Javier Casado

Nitric oxide reduces adenosine transporter ENT1 gene (SLC29A1) promoter activity in human fetal endothelium from gestational diabetes.

Human umbilical vein endothelial cells (HUVEC) from gestational diabetes exhibit reduced adenosine uptake and increased nitric oxide (NO) synthesis. Adenosine transport via human equilibrative nucleoside transporters 1 (hENT1) is reduced by NO by unknown mechanisms in HUVEC. We examined whether gestational diabetes‐reduced adenosine transport results from lower hENT1 gene (SLC29A1) expression. HUVEC from gestational diabetes exhibit reduced SLC29A1 promoter activity when transfected with pGL3‐hENT1−2154 compared with pGL3‐hENT1−1114 constructs, an effect blocked by NG‐nitro‐L‐arginine methyl ester (L‐NAME, NOS inhibitor), but unaltered by S‐nitroso‐N‐acetyl‐L,D‐penicillamine (SNAP, NO donor). In cells from gestational diabetes transfected with pGL3‐hENT1−2154, L‐NAME increased, but SNAP did not alter promoter activity and hENT1 expression. However, in cells from normal pregnancies L‐NAME increased, but SNAP reduced promoter activity and hENT1 expression. Adenovirus‐silenced eNOS expression increased hENT1 expression and activity in cells from normal or gestational diabetic pregnancies. Thus, reduced adenosine transport may result from downregulation of SLC29A1 expression by NO in HUVEC from gestational diabetes. These findings explain the accumulation of extracellular adenosine detected in cultures of HUVEC from gestational diabetes. In addition, fetal endothelial dysfunction could be involved in the abnormal fetal development and growth seen in gestational diabetes. J. Cell. Physiol. 208: 451–460, 2006.

Distribution of CNT2 and ENT1 transcripts in rat brain: selective decrease of CNT2 mRNA in the cerebral cortex of sleep-deprived rats

Nucleoside transport processes regulate the levels of adenosine available to modulate neurotransmission, vascular tone and other physiological events. However, although equilibrative transporter transcripts or proteins have been mapped in the central nervous system of rats and humans, little is known about the presence and distribution of the complete family of nucleoside transporters in brain. In this study, we analysed the distribution of the transcript encoding the high affinity adenosine‐preferring concentrative transporter CNT2 in the rat central nervous system and compared it with that of the equilibrative transporter ENT1. Furthermore, we evaluated the changes in expression of these two transporters in a situation of increased extracellular levels of adenosine, such as sleep deprivation. CNT2 mRNA was widespread in rat brain, although most prevalent in the amygdala, the hippocampus, specific neocortical regions and the cerebellum. The distribution of CNT2 mRNA only partially overlapped that of ENT1. Most of the cells labelled were neurones. Total sleep deprivation dramatically diminished the amounts of CNT2 mRNA, whereas ENT1 mRNA remained unchanged. This specific decrease in CNT2 transcript suggests a new physiological role for the transporter in the modulation of extracellular adenosine levels and the sleep/wakefulness cycle.

The role of system A for neutral amino acid transport in the regulation of cell volume

System A is a secondary active, sodium dependent transport system for neutral amino acids. Strictly coupled with Na,KATPase, its activity determines the size of the intracellular amino acid pool, through a complex network of metabolic reaction and exchange fluxes. Many hormones and drugs affect system A activity in specific cell models or tissues. In all the cell models tested thus far the activity of the system is stimulated by amino acid starvation, cell cycle progression, and the incubation under hypertonic conditions. These three conditions produce marked alterations of cell volume. The stimulation of system A activity plays an important role in cell volume restoration, through an expansion of the intracellular amino acid pool. Under normal conditions, system A substrates represent a major fraction of cell compatible osmolytes, organic compounds that exert a protein stabilizing effect. It is, therefore, likely that the activation of system A represents a portion of a more complex response triggered by exposure to stresses of various nature. Since system A transporters have been recently cloned, the molecular bases of these regulatory mechanisms will probably be elucidated in a short time.

Up-regulation of system A activity in the regenerating rat liver

System A activity for neutral amino acid transport, measured as the MeAIB‐sensitive Na+‐dependent l‐alanine uptake, is induced 6 h after partial hepatectomy in plasma membrane vesicles from rat livers. Other Na+‐dependent transporters, like system ASC (MeAIB‐insensitive Na+‐dependent l‐alanine transport) and the nucleoside carrier show similar inductions. Up‐regulation of system A is not explained by changes in the dissipation rate of the Na+ transmembrane gradient, as deduced from uptake measurements performed in the presence of monensin. To determine whether induced system A shared any similarity with the activity found in hepatoma cell lines, we analyzed the N‐ethylmaleimide (NEM) sensitivity of system A in both regenerating and control rat liver plasma membrane vesicles. NEM treatment was equally effective in inhibiting system A in both experimental groups. Thus, during the prereplicative phase of liver growth, a transport activity similar to basal system A is up‐regulated in liver parenchymal cells, by a stable mechanism that does not involve changes in the Na+ transmembrane gradient.

Rat splanchnic net oxygen consumption, energy implications.

1. The blood flow, PO2, pH and PCO2 have been estimated in portal and suprahepatic veins as well as in hepatic artery of fed and overnight starved rats given an oral glucose load. From these data the net intestinal, hepatic and splanchnic balances for oxygen and bicarbonate were calculated. The oxygen consumption of the intact animal has also been measured under comparable conditions. 2. The direct utilization of oxygen balances as energy equivalents when establishing the contribution of energy metabolism of liver and intestine to the overall energy expenses of the rat, has been found to be incorrect, since it incorporates the intrinsic error of interorgan proton transfer through bicarbonate. Liver and intestine produced high net bicarbonate balances in all situations tested, implying the elimination (by means of oxidative pathways, i.e. consuming additional oxygen) of high amounts of H+ generated with bicarbonate. The equivalence in energy output of the oxygen balances was then corrected for bicarbonate production to 11‐54% lower values. 3. Intestine and liver consume a high proportion of available oxygen, about one‐half in basal (fed or starved) conditions and about one‐third after gavage, the intestine consumption being about 15% in all situations tested and the liver decreasing its oxygen consumption with gavage.

Up-regulation of liver system A for neutral amino acid transport in euglycemic hyperinsulinemic rats.

To determine the role of insulin on the in vivo modulation of liver system A activity, we used the euglycemic hyperinsulinemic clamp coupled to the measurement of solute uptakes into plasma membrane vesicles partially purified from livers of hyperinsulinemic rats and their saline-infused controls. The clamp was performed in chronically catheterized rats, either in the fasted state, 24 h after surgery (Group I), or after 3 days of recovery (Group II). System A activity, measured as the MeAIB-inhibitable L-alanine uptake, was selectively induced by hyperinsulinemia, although the effect was much greater in Group II than in Group I rats (137% vs. 24% over the basal values, respectively). This might be explained by the higher basal levels found in those liver plasma membrane vesicles from Group I fasted animals. Hyperinsulinemia also decreased blood amino acids but to a similar extent in both experimental groups. This suggests that amino acid depletion by itself may not cause up-regulation of system A. Other transport activities involved in neutral amino acid transport (Systems ASC, N and L) were not modified by the clamp. The induction of system A cannot be explained by changes in the dissipation rate of the Na+ transmembrane gradient, because the differences between insulin- and saline-infused rats remained even when the electrochemical Na+ gradient was disrupted in the presence of monensin. Thus, hyperinsulinemia might induce an increase in the number of transporters inserted into the plasma membrane.

Hepatic uptake of gluconeogenic substrates in late-pregnant and mid-lactating rats

Lactate uptake by liver is markedly increased in late-pregnant and mid-lactating rats without concomitant changes in its availability. Glycerol contribution to the liver 3-C unit uptake is only significant at term gestation (50% of lactate uptake) but almost negligible at mid-lactation (10% of lactate uptake). Pyruvate is only taken up by the liver of 15-day lactating rats. As a general trend, the livers of either pregnant or lactating rats are provided with an enhanced capacity to take up gluconeogenic substrates.

Intestinal handling of a glucose gavage by the rat

An oral gavage of either 3, 1 or 0.1 mmoles of 14C-labelled glucose was given to rats under standard feeding conditions or food deprived for 24 hr. The fate of the glucose label was determined at 10, 15, 30 and 60 min after gavage; at 60 min 40% of the glucose was absorbed in fed rats (60% in food deprived). The portal vein blood flows were determined and the levels of glucose, lactate, alanine and pyruvate, and their radioactivity, as well as that of CO, were measured in both portal and arterial blood.The net computed glucose and 3-carbon carriers (lactate, alanine and pyruvate) actually released into the portal system by the intestine was lower than the amount of glucose taken up from the intestinal lumen in one hour. Oxidation to 14CO2 accounted for a 12–15% of the absorbed glucose. The size of the gavage deeply affected the proportion of glucose released into the portal blood (c. 50% with a 3 mmoles gavage and practically nil with a 0.1 mmoles gavage), but it affected much less the generation of lactate and other 3 C carriers. In fed rats, the net intestinal balance of non-radioactive glucose was negative, and that of lactate positive; when radioactive glucose was considered, the pattern was inverted. In starved rats, both glucose and lactate were released in large proportions by the intestine, but alanine efflux was lower.It can be concluded that the intestine consumes a considerable proportion of glucose in the fed state. Glucose handling by the intestine is compartmentalized in two functional circuits: glucose is taken up from the arterial blood and used for intestinal metabolism and lactate production, luminal glucose is absorbed mainly unaltered and transferred to the portal blood. Thus, the generation of lactate is mainly related to the availability of arterial glucose. In addition to the release of the ingested glucose as 3 C carriers or glucose, an extraportal pathway for glucose transfer into the bloodstream is postulated.

Intestinal and hepatic nitrogen balance in the rat after the administration of an oral protein load

The fate of a small oral dose of protein given to overnight-starved rats was studied. After 3 h, 62% of the protein amino acids had been absorbed. Most of the absorbed N went into the bloodstream through the portal in the form of amino acids, but urea and ammonia were also present. About one-quarter of all absorbed N was carried as lymph amino acids. The liver was able to take all portal free ammonia and a large proportion of portal amino acids, releasing urea. The hepatic N balance was negative, indicating active proteolysis and net loss of liver protein.

In the rat, intestinal lymph carries a significant amount of ingested glucose into the bloodstream

Intestinal lymph flow in rats receiving an oral load of 3 mmoles of glucose was determined by measuring the amount of tritiated gavage water entering via the portal route [computed differential arterio-venous tritium water concentrations multiplied by portal blood flow) and measuring the total amount of tritium transferred from the intestinal lumen to the rest of the rat in a given time [from the blood tritium specific activity and the water space (795 ml.kg-1) of the rat]. The approximate figure obtained (79.8 ml.kg-1.h) was used for the evaluation of the amount of glucose (from 14C-labelled glucose in the gavage) transfer via intestinal lymph from the label present in the lymph and its flow. Lymph glucose concentration (c. 11 mM) and specific radioactivity were higher than those of blood. The amount of glucose actually taken up from the intestinal lumen was determined measuring the remaining label in the alimentary canal. The portal contribution to label distribution was measured as for tritium water transfer. Most glucose label passed through in the form of glucose, with much smaller proportions as CO2 and even smaller as lactate and other labelled compounds. The results suggest that the contribution of lymph circulation to dietary glucose incorporation into the bloodstream may be significant, amounting to values higher than 10% of the glucose actually incorporated.