Stimulation of glycogenolysis and vasoconstriction in the perfused rat liver by the thromboxane A2 analogue U-46619

Infusion of the thromboxane A2 analogue U-46619 into isolated perfused rat livers resulted in dose-dependent increases in glucose output and portal vein pressure, indicative of constriction of the hepatic vasculature. At low concentrations, e.g. less than or equal to 42 ng/ml, glucose output occurred only during agonist infusion; whereas at concentrations greater than or equal to 63 ng/ml, a peak of glucose output also was observed upon termination of agonist infusion coincident with relief of hepatic vasoconstriction. Effluent perfusate lactate/pyruvate and beta-hydroxybutyrate/acetoacetate ratios increased significantly in response to U-46619 infusion. Hepatic oxygen consumption increased at low U-46619 concentrations (less than or equal to 20 ng/ml) and became biphasic with a transient spike of increased consumption followed by a prolonged decrease in consumption at higher concentrations. Increased glucose output in response to 42 ng/ml U-46619 was associated with a rapid activation of glycogen phosphorylase, slight increases in tissue ADP levels, and no increase in cAMP. At 1000 ng/ml, U-46619 activation of glycogen phosphorylase was accompanied by significant increases in tissue levels of AMP and ADP, decreases in ATP, and slight increases in cAMP. In isolated hepatocytes, U-46619 did not stimulate glucose output or activate glycogen phosphorylase. Reducing the perfusate calcium concentration from 1.25 to 0.05 mM resulted in a marked reduction of the glycogenolytic response to U-46619 (42 ng/ml) with no efflux of calcium from the liver. U-46619-induced glucose output and vasoconstriction displayed a similar dose dependence upon the perfusate calcium concentration. Thus, U-46619 exerts a potent agonist effect on glycogenolysis and vasoconstriction in the perfused rat liver. The present findings support the concept that U-46619 stimulates hepatic glycogenolysis indirectly via vasoconstriction-induced hypoxia within the liver.     

Stimulation of hepatic glycogenolysis by acetylglyceryl ether phosphorylcholine

1-O-Alkyl-2-acetyl-sn-glyceryl 3-phosphorylcholine or acetylglyceryl ether phosphorylcholine (AGEPC) stimulated glycogenolysis in perfused livers from fed rats at concentrations as low as 10(-11) M. At the lower AGEPC concentrations, e.g. 2 X 10(-10) M, a single transient phase of enhanced hepatic glucose output was elicited upon infusion of this agonist. At higher concentrations, e.g. 2 X 10(-8) M, a sharp transient spike of glucose output was observed, followed by a stable elevated steady state rate of glucose output until the AGEPC infusion was terminated. Increased rates of lactate and acetoacetate output and a diminished hepatic oxygen consumption were characteristic of the response of the livers to AGEPC at 2 X 10(-10) M. Neither alpha- nor beta-adrenergic antagonists blocked the glycogenolytic response of AGEPC. Repeated infusion of AGEPC led to homologous desensitization of the response, but the response of the liver to the alpha-adrenergic agonist, phenylephrine, or to glucagon, subsequent to AGEPC stimulation, was unaffected. Increasing the period of perfusion between successive additions of AGEPC, from 7 to 30 min, resulted in an increased glycogenolytic response to this agonist. When the perfusate calcium concentration was reduced from 1.25 to 0.05 mM, the glycogenolytic response to AGEPC was markedly diminished; calcium efflux from the liver following stimulation with AGEPC was not observed. The data presented in this study illustrate a potent agonist effect of AGEPC on the glycogenolytic system in the rat liver.     

Mobilization of hepatic calcium pools by platelet activating factor

In the perfused rat liver, platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), infusion produces an extensive but transient glycogenolytic response which at low AGEPC concentrations (i.e., 10(-11) M) is markedly dependent upon the perfusate calcium levels. The role of calcium in the glycogenolytic response of the liver to AGEPC was investigated by assessing the effect of AGEPC on various calcium pools in the intact liver. Livers from fed rats were equilibrated with 45Ca2+, and the kinetics of 45Ca2+ efflux were determined in control, AGEPC-stimulated, and phenylephrine-stimulated livers during steady-state washout of 45Ca2+. AGEPC treatment had only a slight if any effect on the pattern of steady-state calcium efflux from the liver, as opposed to major perturbations in the pattern of calcium efflux effected by the alpha-adrenergic agonist phenylephrine. Infusion of short pulses of AGEPC during the washout of 45Ca2+ from labeled livers caused a transient release of 45Ca2+ which was not abolished at low calcium concentrations in the perfusate. Moreover, there occurred no appreciable increase in the total calcium content in the liver perfusate at either high or low concentrations of calcium in the perfusion fluid. Infusion of latex beads, which are removed by the reticuloendothelial cells, caused the release of hepatic 45Ca2+ in a fashion similar to the case with AGEPC. Our findings indicate that AGEPC does not perturb a major pool of calcium within the liver as occurs upon alpha-adrenergic stimulation; it is likely that AGEPC mobilizes calcium from a smaller yet very important pool, very possibly from nonparenchymal cells in the liver.     

Stimulation of uric acid release from the perfused rat liver by platelet activating factor or potassium.

The stimulation of hepatic glycogenolysis by platelet activating factor (AGEPC) or increased perfusate potassium concentration ([K+]o), but not phenylephrine, causes a transient increase in uric acid release into the effluent perfusate of perfused rat livers. Uric acid was identified in chromatograms of perfusate samples using reversed-phase h.p.l.c., which show a peak which co-elutes with authentic uric acid, and by the fact that the A293 of perfusate samples decreases in the presence of uricase. Uric acid release is dose-dependent with respect to both AGEPC and [K+]o, and is blocked completely by prior exposure of the perfused liver to 5 mM-allopurinol, a specific inhibitor of xanthine oxidase (XOD). Allopurinol inhibits the increase in portal vein pressure induced by AGEPC, increased [K+]o or phenylephrine; the inhibitory effect increases with increasing concentrations of the agents. Also, allopurinol inhibits the second phase of O2 uptake and glucose release characteristic of concentrations of AGEPC or increased [K+]o equal to or greater than their reported half-maximal concentration for glucose release. The ratio of xanthine dehydrogenase (XDH) to XOD activity in extracts of freeze-clamped perfused livers is not affected by treatment of the livers with AGEPC or increased [K+]o. The results suggest that uric acid production may be an indicator of ischaemia within localized hepatic sinusoids, and that allopurinol partially protects the hepatocyte from the effects of AGEPC or increased [K+]o by inhibiting XOD-dependent superoxide production. We propose that the second phase of the glycogenolytic response to these agents results from ischaemia and subsequent reperfusion. Activation of XOD in vivo and hence O2-derived free radical production may be involved in the response of the liver to vasoactive agonists under a variety of pathophysiological conditions.     

Effects of beta-adrenergic stimulation on 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine-mediated vasoconstriction and glycogenolysis in the perfused rat liver

The beta-adrenergic agonist isoproterenol inhibited the glycogenolytic response of platelet-activating factor (AGEPC, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) in perfused livers derived from fed rats. AGEPC-stimulated hepatic vasoconstriction, measured by increases in portal vein pressure, also was inhibited by prior isoproterenol infusion. Isoproterenol-mediated inhibition of these hepatic responses to AGEPC was not apparent when isoproterenol (10 microM) was coinfused with the beta-receptor antagonist propranolol (75 microM) or when isoproterenol was replaced with the alpha-adrenergic agonist phenylephrine (10 microM). alpha-Agonist-induced glycogenolysis and vasoconstriction in the perfused liver was unaffected by isoproterenol infusion. Glucagon (2.3 nM) had no effect on the glycogenolytic or vasoconstrictive responses of the liver to AGEPC despite the fact that glucagon increased hepatic cAMP levels to a far greater extent than isoproterenol. Additionally, inhibition of the hepatic responses to AGEPC by isoproterenol occurred in perfused livers from mature rats (i.e. greater than 300 g) in which liver parenchymal cells lack functional beta-adrenergic receptors. The data presented in this study illustrate a specific inhibition of AGEPC-induced hepatic glycogenolysis and vasoconstriction by beta-adrenergic stimulation of the perfused liver. This inhibition appears to be mediated by interaction of isoproterenol with nonparenchymal cells within the liver. These findings are consistent with the concept that AGEPC stimulates hepatic glycogenolysis by an indirect mechanism involving hepatic vasoconstriction.     

Effects of nitric oxide on platelet-activating factor- and alpha-adrenergic-stimulated vasoconstriction and glycogenolysis in the perfused rat liver.

Effects of nitric oxide (NO) on hemodynamic and glycogenolytic responses to platelet-activating factor (PAF) and phenylephrine were investigated in perfused livers derived from fed rats. Infusion of NO (34 microM) into perfused livers inhibited PAF (0.22 nM)-induced increases in hepatic glucose output and portal pressure approximately 90 and 85%, respectively, and abolished effects of PAF on hepatic oxygen consumption. NO attenuated PAF-stimulated increases in glucose output and portal pressure, the latter indicative of hepatic vasoconstriction, with a similar dose dependence with an IC50 of approximately 8 microM. In contrast to its effects on PAF-induced responses in the perfused liver, NO inhibited increases in hepatic portal pressure in response to phenylephrine (10 microM) approximately 75% without altering phenylephrine-stimulated glucose output and oxygen consumption. Similarly, infusion of NO into perfused livers significantly inhibited increases in hepatic portal pressure but not in glucose output in response to a submaximal concentration of phenylephrine (0.4 microM). Like NO, sodium nitroprusside (83 microM) significantly inhibited hemodynamic but not glycogenolytic responses to phenylephrine in perfused livers. However, PAF (0.22 nM)-stimulated alterations in hepatic portal pressure, glucose output, and oxygen consumption were unaffected by infusion of sodium nitroprusside (83 microM) into perfused livers. These results provide the first evidence for regulatory effects of NO in the perfused liver and support the contention that PAF, unlike phenylephrine, stimulates glycogenolysis by mechanisms secondary to hepatic vasoconstriction. These observations raise the intriguing possibility that NO may act in liver to regulate hemodynamic responses to vasoactive mediators.     

Activation of hepatic glycogenolysis by phagocytic stimulation

Infusion of latex beads into isolated perfused rat livers transiently increased glucose output, perfusate lactate/pyruvate ratio and portal vein pressure, mimicking hepatic effects of heat-aggregated IgG (HAG). Indomethacin attenuated hepatic responses to latex beads, and extracellular calcium was required for full expression of hepatic responses. Prior infusion of HAG inhibited the glycogenolytic response to latex beads, supporting a common mechanism of action for the two agents.     

Stimulation of glycogenolysis by adenine nucleotides in the perfused rat liver.

Infusion of adenine nucleotides and adenosine into perfused rat livers resulted in stimulation of hepatic glycogenolysis, transient increases in the effluent perfusate [3-hydroxybutyrate]/[acetoacetate] ratio, and increased portal vein pressure. In livers perfused with buffer containing 50 microM-Ca2+, transient efflux of Ca2+ was seen on stimulation of the liver with adenine nucleotides or adenosine. ADP was the most potent of the nucleotides, stimulating glucose output at concentrations as low as 0.15 microM, with half-maximal stimulation at approx. 1 microM, and ATP was slightly less potent, half-maximal stimulation requiring 4 microM-ATP. AMP and adenosine were much less effective, doses giving half-maximal stimulation being 40 and 20 microM respectively. Non-hydrolysed ATP analogues were much less effective than ATP in promoting changes in hepatic metabolism. ITP, GTP and GDP caused similar changes in hepatic metabolism to ATP, but were 10-20 times less potent than ATP. In livers perfused at low (7 microM) Ca2+, infusion of phenylephrine before ATP desensitized hepatic responses to ATP. Repeated infusions of ATP in such low-Ca2+-perfused livers caused homologous desensitization of ATP responses, and also desensitized subsequent Ca2+-dependent responses to phenylephrine. A short infusion of Ca2+ (1.25 mM) after phenylephrine infusion restored subsequent responses to ATP, indicating that, during perfusion with buffer containing 7 microM-Ca2+, ATP and phenylephrine deplete the same pool of intracellular Ca2+, which can be rapidly replenished in the presence of extracellular Ca2+. Measurement of cyclic AMP in freeze-clamped liver tissue demonstrated that adenosine (150 microM) significantly increased hepatic cyclic AMP, whereas ATP (15 microM) was without effect. It is concluded that ATP and ADP stimulate hepatic glycogenolysis via P2-purinergic receptors, through a Ca2+-dependent mechanism similar to that in alpha-adrenergic stimulation of hepatic tissue. However, adenosine stimulates glycogenolysis via P1-purinoreceptors and/or uptake into the cell, at least partially through a mechanism involving increase in cyclic AMP. Further, the hepatic response to adenine nucleotides may be significant in regulating hepatic glucose output in physiological and pathophysiological states.     

Platelet-activating factor-stimulated hepatic glycogenolysis is not mediated through cyclooxygenase-derived metabolites of arachidonic acid

Platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)) is a potent lipid mediator which stimulates hepatic glycogenolysis, causes hepatic vasoconstriction, and stimulates the production of cyclooxygenase-derived metabolites of arachidonic acid, primarily prostaglandin (PG) D2 in the perfused liver. Following infusion of platelet-activating factor (1 nM) in the perfused rat liver the production of PGD2, measured in the effluent perfusate, increased 4-fold after only 2 min. Infusion of the cyclooxygenase inhibitor, ibuprofen (50 microM), abolished the stimulated production of PGD2 and thromboxane B2 in response to AGEPC without significantly affecting the hepatic glycogenolytic or vasoconstrictive responses to AGEPC. Contrary to previous reports, these observations do not support the suggestion that cyclooxygenase-derived metabolites mediate directly either the glycogenolytic or the vasoactive effects of AGEPC in the perfused rat liver.     

Hepatocyte heterogeneity in response to icosanoids. The perivenous scavenger cell hypothesis

1. The metabolic and hemodynamic effects of prostaglandin F2 alpha, leukotriene C4 and the thromboxane A2 analogue U-46619 were studied during physiologically antegrade (portal to hepatic vein) and retrograde (hepatic to portal vein) perfusion and in a system of two rat livers perfused in sequence. 2. The stimulatory effects of prostaglandin F2 alpha (3 microM) on hepatic glucose release, perfusion pressure and net Ca2+ release were diminished by 77%, 95% and 64%, respectively, during retrograde perfusion when compared to the antegrade direction, whereas the stimulation of 14CO2 production from [1-14C]glutamate by prostaglandin F2 alpha (which largely reflects the metabolism of perivenous hepatocytes) was lowered by only 20%. Ca2+ mobilization and glucose release from the liver comparable to that seen during antegrade perfusion could also be observed in retrograde perfusions; however, higher concentrations of the prostaglandin were required. 3. The glucose, Ca2+ and pressure response to leukotriene C4 (20 nM) or the thromboxane A2 analogue U-46619 (200 nM) of livers perfused in the antegrade direction were diminished by about 90% during retrograde perfusion. Sodium nitroprusside (20 microM) decreased the pressure response to leukotriene C4 (20 nM) and U-46619 (200 nM) by about 40% and 20% in antegrade perfusions, respectively, but did not affect the maximal increase of glucose output. 4. When two livers were perfused antegradely in series, such that the perfusate leaving the first liver (liver I) entered a second liver (liver II), infusion of U-46619 at concentrations below 200 nM to the influent perfusate of liver I increased the portal pressure of liver I, but not of liver II. At higher concentrations of U-46619 there was also an increase of the portal pressure of liver II and with concentrations above 800 nM the pressure responses of both livers were near-maximal [19.6 +/- 0.8 (n = 7) cm H2O and 16.5 +/- 1.1 (n = 8) cm H2O for livers I and II, respectively]. There was a similar behaviour of glucose release from livers I and II in response to U-46619 infusion. When liver I was perfused in the retrograde direction, a significant pressure or glucose response of liver II (antegrade perfusion) could not be observed even with U-46619 concentrations up to 1000 nM. 5. Similarly, the perfusion pressure increase and glucose release induced by leukotriene C4 (10 nM) observed with liver II was only about 20% of that seen with liver I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of glycogenolysis and vasoconstriction by adenosine and adenosine analogues in the perfused rat liver.

Infusion of adenosine into perfused rat livers resulted in transient increases in glucose output, portal-vein pressure, the effluent perfusate [lactate]/[pyruvate] ratio, and O2 consumption. 8-Phenyltheophylline (10 microM) inhibited adenosine responses, whereas dipyridamole (50 microM) potentiated the vasoconstrictive effect of adenosine. The order of potency for adenosine analogues was: 5'-N-ethylcarboxamidoadenosine (NECA) greater than L-phenylisopropyladenosine greater than cyclohexyladenosine greater than D-phenylisopropyladenosine greater than 2-chloroadenosine greater than adenosine, consistent with adenosine actions modulated through P1-purine receptors of the A2-subtype. Hepatic responses exhibited homologous desensitization in response to repeated infusion of adenosine. Adenosine effects on the liver were attenuated at lower perfusate Ca2+ concentrations. Indomethacin decreased hepatic responses to both adenosine and NECA. Whereas adenosine stimulated glycogen phosphorylase activity in isolated hepatocytes, NECA caused no effect in hepatocytes. The response to adenosine in hepatocytes was inhibited by dipyridamole (50 microM), but not 8-phenyltheophylline (10 microM). The present study indicates that, although adenosine has direct effects on parenchymal cells, indirect effects of adenosine, mediated through the A2-purinergic receptors on another hepatic cell type, appear to play a role in the perfused liver.     

Acetylglyceryl ether phosphorylcholine. A potent activator of hepatic phosphoinositide metabolism and glycogenolysis

Acetylglyceryl ether phosphorylcholine (AGEPC), or 1-O-Alkyl-2-acetyl-sn-glyceryl 3-phosphorylcholine, has been shown to have a dramatic influence on phosphoinositide metabolism in isolated rat hepatocytes and upon glycogenolysis in the intact perfused rat liver. Addition of 5 X 10(-10) M AGEPC to 32Pi-labeled rat hepatocytes resulted in up to a 30 to 40% decrease in the [32Pi]phosphatidylinositol 4,5-bisphosphate within 10 s. The 32P content of phosphatidylinositol 4-phosphate decreased approximately 25% within 60 s, while a 5 to 8% decrease in [32P]phosphatidylinositol was observed only after 2 to 5 min of incubation of hepatocytes with AGEPC. Infusion of AGEPC (2 X 10(-10) M) into perfused livers resulted in a 3-fold increase in the glucose output in the effluent perfusate within 2 min. Interestingly, when a 500-fold higher concentration, i.e. 1 X 10(-7) M, of 1-O-alkyl-sn-glyceryl 3-phosphorylcholine or the stereoisomer 3-O-alkyl-2-acetyl-sn-glyceryl 1-phosphorylcholine was infused, no increase in the hepatic glucose output was seen. These observations lead to the conclusion that AGEPC exerts a potent influence on the polyphosphoinositide metabolism and glycogenolysis in rat liver and establishes the liver as an ideal system in which to conduct a detailed inquiry into the biochemical mechanism(s) responsible for the biological action of this unusual phospholipid.     

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.     

Role of extracellular calcium and calcium efflux in the activation of hepatic glycogenolysis by calcium-dependent hormones

The role of extracellular calcium in the glycogenolytic effects of calcium-dependent hormones was examined in a rat liver perfusion system. Decreasing the perfusate CaCl2 concentration resulted in a concentration-dependent inhibition of glucose output by maximal concentrations of vasopressin (20 nM) and angiotensin II (10 nM), but not of glucagon (1.4 nM), cyclic AMP (100 microM), dibutyryl cyclic AMP (10 microM) or phenylephrine (5 microM). However, the effect of phenylephrine was inhibited when livers were perfused with CaCl2-free perfusate containing 0.5 mM EGTA in a duration-dependent manner. These effects were exerted through the inhibition of the maximal response of each hormone, and were associated with a parallel decrease in phosphorylase activation but not with changes in tissue cyclic AMP concentrations. When livers were preloaded with 45Ca for 45 min and then washed for either 15 min or 45 min, these hormones elicited a rapid and transient 45Ca efflux regardless of the perfusate calcium concentration. The sequential perfusion of two hormones resulted in the loss of 45Ca efflux by the second hormone. These results suggest that the glycogenolytic effects of vasopressin and angiotensin II depend on the extracellular calcium and that of phenylephrine primarily on the cellular calcium. It was also demonstrated that these calcium-dependent hormones mobilize calcium from the same pools. However, the mobilization of cellular calcium does not necessarily correlate directly with the glycogenolytic actions of vasopressin and angiotensin II.     

Presinusoidal vessels predominantly contract in response to norepinephrine, histamine, and KCl in rabbit liver.

In rabbit livers, it is not well known which segments of the hepatic vasculature are predominantly contracted by various vasoconstrictors. We determined effects of histamine, norepinephrine, and KCl on hepatic vascular resistance distribution in isolated rabbit livers perfused via the portal vein with 5% albumin-Krebs solution at a constant flow rate. Hepatic capillary pressure was measured by double vascular occlusion pressure (Pdo) and was used to determine portal (Rpv) and hepatic venous (Rhv) resistances. A bolus injection of either histamine or norepinephrine dose-dependently increased portal venous pressure but not Pdo, resulting in a dose-dependent increase in Rpv and no changes in Rhv. KCl (50 mM), when injected in anterogradely perfused livers, contracted the presinusoidal vessels selectively with liver weight loss. Although KCl significantly increased Rhv in retrogradely perfused livers, the increase in Rpv by 400% of baseline predominated over the increase in Rhv by 85% of baseline. In the retrogradely perfused livers, KCl produced an initial liver weight loss followed by a profound weight gain. We conclude that histamine and norepinephrine selectively contract the presinusoidal vessels. The results on KCl effects suggest that this selective presinusoidal constriction might be possibly due to predominant distribution of functionally active vascular smooth muscle in the presinusoidal vessels rather than the hepatic vein in rabbit livers.     

Roles of anandamide in the hepatic microcirculation in cirrhotic rats

Cannabinoids have been reported to participate in the pathogenesis of peripheral vasodilatation in cirrhosis. However, their roles in increased intrahepatic resistance (IHR) in cirrhotic livers are unknown. We aimed to investigate the effects of cannabinoids in the hepatic microcirculation of cirrhotic rats produced by bile duct ligation. In isolated liver perfusion, portal perfusion pressure (PPP) and the production of eicosanoids in the perfusate were measured. In addition, various hepatic protein levels [cyclooxygenase (COX) isoform and 5-lipoxygenase (5-LOX)] were also determined. Finally, concentration-response curves for PPP and the corresponding production of eicosanoids in response to anandamide (1.44 x 10(-10)-1.44 x 10(-3) M) after indomethacin (COX inhibitor), piriprost (5-LOX inhibitor), or furegrelate (thromboxane A(2) synthase inhibitor) preincubation were obtained. The study showed that cirrhotic livers had significantly higher levels of PPP, COX-2 and 5-LOX protein expression, and production of thromboxane B(2) (TXB(2)) and cysteinyl leukotrienes (Cys-LTs) than normal livers. Anandamide induced a dose-dependent increase in PPP in both normal and cirrhotic livers. The anandamide-induced increase in PPP was found concomitantly with a significant increase in TXB(2) and Cys-LT production in the perfusate. In response to anandamide administration, cirrhotic livers exhibited a significantly greater increase in IHR and production of TXB(2) and Cys-LTs than normal livers. Indomethacin and furegrelate, but not piriprost, significantly ameliorated the anandamide-induced increase in IHR in cirrhotic livers. In conclusion, anandamide plays, in part, an important role in increased IHR of cirrhotic livers. The anandamide-induced increase in IHR in cirrhotic livers may be mediated by increased COX-derived eicosanoid (mainly thromboxane A(2)) production.     

Aggravation of chemically-induced injury in perfused rat liver by extracellular ATP

The effects of purinergic receptor agonists on acute liver damage and hemodynamics were studied using chemically-induced liver injury. Rat livers were perfused in situ 24 h after treatment with D-galactosamine (800 mg/kg, i.p.). In these livers, infusion of ATP (50 microM) into the portal vein caused a rapid increase in the leakage of LDH and AST from perfused liver in a dose dependent manner, accompanied with flow reduction. The similar but less effective responses were also observed by the infusion of ADP. Infusion of adenosine, a P1-receptor agonist, induced only minimal changes of liver damage and flow rate. The ATP-induced changes were almost completely suppressed by P2-receptor antagonist, suramin, but not affected by P1-receptor antagonist, 8-phenyltheophylline. Pretreatment of rats with gadolinium chloride, which depletes Kupffer cells, did not inhibit the potentiation of liver damage caused by ATP, whereas hemodynamic effects of ATP were significantly attenuated by gadolinium. These results indicate that extracellular ATP aggravates acute liver injury mediated by P2-type purinergic receptors.     

Endothelin, a potent peptide agonist in the liver

Endothelin, a peptide mediator produced by vascular endothelial cells, caused sustained vasoconstriction of the portal vasculature in the perfused rat liver. The vasoactive effect of endothelin was accompanied by increased glycogenolysis and alterations in hepatic oxygen consumption. The endothelin-induced increase in the portal pressure was concentration-dependent with an EC50 of 1 nM. Endothelin-induced hepatic glycogenolysis was dose-dependent but exhibited a different EC50 than for the vasoconstrictive effects of endothelin. Hepatic vasoconstriction and glycogenolysis following endothelin infusion were inhibited when Ca2+ was removed from the perfusion medium. The endothelin-induced responses in the liver were not altered by prior infusion of phenylephrine (alpha-adrenergic agonist), isoproterenol (beta-adrenergic agonist), angiotensin II, glucagon, platelet-activating factor, or the platelet-activating factor antagonist, BN52021. However, repeated infusion of endothelin resulted in desensitization of the glycogenolytic response but was without a significant effect on hepatic vasoconstriction. Endothelin also stimulated metabolism of inositol phospholipids in isolated hepatocytes and Kupffer cells in primary culture. The present experiments demonstrate, for the first time, that endothelin is a very potent agonist in the liver eliciting both a sustained vasoconstriction of the hepatic vasculature and a significant increase in hepatic glucose output.     

Regulation of hepatic parenchymal and non-parenchymal cell function by the diadenine nucleotides Ap3A and Ap4A

The diadenine nucleotides diadenosine 5',5"-P1,P3-triphosphate (Ap3A) and diadenosine 5',5"-P1,P4-tetraphosphate (Ap4A) can be released from platelets and were shown to act as long-lived signal molecules. Accordingly, we studied their potential effect on hepatic metabolism. In isolated perfused rat liver, Ap3A and Ap4A increase the portal pressure, lead to a transient net release of Ca2+, complex net K+ movement across the liver plasma membrane and stimulate hepatic glucose output and 14CO2 production from [1-14C]glutamate. These responses resemble that obtained with extracellular ATP. This and studies on the additivity of ATP and Ap4A effects suggest similar mechanisms mediating the ATP and diadenine nucleotide effects in the liver. Ap3A and Ap4A increased the activity of glycogen phosphorylase a in isolated hepatocyte suspensions by about 100%, pointing to a direct effect of these nucleotides on hepatic parenchymal cells. A response of hepatic non-parenchymal cells to diadenine nucleotide infusion is suggested by a marked stimulation of thromboxane and prostaglandin D2 release from perfused liver. Studies with the thromboxane A2 receptor antagonist BM 13.177 (20 microM) show that the pressure and glucose response to the diadenine nucleotides is partially mediated by this thromboxane formation. Studies with retrograde and sequential liver perfusions suggest a less efficient degradation of the diadenine nucleotides during a single liver passage compared to extracellular ATP. The data suggest that Ap3A and Ap4A are potential regulators of hepatic hemodynamics and metabolism, involving complex interactions between hepatic parenchymal cells and hepatic non-parenchymal cells, including eicosanoids as signal molecules.     

Stimulation of hepatic glycogenolysis by phorbol 12-myristate 13-acetate.

In isolated perfused rat livers, infusion of phorbol 12-myristate 13-acetate (PMA) (150 nM) resulted in a 3-fold stimulation of the rate of glucose production. This response was maximal at a perfusate PMA concentration of 150 nM, and was significantly diminished at higher concentrations of PMA (e.g. 300 nM). Stimulation of glycogenolysis by PMA was greatly decreased in livers perfused with Ca2+-free medium. PMA infusion into livers perfused in the absence of Ca2+ did not result in Ca2+ efflux from the livers. Additionally, in hepatocytes isolated from livers of fed rats, neither PMA nor 1-oleoyl-2-acetyl-rac-glycerol stimulated the rate of glucose production. Although indomethacin has been demonstrated to block PMA-stimulated hepatic glycogenolysis [Garcia-Sainz & Hernandez-Sotomayor (1985) Biochem. Biophys. Res. Commun. 132, 204-209], infusion of PMA into perfused rat livers did not alter the rates of production of either prostaglandin E2 or 6-oxo-prostaglandin F1 alpha in the livers. These data, along with the observed increases in the perfusion pressure and decrease in O2 consumption in isolated perfused livers suggest that phorbol-ester-stimulated glycogenolysis is not a consequence of a direct effect of phorbol ester on liver parenchymal cells.