Importance of Residual Water Permeability on the Excretion of Water during Water Diuresis in Rats

When the concentration of sodium (Na+) in arterial plasma (PNa) declines sufficiently to inhibit the release of vasopressin, water will be excreted promptly when the vast majority of aquaporin 2 water channels (AQP2) have been removed from luminal membranes of late distal nephron segments. In this setting, the volume of filtrate delivered distally sets the upper limit on the magnitude of the water diuresis. Since there is an unknown volume of water reabsorbed in the late distal nephron, our objective was to provide a quantitative assessment of this parameter. Accordingly, rats were given a large oral water load, while minimizing non-osmotic stimuli for the release of vasopressin. The composition of plasma and urine were measured. The renal papilla was excised during the water diuresis to assess the osmotic driving force for water reabsorption in the inner medullary collecting duct. During water diuresis, the concentration of creatinine in the urine was 13-fold higher than in plasma, which implies that ~8% of filtered water was excreted. The papillary interstitial osmolality was 600 mOsm/L > the urine osmolality. Since 17% of filtered water is delivered to the earliest distal convoluted tubule micropuncture site, we conclude that half of the water delivered to the late distal nephron is reabsorbed downstream during water diuresis. The enormous osmotic driving force for the reabsorption of water in the inner medullary collecting duct may play a role in this reabsorption of water. Possible clinical implications are illustrated in the discussion of a case example

The Role of the Mitochondrial Citrate Transporter in the Regulation of Fatty Acid Synthesis: Effect of Fasting and Diabetes

The Michaelis constant (Km) for citrate for the hepatic mitochondrial citrate transporter of fed rats is 239 μM. This Km increases approximately twofold in mitochondria of fasted or diabetic rats. The long-chain fatty acyl-CoA content was approximately twofold higher in mitochondria from fasted and diabetic rats and could account for this increased Km. The role of regulation of the mitochondrial citrate transporter in lipogenesis is discussed. </jats:p

Relative rates of appearance of nitrogen and sulphur: implications for postprandial synthesis of proteins

The purpose of this study was to gain insights on the temporal fate of proteins based on the rate of appearance of waste products of nitrogen (urea) and sulphur (sulphate) metabolism. Urine was collected every 2 h from 25 normal subjects to measure the rates of excretion of urea, creatinine, and sulphate throughout the 24-h cycle. Samples of blood and urine were also obtained over a 4-h period from 10 subjects who consumed a mixed meal containing 0.4 g protein/kg body weight to obtain information on the relative rates of degradation of amino acids with and without sulphur in a noninvasive fashion. The daily excretion (mean ± SEM) of urea, creatinine, and sulphate was 396 ± 28, 14 ± 0.4, and 15 ± 0.6 mmol, respectively; the molar sulphate/nitrogen (S/N) ratio was 2.0 ± 0.1%. There were relatively minor (&lt;20%) excursions in the rate of excretion of urea and creatinine in any 2-h period as compared with the corresponding 24-h rate; the concentrations of urea and creatinine in plasma also varied &lt;20% throughout the day. Only 23% of the nitrogen in protein in the standard meal appeared as urea in the 210 min after this meal was consumed. The small changes in the rate of appearance of urea and creatinine imply that the oxidation of amino acids was spread out over the day. In contrast to urea and creatinine, the rate of appearance of sulphate underwent a greater variation; in general, there was a nadir just after breakfast and a peak overnight (7.0 ± 0.6 and 14.3 ± 1.6 μmol/min, respectively), and a S/N appearance rate that rose from 1.3 to 2.6%, suggesting specific retention of sulphur-containing compounds during the day, with catabolism occurring many hours later. When there was a low intake of protein for 40 h, the S/N appearance rate changed appreciably: the mean value fell in the last 24 h from 1.8 ± 0.1 to 1.2 ± 0.1% when carbohydrate was consumed, whereas it was 2.3 ± 0.1% during a 40-h fast. We conclude that most of the amino acids of dietary origin are not catabolized directly after a meal in normal subjects and that sulphur in proteins or other compounds is retained for longer periods following meals. During a 40-h fast, net protein catabolism includes proteins rich in of sulphur-containing amino acids, whereas the converse occurs when subjects consumed the low protein – high carbohydrate diet.Key words: amino acids, creatinine, diurnal variation, nitrogen balance, protein turnover, sulphate, urea. </jats:p

Effect of palmitoyl-CoA and β-oxidation of fatty acids on the kinetics of mitochondrial citrate transporter

The kinetics of the hepatic mitochondrial citrate transporter were studied using 1,2,3-benzene tricarboxylate and the inhibitor-stop technique at 8 °C. The apparent Km for this transporter was 250 μM and the maximum velocity was 2 nmol of citrate transported per minute per milligram of mitochondrial protein. This apparent Km was increased when hepatic mitochondria were preincubated with both L-palmitoylcarnitine and CoA-SH but not with either alone. This rise in apparent Km was accompanied by a rise in the acid insoluble CoA-SH content. Removal of mitochondrial acid insoluble CoA by 'defatted albumin' resulted in a parallel decrease in the apparent Km. The apparent Km for the citrate transporter was increased after coupled β-oxidation of L-palmitoylcarnitine or octanoate without a detectable increase in acid insoluble CoA. Inhibition of β-oxidation of L-palmitoylcarnitine by the D-derivative prevented the rise in the apparent Km. Preincubation with ATP resulted in an increase in this apparent Km. When L-palmitoylcarnitine oxidation occurred without ATP accumulation (hexokinase, glucose, ADP, and inorganic phosphate) the apparent Km for the citrate transporter increased two- to threefold.Therefore, the apparent Km for the citrate transporter varied directly with the acid insoluble CoA content. In addition, this Km was increased as a result of β-oxidation of fatty acids but the mechanism was not solely attributable to a rise in acid insoluble CoA or ATP. The physiological implications of these findings are discussed. </jats:p

Ammoniagenesis in kidney cortex mitochondria of the rat: role of the mitochondrial dicarboxylate anion transporter

Since glutamine enters rat kidney mitochondria without exchange for an anion, the exit of its carbon skeleton must involve the dicarboxylate anion transporter (malate – inorganic phosphate) for ammoniagenesis to proceed. Therefore, this important mitochondrial anion transporter was studied in isolated renal cortex mitochondria. The phosphate concentration required for half-maximal rates of malate exit from renal cortex mitochondria of normal rats was 1.0 mM. This value was not decreased in renal cortex mitochondria from rats with chronic metabolic acidosis. The maximum velocity of the dicarboxylate transporter was not increased in renal cortex mitochondria from these acidotic rats. These kinetic parameters were similar in liver mitochondria. There was no acute activation of the dicarboxylate carrier when the incubation medium pH was lowered. Thus, there is no demonstrable activation of the dicarboxylate anion transporter in kidney cortex mitochondria of the rat with chronic metabolic acidosis. The significance of these results with respect to the regulation of renal ammoniagenesis is discussed. </jats:p

Properties of the citrate transporter in rat heart: implications for regulation of glycolysis by cytosolic citrate

The efflux of [14C]citrate from rat heart mitochondria was significantly greater with L-malate as the extramitochondrial substrate as compared with [12C]citrate, isocitrate or phosphoenolpyruvate. The concentration of L-malate required for half-maximal rate of efflux of citrate was 0.45 mM and the maximum velocity was 0.36 nmol min−1 mg−1 mitochondrial protein at 23 °C. This citrate transporter was inhibited by 1,2,3-benzenetricarboxylate and palmitoyl-CoA but not to the same extent as these compounds inhibit the tricarboxylate carrier in rat liver mitochondria. The apparent inability of these mitochondria to transport citrate in the inward direction necessitates the presence of a cytosolic citrate removal pathway. We propose that the enzymes of this pathway in rat heart could be ATP citrate (pro-3S)-lyase (EC 4.1.3.a) and carnitine acetyltransferase (EC 2.3.1.7), both of which we demonstrate to have adequate activity in both the fed and fasted state.An hypothesis has been put forward to account for the inhibition of rat heart phosphofructokinase by citrate in the fasted state incorporating these properties of the citrate transporter and ATP citrate (pro-3S)-lyase. </jats:p

Is accelerated oxidation of lactate required for dichloroacetate to lower the level of lactate in blood?

We examined mechanisms by which dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDH), led to a decrease in the concentration of lactate in blood in a unique "metabolic setting," where the concentration of lactate in blood was 5.4 ± 0.5 mmol/L. Elevated levels of lactate were induced in anaesthetized rabbits by the administration of a large dose of insulin. The rate of consumption of oxygen was 1.2 ± 0.1 mmol/min, the respiratory quotient was close to unity, and close to half of the PDH was in its active form; therefore, virtually all ATP synthesis should require flux through PDH. Hence, we predicted that DCA should not cause a significant decrease in the concentration of lactate in blood in this model. In contrast, if DCA was effective, new insights could be obtained into its mechanisms of action, at least in this setting. During steady-state hyperlactatemia, DCA was given as its sodium salt, 2 mmol/kg (n = 10); a control group (n = 5) received equimolar NaCl. Forty minutes later, the level of lactate in blood in the DCA group was 1.3 ± 0.2 mmol/L, significantly lower than in the NaCl group (4.2 ± 0.6 mmol/L). To determine the organ(s) responsible for removing lactate, arteriovenous differences were measured in organs drained by the jugular, femoral, and hepatic veins. There was no net uptake of lactate in these drainage beds after DCA was administered. From a quantitative analysis of the rate of removal of lactate and the rate of consumption of oxygen, it seems unlikely that the majority of the decrease in lactate could be directly attributed to an increase in its oxidation.Key words: lactic acidosis, dichloroacetate, pyruvate dehydrogenase, metabolism. </jats:p