Rabbit Kidney-cortex Tubules Research Articles

DHEA-induced modulation of renal gluconeogenesis, insulin sensitivity and plasma lipid profile in the control- and dexamethasone-treated rabbits. Metabolic studies.

In view of antidiabetic and antiglucocorticoid effects of dehydroepiandrosterone (DHEA) both invitro and invivo studies were undertaken: (i) to elucidate the mechanism of action of both dexamethasone phosphate (dexP) and DHEA on glucose synthesis in primary cultured rabbit kidney-cortex tubules and (ii) to investigate the influence of DHEA on glucose synthesis, insulin sensitivity and plasma lipid profile in the control- and dexP-treated rabbits. Data show, that in cultured kidney-cortex tubules dexP significantly stimulated gluconeogenesis by increasing flux through fructose-1,6-bisphosphatase (FBPase). DexP-induced effects were dependent only upon glucocorticoid receptor. DHEA decreased glucose synthesis via inhibition of glucose-6-phosphatase (G6Pase) and suppressed the dexP-induced stimulation of renal gluconeogenesis. Studies with the use of inhibitors of DHEA metabolism in cultured renal tubules showed for the first time that DHEA directly affects renal gluconeogenesis. However, in view of analysis of glucocorticoids and DHEA metabolites levels in urine, it seems likely, that testosterone may also contribute to DHEA-evoked effects. In dexP-treated rabbits, plasma glucose level was not altered despite increased renal and hepatic FBPase and G6Pase activities, while a significant elevation of both plasma insulin and HOMA-IR was accompanied by a decline of ISI index. It thus appears that increased insulin levels were required to maintain normoglycaemia and to compensate the insulin resistance. DHEA alone affected neither plasma glucose nor lipid levels, while it increased insulin sensitivity and diminished both renal and hepatic G6Pase activities. Surprisingly, DHEA co-administrated with dexP did not alter insulin sensitivity, while it partially suppressed the dexP-induced elevation of renal G6Pase activity and plasma cholesterol and triglyceride contents. As (i) gluconeogenic pathway in rabbit is similar to that in human, and (ii) DHEA counteracts several dexP-evoked effects, it seems likely, that its supplementation might be beneficial to patients treated with glucocorticoids.

Proinsulin C-peptide potentiates the inhibitory action of insulin on glucose synthesis in primary cultured rabbit kidney-cortex tubules: Metabolic studies

Effects of equimolar concentrations of proinsulin C-peptide and insulin on glucose synthesis were studied in primary cultures of rabbit kidney-cortex tubules grown in the presence of alanine, glycerol, and octanoate. The rhodamine-labeled C-peptide entered renal tubular cells and localized in nuclei, both in the presence and absence of insulin; preincubations with the unlabeled compound inhibited internalization. C-peptide did not affect glucose formation when added alone but potentiated the inhibitory action of insulin by about 20% due to a decrease in flux through glucose-6-phosphate isomerase (GPI) and (or) glucose-6-phosphatase (G6Pase). GPI inhibition was caused by: (i) increased intracellular contents of fructose-1,6-bisphosphate and fructose-1-phosphate, inhibitors of the enzyme and (ii) reduced level of the phosphorylated GPI, which exhibits higher enzymatic activity in the presence of casein kinase 2. A decrease in flux through G6Pase, due to diminished import of G6P by G6P-transporter from the cytoplasm into endoplasmic reticulum lumen, is also suggested. The data show for the first time that in the presence of insulin and C-peptide, both GPI and G6P-ase may act as regulatory enzymes of renal gluconeogenic pathway.

PPAR-γ-independent inhibitory effect of rosiglitazone on glucose synthesis in primary cultured rabbit kidney-cortex tubules

Therapeutic effect of rosiglitazone has been reported to result from an improvement of insulin sensitivity and inhibition of glucose synthesis. As the latter process occurs in both liver and kidney cortex the aim of this study was to elucidate the rosiglitazone action on glucose formation in both tissues. Primary cultured cells of both liver and kidney cortex grown in defined medium were use throughout. To identify the mechanism responsible for drug-induced changes, intracellular gluconeogenic intermediates and enzyme activities were determined. In contrast to hepatocytes, the administration of a 10 micromol/L concentration of rosiglitazone to renal tubules resulted in about a 70% decrease in the rate of gluconeogenesis, accompanied by an approximately 75% decrease in alanine utilization and a 35% increase in lactate synthesis. The effect of rosiglitazone was not abolished by GW9662, the PPAR-gamma irreversible antagonist, indicating that this action is not dependent on PPAR-gamma activation. In view of rosiglitazone-induced changes in gluconeogenic intermediates and a diminished incorporation of 14CO2 into pyruvate, it is likely that the drug causes a decline in flux through pyruvate carboxylase and (or) phosphoenolpyruvate carboxykinase. It is likely that the hypoglycemic action of rosiglitazone is PPAR-gamma independent and results mainly from its inhibitory effects on renal gluconeogenesis.

Differential effects of selegiline on glucose synthesis in rabbit kidney-cortex tubules and hepatocytes: In vitro and in vivo studies

The action of selegiline, a selective and irreversible inhibitor of monoamine oxidase B, commonly applied in the therapy of Parkinson's disease, on glucose formation was investigated in isolated rabbit hepatocytes and kidney-cortex tubules, maintaining the whole body glucose homeostasis via gluconeogenic pathway activity. An intensive hepatic metabolism of selegiline resulted in formation of selegiline- N-oxide, desmethylselegiline, methamphetamine and amphetamine, whereas during slow degradation of the drug in freshly isolated renal tubules selegiline- N-oxide was mainly produced. At 100 μM concentration selegiline markedly diminished glucose synthesis in isolated renal tubules incubated with dihydroxyacetone or alanine + glycerol + octanoate (by about 60 and 30%, respectively), while at 5 μM concentration a similar degree of inhibition was achieved in renal tubules grown in primary culture under the same conditions (about 40 and 60%, respectively). Moreover, desmethylselegiline and selegiline- N-oxide considerably diminished glucose production in renal tubules whereas selegiline and its metabolites did not affect gluconeogenesis in hepatocytes. Contrary to control animals, following selegiline administration to alloxan-diabetic rabbits for 8 days (10 mg kg −1 body wt. daily) the blood glucose and serum creatinine levels were significantly diminished, suggesting a decrease in renal gluconeogenesis and improvement of kidney functions. Since in renal tubules selegiline induced a decline in the intracellular levels of gluconeogenic intermediates and ATP content accompanied by a decrease in oxygen consumption in both kidney-cortex and hepatic mitochondria it seems possible that its inhibitory action on renal gluconeogenesis might result from an impairment of mitochondrial function, while an intensive selegiline metabolism in hepatocytes causes decrease of its concentration and in consequence no inhibition of gluconeogenesis. In view of these observations it is likely that an increased risk of selegiline-induced hypoglycemia might be expected particularly in patients exhibiting an impairment of liver function and following transdermal administration of this drug, i.e. under conditions of increased serum selegiline concentrations.

Differential effects of selenium compounds on glucose synthesis in rabbit kidney-cortex tubules and hepatocytes. In vitro and in vivo studies

Although selenium is taken with diet mainly as selenoamino acids, its hypoglycaemic action on hepatic gluconeogenesis has been studied with the use of inorganic selenium derivatives. The aim of the present investigation was to compare relative efficacies of inorganic and organic selenium compounds in reducing glucose synthesis in hepatocytes and renal tubules, significantly contributing to the glucose homeostasis. In contrast to hepatocytes, both selenite and methylselenocysteine inhibited renal gluconeogenesis by about 40–45% in control rabbits. Selenate did not affect this process, whereas selenomethionine inhibited gluconeogenesis by about 20% in both hepatocytes and renal tubules. In contrast to methylselenocysteine, selenite decreased intracellular ATP content, glutathione reduced/glutathione oxidized (GSH/GSSG) ratio and pyruvate carboxylase, PEPCK and FBPase activities, while methylselenocysteine diminished PEPCK activity due to elevation of intracellular 2-oxoglutarate and GSSG, inhibitors of this enzyme. Experiments in vivo indicate that in 3 of 9 alloxan-diabetic rabbits treated for 14 days with methylselenocysteine (0.182 mg/kg body weight) blood glucose level was normalized, whereas in all diabetic rabbits plasma creatinine and urea levels decreased from 2.52 ± 0.18 and 87.4 ± 9.7 down to 1.63 ± 0.11 and 39.0 ± 2.8, respectively. In view of these data selenium supplementation might be beneficial for protection against diabetes-induced nephrotoxicity despite selenium accumulation in kidneys and liver.

Contribution of l-3,4-dihydroxyphenylalanine metabolism to the inhibition of gluconeogenesis in rabbit kidney-cortex tubules

The circulating l-3,4-dihydroxyphenylalanine, the drug of choice in the therapy of Parkinson's disease (PD), is efficiently extracted by kidney and converted to dopamine, known to control several renal functions. As: (i) in addition to liver, kidney is an important source of glucose in mammals and (ii) the action of this drug on renal gluconeogenesis has not yet been studied, the aim of the present investigation was to estimate the influence of l-3,4-dihydroxyphenylalanine metabolism on glucose formation in isolated kidney-cortex tubules incubated with various gluconeogenic substrates. The data indicate that a rapid intracellular degradation of l-3,4-dihydroxyphenylalanine and tyramine (at 100 and 200 μM concentrations) is accompanied by 25–40% decrease in glucose production from pyruvate, alanine + glycerol + octanoate and dihydroxyacetone due to augmented generation of hydrogen peroxide via monoamine oxidase B, resulting in a decline of glutathione redox state by 40%. Moreover, following inhibition of monoamine oxidase B by deprenyl or substitution of pyruvate by aspartate + glycerol + octanoate both l-3,4-dihydroxyphenylalanine and tyramine affect neither the rate of gluconeogenesis nor glutathione redox state. In view of: (i) l-3,4-dihydroxyphenylalanine- and tyramine-induced changes in intracellular levels of gluconeogenic intermediates, and (ii) a significant decline of phosphoenolpyruvate carboxykinase activity by 500 μM oxidized glutathione, it is likely that l-3,4-dihydroxyphenylalanine- and tyramine-evoked disturbances in the glutathione redox state might diminish flux through phosphoenolpyruvate carboxykinase and in consequence decrease glucose formation in renal tubules, suggesting a new potential side-action of l-3,4-dihydroxyphenylalanine treatment.

Diabetes-induced changes in glucose synthesis, intracellular glutathione status and hydroxyl free radical generation in rabbit kidney-cortex tubules.

Diabetes-induced changes in glucose formation, intracellular and mitochondrial glutathione redox states as well as hydroxyl free radicals (HFR) generation have been investigated in rabbit kidney-cortex tubules. In contrast to renal tubules of control animals, diabetes-evoked increase in glucose formation in the presence of either aspartate+glycerol+octanoate or malate as gluconeogenic precursors (for about 50%) was accompanied by a diminished intracellular glutathione reduced form (GSH)/glutathione oxidised one (GSSG) ratio by about 30-40%, while the mitochondrial GSH/GSSG ratio was not altered. However, a relationship between the rate of gluconeogenesis and the intracellular glutathione redox state was maintained in renal tubules of both control and diabetic rabbits, as concluded from measurements in the presence of various gluconeogenic precursors. Moreover, diabetes resulted in both elevation of the glutathione reductase activity in rabbit kidney-cortex and acceleration of renal HFR generation (by about 2-fold). On the addition of melatonin, the hormone exhibiting antioxidative properties, the control values of HFR production were restored, suggesting that this compound might be beneficial during diabetes therapy. In view of the data, it seems likely that diabetes-induced increase in HFR formation in renal tubules might be responsible for a diminished intracellular glutathione redox state despite elevated glutathione reductase activity and accelerated rate of gluconeogenesis, providing glucose-6-phosphate for NADPH generation via pentose phosphate pathway.

Relationship between gluconeogenesis and glutathione redox state in rabbit kidney-cortex tubules

The intracellular glutathione redox state and the rate of glucose formation were studied in rabbit kidney-cortex tubules. In the presence of substrates effectively utilized for glucose formation, ie, aspartate + glycerol + octanoate, alanine + glycerol + octanoate, malate, or pyruvate, the intracellular reduced glutathione/oxidized glutathione (GSH/GSSG) ratios were significantly higher than those under conditions of negligible glucose production. Changes in the intracellular GSH/GSSG ratio corresponded to those in glucose-6-phosphate content and reduced nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) ratio obtained from malate/pyruvate measurements. Gluconeogenesis stimulation by extracellular adenosine triphosphate (ATP) or inosine caused an elevation of the intracellular GSH/GSSG and NADPH/NADP+ ratios, as well as glucose-6-phosphate level. Surprisingly, in the presence of 5 mmol/L glucose, both the intracellular GSH/GSSG and NADPH/NADP+ ratios and glucose-6-phosphate content were almost as low as under conditions of negligible glucose synthesis. L-buthionine sulfoximine (BSO)-induced decline in both the intracellular glutathione level and redox state resulted in inhibition of gluconeogenesis accompanied by accumulation of phosphotrioses and a decrease in fructose-1,6-bisphosphate content, while cysteine precursors altered neither GSH redox state nor the rate of glucose formation. In view of the data, it seems likely that: (1) intensive gluconeogenesis rather than extracellular glucose is responsible for maintaining a high intracellular GSH/GSSG ratio due to effective glucose-6-phosphate delivery for NADPH generation via the pentose phosphate pathway; (2) a decline in the intracellular glutathione level and/or redox state causes a decrease in glucose synthesis resulting from a diminished flux through aldolase; (3) induced by cysteine precursors, elevation of the intracellular GSH level does not affect the rate of glucose formation, probably due to no changes in the intracellular GSH/GSSG ratio.

Purinergic regulation of glucose and glutamine synthesis in isolated rabbit kidney–cortex tubules

The effects of extracellular purinergic agonists and their breakdown products on glucose and glutamine synthesis in rabbit kidney–cortex tubules incubated with aspartate+glycerol or alanine+glycerol+octanoate were investigated. A rapid extracellular degradation of ATP was accompanied by an accumulation of AMP, inosine, and hypoxanthine. Extracellular ATP and its breakdown products accelerated glucose synthesis in renal tubules, while ammonium released from adenine-containing compounds enhanced glutamine synthesis and diminished the degree of gluconeogenesis stimulation. In contrast to AMP and inosine, ATP evoked calcium signals, while both ATP and inosine decreased intracellular cAMP content and accelerated the flux through fructose-1,6-bisphosphatase as concluded from changes in gluconeogenic intermediates. Since (i) the activity of partially purified renal fructose-1,6-bisphosphatase was increased upon protein phosphatase-1 treatment and decreased following treatment of previously dephosphorylated enzyme with protein kinase A catalytic subunit and (ii) both 8-bromoadenosine 3 ′,5 ′-cyclic monophosphate and 8-(4-chlorophenyltio)-cAMP inhibited renal glucose synthesis, it seems likely that in rabbit renal tubules ATP and inosine stimulate gluconeogenesis via cAMP decrease, which favors the appearance of a more active, dephosphorylated form of fructose-1,6-bisphosphatase, a key gluconeogenic enzyme.

Glycerol and Lactate Induce Reciprocal Changes in Glucose Formation and Glutamine Production in Isolated Rabbit Kidney-Cortex Tubules Incubated with Aspartate

In renal tubules isolated from fed rabbits, 1 mM aspartate is mainly utilized for production of glutamine, glutamate, alanine, and serine, while it is not used for glucose synthesis. However, the addition of either 2 mM glycerol or 2 mM lactate, which are poor gluconeogenic substrates in renal tubules, results in acceleration of both glucose formation and incorporation of [ 14C]aspartate into glucose by several fold, accompanied by about a twofold decrease in glutamine synthesis and marked accumulation of glutamate and alanine. Ammonium release in renal tubules incubated with aspartate in the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, is also decreased on the addition of glycerol and lactate by about two- and threefold, respectively. Since intracellular [glyceraldehyde 3-phosphate]/[3-phosphoglycerate] [glycerol 3-phosphate]/[dihydroxyacetone phosphate], [lactate]/[pyruvate], and intramitochondrial [glutamate]/[2-oxoglutarate] × [NH + 4] ratios are increased in comparison with control values determined with aspartate alone, it is likely that the stimulatory effect of lactate and glycerol on glucose formation from aspartate may be due to (i) an increased availability of reducing equivalents in the cytosol resulting in an enhancement of glyceraldehyde-3-phosphate dehydrogenase activity and (ii) elevation of the mitochondrial NADH/NAD + ratio causing a decrease in glutamate dehydrogenase activity resulting in a diminished glutamine synthesis and enhanced provision of carbon skeleton of aspartate for gluconeogenesis. Stimulation of glucose formation in the presence of 1 mM aspartate + glycerol is not related to cell volume changes. However, an increase for about 30% of intracellular water space induced by 10 mM aspartate + glycerol is accompanied by both diminished gluconeogenesis and enhanced glutamine synthesis, compared with values measured with 1 mM aspartate plus glycerol.

Differential in vivo and in vitro effect of gentamicin on glutamate synthesis and glutamate deamination in rabbit kidney-cortex tubules and mitochondria

The effect of gentamicin on both glutamate synthesis and glutamate deamination was studied in kidney-cortex mitochondria and tubules isolated from both control and gentamicin-treated animals. In kidney-cortex mitochondria which were permeabilized in order to make a free access of substrates and antibiotic to the glutamate dehydrogenase, gentamicin appeared to be a very potent inhibitor of glutamate synthesis, resulting in about 60% decrease of the enzyme activity at 5 mm concentration. Other aminoglycoside antibiotics decreased the enzymatic activity, in the following order: gentamicin>neomycin=tobramycin=kanamycin> biodacyna>amikacin>streptomycin. This, in principle, corresponds to their known nephrotoxic potential observed in vivo. The inhibitory action of antibiotics was abolished by neither ADP nor leucine, allosteric activators of glutamate dehydrogenase. Surprisingly, gentamicin did not decrease the rate of ammonia formation from glutamate when added to both renal tubules and mitochondria isolated from control rabbits. This indicates that the antibiotic exerts its inhibitory effect on glutamate dehydrogenase activity in the direction of glutamate synthesis only. In contrast, the rate of both glutamate deamination and glutamate synthesis was about 40% lower in renal tubules and mitochondria isolated from kidney-cortex of animals which were given antibiotics for 10 days. In view of these results it seems that (i) the depression of ammoniagenesis in gentamicin-treated animals may be due to a decrease of glutamate dehydrogenase content and (ii) under conditions in vitro the aminoglycoside inhibits the enzyme activity in the direction of glutamate synthesis while it does not affect the glutamate deamination.

Calcium-Dependent Effect of Gentamicin on Glucose Formation in Isolated Rabbit Kidney-Cortex Tubules

In contrast to the inhibition by gentamicin of glucose production from propionate, pyruvate and lactate in renal tubules incubated at 2.5 mM Ca2+, this antibiotic does not affect gluconeogenesis from propionate and lactate, and significantly stimulates this process from other substrates at 0.5 mM Ca2+. This may be due to the gentamicin-induced increase of the cytosolic manganese content (from 1.7 to 2.7 nmol/mg protein), resulting in a stimulation of cytosolic phosphoenolpyruvate carboxykinase activity. At 2.5 mM Ca2+ the cytosolic Mn content (2.7 nmol/mg protein) seems to be high enough to accomplish activation of the enzyme.

Utilization of alanine for glucose formation in isolated rabbit kidney-cortex tubules

In kidney cortex tubules isolated from fed rabbits L-alanine is not utilized as glucose precursor, when added as a sole substrate. However, this amino acid decreases gluconeogenesis from low (up to 1 mM) 2-oxoglutarate concentrations and stimulates this process at higher (2.5–10 mM) ketoacid contents in the suspension medium. Aminooxyacetate, an inhibitor of aminotransferases, abolishes both inhibitory and stimulatory effects of L-alanine on glucose formation. The addition of 2-oxoglutarate increases the incorporation of L-[U- 14C]alanine to glucose from 8- to 123-fold, depending upon the ketoacid and alanine concentrations used. In contrast, nonlabelled L-alanine decreases the incorporation of low [U- 14C)2-oxoglutarate concentrations into glucose, while it does not affect contribution of 5 mM ketoacid to gluconeogenesis. The data indicate that (i) in the presence of 2-oxoglutarate L-alanine is utilized as glucose precursor in rabbit renal tubules and (ii) this amino acid may decrease the contribution of low extracellular concentrations of the ketoacid to gluconeogenesis.

Effect of various aminoglycoside antibiotics on glucose formation in isolated rabbit kidney cortex tubules

The effect of six aminoglycoside antibiotics on the rate of gluconeogenesis was studied in isolated rabbit kidney-cortex tubules incubated with various substrates. All antibiotics studied did not affect glucose formation from both malate and 2-oxoglutarate. The rank order of the drug-induced inhibition of glucose formation from lactate, pyruvate and propionate was the following: neomycin = gentamicin > tobramycin > kanamycin = amikacin > streptomycin. This in principle corresponds to their ability to diminish pyruvate carboxylation in isolated kidneycortex mitochondria, as well as to their known nephrotoxic potential observed in vivo. Aminoglycoside antibiotics decreased the respiration of tubule. suspension incubated with various substrates. However, the rank order of the inhibition by antibiotics of oxygen uptake was different from that observed for gluconeogenesis, suggesting that the rate of energy generation does not limit glucose formation under conditions studied.

Effect of glycerol on gluconeogenesis in isolated rabbit kidney cortex tubules

In renal tubules isolated from fed rabbits glycerol is not utilized as a glucose precursor, probably due to the rate-limiting transfer of reducing equivalents from cytosol to mitochondria. Pyruvate and glutamate stimulated an incorporation of [ 14C]glycerol to glucose by 50- and 10-fold, respectively, indicating that glycerol is utilized as a gluconeogenic substrate under these conditions. Glycerol at concentration of 1.5 mM resulted in an acceleration of both glucose formation and incorporation of [ 14C]pyruvate and [ 14C]glutamate into glucose by 2- and 9-fold, respectively, while it decreased the rates of these processes from lactate as a substrate. In the presence of fructose, glycerol decreased the ATP level, limiting the rate of fructose phosphorylation and glucose synthesis. As concluded from the ‘cross-over’ plots, the ratios of both 3-hydroxybutyrate/acetoacetate and glycerol 3-phosphate/dihydroxyacetone phosphate, as well as from experiments performed with methylene blue and acetoacetate, the stimulatory effect of glycerol on glucose formation from pyruvate and glutamate may result from an acceleration of fluxes through the first steps of gluconeogenesis as well as glyceraldehyde-3-phosphate dehydrogenase. As inhibition by glycerol of gluconeogenesis from lactate is probably due to a marked elevation of the cytosolic NADH/NAD + ratio resulting in a decline of flux through lactate dehydrogenase.

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.

Renal glycerol metabolism and the distribution of glycerol kinase in rabbit nephron

Glycerol and dihydroxyacetone are metabolized by rabbit kidney-cortex tubules, isolated by collagenase treatment. Half-maximal concentrations of both substrates were determined with regard to uptake rates and product formations. Maximal uptake rates were 643 and 329 mumol/h per g of protein for dihydroxyacetone and glycerol respectively. Glucose and lactate were found as major metabolic products. Glycerol kinase, the enzyme catalysing the first step in renal glycerol and dihydroxyacetone metabolism, was measured radiochemically as described by Newsholme, Robinson & Taylor [(1967) Biochim, Biophys. Acta 132, 338-346] and adapted for studies of the localization of this enzyme along the different structures of rabbit nephron. The results show that glycerol kinase is located exclusively in the proximal segments, i.e. the proximal convoluted tubules and the pars recta, but is negligible in the other structures studied. The activities were close to the maximal dihydroxyacetone uptake rates measured in tubule suspensions.

Parathyroid hormone and adenosine-3',5'-monophosphate acutely increase phospholipids of the phosphatidate-polyphosphoinositide pathway in rabbit kidney cortex tubules in vitro by a cycloheximide-sensitive process.

Parathyroid hormone (PTH) rapidly increased to concentrations of phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide during incubations of rabbit kidney cortical tubules in vitro. These effects were preceded by increases in cAMP, which also induced virtually identical increases in these phospholipids. Pretreating the tubules with cycloheximide inhibited these phospholipid effects of PTH and cAMP. These findings are similar to those reported for ACTH and cAMP in the adrenal cortex. Hormones that utilize cAMP as their "second messenger" may influence membrane structure and function via stimulation of the phosphatidate-polyphosphoinositide pathway.

Effects of probenecid on transport and metabolism of cyclic AMP by isolated rabbit renal tubules

The effects of probenecid on the transport and metabolism of cyclic [ 14C]-AMP were studied in isolated rabbit kidney cortex tubules. Incubation in a medium with 10–400 μM probenecid for 30 min caused a 30–70% decrease in the tubular uptake of labeled material from a medium containing 0.1 mM cyclic [ 14C]AMP. The radioactivity in the tubules, after 30 min incubation, with or without probenecid, was mostly in the form of inosine and hypoxanthine. The disappearance of external cyclic [ 14C]AMP was retarded by probenecid and the concentration ratio of cyclic AMP to inosine + hypoxanthine was increased. Cyclic AMP phosphodiesterase activities, from both the soluble and particulate fractions of the kidney, were inhibited by probenecid. These findings indicate that the changes caused by probenecid on the renal disposal of extracellular cyclic AMP can be accounted for by a decrease in the accumulation of the products of cyclic AMP metabolism secondary to inhibition of extracellular cyclic AMP phosphodiesterase activity.