Metabolism of isolated kidney tubules. Interactions between lactate, glutamine and oleate metabolism.

Kidney-cortex tubule suspensions were prepared by collagenase treatment of kidney cortex from fed and starved rats. This preparation, consisting mainly of proximal convoluted tubules was incubated with three major renal substrates, L-lactate, glutamine and oleate to study the dose dependence of substrate uptake rates from medium substrate combinations. All three substances, when added at near physiological concentrations, modified the uptake rate and fate of the other substrates. In accordance with previous observations, oleate inhibited lactate uptake, and lactate decreased glutamine metabolism. Glutamine on the other hand led to a marked increase in lactate uptake. Both, glutamine and lactate increased oleate metabolism. Glucose was the main product of lactate and glutamine metabolism, lactate being preferentially taken up for this process. Oleate led to a net synthesis of triglycerides in the tubules, which was stimulated by the addition of lactate and glutamine. More than 75% of the oleate taken up was recovered as triglycerides. In the absence of fatty acids, triglyceride content of tubules decreased. The results indicate that oleate is taken up in preference to lactate and glutamine when all three substrates are offered to the tubule. Glucose and triglycerides are the main metabolic products of tubular substrate metabolism. Whereas glucose is released into the medium, triglycerides are stored in the tubule cell.     

Relationship of energy production to gluconeogenesis in renal cortical tubules.

Isolated tubules prepared by collagenase treatment of rat renal cortex retained their ultrastructural integrity and responded to added lactate and succinate with an increase in gluconeogenesis and respiration. Inhibition of the mitochondrial respiratory chain with rotenone, or energy conservation sites with oligomycin caused a marked reduction in respiration and ATP content thereby completely inhibiting net gluconeogenesis. Dissociation of gluconeogenesis from respiration was accomplished with quinolinic acid and hydrazine, inhibitors of gluconeogenesis. At 5 times 10(-3) M quinolinic acid, gluconeogenesis from succinate was inhibited approximately 50% and from lactate nearly 100%. This concentration of quinolinic acid did not affect oxygen uptake or the ATP content of tubules in the presence or absence of substrate. Hydrazine at 10(-3) M resulted in approximately 75% inhibition of glucose formation from succinate and complete inhibition from lactate without interfering with respiration or ATP content. The increased mitochondrial energy generation, as manifested by accelerated respiration was independent of gluconeogenesis. The unchanging cell ATP concentration with a higher respiratory rate upon addition of exogenous substrate bespeaks increased ATP turnover. ATP utilization for the substrate-induced enhancement of gluconeogenesis could not account for the increment in ATP hydrolysis.     

Effects of added adenine nucleotides on renal carbohydrate metabolism

1. The regulatory effects that adenine nucleotides are known to exert on enzymes of glycolysis and gluconeogenesis were demonstrated to operate in kidney-cortex slices and in the isolated perfused rat kidney by the addition of exogenous ATP, ADP and AMP to the incubation or perfusion media. 2. Both preparations rapidly converted added ATP into ADP and AMP, and ADP into AMP; added AMP was rapidly dephosphorylated. AMP formed from ATP was dephosphorylated at a lower rate than was added AMP, especially when the initial ATP concentration was high (10mm). Deamination of added AMP occurred more slowly than dephosphorylation of AMP. 3. Gluconeogenesis from lactate or propionate by rat kidney-cortex slices, and from lactate by the isolated perfused rat kidney, was inhibited by the addition of adenine nucleotides to the incubation or perfusion media. In contrast, oxygen consumption and the utilization of propionate or lactate by slices were not significantly affected by added ATP or AMP. 4. The extent and rapidity of onset of the inhibition of renal gluconeogenesis were proportional to the AMP concentration in the medium and the tissue, and were not due to the production of acid or P(i) or the formation of complexes with Mg(2+) ions. 5. Glucose uptake by kidney-cortex slices was stimulated 30-50% by added ATP, but the extra glucose removed was not oxidized to carbon dioxide and did not all appear as lactate. Glucose uptake, but not lactate production, by the isolated perfused kidney was also stimulated by the addition of ATP or AMP. 6. In the presence of either glucose or lactate, ATP and AMP greatly increased the concentrations of C(3) phosphorylated intermediates and fructose 1,6-diphosphate in the kidney. There was a simultaneous rise in the concentration of malate and fall in the concentration of alpha-oxoglutarate. 7. The effects of added adenine nucleotides on renal carbohydrate metabolism seem to be mainly due to an increased concentration of intracellular AMP, which inhibits fructose diphosphatase and deinhibits phosphofructokinase. This conclusion is supported by the accumulation of intermediates of the glycolytic pathway between fructose diphosphate and pyruvate. 8. ATP or ADP (10mm) added to the medium perfusing an isolated rat kidney temporarily increased the renal vascular resistance, greatly diminishing the flow rate of perfusion medium for a period of several minutes.     

Effect of valproate on lactate and glutamine metabolism by rat renal cortical tubules

The metabolic effects of sodium valproate (VPA) on rat renal cortical tubules have been examined. When 1 or 5 mM lactate was used as substrate in the incubation medium, VPA decreased markedly the lactate uptake by the tubules. When 1 or 5 mM glutamine was used, the addition of VPA accelerated glutamine uptake, ammoniagenesis, but also stimulated markedly the accumulation of lactate and pyruvate produced from glutamine. VPA had a dose-dependent inhibitory effect on gluconeogenesis from both glutamine and lactate. With 5 mM glutamine, VPA also induced a significant accumulation of glutamate in the medium. The oxygen consumption by the tubules was diminished by 40% following VPA addition. It is concluded that VPA modifies the metabolism of rat cortical tubules by interfering with the oxidation of natural substrates and stimulates in this fashion the production of ammonia by kidney tubules.     

Effect of Adenine Nucleotides on the Respiration of Carrot Root Slices

Sodium pyruvate and dinitrophenol stimulated O(2) uptake of freshly cut phloem parenchyma from carrot roots by 63 and 120% at optimal concentrations, indicating that production of pyruvate by glycolysis regulates over-all respiratory rate. Adding 0.5 to 6.7 mm Na(3)ADP and Na(3)ATP to slices rapidly stimulates respiration rate by 20 to 85%. The effect is greater at the lower end of this concentration range and is not due to change in pH or active cation uptake. It is suggested that treating tissue with both nucleotides stimulates pyruvate kinase, the rate-limiting step in respiration of freshly cut slices, by increasing the concentration of endogenous ADP. Adenosine diphosphate continued to stimulate O(2) uptake until the peak of induced respiration, but ATP inhibited respiration during development and decline of this peak. Absence of respiratory stimulation by NaH(2)PO(4) and of respiratory inhibition by added nucleosides confirms that inorganic phosphate is not a limiting factor of respiration in freshly cut slices. The stimulation of respiration rate of these slices by dinitrophenol is consistent with results from experiments in which ADP and ATP were applied to the tissue.     

Properties of mitochondria isolated from cyanide-sensitive and cyanide-stimulated cultures of Acanthamoeba castellanii

1. Mitochondria isolated from cultures of Acanthamoeba castellanii exhibit respiratory control and oxidize alpha-oxoglutarate, succinate and NADH with ADP:O ratios of about 2.4, 1.4 and 1.25 respectively. 2. Mitochondria from cultures of which the respiration was stimulated up to 50% by 1mm-cyanide (type-A mitochondria) and from cyanide-sensitive cultures (type-B mitochondria) had similar respiratory-control ratios and ADP:O ratios. 3. State-3 rates of respiration were generally more cyanide-sensitive than State-4 rates, and the respiration of type-A mitochondria was more cyanide-resistant than that of type-B mitochondria. 4. Salicylhydroxamic acid alone had little effect on respiratory activities of either type of mitochondria, but when added together with cyanide, irrespective of the order of addition, inhibition was almost complete. 5. Oxidation of externally added NADH by type-A mitochondria was mainly via an oxidase with a low affinity for oxygen (K(m)[unk]15mum), which was largely cyanide-sensitive and partially antimycin A-sensitive; this electron-transport pathway was inhibited by ADP. 6. Cyanide-insensitive but salicylhydroxamic acid-sensitive respiration was stimulated by AMP and ADP, and by ATP after incubation in the presence of MgCl(2). 7. Addition of rotenone to mitochondria oxidizing alpha-oxoglutarate lowered the ADP:O ratios by about one-third and rendered inhibition by cyanide more complete. 8. The results suggest that mitochondria of A. castellanii possess branched pathways of electron transport which terminate in three separate oxidases; the proportions of electron fluxes via these pathways vary at different stages of growth.     

Inhibitory effect of gentamicin on gluconeogenesis from pyruvate, propionate, and lactate in isolated rabbit kidney-cortex tubules

The effect of gentamicin on glucose production in isolated rabbit renal tubules was studied with lactate, propionate, malate, 2-oxoglutarate, and succinate as substrates. This antibiotic at 5 mM concentration inhibited gluconeogenesis from lactate by about 60% and that from either pyruvate or propionate by about 30%. In contrast, it did not alter the rate of glucose formation from other substrates studied. The rate of gluconeogenesis was higher at 1 mM propionate than at increasing concentrations of this substrate and was stimulated in the presence of 1 mM carnitine. However, the addition of carnitine did not affect the degree of inhibition of glucose formation by gentamicin. Since the mitochondrial free coenzyme A level was significantly lower in the presence of 10 than 1 mM propionate and increased on the addition of carnitine to the reaction medium, the inhibitory effect of propionate concentrations above 1 mM on gluconeogenesis in rabbit renal tubules may be due to a depletion of the free mitochondrial coenzyme A level, resulting in an inhibition of the mitochondrial coenzyme A-dependent reactions. In intact rabbit kidney cortex mitochondria incubated in State 4 as well as in Triton X-100-treated mitochondria, 5 mM gentamicin inhibited by about 30-40% the incorporation of 14CO2 into both pyruvate and propionate. The results indicate that the inhibitory effect of gentamicin on glucose formation in isolated kidney tubules incubated with lactate, pyruvate, or propionate is likely due to a decrease of the rate of carboxylation reactions.     

Studies on brain-cortex slices: The influence of various inhibitors on the retention of potassium ions and amino acids with glucose or pyruvate as substrate

1. The rate of appearance of (14)CO(2) from [6-(14)C]glucose and [3-(14)C]pyruvate was measured. Pyruvate is oxidized to carbon dioxide twice as fast as glucose, although the oxygen uptake is almost the same with each substrate. 2. The presence of 30mum-2,4-dinitrophenol increases the output of (14)CO(2) from [6-(14)C]glucose sixfold whereas the oxygen uptake is not quite doubled. Similar results are obtained with 0.1m-potassium chloride. The stimulating action of these two agents on the output of (14)CO(2) from [3-(14)C]pyruvate is much less than on that from [6-(14)C]glucose. 3. The effects of oligomycin, ouabain and triethyltin on the respiration of control and stimulated brain-cortex slices were studied. Triethyltin (1.3mum) inhibited the oxidation of [6-(14)C]glucose more than 70%, but did not inhibit the oxidation of[3-(14)C]pyruvate. [3-(14)C]pyruvate. 4. The production of lactic acid by brain-cortex slices incubated with glucose is twice as great as that with pyruvate. Lactic acid increases two and a half times in the presence of either triethyltin or oligomycin when the substrate is glucose, but is no different from the control when the substrate is pyruvate. 5. With kidney slices the production of lactic acid from glucose is very low. It is increased by oligomycin but not by triethyltin. 6. The results are discussed in terms of the oxidation of the extramitochondrial NADH(2) produced during glycolysis.     

Contribution of pH-sensitive metabolic processes to pH homeostasis in isolated rat kidney tubules.

The metabolism of isolated rat kidney tubules suspended in calcium-free physiological saline buffered with phosphate was found to be sensitive to changes in the pH of the suspending medium. Lowering the pH from 7.8 to 6.4 brought about increases in the rates of oxidation of added succinate, glutamate or glutamine as well as in the production of glucose from lactate, glutamine, succinate and fructose. The cellular ATP level was also higher in tubules incubated at pH 6.4 In contrast, the utilization of added glucose was greater at pH 7.8 than at pH 6.4, a substantial amount of lactate being produced at the higher pH. When glucose and either lactate or glutamine were provided as co-substrates glucose was the preferred fuel at pH 7.8 but the alternative substrate was the more readily utilized at pH 6.4. As a consequence of the metabolic activities of the tubules the pH of the suspending medium changed, utilization of lactate, glutamate or glutamine causing a rise in pH while conversion of glucose to lactate caused a fall in pH. In cases where two substrates were metabolized concurrently over a period of 3 h the extracellular pH tended towards a plateau level of approximately pH 7.4. It is proposed that pH-sensitive metabolism in isolated kidney tubules contributes to pH homeostasis in the cellular environment.     

Contribution of pH-sensitive metabolic processes to pH homeostasis in isolated rat kidney tubules

The metabolism of isolated rat kidney tubules suspended in calcium-free physiological saline buffered with phosphate was found to be sensitive to changes in the pH of the suspending medium. Lowering the pH from 7.8 to 6.4 brought about increases in the rates of oxidation of added succinate, glutamate or glutamine as well as in the production of glucose from lactate, glutamine, succinate and fructose. The cellular ATP level was also higher in tubules incubated at pH 6.4. In contrast, the utilization of added glucose was greater at pH 7.8 than at pH 6.4, a substantial amount of lactate being produced at the higher pH. When glucose and either lactate or glutamine were provided as co-substrates glucose was the preferred fuel at pH 7.8 but the alternative substrate was the more readily utilized at pH 6.4. As a consequence of the metabolic activities of the tubules the pH of the suspending medium changed, utilization of lactate, glutamate or glutamine causing a rise in pH while conversion of glucose to lactate caused a fall in pH. In cases where two substrates were metabolized concurrently over a period of 3 h the extracellular pH tended towards a plateau level of approximately pH 7.4. It is proposed that pH-sensitive metabolism in isolated kidney tubules contributes to pH homeostasis in the cellular environment.     

Gluconeogenic and lipogenic properties of isolated kidney tubules from the domestic fowl (Gallus domesticus)

Isolated kidney tubules synthesize glucose actively from fructose, lactate, glycerol and pyruvate and, to a lesser extent, from a variety of amino acids. Ethanol stimulated gluconeogenesis from pyruvate and inhibited it from lactate. The aminotransferase inhibitor, aminooxyacetate, greatly reduced synthesis from lactate but not from pyruvate. Quinolinate inhibited gluconeogenesis from both precursors, indicating an active role for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in the gluconeogenic pathway. Incorporation of lactate or glucose into triglycerides was relatively low, and since no fatty acid synthase (FAS) activity could be detected, probably represented chain elongation or reesterification.     

The supply of metabolic substrate from glia to photoreceptors in the retina of the honeybee drone

1. The drone retina is composed essentially of only two types of cells: a population of identical photoreceptor cells occupying 38% of the volume is embedded in a syncytium of glia (called outer pigment cells). Nearly all the mitochondria are in the photoreceptors. 2. A retinal slice consumes 18 microliter O2 (ml tissue)-1 min-1 in the dark for up to 6 h, even without exogenous substrate; in 6 h this would require the equivalent of 127 mM glucose in the photoreceptors or 8.7 mg glycogen (ml tissue)-1. 3. Freshly dissected retinas contain about 45 mg glycogen (ml tissue)-1, but this appears, from electron micrographs and from the PAS reaction, to be exclusively in the glia. After superfusion with substrate-free Ringer solution for 30 min, slices of retina contained less than 20 microM glucose. It therefore appears that to sustain respiration, carbohydrate substrate must be transferred from the glia to the photoreceptors. 4. Even after 6 h superfusion with substrate-free Ringer solution O2 consumption (QO2) was not increased by exogenous glucose, pyruvate, trehalose or lactate, nor decreased by 2-deoxy-D-glucose. QO2 was increased 2-3 fold by either light stimulation or (for at least 20 min) by 50 microM dinitrophenol. 5. QO2 was only slightly reduced when Na-dependent glucose transport was inhibited either by reduction of extracellular [Na+], or the presence of phlorizin. 6. It is suggested that drone retinal function does not require the uptake of glucose by the photoreceptors, but that the glia do take up glucose.     

Respiratory Response of Acer pseudoplatanus Cells to Pyruvate and 2,4-Dinitrophenol

The endogenous respiration rate of unstarved cultured cells of Acer pseudo-platanus L. is markedly stimulated by 2,4-dinitrophenol. Pyruvate is also stimulatory but to a lesser degree than dinitrophenol. Exogenously supplied sugars cause no short-term stimulation. Pyruvate does not enhance the elevated rate of O₂ uptake in the presence of dinitrophenol but does cause additional CO₂ evolution. The endogenous concentration of pyruvate is elevated in the presence of dinitrophenol. These observations suggest that the rate of O₂ uptake by the unstarved intact cells is limited by the rate of glycolysis and that rate of glycolysis is regulated by the intracellular concentration of adenine nucleotides or inorganic phosphate. Dinitrophenol stimulation of endogenous respiration is due in part to an indirect acceleration of glycolysis but also to a more direct facilitation of oxidation in the presence of excess mitochondrial substrate.     

The metabolic fate of lactate in renal cortical tubules

1. Isolated kidney cortex tubules prepared from fed rats and incubated with near-physiological concentrations of [¹⁴C]lactate decrease the specific radioactivity of the added lactate. This effect may be attributable to at least two mechanisms; formation of lactate from endogenous precursors, or entry of unlabelled carbon into the lactate pool as a result of substrate cycling, via phosphoenolpyruvate, pyruvate and oxaloacetate, together with equilibration of the oxaloacetate pool with malate and fumarate. Such substrate cycling could occur within a single cell, or between two populations of different cells, one glycolytic and the other gluconeogenic. These possibilities have been investigated by using metabolic inhibitors or alternative metabolic substrates. 2. Tubules from fed rats produced a fall in specific radioactivity of 14.4% when incubated for 40min with 2mm-lactate alone. A mathematical treatment of this result is presented, which allows the rate of fall in specific radioactivity to be expressed as the addition of unlabelled lactate to the pool. This corresponds to a rate of formation of unlabelled lactate of 121±22μmol/h per g dry wt., a rate close to that of gluconeogenesis. In tubules from fasting rats, there was no reduction of the specific radioactivity of lactate, indicating that fasting for 24h suppresses production of unlabelled-lactate carbon. 3. Addition of 2mm-fumarate resulted in a significantly greater decrease in the specific radioactivity of lactate, but aspartate (2mm), malate (2mm) and glucose (5mm) were without effect. Total inhibition of gluconeogenesis with 3-mercaptopicolinate did not prevent the fall in specific radioactivity of lactate observed in tubules from fed-rat kidney, thereby excluding significant activity of the substrate cycle pyruvate→oxaloacetate→phosphoenolpyruvate→pyruvate. 4. The capacity of pyruvate kinase under the test conditions in tubules prepared from kidneys of fed or starved rats was at least ten times higher than the observed rate of production of lactate, so that failure to observe recycling of lactate in starved-rat tubules indicates suppression of pyruvate kinase activity. 5. The endogenous glycogen and glucose content of isolated renal cortex tubules is too low to account for the dilution of label of lactate. Endogenous concentrations of glycerol and amino acids were also very low. As for glycogen, the possibility that very rapid turnover of these metabolites, in fed rats but not in starved rats, may account for formation of unlabelled lactate cannot be excluded. 6. It is concluded that substrate cycling via phosphoenolpyruvate does not occur to any significant extent in either fed or starved-rat kidney. In fed rats recycling of lactate carbon does occur and the rate of this reaction is similar to the rate of gluconeogenesis at physiological concentrations of lactate. The present results favour participation of oxaloacetate decarboxylase rather than `malic' enzyme in this cycle.     

Calcium uptake by bovine epididymal spermatozoa is regulated by the redox state of the mitochondrial pyridine nucleotides

Immature caput epididymal sperm accumulate calcium from exogenous sources at a rate 2- to 4-fold greater than mature caudal sperm. Calcium accumulation by these cells, however, is maximal in the presence of lactate as external substrate. This stimulation of calcium uptake by optimum levels of lactate (0.8-1.0 mM) is about 5-fold in caput and 2-fold in caudal sperm compared to values observed with glucose as substrate. Calcium accumulation by intact sperm is almost entirely mitochondrial as evidenced by the inhibition of uptake by rotenone, antimycin, and ruthenium red. The differences in the ability of the various substrates in sustaining calcium uptake appeared to be related to their ability to generate NADH (nicotinamide adenine dinucleotide). Previous reports have documented that mitochondrial calcium accumulation in several somatic cells is regulated by the oxidation state of mitochondrial NADH. A similar situation obtains for bovine epididymal sperm since calcium uptake sustained by site III oxidation of ascorbate in the presence of tetramethyl phenylenediamine and rotenone was also stimulated by NADH-producing substrates, including lactate, and inhibited by substrates generating NAD+ (nicotinamide adenine dinucleotide, oxidized form). Further, calcium uptake by digitonin-permeabilized sperm in the presence of succinate was stimulated when NADH oxidation was inhibited by rotenone. The compounds alpha-keto butyric, valeric, and caproic acids, which generate NAD+, inhibited the maximal calcium uptake observed in the presence of succinate and rotenone, and the hydroxy acids lactate and beta-hydroxybutyrate reversed this inhibition. These results document the regulation of sperm calcium accumulation by the physiological substrate lactate, emphasize the importance of mitochondria in the accumulation of calcium by bovine epididymal sperm, and suggest that the mitochondrial location of the isozyme LDH-X in mammalian sperm may be involved in the regulation of calcium accumulation.     

Regulation of glucose formation from lactate and pyruvate in isolated tubules of chicken kidney.

1. Isolated kidney tubules from chicken have been used to study the actions of ethanol, ouabain and aminooxyacetate on glucose formation from lactate and pyruvate. 2. In kidney tubules from well-fed chickens the rate of glucose production from lactate was higher than from pyruvate. Ethanol (10 mM) and ouabain (0.1 mM) were found to increase glucose formation from pyruvate but not from lactate. 3. It is concluded that in the presence of ethanol the fluxes of pyruvate through pyruvate dehydrogenase are in favour of the pyruvate carboxylase reaction restricted. 4. Glucose formation from lactate is decreased by aminooxyacetate (0.1 mM) and ouabain (0.1 mM). 5. Aminooxyacetate inhibited glucose formation from lactate, although chicken phosphoenolpyruvate carboxykinase is located intramitochondrially. 6. The results indicate that the effect of aminooxyacetate like that of ouabain is caused by the restricted formation of pyruvate.     

Metabolism of propionate by sheep-liver mitochondria: Effects of α-oxoglutarate, adenosine triphosphate, sodium chloride and potassium chloride

1. A study has been made of the effects of ATP and alpha-oxoglutarate on the rate of metabolism of propionate by whole mitochondria from sheep liver, and by mitochondria disrupted with ultrasonic energy or by freezing and thawing. Whole mitochondria metabolized propionate aerobically; the rate was increased and stabilized by 0.5mm-ATP, and increased at least a further 50% by 1.67mm-alpha-oxoglutarate. 2. Anaerobically, externally added ATP at high concentrations permitted slow consumption of propionate. 3. In the presence of 1.3mm-ATP, but in the absence of alpha-oxoglutarate, there was no significant lag phase in the removal of propionate by whole mitochondria, and the rate declined at concentrations below 2mm. In the additional presence of 1.67mm-alpha-oxoglutarate or -glutamate, propionate was removed at linear rates until the residual propionate concentration was about 0.1mm. 4. Maximum rates of metabolism of propionate by whole mitochondria with 1.3mm-ATP occurred with alkali-metal chloride concentrations of 65-95mm and with K(+)/Na(+) ratios 5-10, both in the presence and absence of alpha-oxoglutarate. 5. With disrupted mitochondria stimulatory effects of alpha-oxoglutarate were obtained only aerobically, only with propionate and not propionyl-CoA as substrate, and only when sufficient mitochondrial structure remained to permit unsupplemented metabolism of propionate to occur. 6. In the presence of ATP and CoA, disrupted mitochondria fixed [2-(14)C]propionate at a rate adequate to explain the rate with whole mitochondria stimulated with ATP and alpha-oxoglutarate. 7. With both whole and partially disrupted mitochondria in the absence of ATP, the rate of metabolism of propionate was inhibited by about 80% by 3.3mm-AMP. The inhibition was partly overcome by alpha-oxoglutarate plus CoA. 8. It is concluded that the ultimate effect of alpha-oxoglutarate was to increase the rate of supply of ATP within the mitochondria. Reasons are given why it is premature to conclude that the extra ATP arose entirely from the oxidation of alpha-oxoglutarate itself.     

The influence of renal function on lactate and glucose metabolism.

The relationship of lactate metabolism to renal function was studied in the isolated perfused rat kidney. A new radioisotopic method has been developed that enables the simultaneous measurement of lactate production and consumption in the presence of physiological concentrations of both lactate and glucose. In kidneys from fed rats, when glucose was absent, lactate production was only 12 mumol/h per g dry wt, and in kidneys from starved rats there was no lactate production, indicating that neither the phosphoenolpyruvate/pyruvate substrate cycle nor other analogous cycles for the recycling of lactate carbon are operating in the intact kidney cortex. Lactate production from glucose occurred at a high rate, at the same time as lactate consumption, demonstrating that lactate recycling between renal cortex and medulla can occur in the intact kidney. Lactate production from glucose correlated with glomerular filtration rate (P less than 0.001), urine flow rate (P less than 0.01) and sodium reabsorption (P less than 0.05). There was significant basal lactate production at zero glomerular filtration rate. Lactate consumption was not correlated with any renal function. When Na+ reabsorption was inhibited with the diuretic frusemide, or when filtration was entirely prevented (the 'non'-filtering kidney'), lactate production was decreased by 39% and 50% respectively. Basal lactate production determined in this way was the same as that calculated above by linear regression. Prevention of filtration, but not the addition of frusemide, significantly inhibited lactate consumption. It is concluded that glycolysis is required for medullary Na+ transport, and that some different transport function(s) require lactate oxidation.     

Accumulation and conversion of sugars by developing wheat grains. 3. Non-diffusional uptake of sucrose, the substrate preferred by endosperm slices

Abstract. Starch synthesis by developing wheat endosperm slices incubated in liquid media was more rapid, at optimum concentration, from sucrose as external substrate than from glucose and/or fructose. Fructose inhibited conversion of sucrose or glucose. The results are consistent with the hypothesis that sucrose is not hydrolysed in the apoplast before uptake.
Besides a diffusional influx and efflux of labelled sucrose there was a non-diffusional influx; it was inhibited by dinitrophenol, potassium arsenate, potassium iodide, and parachloromercuribenzene sulphonate (PCMBS). PCMBS inhibited both uptake and conversion of label from 150 molm⁻³¹⁴C-sucrose by 75%. Uptake and conversion of sucrose were stimulated by lowering pH and by fusicoccin, a promoter of proton extrusion.
Extracellular solutes like raffinosc and polyethylene glycol stimulated net uptake of label from ¹⁴C-sucrose — the larger molecule being more effective — this being due to a non-specific inhibition of diffusional efflux. At too high an osmotic concentration such solutes reduced net uptake; the larger the molecule the lower this transitional concentration.
In conclusion, wheat endosperm is better equipped to convert apoplastic sucrose rather than the hydrolysis products to starch; active loading of sucrose possibly involves proton co-transport; and large molecules in the extracellular solution reduce the diffusional elllux of loaded substrate.     

Substrate-dependent effect of 1-34 human parathyroid hormone fragment, dibutyryl cAMP and cAMP on gluconeogenesis in rabbit renal tubules

In the presence of 0.5 mM extracellular Ca2+ concentration both 1-34 human parathyroid hormone fragment (0.5 micrograms/ml) as well as 0.1 mM dibutyryl cAMP stimulated gluconeogenesis from lactate in renal tubules isolated from fed rabbits. However, these two compounds did not affect glucose synthesis from pyruvate as substrate. When 2.5 mM Ca2+ was present the stimulatory effect of the hormone fragment on gluconeogenesis from lactate was not detected but dibutyryl cAMP increased markedly the rate of glucose formation from lactate, dihydroxyacetone and glutamate, and inhibited this process from pyruvate and malate. Moreover, dibutyryl cAMP was ineffective in the presence of either 2-oxoglutarate or fructose as substrate. Similar changes in glucose formation were caused by 0.1 mM cAMP. As concluded from the 'crossover' plot the stimulatory effect of dibutyryl cAMP on glucose formation from lactate may result from an acceleration of pyruvate carboxylation due to an increase of intramitochondrial acetyl-CoA, while an inhibition by this compound of gluconeogenesis from pyruvate is likely due to an elevation of mitochondrial NADH/NAD+ ratio, resulting in a decrease of generation of oxaloacetate, the substrate of phosphoenolpyruvate carboxykinase. Dibutyryl cAMP decreased the conversion of fracture 1,6-bisphosphate to fructose 6-phosphate in the presence of both substrates which may be secondary to an inhibition of fructose 1,6-bisphosphatase.