Alteration of purinergic neurotransmission in isolated atria of streptozotocin-induced diabetic rats.

Cardiac dysfunctions are described in diabetes. However, the role of purinergic neurotransmission in diabetes-related cardiovascular diseases is unknown. The purpose of this study was to evaluate the purinergic neurotransmission in isolated atria from streptozotocin-induced diabetic rats. The animals were grouped as control and diabetic with 30 days (D30) and 60 days (D60) after streptozotocin-induced diabetes. The isolated left and right atria were used in functional experiments. The effects of adenosine triphosphate, uridine diphosphate, and adenosine were evaluated on atrial inotropism and chronotropism. The antagonists 8-cyclopentyl-1,3-dipropylxanthine and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate were also used, as blockers of P1 and P2 receptors, respectively. A negative inotropic effect followed by a positive inotropic effect was induced by adenosine triphosphate in isolated atria. This negative inotropic effect was decreased by 25% in left atria of D30. Additionally, the apparent affinity for adenosine was diminished in left atria of D30, suggesting changes in P1 receptor function. No changes were found in the right atria of D30 stimulated by adenosine. The left atria and right atria stimulated by uridine diphosphate showed an increased inotropic effect of 92% and 17%, respectively. No changes were observed in left and right atria of D30 stimulated by uridine diphosphate. Our data showed the involvement of purinergic neurotransmission in diabetes-related cardiovascular changes.

Transformation and action of extracellular NAD+ in perfused rat and mouse livers.

AIM: Transformation and possible metabolic effects of extracellular NAD+ were investigated in the livers of mice (Mus musculus; Swiss strain) and rats (Rattus novergicus; Holtzman and Wistar strains). METHODS: The livers were perfused in an open system using oxygen-saturated Krebs/Henseleit-bicarbonate buffer (pH 7.4) as the perfusion fluid. The transformation of NAD+ was monitored using high-performance liquid chromatography. RESULTS: In the mouse liver, the single-pass metabolism of 100 micromol/L NAD+ was almost complete; ADP-ribose and nicotinamide were the main products in the outflowing perfusate. In the livers of both Holtzman and Wistar rats, the main transformation products were ADP-ribose, uric acid and nicotinamide; significant amounts of inosine and AMP were also identified. On a weight basis, the transformation of NAD+ was more efficient in the mouse liver. In the rat liver, 100 micromol/L NAD+ transiently inhibited gluconeogenesis and oxygen uptake. Inhibition was followed by a transient stimulation. Inhibition was more pronounced in the Wistar strain and stimulation was more pronounced in the Holtzman strain. In the mouse liver, no clear effects on gluconeogenesis and oxygen uptake were found even at 500 micromol/L NAD+. CONCLUSION: It can be concluded that the functions of extracellular NAD+ are species-dependent and that observations in one species are strictly valid for that species. Interspecies extrapolations should thus be made very carefully. Actually, even variants of the same species can demonstrate considerably different responses.

Transformation and actions of extracellular NADP(+) in the rat liver.

The possible actions and transformation of extracellular NADP(+) in the rat liver have not yet been studied. Considering the various effects of its analogue NAD(+) in the liver, however, effects of NADP(+) can equally be expected. In the present work, this question was approached in the isolated perfused rat liver to get a preliminary picture of the action of extracellular NADP(+) in this organ. NADP(+) (100 microM) produced transient increases in the portal perfusion pressure. Glucose release (glycogenolysis) and lactate production from endogenous glycogen were transiently increased in antegrade and retrograde perfusion. Oxygen uptake was stimulated after a transient inhibition in antegrade perfusion, which was practically absent in retrograde perfusion. Pyruvate production was transiently inhibited. In the absence of Ca(2+), all of these effects were no longer observed. Bromophenacyl bromide, an inhibitor of eicosanoid synthesis, almost abolished all effects. Suramin, a non-specific purinergic P2(YX) antagonist, also inhibited the action of NADP(+). Single pass transformation of 75 microM NADP(+) was equal to 92%. Besides nicotinamide, at least two additional transformation products were detected: 2'-phospho-ADP-ribose and a non-identified component, the former being more important (67% of the transformed NADP(+)). Nicotinic acid adenine dinucleotide phosphate (NAADP) was not found in the outflowing perfusate. It was concluded that NADP(+), like NAD(+), acts on perfusion pressure and glycogen catabolism in the liver mainly via eicosanoid synthesis mediated by purinergic P2(YX) receptors.

Transformation products of extracellular NAD(+) in the rat liver: kinetics of formation and metabolic action.

The perfused rat liver responds in several ways to NAD(+) infusion (20-100 microM). 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 microM 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.

Low doses of tumour necrosis factor alpha and interleukin 1beta diminish hepatic gluconeogenesis from alanine in vivo.

Previous reports have attributed a stimulating action on hepatic gluconeogenesis to tumour necrosis factor alpha (TNFalpha) administered to rats at high doses (250 mug/kg). However, in adjuvant-induced arthritic rats, which present TNFalpha and other interleukins in the circulation, hepatic gluconeogenesis is diminished. The same occurs in some types of experimental cancer models as, for example, rats bearing the Walker-256 tumour. The present work represents an attempt of reproducing in rats gluconeogenesis inhibition by interleukins using low instead of high doses of both TNFalpha and interleukin 1beta (IL1beta). TNFalpha and IL1beta at doses of up to 10 mug/kg were given endovenously to rats and, after six hours, gluconeogenesis from alanine and several related parameters were evaluated in the isolated haemoglobin-free perfused rat liver. Livers from rats injected with TNFalpha and IL1beta, either alone or in combination, presented diminished gluconeogenesis. The degrees of inhibition caused by TNFalpha+IL1beta, TNFalpha and IL1beta were, respectively, 48.5, 38.8 and 30.4%. TNFalpha also diminished oxygen uptake. No action on urea and ammonia production was found. Possibly, both TNFalpha and IL1beta contribute to the decreased rates of hepatic gluconeogenesis that were found in rats with arthritis, sepsis and some kinds of cancer, but not to the decreased rates of ureagenesis.

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.

The action of extracellular NAD+ on Ca2+ efflux, hemodynamics and some metabolic parameters in the isolated perfused rat liver.

The action of NAD+ on hemodynamics and metabolism of the isolated perfused rat liver was investigated. Extracellular NAD+ (20-100 microM) stimulated glycogen breakdown (glucose release) and inhibited oxygen uptake. Lactate production was predominantly increased, and pyruvate production was predominantly inhibited. NAD+ also increased the portal perfusion pressure. All metabolic effects were strictly Ca2+-dependent. The effects were absent when Ca2+ was excluded, and reintroduction of the cation restored the effects. In preloaded livers, NAD+ accelerated 45Ca2+ efflux. The action of NAD+ was sensitive to three inhibitors of eicosanoid synthesis, suggesting that this action is mediated by these compounds, which are known to be produced and released by Kupffer and endothelial cells. It is impossible to infer from the available data if NAD+ exerts all these effects by itself or if they are caused by one or more of its extracellular hydrolysis products. Nicotinamide was ineffective and can be excluded, but especially cyclic ADP-ribose and ADP-ribose are possibilities that should be considered in future work.

Kinetic properties of the glucose 6-phosphatase of the liver from arthritic rats.

According to previous reports, adjuvant-induced arthritic rats present reduced activities of the hepatic glucose 6-phosphatase. A kinetic study was done in order to characterize this phenomenon. Microsomes were isolated from livers of arthritic and control rats (Holtzman strain) and the glucose 6-phosphatase was measured at various temperatures (13-37 degrees C) and glucose 6-phosphate concentrations. Irrespective of the temperature, the enzyme from arthritic rats presented a reduction of both V(max) and K(M). Detergent treatment of liver microsomes from control rats increased the activity, but no increase was found when microsomes from arthritic rats were treated in the same way. The mannose 6-phosphatase activity of detergent-treated microsomes from arthritic rats was only 25% of the activity found with detergent-treated microsomes from control rats. Without detergent treatment, the mannose 6-phosphatase activities of both control and arthritic rats were minimal. The activation energy, derived from V(max), was not changed by arthritis. In vivo arthritic rats presented higher hepatic glucose 6-phosphate concentrations, a phenomenon that is consistent with a reduced activity of glucose 6-phosphatase. It was concluded that in arthritic rats, the hydrolase is probably reduced, without a similar change in the translocase activity.