Developmental changes of purinergic control of intestinal motor activity during metamorphosis in the African clawed frog, Xenopus laevis

Little is known about the purinergic regulation of intestinal motor activity in amphibians. Purinergic control of intestinal motility is subject to changes during development in mammals. The aim of this study was to investigate purinergic control of intestinal smooth muscle in the amphibian Xenopus laevis and explore possible changes in this system during the developmental phase of metamorphosis. Effects of purinergic compounds on mean force and contraction frequency in intestinal circular muscle strips from prometamorphic, metamorphic, and juvenile animals were investigated. Before metamorphosis, low concentrations of ATP reduced motor activity, whereas the effects were reversed at higher concentrations. ATP-induced relaxation was not inhibited by the P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) but was blocked by the ecto-nucleotidase inhibitor 6-N,N-diethyl-d-beta,gamma-dibromomethylene ATP (ARL67256), indicating that an ATP-derived metabolite mediated the relaxation response at this stage. Adenosine induced relaxation before, during, and after metamorphosis, which was blocked by the A(1)-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The stable ATP-analog adenosine 5'-[gamma-thio]-triphosphate (ATPgammaS) and 2-methylthioATP (2-MeSATP) elicited contractions in the circular muscle strips in prometamorphic tadpoles. However, in juvenile froglets, 2-MeSATP caused relaxation, as did ATPgammaS at low concentrations. The P2Y(11)/P2X(1)-receptor antagonist NF157 antagonized the ATPgammaS-induced relaxation. The P2X-preferring agonist alpha-beta-methyleneadenosine 5'-triphosphate (alpha-beta-MeATP) evoked PPADS-sensitive increases in mean force at all stages investigated. This study demonstrates the existence of an adenosine A(1)-like receptor mediating relaxation and a P2X-like receptor mediating contraction in the X. laevis gut before, during, and after metamorphosis. Furthermore, the development of a P2Y(11)-like receptor-mediated relaxation during metamorphosis is shown.     

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.     

Skeletal muscle hexokinase: regulation in mammalian hibernation

The perfused rat liver responds in several ways to NAD⁺ infusion (20–100 μM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption and gluconeogenesis are some of the effects that were observed. Extracellular NAD⁺ is also extensively transformed in the liver. The purpose of the present work was to determine the main products of extracellular NAD⁺ transformation under various conditions and to investigate the possible contribution of these products for the metabolic effects of the parent compound. The experiments were done with the isolated perfused rat liver. The NAD⁺ transformation was monitored by HPLC. Confirming previous findings, the single-pass transformation of 100 μM NAD⁺ ranged between 75% at 1.5 min after starting infusion to 95% at 8 min. The most important products of single-pass NAD⁺ transformation appearing in the outflowing perfusate were nicotinamide, ADP-ribose, uric acid, and inosine. The relative proportions of these products presented some variations with the time after initiation of NAD⁺ infusion and the perfusion conditions, but ADP-ribose was always more abundant than uric acid and inosine. Cyclic ADP-ribose (cADP-ribose) as well as adenosine were not detected in the outflowing perfusate. The metabolic effects of ADP-ribose were essentially those already described for NAD⁺. These effects were sensitive to suramin (P2_XY purinergic receptor antagonist) and insensitive to 3,7-dimethyl-1-(2-propargyl)-xanthine (A2 purinergic receptor antagonist). Inosine, a known purinergic A3 agonist, was also active on metabolism, but uric acid and nicotinamide were inactive. It was concluded that the metabolic and hemodynamic effects of extracellular NAD⁺ are caused mainly by interactions with purinergic receptors with a highly significant participation of its main transformation product ADP-ribose.     

31P nuclear magnetic resonance of metabolic changes associated with cyanide intoxication in the perfused rat liver.

Metabolic changes associated with cyanide intoxication were observed for the first time in perfused rat liver using ³¹P nuclear magnetic resonance (NMR) at 60.7 MHz. Well-oxygenated control livers showed strong ATP peaks and little discernable internal orthophosphate (Pi). Perfusion with 2 mM cyanide eliminated the observable ATP peaks and caused internal Pi to increase. Despite clear evidence for ATP hydrolysis, resonances from cytoplasmic ADP were conspicuously absent. Resumption of perfusion with cyanide-free buffer caused a dramatic return of the ATP peaks with a concomitant fall in internal Pi. These metabolic changes are consistent with reversible binding of cyanide to mitochondrial cytochromes and their observation by ³¹P NMR indicates the potential of this method for studying metabolism in whole, perfused rat liver under physiologic conditions.     

Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells

Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X or P2Y receptors. Here we show that allergen challenge causes acute accumulation of ATP in the airways of asthmatic subjects and mice with experimentally induced asthma. All the cardinal features of asthma, including eosinophilic airway inflammation, Th2 cytokine production and bronchial hyper-reactivity, were abrogated when lung ATP levels were locally neutralized using apyrase or when mice were treated with broad-spectrum P2-receptor antagonists. In addition to these effects of ATP in established inflammation, Th2 sensitization to inhaled antigen was enhanced by endogenous or exogenous ATP. The adjuvant effects of ATP were due to the recruitment and activation of lung myeloid dendritic cells that induced Th2 responses in the mediastinal nodes. Together these data show that purinergic signaling has a key role in allergen-driven lung inflammation that is likely to be amenable to therapeutic intervention.     

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

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

Effects of fructose on the energy metabolism and acid-base status of the perfused starved-rat liver. A 31phosphorus nuclear magnetic resonance study.

Fructose metabolism has been studied with 31P n.m.r. in perfused livers from rats starved for 48h. The time course of changes in liver ATP, Pi and sugar phosphate (fructose l-phosphate) concentrations, and intracellular pH were followed in each perfusion after infusion of fructose to give an initial concentration of either 5mM or 10mM. Rapid falls in the concentrations of ATP and Pi and intracellular pH occurred after infusion of fructose, reaching a minimum after 4-5 min, which was lower in the 10mM group than in the 5mM group. These changes were accompanied by a rapid rise in fructose 1-phosphate, reaching a plateau also after 4-5 min. At both concentrations of fructose, after the early falls, some recovery of ATP, Pi and intracellular pH occurred; this was complete for Pi and intracellular pH in the 5mM-fructose experiments (within 12-30 min). Complete restoration of ATP to the pre-fructose value was not achieved in either the 5mM of 10mM groups. Measurements of the uptake of lactate by the liver indicated that the fall in intracellular pH was caused primarily by production of protons accompanying the formation of lactate from fructose with possibly a transient contribution generated during the rise in fructose 1-phosphate.     

Calcium antagonists and heat-induced hepatic injury

Several laboratories have demonstrated the value of the isolated perfused rat liver as a suitable model for heat-induced hepatic injury in vivo. Membrane changes caused by perfusion of rat livers at 42 degrees C for 90 min were similar to those induced by toxic chemicals or hypoxia. In an evaluation of several categories of drugs reported to reduce cell injury, calcium antagonists (nifedipine, dantrolene, and verapamil), were evaluated for their therapeutic potential for heat injury. Isolated rat livers were perfused at 42 degrees C for 90 min with and without calcium antagonists. Livers were also perfused at 37 degrees C. Potassium and transaminase leakage, bile production and ultrastructure were used to evaluate their responses. Neither of the three calcium antagonists significantly improved any of the functional parameters measured. However, dantrolene produced dilated or vesicular rough endoplasmic reticulum in the heated livers. These changes suggest selective intracellular action on endoplasmic reticulum of heated livers. Ring-shaped mitochondria and vesicular endoplasmic reticulum were observed in the heated, verapamil-treated livers, but these could not be quantitatively distinguished from controls. Nifedipine did not appear to alter intracellular membranes, but did increase bile production.     

P2X7 receptor-mediated purinergic signaling promotes liver injury in acetaminophen hepatotoxicity in mice

Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice and is triggered by stimulation of immune cells. The purinergic receptor P2X7 is upstream of the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in immune cells and is activated by ATP and NAD that serve as damage-associated molecular patterns. APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7-/-), and in wild-type mice. P2X7-/- mice had significantly decreased APAP-induced liver necrosis. In addition, APAP-poisoned mice were treated with the specific P2X7 antagonist A438079 or etheno-NAD, a competitive antagonist of NAD. Pre- or posttreatment with A438079 significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury in wild-type but not P2X7-/- mice. Pretreatment with etheno-NAD also significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ecto-NTPDase CD39 (CD39-/-) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild-type mice. CD39-/- mice had increased APAP-induced hemorrhage and mortality, whereas apyrase also decreased APAP-induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7-dependent inflammasome activation. P2X7 was required for ATP-stimulated IL-1β release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAP-induced hepatotoxicity.     

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

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

Zymosan-induced changes in glucose release and fatty acid oxidation in the perfused rat liver

The aim of the present study was to investigate the actions of zymosan on glucose release and fatty acid oxidation in perfused rat livers and to determine if Kupffer cells and Ca2+ ions are implicated in these actions. Zymosan caused stimulation of glycogenolysis in livers from fed rats. In livers from fasted rats zymosan caused gradual inhibition of glucose production and oxygen consumption from lactate plus pyruvate. Ketogenesis, oxygen consumption, and [14C-]-CO2 production were inhibited by zymosan when the [1-14C]-palmitate was supplied exogenously. However, ketogenesis and oxygen consumption from endogenous sources were not inhibited. An interference with substrate-uptake by the liver may be the cause of the changes in gluconeogenesis and oxidation of fatty acids from exogenous sources. The pretreatment of the rats with gadolinium chloride and the removal of Ca2+ ions did not suppress the effects of zymosan on glucose release, a finding that argues against the participation of Kupffer cells or Ca2+ ions in the liver responses. The hepatic metabolic changes caused by zymosan could play a role in the systemic metabolic alterations reported to occur after in vivo zymosan administration.     

Mechanism of action of cysteinyl leukotrienes on glucose and lactate balance and on flow in perfused rat liver. Comparison with the effects of sympathetic nerve stimulation and noradrenaline

Rat livers were perfused at constant pressure via the portal vein with media containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate. 1. Leukotrienes C4 and D4 enhanced glucose and lactate output and reduced perfusion flow to the same extent and with essentially identical kinetics. They both caused half-maximal alterations (area under the curve) of carbohydrate metabolism at a concentration of about 1 nM and of flow at about 5 nM. The leukotriene-C4/D4 antagonist CGP 35949 B inhibited the metabolic and hemodynamic effects of 5 nM leukotrienes C4 and D4 with the same efficiency, causing 50% inhibition at about 0.1 microM. 2. Leukotriene C4 elicited the same metabolic and hemodynamic alterations with the same kinetics as leukotriene D4 in livers from rats pretreated with the gamma-glutamyltransferase inhibitor, acivicin. 3. The calcium antagonist, nifedipine, at a concentration of 50 microM did not affect the metabolic and hemodynamic changes caused by 5 nM leukotriene D4. The smooth-muscle relaxant, nitroprussiate, at a concentration of 10 microM reduced flow changes, without significantly affecting the metabolic alterations. 4. Leukotriene D4 not only reduced flow; it also caused an intrahepatic redistribution of flow, restricting some areas from perfusion. Thus, leukotrienes increased glucose and lactate output directly in the accessible parenchyma and, in addition, indirectly by washout from restricted areas during their reopening upon termination of application. 5. The phospholipase A2 inhibitor, bromophenacyl bromide, but not the cyclooxygenase inhibitor, indomethacin, at a concentration of 20 microM reduced the metabolic and hemodynamic effects of 5 mM leukotriene D4. 6. Stimulation of the sympathetic hepatic nerves with 2-ms rectangular pulses at 20 Hz and infusion of 1 microM noradrenaline increased glucose and lactate output and decreased flow, similar to 10 nM leukotrienes C4 and D4. The kinetics of the metabolic and hemodynamic changes caused by the leukotrienes differed, however, from those due to nerve stimulation and noradrenaline. 7. The leukotriene-C4/D4 antagonist, CGP 35949 B, even at very high concentrations (20 microM) inhibited the metabolic and hemodynamic alterations caused by nerve stimulation or noradrenaline infusion only slightly and unspecifically.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential effect of glucagon on gluconeogenesis in periportal and pericentral regions of the liver lobule.

The effect of glucagon on gluconeogenesis was measured in periportal and pericentral regions of the liver lobule by monitoring changes in rates of O2 uptake on the surface of the perfused liver with miniature O2 electrodes after infusion of lactate. When lactate (2 mM) was infused into livers from starved rats perfused in the anterograde direction, O2 uptake was increased 2.5-fold more in periportal than in pericentral regions, reflecting increased energy demands for glucose synthesis. Under these conditions, glucagon infusion in the presence of lactate increased O2 uptake exclusively in periportal regions of the liver lobule. Thus, when perfusion is in the physiological anterograde direction, the metabolic actions of glucagon predominate in periportal regions of the liver lobule under gluconeogenic conditions in the starved state. When livers were perfused in the retrograde direction, however, glucagon stimulated O2 uptake exclusively in pericentral regions. Thus glucagon only stimulates gluconeogenesis in 'upstream' regions of the liver lobule irrespective of the direction of flow.     

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

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

The relation between inhibition of glycolysis and stimulation of oxygen uptake due to glucagon in livers from rats in different metabolic conditions

The relation between the effects of glucagon on oxygen consumption and glycolysis in livers from rats under different metabolic conditions was examined. Respiration of substrate-free perfused livers with different glycolytic fluxes, induced by changes in the pattern of food intake, responds differently to the infusion of 1 nM glucagon. The increases in oxygen uptake caused by 1 nM glucagon correlate reasonably well with the absolute decreases in glycolysis. The degree of inhibition of glycolysis is approximately constant (58 per cent) for all metabolic conditions. When no recovery of glycolysis occurs upon cessation of glucagon infusion, the same happens with oxygen consumption, which remains stimulated. It is concluded that in livers with no appreciable biosynthetic activities, the action of glucagon on respiration and glycolysis may be interpreted in terms of an interaction of interpreted in terms of an interaction of cytosolic and mitochondrial ATP generating processes.     

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.     

Response of normal and reperfused livers to glucagon stimulation: NMR detection of blood flow and high-energy phosphates

The effects of glucagon on blood flow and high-energy phosphates in control and in rat livers damaged by ischemia were studied using in vivo nuclear magnetic resonance (NMR) spectroscopy. Normal livers and livers which had been made ischemic for 20, 40, and 60 min followed by 60 min of reperfusion were studied. Ischemia led to a loss in adenosine triphosphate (ATP) within 30 min. Reperfusion after 20 min of ischemia led to complete recovery of ATP. 60 min of reperfusion after 40 or 60 min of ischemia led to only a 76% and 48% recovery of ATP, respectively. Glucagon, at doses up to 2.5 mg/kg body weight, caused no changes in the inorganic phosphate (Pi) to ATP ratio in normal livers as measured by ³¹P-NMR spectroscopy. In livers which had been made ischemic for 20, 40, or 60 min, glucagon caused an increase in the Pi/ATP ratio of 18%, 40%, and 40%, respectively. ¹⁹F-NMR detection of the washout of trifluoromethane from liver was used to measure blood flow. Glucagon-stimulated flow in the normal liver in a dose-dependent manner, with 2.5 mg glucagon/kg body weight leading to a 95% increase in flow. Ischemia for 20, 40, and 60 min followed by 60 min of reperfusion led to hepatic blood flows which were 63%, 68%, and 58% lower than control liver. In reperfused livers, blood flow after glucagon-stimulation was reduced to 56%, 43%, and 48% of control glucagon-stimulated flow after 20, 40, and 60 min of ischemia. These results indicate that ischemia followed by reperfusion leads to deceases in hepatic blood flow prior to alterations in ATP and the response of the liver to glucagon is altered in the reperfused liver.     

Altered neurogenic and mechanical responses to acetylcholine, ATP and substance P in detrusor from rat with outlet obstruction

The well-known side effects of anticholinergic compounds used to treat urinary incontinence caused by detrusor overactivity have addressed the interest on other pharmacological intervention. The purpose of the present work was to investigate the possible changes in purinergic and cholinergic components of parasympathetic neurotransmission in obstructed rat bladders with detrusor overactivity, and to examine the effect of the association of suramin, atropine and indomethacin on nerve-mediated responses to electrical field stimulation (EFS). Mechanical responses to exogenous acetylcholine, ATP and substance P were also evaluated. Altered sensitivities to acetylcholine and to the sensory neurotransmitter substance P, but unchanged sensitivity to the stable ATP analogue alpha,beta-methyleneATP were observed in bladders from obstructed rats. Suramin and atropine inhibited purinergic and cholinergic components of the neurogenic responses evoked by EFS in detrusor strips from control and obstructed rats. Interestingly, suramin enhanced the antagonistic effect of atropine on neurogenic responses of detrusor strips at all frequencies of stimulation tested. Our results suggest that the association between an antimuscarinic drug and an antagonist of P2X purinoceptors such as suramin might be helpful to reduce the therapeutic dosage of the antimuscarinic drug, along with its side effects. This approach may be of interest in the therapy of patients with bladder incontinence caused by detrusor overactivity, which do not even respond to a maximal dosage of antimuscarinic drug.     

Involvement of platelet-activating factor and leukotrienes in anaphylactic segmental venoconstriction in ovalbumin sensitized guinea pig livers

The hepatic anaphylactic venoconstriction is partly involved in anaphylactic hypotension, and is characterized by significant post-sinusoidal constriction and liver congestion in guinea pigs. We determined what chemical mediators are involved in anaphylaxis-induced segmental venoconstriction and liver congestion in perfused livers isolated from ovalbumin sensitized guinea pigs. Livers were perfused portally and recirculatingly at constant flow with diluted blood. The sinusoidal pressure was measured by the double occlusion pressure (Pdo), and was used to determine the pre-sinusoidal (Rpre) and post-sinusoidal (Rpost) resistances. An antigen injection increased both the portal vein pressure and Pdo, resulting in 4.1- and 2.3-fold increases in Rpre and Rpost, respectively. Hepatic congestion was observed as reflected by liver weight gain. Pretreatment with TCV-309 (10microM, platelet-activating factor (PAF) receptor antagonist) or ONO-1078 (100microM, human cysteinyl-leukotriene (Cys-LT) receptor 1 antagonist), but not indomethacin (10microM, cyclooxygenase inhibitor), ketanserin (10microM, serotonin receptor antagonist), or diphenhydramine (100microM, histamine H1 antagonist), significantly attenuated this anaphylactic hepatic venoconstriction. Anaphylaxis-induced increases in Rpre and Rpost were significantly inhibited by TCV-309 (by 48%) and ONO-1078 (by 36%), respectively. Combined TCV-309 and ONO-1078 pretreatment exerted additive inhibitory effects on anaphylactic hepatic venoconstriction. Anaphylactic hepatic weight gain was converted to weight loss when post-sinusoidal constriction was attenuated. It is concluded that anaphylaxis-induced pre-sinusoidal constriction is mainly caused by PAF and the post-sinusoidal constriction by Cys-LTs in guinea pig livers.     

Zymosan-activated plasma-mediated thromboxane production by the perfused rabbit liver and isolated hepatocytes: involvement of calcium

The present study investigates the mechanism of zymosan-activated plasma (ZAP)-mediated eicosanoid production by the isolated, perfused rabbit liver and described ZAP-mediated eicosanoid stimulation in cultured hepatocytes. Perfused livers receiving untreated plasma demonstrated no significant changes in portal venous pressure or the rates of release of lactic dehydrogenase or acid phosphatase activity (indicators of cellular injury). The control group livers demonstrated stable rates of release for 6-keto PGF1 alpha and thromboxane B2 (TXB2). In contrast, the infusion of ZAP alone resulted in a rapid but transient release of TXB2 from the livers. No significant changes in perfusion pressure or enzyme release were observed following ZAP administration. Perfusion of livers with a calcium-free buffer decreased the basal rates of both 6-keto PGF1 alpha and TXB2 production and significantly, but not completely, attenuated the ZAP-mediated increase in hepatic TXB2 production. Perfusion of livers with nifedipine (3 microM) had no effect on ZAP-mediated TXB2 production in this model. Isolated hepatocytes responded to ZAP-treatment with significant increases in TXB2 production. These data suggest that activated fluid phase complement components induce thromboxane production by specific cells within the liver and that this stimulation is partially dependent upon the release of intracellular calcium but independent of complement-mediated cellular injury.