Diuretic effect of intraventricular and intravenous infusions of noradrenaline in conscious sheep

Infusion of noradrenaline at rates between 32-160 nmol.min-1 for 30 min into one lateral cerebral ventricle of conscious sheep caused a diuresis which was accompanied by negative solute-free water reabsorption and which lasted for 90-120 min. The range of noradrenaline infusion rates used reflects differences between individual animals in the rate of infusion necessry to cause diuresis. Intracerebroventricular (ICV) infusion of noradrenaline at half the diuretic rate caused no significant changes in urine flow. The diuresis induced by ICV noradrenaline infusion was prevented by concurrent ICV administration of the alpha-adrenergic antagonist, phentolamine, but was not prevented by concurrent ICV administration of the beta antagonist, propranolol, or by concurrent intravenous infusion of phentolamine. Intravenous infusion of noradrenaline at rates that were diuretic by ICV infusion caused a diuresis of approximately 30 min duration which coincided with the period of intravenous noradrenaline infusion. This diuresis was prevented by concurrent intravenous infusion of phentolamine. These results were interpreted as indicating that the higher rates of ICV infusion of noradrenaline caused the prolonged water diuresis by acting at a site in the brain and, thereby, inhibiting the release of endogenous vasopressin. ICV infusion of noradrenaline at all rates was followed by a reduction in mean arterial blood pressure and pulse pressure with variable changes in heart rate and by depression of the rates of renal clearance of PAH, potassium and total solute.     

Cerebrovascular effects of intravenous dopamine infusions in fetal sheep.

Preterm infants are often treated with intravenous dopamine to increase mean arterial blood pressure (MAP). However, there are few data regarding cerebrovascular responses of developing animals to dopamine infusions. We studied eight near-term and eight preterm chronically catheterized unanesthetized fetal sheep. We measured cerebral blood flow and calculated cerebral vascular resistance (CVR) at baseline and during dopamine infusion at 2.5, 7.5, 25, and 75 microg x kg(-1) x min(-1). In preterm fetuses, MAP increased only at 75 microg x kg(-1) x min(-1) (25 +/- 5%), whereas in near-term fetuses MAP increased at 25 microg x kg(-1) x min(-1) (28 +/- 4%) and further at 75 microg x kg(-1) x min(-1) (51 +/- 3%). Dopamine infusion was associated with cerebral vasoconstriction in both groups. At 25 microg x kg(-1) x min(-1), CVR increased 77 +/- 51% in preterm fetuses and 41 +/- 11% in near-term fetuses, and at 75 microg x kg(-1) x min(-1), CVR increased 80 +/- 33% in preterm fetuses and 83 +/- 21% in near-term fetuses. We tested these responses to dopamine in 11 additional near-term fetuses under alpha-adrenergic blockade (phenoxybenzamine, n = 5) and under dopaminergic D(1)-receptor blockade (SCH-23390, n = 6). Phenoxybenzamine completely blocked dopamine's pressor and cerebral vasoconstrictive effects, while D(1)-receptor blockade had no effect. Therefore, in unanesthetized developing fetuses, dopamine infusion is associated with cerebral vasoconstriction, which is likely an autoregulatory, alpha-adrenergic response to an increase in blood pressure.     

Effects of prolonged intravenous infusions of adrenaline on glucose utilization, plasma metabolites, hormones and milk production in lactating sheep

Lactating ewes received continuous intravenous infusions of adrenaline (0.05 micrograms/kg liveweight) for 4 days. Prior to, during and after adrenaline infusions, milk yield and composition were monitored. Plasma concentrations of metabolites and hormones were measured each day and glucose biokinetics were measured in non-steady state at the start and end of adrenaline infusions. During adrenaline infusion, milk yield and content of solids-not-fat decreased and milk fat content was reduced on the first day of infusion. Plasma glucose was raised throughout the period of adrenaline infusion, plasma lactate increased over the first 4 h from the start of infusion and plasma non-esterified fatty acids increased for 2 h at the start of infusion and tended to increase during the first 2-3 h after withdrawal of adrenaline. Plasma growth hormone remained relatively stable except for a marked increase at 30 min after withdrawal of adrenaline. At the start and immediately after withdrawal of adrenaline infusion plasma insulin was increased approximately twofold. Glucose production, but not utilization, increased at the start of infusions. Immediately after withdrawal of adrenaline glucose utilization increased 2.5-fold with a smaller response in glucose production. There was essentially no change in glucose clearance during adrenaline infusion but a marked increase occurred after withdrawal of adrenaline.     

Chronic metabolic effects of ammonia in mouse brain.

Chronic ammonia toxicity in experimental mice was induced by exposing them for 2 and 5 days to 5 % (v/v) ammonia solution. The enzymes concerned with glutamate metabolism (aspartate-, alanine- and tyrosine aminotransferases, glutamate dehydrogenase and glutamine synthetase) and (Na+ + K+)-ATPase were estimated in the three regions of brain (cerebellum, cerebral cortex and brain stem) and in liver. Glutamate, aspartate, alanine, glutamine and GABA, RNA and protein were also estimated in the three regions of brain and liver. A significant rise in the activity of (Na+ + K+)-ATPase in all the three regions of brain along with a fall in the activity of alanine aminotransferase was noticed. Changes in the activities of other enzymes were also observed. A significant increase in alanine and a decrease in glutamic acid was observed while no change was observed in the content of other amino acids belonging to the glutamate family. As a result of this, changes in the ratios of glutamate/glutamine and glutamate + aspartate/GABA was observed. The results indicated that the brain was in a state of more depression and less of excitation. Under these conditions the liver tissue was showing a profound rise in the activity of the enzymes of glutamate metabolism. The results are further discussed.     

Intravascular infusions of plasma into fetal sheep cause arterial and venous hypertension.

Fetal volume control is driven by an equilibrium between fetal and maternal hydrostatic and oncotic pressures in the placenta. Renal contributions to blood volume regulation are minor because the fetal kidneys cannot excrete fluid from the fetal compartment. We hypothesized that an increase in fetal plasma protein would lead to an increase in plasma oncotic pressure, resulting in an increase in fetal arterial and venous pressures and decreased angiotensin levels. Plasma or lactated Ringer solution was infused into each of five twin fetuses. After 7 days, fetal protein concentration was 71.2 +/- 4.2 g/l in the plasma-infused fetuses compared with 35.7 +/- 6.3 g/l in the lactated Ringer-solution-infused fetuses. Arterial pressure was 68.0 +/- 3.6 compared with 43.4 +/- 1.9 mmHg in the lactated Ringer solution-infused fetuses (P < 0.0003), whereas venous pressure was 4.8 +/- 0.3 mmHg in the plasma-infused fetuses compared with 3.3 +/- 0.4 mmHg in the lactated Ringer solution-infused fetuses (P < 0.036). Six fetuses were studied on days 0, 7, and 14 of plasma protein infusion. Fetal protein concentration increased from 31.1 +/- 1.5 to 84.8 +/- 3.8 g/l after 14 days (P < 0.01), and arterial pressure increased from 43.1 +/- 1.8 to 69.1 +/- 4.1 mmHg (P < 0.01). Venous pressure increased from 3.0 +/- 0.4 to 6.2 +/- 1.3 mmHg (P < 0.05). Fetal heart rate did not change. Angiotensin II concentration decreased, from 24.6 +/- 5.6 to 2.9 +/- 1.3 pg/l, after 14 days (P < 0.01). Fetal plasma infusions resulted in fetal arterial and venous hypertensions that could not be corrected by reductions in angiotensin II levels.     

Biochemical aspects of renal ammonia formation in metabolic acidosis.

In omnivorous creatures, the diet is acidogenic, especially as a result of the meat content, which gives rise to phosphoric and sulphuric acids, i.e., to metabolic acidosis. In the short term, metabolic acids are buffered by tissue proteins and bicarbonate (the 'alkali reserve'). In the longer term, acid must be excreted, or neutralized with base which is also generated from the diet, by conversion of dietary amino-nitrogen to ammonia. The final steps of this process occur in the kidney, which converts circulating glutamine to ammonia, and to carbon products such as glucose and carbon dioxide, by metabolic reactions which adapt during acidosis to generate more ammonia and maintain an increased renal ammonia content. The complex mechanisms which govern the formation of ammonia, glucose and carbon dioxide from glutamine, involving the reactions of amino acids, the tricarboxylic acid cycle, and gluconeogenesis, are reviewed.     

The metabolic fate of 13N-labeled ammonia in rat brain.

13N-labeled ammonia was used to study the cerebral uptake and metabolism of ammonia in conscious rats. After infusion of physiological concentrations of [13N]ammonia for 10 min via one internal carotid artery, the relative specific activities of glutamate, glutamine (alpha-amino), and glutamine (amide) in brain were approximately 1:5:400, respectively. The data are consistent with the concept that ammonia, entering the brain from the blood, is metabolized in a small pool of glutamate that is both rapidly turning over and distinct from a larger tissue glutamate pool (Berl, S., Takagaki, G., Clarke, D.D., and Waelsch, H. (1962) J. Biol. Chem. 237, 2562-2569). Analysis of 13N-metabolites, after infusion of [13N]ammonia into one lateral cerebral ventricle, indicated that ammonia entering the brain from the cerebrospinal fluid is also metabolized in a small glutamate pool. Pretreatment of rats with methionine sulfoximine led to a decrease in the label present in brain glutamine (amide) following carotid artery infusion of [13N]ammonia. On the other hand, 13N activity in brain glutamate was greater than that in the alpha-amino group of glutamine, i.e. following methionine sulfoximine treatment the expected precursor-product relationship was observed, indicating that the two pools of glutamate in the brain were no longer metabolically distinct. The amount of label recovered in the right cerebral hemisphere, 5 s after a rapid bolus injection of [13N]ammonia via the right common carotid artery, was found to be independent of ammonia concentration within the bolus over a 1000-fold range. This finding indicates that ammonia enters the brain from the blood largely by diffusion. In normal rats that were killed by a freeze-blowing technique 5 s after injection of an [13N]ammonia bolus, approximately 60% of the label recovered in brain had already been incorporated into glutamine, indicating that the t1/2 for conversion of ammonia to glutamine in the small pool is in the range of 1 to 3 s or less. The data emphasize the importance of the small pool glutamine synthetase as a metabolic trap for the detoxification of blood-borne and endogenously produced brain ammonia. The possibility that the astrocytes represent the anatomical site of the small pool is considered.     

Contribution of branched-chain amino acids to uteroplacental ammonia production in sheep.

The uteroplacental tissues are a principal site of ammonia production for the conceptus. The goal of this study was to examine the effect of the composition of maternal amino acid (AA) infusate on uteroplacental ammonia production. Seven pregnant ewes (126 +/- 1. 4 days gestation) were infused through the maternal femoral vein (duration 3.5 h, rate 240 ml per hour) with three solutions of AAs. The first infusate was comparable to commercial parenteral nutrition preparations, the second infusate contained the same solution without branched-chain AAs (BCAAs), and the third infusate contained only BCAAs. Blood samples were simultaneously collected from the maternal artery, uterine vein, fetal artery, and umbilical vein to determine plasma AA concentrations and whole blood ammonia concentrations, before (control) and 2 h after (experimental) the start of infusion. Uterine and umbilical blood flows were measured using the ethanol steady-state diffusion method. Results showed that fetal arterial and venous ammonia concentrations increased significantly after infusions with all AAs or only BCAAs, but not without BCAAs. Uteroplacental ammonia production increased in response to each of the three infusates. However, this increase was much greater when the BCAAs were present in infusates. We conclude that there is a significant contribution of BCAAs to the uteroplacental ammonia production. Maternal AA infusions containing BCAAs can result in increased fetal blood ammonia concentrations.     

The effect of adrenal denervation on the metabolic effects of hyperammonemia in sheep

The metabolic effect of intravenous infusion of ammonium chloride (60 mumol/(kg body weight.min] was compared in five sheep before and after adrenal denervation. Adrenal denervation completely abolished the hyperglycemic effect of ammonium chloride, diminished the rise of pyruvate and lactate concentration, and failed to influence the lipolytic effect of NH4Cl. It is suggested that the metabolic effects of ammonia are in a different degree related to the action of ammonia on the central nervous system and (i) the hyperammonemic effect of ammonia completely depends on the neurogenic increase of adrenal medullary hormones; (ii) the rise of blood lactate and pyruvate level observed during hyperammonemia is only partially mediated by adrenaline; and (iii) the lipolytic effect of ammonia ion does not depend on the nerve-controlled secretion of adrenal medullary hormones.     

Response of the regional lymphatic system of the sheep to acute stress and adrenaline.

An acute pain stimulus resulted in elevated lymph flow and output of cells from the popliteal lymph node of the sheep in the first 15 min after the stress. Efferent lymph flow increased by an average of 93% above the mean resting flow and cell output rose by an average of 170% during this period, but by 30 min after the stress, values for both lymph flow and cell output had returned to normal. The cell content of the efferent lymph was significantly higher in the first 15 min after the acute stress and it is suggested that there is a sizeable pool of lymphocytes within the resting popliteal node which can be mobilized into the lymph by an acute stress. A single intravenous injection of 1 mg adrenaline the efferent lymph flow in all the sheep examined but gave rise to an increased cell output in only 50% of the sheep. This indicated that there may be other factors, possibly hormonal, involved in the movement of the pool of lymphocytes out of the regional lymph node following acute stress. Both acute pain stress and adrenaline resulted in an increased afferent popliteal lymph flow and output of cells from the regional tissues in the first 15 min after administration. The results are suggestive of a small pool of lymphocytes in the regional tissues which may be readily mobilized by either acute stress or adrenaline. Part of the increases in efferent and afferent lymph flow observed following acute stress and adrenaline appeared to be due to an increased lymph formation, presumably as a result of an increased capillary pressure. Nevertheless, it is considered that the greater part of the increased flow of lymph from both regions resulted from an accelerated movement of performed lymph.     

Cerebral metabolic and pressure-flow responses during sustained hypoxia in awake sheep.

Conscious sheep (n = 6), exposed to 3.5 h of normobaric hypoxia (arterial PO2 = 40 Torr) while allowed varying arterial PCO2, showed striking early increments of cerebral blood flow (CBF; +200-250%, by radiolabeled microspheres) and decrements of cerebral vascular resistance (CVR) in association with an early temporary elevation of cerebral O2 consumption (CMRO2; +25-60%). After 2 h, CMRO2 returned to normoxic levels, while CBF declined to a lower but still elevated level (+150%). CBF/CMRO2 increased twofold, while cerebral fractional extraction of O2 was unchanged. Mean arterial pressure was unchanged, but cerebral venous pressure rose (+11 mmHg) in a stable fashion such that cerebral perfusion pressure declined by 13%. Cerebral venous hematocrit and hemoglobin concentration were both elevated (+2.2-2.7% Hct units; +1.0-1.3 g/dl, respectively) above the corresponding arterial values between 150 and 210 min of hypoxia, suggesting venous hemoconcentration in possible association with a transcapillary fluid shift. CBF, and especially CVR, were well correlated with arterial O2 content.     

Acute metabolic effects of ammonia in mouse brain

Acute effects of intraperitoneal administration of ammonium chloride (200 mg/kg) on Na⁺,K⁺-ATPase and amino acid content of the glutamate family (glutamate, aspartate, alanine, glutamine, and GABA), as well as the enzymes involved in the metabolism of these amino acids, have been studied in the different regions of brain and liver in mice. A significant increase in the activity of Na⁺,K⁺-ATPase was observed in the cerebellum, cerebral cortex, and brain stem. A similar increase in the activity of glutamate dehydrogenase was observed in the brain stem, while a moderate increase in the activity of this enzyme was observed in the cerebral cortex and liver in the mice treated with ammonium chloride. In all three regions of brain, a 50% decrease was observed in the activity of alanine aminotransferase, while the activity of aspartate aminotransferase significantly rose in the brain stem. The activity of glutamine synthetase did not change much in the three regions of brain, and a significant fall was registered in the liver. The activity of tyrosine aminotransferase showed a rise in the cerebellum, brain stem, and in liver. Not much change was observed in the protein content in either brain or liver, whereas there was a 1.5-fold increase in the total RNA content in the liver of the animals treated with ammonium chloride. Under the experimental conditions, there was an increase only in the content of glutamine, of all the amino acids tested, in the cerebral cortex and liver. Similar results were obtained with homogenates of tissues enriched with ammonium chloride (in vitro) for the enzyme systems studied. These results are discussed, and the probable metabolic and functional significance of ammonia in brain is indicated.     

Effect of lactate and beta-hydroxybutyrate infusions on brain metabolism in the fetal sheep

Brain uptake of substrates other than glucose has been demonstrated in neonatal but not fetal animals in vivo. This study was undertaken to investigate the ability of the fetal sheep brain to use potential alternative substrates when they were provided in increased amounts. Brain substrate uptake was measured in chronically catheterised fetal sheep during 2-h infusions of neutralised lactate (n = 12) or beta-hydroxybutyrate (n = 12). Despite large increases in fetal arterial lactate and beta-hydroxybutyrate during the respective infusions, no significant uptake of either substrate was demonstrated. However during both types of infusion, the brain arterio-venous difference for glucose decreased 30% (P less than 0.05). Since the brain arterio-venous difference for oxygen was unchanged, and blood flow to the cerebral hemispheres (measured in 11 studies) was also unchanged, the infusions appeared to cause a true decrease in brain glucose uptake. This decrease paralleled the rise in lactate concentration during lactate infusions, and the rise in lactate and butyrate concentrations during the butyrate infusions. Both substrates have metabolic actions that may inhibit brain glucose uptake. We speculate that the deleterious effects of high lactate and ketone states in the perinatal period may in part be due to inhibition of brain glucose uptake.