Bivascularly Perfused Rat Liver Research Articles

Escherichia coli (Lilly) and Saccharomyces cerevisiae (Novo) rDNA Glucagon: An Assessment of Their Actions When Supplied Selectively to Periportal Cells in the Bivascularly Perfused Rat Liver

The actions of Eli Lilly-rDNA glucagon and Novo Nordisk-rDNA glucagon on glycogen catabolism and related parameters were investigated using the bivascularly perfused rat liver. The technique allows glucagon to be supplied to a selective portion of the hepatic periportal region (≈39%) when the former is infused into the hepatic artery in retrograde perfusion. Both glucagon preparations were equally effective in influencing metabolism (glucose output, glycolysis and O2 uptake) when supplied to all cells along the liver sinusoids. When only a selective periportal region of the liver was supplied with the hormone, however, the action of Novo Nordisk-rDNA glucagon was proportional to the accessible cell space, whereas the action of Eli Lilly-rDNA glucagon greatly exceeded the action that was expected for the accessible space. Chromatographically, both rDNA preparations were not pure, but their impurities were not the same. The impurities in Eli Lilly-rDNA glucagon resembled those found in the similarly acting pancreatic Eli Lilly glucagon. It was concluded that the space-extrapolating action of Eli Lilly-rDNA glucagon is caused by a yet-to-be-identified impurity. The hypothesis was raised that an impurity in certain glucagon preparations can enhance cell-to-cell propagation of the glucagon signal, possibly via gap junctional communication.

Effects of Arterially Infused Hydroalcoholic <i>Agaricus blazei</i> Extracts on Perfusion Pressure and Oxygen Uptake in the Bivascularly Perfused Rat Liver

In a preceding work we have reported experiments showing that an hydroalcoholic exctract of Agaricus blazei is able to exert purinergic effects in the isolated perfused rat liver when it is infused into the portal vein in monovascular perfusion (entry: portal vein; exit: hepatic vein). In the present communication we are presenting and discussing experiments done with the bivascularly perfused rat liver (entry: portal vein + hepatic artery; exit: hepatic vein) in order to verify if the hemodynamic effects also occur in the arterial bed. It was found that the A. blazei extract is also active when infused into the hepatic arterial bed, with differences in both sensitivity and nature of the effects on either perfusion pressure or oxygen consumption. Constriction of the arterial bed required much higher concentrations of the extract than the portal bed. The kinetics of the response was also different, with a biphasic instead of a monophasic response. These results provide a promising starting point for future studies aiming to bring to light more mechanistic details about these and possibly other effects.

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

BackgroundGlutaminase 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.MethodsZonation 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).ResultsAmmonia 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 14CO2 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.ConclusionsIn 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.

Effect of glucagon on glucose output from bivascularly perfused rat liver.

Glucose output was investigated in bivascularly perfused rat liver. Flow rate was 1 ml/min/g from hepatic artery and 2 ml/min/g from portal vein. Basal glucose output (130 +/- 30 microM/min) from bivascularly perfused liver was significantly (p less than 0.02) higher than that (80 +/- 20 microM/min) from usual monovascularly perfused (3 ml/min/g from portal vein) liver. However, glucagon-induced (1 ng/ml) increment in glucose output from bivascularly perfused liver 589 +/- 127 microM/30 min) was not significantly different from that (630 +/- 127 microM/30 min) of monovascularly perfused liver. These results suggest that glucagon-dependent glucose output was similar in monovascularly and bivascularly perfused rat liver, and that arterial blood supply may have some role in glycogenolysis in the perfused rat liver.

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 ± 0.22 mM) when compared to downstream localized regions (inhibition constant = 8.64 ± 2.73 mM). 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.

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.

Liver parenchyma heterogeneity in the response to extracellular NAD+

The perfused rat liver responds intensely to NAD+ infusion (20-100 microM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption are some of the effects that were observed. The aim of the present work was to investigate the distribution of the response to extracellular NAD+ along the hepatic acinus. The bivascularly perfused rat liver was used. Various combinations of perfusion directions (antegrade and retrograde) and infusion routes (portal vein, hepatic vein and hepatic artery) were used in order to supply NAD+ to different regions of the liver parenchyma, also taking advantage of the fact that its extracellular transformation generates steep concentration gradients. Oxygen uptake was stimulated by NAD+ in retrograde perfusion (irrespective of the infusion route) and transiently inhibited in antegrade perfusion. This indicates that the signal causing oxygen uptake inhibition is generated in the periportal area. The signal responsible for oxygen uptake stimulation is homogenously distributed. Stimulation of glucose release was more intense when NAD+ was infused into the portal vein or into the hepatic artery, indicating that stimulation of glycogenolysis predominates in the periportal area. The increases in perfusion pressure were more pronounced when the periportal area was supplied with NAD+ suggesting that the vasoconstrictive elements responding to NAD+ predominate in this region. The response to extracellular NAD+ is thus unequally distributed in the liver. As a paracrine agent, NAD+ is likely to be released locally. It can be concluded that its effects will be different depending on the area where it is released.

Zonation of alanine metabolism in the bivascularly perfused rat liver

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

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. 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). 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. 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 μM 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 (10×) 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.

Changes in distribution spaces and cell permeability caused by ATP in the rat liver.

Cellular and extracellular volume changes caused by ATP were investigated in the liver as well as the possible formation of diffusion barriers, which could be responsible for some of its metabolic effects. The experimental system was the bivascularly perfused rat liver. [(14)C]Sucrose and [(3)H]water were simultaneously injected into either the portal vein or the hepatic artery. Mean transit times, distribution spaces, variances and linear superimpositions were calculated. In the portal system, ATP reduced the transit time in the great vessels, had little or no effect on sinusoidal and cellular spaces, but impaired the flow-limited distribution of both [(14)C]sucrose and [(3)H]water. In the arterial bed ATP infused into either the portal vein or the hepatic artery produced vasodilation and increased the aqueous extra-sucrose space. These effects were inhibited by Nomega-nitro-L-arginine methyl ester infused into the hepatic artery. Sucrose and extra-sucrose space changes caused in the arterial bed by portally infused ATP are most probably analogous to the transhepatic vasodilation effect already described for the rabbit liver. Impairment of flow-limited distribution of tracers in the sinusoidal bed indicates that ATP induces the formation of permeability barriers, which could be responsible for some of its metabolic effects.

11 beta-hydroxysteroid dehydrogenase type 1 is a predominant 11 beta-reductase in the intact perfused rat liver.

11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD-1), a regulator of intrahepatocellular glucocorticoid activity, is bidirectional in homogenates but catalyses 11 beta-reduction (regenerating glucocorticoid) in intact primary hepatocytes in culture. To examine this discrepancy at the whole-organ level, we examined 11 beta-HSD-1 activity in the intact bivascularly perfused rat liver. On a single pass through male rat liver, 44+/-5% of 11-dehydrocorticosterone (11-DHC) recovered was 11 beta-reduced to corticosterone, whereas 10+/-1% of corticosterone was 11 beta-dehydrogenated to 11-DHC. 11 beta-Reduction was less in female liver (21+/-2%, P<0.01) and was significantly greater with perfusion of all substrate via the portal vein (50+/-3%) than via the hepatic artery (30+/-2%, P<0.05). 11 beta-Reductase activity was not saturated by 11-DHC (10(-)(9)-10(-)(6) M). Perfusion with carbenoxolone (CBX, 10(-)(6)-10(-)(3 )M) did not alter 11 beta-reduction of 11-DHC. In contrast, pretreatment with CBX in vivo (10 mg/day) for 7 days inhibited 11 beta-reductase (19+/-4% conversion, P<0.01). Concentrations of 11-DHC in male rat plasma were 44+/-6 nM. Thus 11 beta-HSD-1 is predominantly an 11 beta-reductase in the intact rat liver and is only inhibited by chronic administration of CBX. The substantial concentrations of plasma 11-DHC as substrate suggest that 11 beta-HSD-1 activity and its potential selective inhibition could modify glucocorticoid action in vivo.

The heterogeneous response of the bivascularly perfused rat liver to adenosine

The heterogeneity of the liver parenchyma in relation to the metabolic response to adenosine was investigated using the bivascularly perfused rat liver in the anterograde and retrograde modes. Adenosine was infused into livers from fed rats according to four experimental protocols: (A) anterograde perfusion, adenosine via the portal vein; (B) anterograde perfusion, adenosine via the hepatic artery; (C) retrograde perfusion, adenosine via the hepatic vein; and (D) retrograde perfusion, adenosine via the hepatic artery. Due to the very pronounced concentration gradients generated by metabolic transformation, the infused adenosine attained maximal concentrations in different regions with each experimental protocol. The sinusoidal mean transit times (t̄ s) were not changed by adenosine in anterograde perfusion, but were increased in retrograde perfusion. It was concluded that the vasoconstrictive elements are localized essentially in the presinusoidal region. Glucose release stimulation presented two kinetic components. The first one was rapid in both onset and decay with a peak around 30 sec; the second one developed more slowly (several minutes). The factors of the first kinetic component are possibly generated in the presinusoidal region or in the first periportal cells. The initial decrease in oxygen consumption seemed to be localized in the region just after the intrasinusoidal confluence of the ramifications of the portal vein and hepatic artery. Indomethacin decreased glucose release stimulation by adenosine in both anterograde and retrograde perfusion only when DMSO was the vehicle. The participation of eicosanoids in the generation of the effects of adenosine seems to be less important than hitherto believed.

Regional heterogeneities in the production of uric acid from adenosine in the bivascularly perfused rat liver.

The heterogeneity of the liver parenchyma in relation to uric acid production from adenosine was investigated using the bivascularly perfused rat liver in the anterograde and retrograde modes. Adenosine was infused in livers from fed rats during 20 min at four different concentrations (20, 50, 100 and 200 microM) according to four experimental protocols as follows: (A) anterograde perfusion, with adenosine infusion into the portal vein; (B) anterograde perfusion, with adenosine in the hepatic artery, (C) retrograde perfusion, with adenosine in the hepatic vein; (D) retrograde perfusion, with adenosine in the hepatic artery. With protocols A, B, and D uric acid production from adenosine was always characterized by initial bursts followed by progressive decreases toward smaller steady-states. With protocol C the initial burst was present only when 200 microM adenosine was infused. The initial bursts in uric acid production were accompanied by simultaneous increases in the ratio of uric acid production/adenosine uptake rate. These initial bursts are thus representing increments in the production of uric acid that are not corresponded by similar increments in the metabolic uptake rates of adenosine. Global analysis of uric acid production revealed that the final steady-state rates were approximately equal for all infusion rates with protocols A, B and C, but smaller with protocol D. This difference, however, can be explained in terms of the differences in accessible cellular spaces, which are much smaller when protocol D is employed. When the analysis was performed in terms of the extra amounts of uric acid produced during the infusion of adenosine, where the initial bursts are also taken into account, different dose-response curves were found for each experimental protocol. These differences cannot be explained in terms of the accessible cell spaces and they are likely to reflect regional heterogeneities. From the various dose-response curves and from the known characteristics of the microcirculation of the rat liver it can be concluded that the initial bursts in uric acid production are generated in periportal hepatocytes. The reason for this heterogeneity could be related to the metabolic effects of adenosine, especially to oxygen uptake inhibition, which is likely to produce changes in the ATP/AMP ratios.

Hepatic heterogeneity in the response to AMP studies in the bivascularly perfused rat liver.

The zonation of the purinergic action of AMP in the hepatic parenchyma was investigated in the bivascularly perfused rat liver by means of anterograde and retrograde perfusion. Livers from fed rats were used and AMP (100 microM) was infused according to four different experimental protocols: (A) anterograde perfusion and AMP infusion via the portal vein; (B) anterograde perfusion and AMP infusion via the hepatic artery; (C) retrograde perfusion and AMP via the hepatic vein; (D) retrograde perfusion and AMP via the hepatic artery. The response of the liver cells was heterogeneous. Oxygen uptake inhibition by AMP predominates in cells situated shortly after the intrasinusoidal confluence of the portal vein and hepatic artery. Oxygen consumption in all other cells seems to be increased by AMP. Glycogenolysis stimulation by AMP (glucose release) was more pronounced in the periportal cells situated in the region of the intrasinusoidal confluence of the portal vein and the hepatic artery. It can be concluded that the heterogenic response of the liver to AMP is similar to the heterogenic response to ATP.

Hepatic heterogeneity in the response to ATP studied in the bivascularly perfused rat liver

The zonation of the purinergic action of ATP in the hepatic parenchyma was investigated in the bivascularly perfused rat liver by means of anterograde and retrograde perfusion. Livers from fed rats were used, and ATP was infused according to four different experimental protocols: (A) anterograde perfusion and ATP infusion via the portal vein; (B) anterograde perfusion and ATP via the hepatic artery; (C) retrograde perfusion and ATP via the hepatic vein; (D) retrograde perfusion and ATP via the hepatic artery. The following metabolic parameters were measured: glucose release, lactate production and oxygen consumption. The hemodynamic effects were evaluated by measuring the sinusoidal mean transit times by means of the indicator-dilution technique. ATP was infused during 20 min at four different rates (between 0.06-0.77 µmol min-1 g liver-1; 20-200 µM) in each of the four experimental protocols.

Hepatic heterogeneity in the response to ATP studied in the bivascularly perfused rat liver.

The zonation of the purinergic action of ATP in the hepatic parenchyma was investigated in the bivascularly perfused rat liver by means of anterograde and retrograde perfusion. Livers from fed rats were used, and ATP was infused according to four different experimental protocols: (A) anterograde perfusion and ATP infusion via the portal vein; (B) anterograde perfusion and ATP via the hepatic artery; (C) retrograde perfusion and ATP via the hepatic vein; (D) retrograde perfusion and ATP via the hepatic artery. The following metabolic parameters were measured: glucose release, lactate production and oxygen consumption. The hemodynamic effects were evaluated by measuring the sinusoidal mean transit times by means of the indicator-dilution technique. ATP was infused during 20 min at four different rates (between 0.06-0.77 micromol min[-1] g liver[-1]; 20-200 microM) in each of the four experimental protocols. The results that were obtained allow several conclusions with respect to the localization of the effects of ATP along the hepatic acini: (1) In retrograde perfusion the sinusoidal mean transit times were approximately twice those observed in anterograde perfusion. ATP increased the sinusoidal mean transit times only in retrograde perfusion (protocols C and D). The effect was more pronounced with protocol D. These results allow the conclusion that the responsive vasoconstrictive elements are localized in a pre-sinusoidal region; (2) All hepatic cells, periportal as well as perivenous, were able to metabolize ATP, so that concentration gradients were generated with all experimental protocols. Extraction of ATP was more pronounced in retrograde perfusion, an observation that can be attributed, partly at least, to the longer sinusoidal transit times. In anterograde perfusion, the extraction of ATP was time-dependent, a phenomenon that cannot be satisfactorily explained with the available data; (3) ATP produced a transient initial inhibition of oxygen uptake when protocols A and B were employed. These protocols are the only ones in which the cells situated shortly after the intrasinusoidal confluence of the portal vein and the hepatic artery were effectively supplied with ATP. The decrease in oxygen consumption was more pronounced at low ATP infusions when protocol B was employed. These observations allow the conclusion that the former phenomenon is localized mainly in cells situated shortly after the intrasinusoidal confluence of the portal vein and hepatic artery. Oxygen consumption in all other cells, especially the proximal periportal ones, is increased by ATP; (4) In agreement with previous data found in the literature, glycogenolysis stimulation by ATP was more pronounced in the periportal region. The cells that respond more intensively are not the proximal periportal ones, but those situated in the region of the intrasinusoidal confluence of the portal vein and the hepatic artery.

Production, Uptake, and Metabolic Effects of Cyclic AMP in the Bivascularly Perfused Rat Liver

Production, uptake, and metabolic effects of cyclic AMP (cAMP) were measured in the bivascularly perfused rat liver in anterograde and retrograde perfusion. Glucagon, cAMP, N 6,2′- O-dibutyryl cAMP and N 6-monobutyryl cAMP were infused into the portal vein (anterograde perfusion), the hepatic vein (retrograde perfusion), or the hepatic artery (anterograde and retrograde perfusion) in order to reach different cell populations. The following results were obtained: (1) cAMP release caused by glucagon was directly proportional to the cell spaces that were accessible via the hepatic artery in anterograde and retrograde perfusion; since the metabolic effects of glucagon were not proportional to the accessible cell spaces, this observation also implies a disproportion between cAMP release and metabolic effects of the hormone; (2) when cAMP and N 6,2′- O-dibutyryl cAMP were given to all liver cells (e.g. when infused into the portal vein), their metabolic effects were qualitatively and quantitatively the same and qualitatively equal to the effects of glucagon; (3) the changes caused by cAMP were a function of the cell spaces that can be reached via the hepatic artery in anterograde and retrograde perfusion; this behaviour contrasts markedly with that of glucagon, whose metabolic effects were practically independent of the accessible cell spaces; and (4) the effects of N 6,2′- O-dibutyryl cAMP and N 6-monobutyryl cAMP were independent of the cell spaces that were accessible via the hepatic artery in anterograde and retrograde perfusion; in this respect their behaviour was equal to that of glucagon. It is apparent that exogenously added cAMP mimicked the metabolic effects of glucagon in the liver only when it was supplied to all liver cells. Since glucagon, N 6,2′- O-dibutyryl cAMP, and N 6-monobutyryl cAMP were able to produce a full response even when given to only 30% of the liver parenchyma, it was concluded that cAMP production under the stimulus of glucagon or in consequence of the metabolic transformation of N 6,2′- O-dibutyryl cAMP and N 6-monobutyryl cAMP occurs in a compartment to which exogenous cAMP has no access. cAMP generated within this compartment is possibly able to diffuse from cell to cell.