of oxidative phosphorylation, which has been equally reported for this drug, however, should stimulate glycogenolysis. The present work aimed to find an answer to the question of how glibenclamide affects glycogen catabolism in the liver of fed rats undergoing substrate- and hormone-free perfusion. The experimental system was the isolated perfused liver of ad libitum fed rats. Metabolites in the outflowing perfusate were assayed enzymatically. Oxygen uptake was measured polarographically. Glibenclamide (25-500 microM) stimulated glucose production and lactate release, with a clear correlation between concentrations and effects. Maximal stimulations were 132 and 127% for lactate production and glucose release, respectively. At low glibenclamide concentrations (up to 100 microM) both oxygen uptake and pyruvate production were stimulated, but at higher concentrations inhibition took place. Uric acid production was stimulated by glibenclamide. All effects of glibenclamide are probably due to decreases in oxidative phosphorylation. Stimulation of glucose release is the opposite of what should be expected for a hypoglycemic drug and it also contrasts with some reports of diminishing effects in the presence of glucagon plus insulin. This means that the stimulatory action on glycogenolysis that was seen as a net effect under the specific conditions of the present work could be counterbalancing inhibitory effects in vivo. This combination of events could eventually diminish the effectiveness of the drug as a hypoglycemic agent in the fed state.

Zonation of alanine metabolism in the bivascularly perfused rat liver.

AIMS/BACKGROUND: Zonation of alanine metabolism was investigated in the bivascularly perfused rat liver, a technique in which a selective area of the periportal region can be reached via the hepatic artery. METHODS: Bivascular liver perfusion was done in both the antegrade and retrograde modes. Predominance of a given metabolic parameter in the periportal or perivenous area was deduced from comparisons of the changes caused by alanine infusion into the hepatic artery in antegrade and retrograde perfusion. RESULTS: Confirming previous notions, glutamine synthesis predominated in the perivenous area, however, the contribution of the periportal area was significant. Gluconeogenesis and the associated extra oxygen consumption were more pronounced in the periportal region. The capacity of urea synthesis in the periportal region was relatively small as indicated by the ratios of urea to glucose production, which were lower in this region. Ammonia in the periportal region was considerably above the mean ammonia production of whole the liver parenchyma. The overflows of pyruvate and lactate were considerably smaller in the periportal region. CONCLUSION: The distribution of alanine metabolism seems to reflect mainly zonation of the fates of the carbon skeleton (mainly gluconeogenesis). The production of glutamine in the periportal area is in agreement with recent reports about the presence of glutamine synthetase in Kupffer and endothelial cells.

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.

Heterogenic response of the liver parenchyma to ethanol studied in the bivascularly perfused rat liver.

Zonation of ethanol oxidation and metabolic effects along the hepatic acini were investigated in the bivascularly perfused liver of fed rats. Ethanol was infused into the hepatic artery in antegrade and retrograde perfusion. Inhibition of glycolysis by ethanol, expressed as micromol min(-1) (ml accessible cell space)(-1), was more pronounced in the retrograde mode; the retrograde/antegrade ratio was equal to 1.63 for an ethanol infusion rate of 37.5 micromol min(-1) g(-1). Stimulation of oxygen uptake by ethanol was more pronounced in the retrograde mode; the retrograde/antegrade ratio was equal to 1.77. Diminution of the citrate cycle caused by ethanol was more pronounced in the retrograde mode; the retrograde/antegrade ratio was equal to 1.46. Transformation of arterially infused ethanol into acetate was more pronounced in retrograde perfusion; the retrograde/antegrade ratio was equal to 1.63. The increments in glucose release (glycogenolysis) caused by ethanol in the antegrade and retrograde modes were similar. It was assumed that the changes caused by arterially infused ethanol in retrograde and antegrade perfusion closely reflect a significant part of the periportal parenchyma and an average over the whole liver parenchyma, respectively. Under such assumptions it can be concluded that, in the perfused liver from fed rats, four related parameters predominate in the periportal region: ethanol oxidation, glycolysis inhibition, oxygen uptake stimulation and citrate cycle inhibition. One of the main causes for this predominance could be the malate/aspartate shuttle, which operates more rapidly in the periportal area and is essential for NADH oxidation.

The action of oxybutynin on haemodynamics and metabolism in the perfused rat liver.

The present study was planned to investigate the possible action of oxybutynin on liver haemodynamics and its influence on metabolic variables. The isolated liver perfused either bivascularly or monovascularly in the non-recirculating system was used for the experiments and Krebs/Henseleit-bicarbonate buffer (pH 7.4) as a perfusion fluid. Oxybutynin (25-200 microM) was infused, the infusion time for each concentration being 14 min. Portally infused oxybutynin increased the perfusion pressure starting at 100 microM. Oxygen uptake was diminished, also starting at 100 microM. Arterially infused oxybutynin also increased the perfusion pressure in the hepatic artery. Lactate and pyruvate releases were considerably diminished by oxybutynin. Glucose release showed a small initial stimulation, then returned to values slightly below the basal ones. Cessation of oxybutynin infusion resulted in progressive stimulation of glucose release. When Ca2+ was omitted all effects of oxybutynin vanished. The hepatic contents of glucose, glucose 6-phosphate and lactate in the presence of 200 microM oxybutynin increased 7.8-, 4.6- and 5.1 times, respectively. The pyruvate content was not changed. The ATP content was diminished by 26.6% in the presence of 200 microM oxybutynin, but the AMP content was increased by 64.3%. The ADP content was not changed. Apparently, upon administration of oxybutynin, a considerable fraction of the liver parenchyma ceased to be irrigated or almost so, which is apparent from the concomitant inhibition of oxygen uptake, pressure increase and inhibition of glucose, lactate and pyruvate release together with the simultaneous intracellular accumulation of glucose, lactate and glucose 6-phosphate.

Perfusion pressure in kidneys of arthritic rats and the influence of L-NAME.

Kidneys of control and arthritic rats were perfused with a hemoglobin-free perfusion fluid with simultaneous monitoring of perfusion pressure and oxygen uptake. The basal values of renal perfusion pressure were 76.3 +/- 4.63 and 59.96 +/- 3.65 mm Hg in control and arthritic rats, respectively. Infusion of 100 microM Nomega-nitro-L-arginine methyl ester (L-NAME) an inhibitor of constitutive and inducible nitric oxide synthase, increased the renal perfusion pressure to 91.6 +/- 5.52 and 106.69 +/- 8.47 mm Hg in control and arthritic rats, respectively. Oxygen uptake of kidneys from control rats was 2.48 +/- 0.74 micromol min(-1) g(-1) and that of the arthritic rats was 2.44 +/- 0.75 micromol min(-1) g(-1). These results are consistent with an increased production of hemodynamically active nitric oxide in kidneys of arthritic rats.

The hemodynamic effects of ATP in retrograde perfusion of the bivascularly perfused rat liver.

AIMS/BACKGROUND: In the sinusoidal bed the distribution of water is flow-limited, but it becomes partly barrier-limited when adenosine triphosphate (ATP) is introduced. This effect could be exerted either directly by ATP or by substances released from presinusoidal regions. Furthermore, portally infused ATP seems to be able to diffuse in the direction of the arterial bed. It is not known if this diffusion route is specific. Answers to these questions can be obtained from indicator-dilution experiments in retrograde perfusion. METHODS: Indicator-dilution experiments, using [14C]sucrose and [3H]water, were conducted. Rat livers were perfused in the retrograde mode (hepatic vein+hepatic artery --> portal vein). RESULTS: When ATP was infused into the hepatic vein, the distribution of [3H]water remained essentially flow-limited. The infusion of ATP into the hepatic artery increased the sucrose and extra-sucrose spaces of the arterial bed, but infusion into the hepatic vein was without effect. CONCLUSIONS: The results indicate that the induction of barrier-limited distribution of [3H]water is not a direct effect of ATP. Furthermore, if the transhepatic diffusion of ATP can occur from presinusoidal regions to the arterial bed, as shown by previous work, a similar diffusion does not occur from postsinusoidal regions.

Inhibition by extracellular ATP of organic anion transport in the perfused rat liver.

The action of extracellular ATP on organic anion transport in the bivascularly perfused rat liver was investigated, using bromosulfophthalein as a model substance. Transport was measured by means of the multiple-indicator dilution technique. The action of portal 100 microM ATP presented the following characteristics: (a) inhibition of bromosulfophthalein single pass extraction; the inhibition degree decreased with increasing bromosulfophthalein doses; (b) diminution of the influx rate coefficients; (c) 86.7% decrease of the maximal activity of the saturable component for bromosulfophthalein transport, but 100% increase of the non-saturable component; (d) diminution of the bromosulfophthalein flow-limited distribution space; (e) no significant alteration of the rate coefficients for metabolic sequestration. The action of ATP on organic anion transport in the intact liver occurred at much lower concentrations (10x) than those previously reported for isolated hepatocytes. This reinforces the suggestion that inhibition of organic anion transport could be a physiologically relevant effect of extracellular ATP.