Intracellular distribution of arylsulphatase activity in adrenal cortical cells of the rat

Summary Arylsulphatase (B) activity has been demonstrated by means of an electronmicroscopic cytochemical method (substrate: p-nitrocatechol sulphate; capturing ion: barium, pH 5.5) in the lysosomes, in many Golgi elements, and occasionally in the endoplasmic reticulum of rat adrenal cortical cells.     

Metabolic effects of valproate on dog renal cortical tubules

The effect of valproate (0.01-10 mM), an antiepileptic drug inducing hyperammonemia in humans, was studied in vitro on a suspension of renal cortical tubules (greater than 85% proximal tubules) obtained from six normal dogs. When these tubules were incubated with 1 mM glutamine, the addition of valproate accelerated glutamine uptake, ammoniagenesis, and the production of alanine, lactate, and pyruvate. With 5 mM glutamine, a rise in glutamate accumulation, a much greater synthesis of alanine, an important aspartate production, and a striking accumulation of lactate and pyruvate were observed. With 1 or 5 mM lactate, lactate utilization and gluconeogenesis were markedly reduced with increasing concentrations of valproate. Oxygen consumption was reduced by only 15-20% by 10 mM valproate. The accelerated glutamine utilization resulting from valproate could not be prevented by aminooxyacetate, an inhibitor of transamination. Valproate also reduced various enzymatic activities, a finding that could not explain its metabolic effects. Four sites of action may explain these various metabolic changes: (i) a stimulation of mitochondrial glutamine transport, (ii) an increase in the flux of glutamate to malate, and (iii) a reduction in the net oxidation of pyruvate and (iv) in the flux through pyruvate carboxylase.     

Improvements in the method for the electron microscopic localization of arylsulphatase activity

Summary The optimal conditions for the demonstration of arylsulphatase activity in the proximal convoluted tubule cells of the rat kidney were studied at light and electron microscopic level. 8-hydroxyquinoline sulphate, p-nitrophenyl sulphate and 2-hydroxy-5-nitrophenylsulphate were used as substrates and barium and lead as capturing ions. The effect of fixation, capturing ions, substrate concentration and pH was studied biochemically. The results of these biochemical studies were then verified histochemically. Finally a recommended method for the light and electron microscopic demonstration of arylsulphatase activity was presented.     

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.     

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.     

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.     

Lipid methylation, hormone action and compensatory hypertrophy in renal cortical tubules

Lipid methylation has been studied in homogenized dog kidney cortical tubules. At pH 9, using S-adenosyl-L-methionine as methyl donor, most of the methyl groups appeared incorporated into phosphatidylcholine, the activity showing an apparent Km of 16 microM. This enzymatic activity was unchanged by the presence of the divalent cations Ca2+ or Mg2+, cAMP or cGMP. In addition, lipid methylation was also unchanged after treatment of intact tubules with angiotensin II, parathyroid hormone or vasopressin. An increased phosphatidylcholine synthesis was observed in the remnant kidney cortical tubules after uninephrectomy through an activation of phosphocholine transferase without detectable modification of lipid methylation. These findings suggest that lipid methylation is not regulated by these biochemical or functional stimuli tested in canine renal cortical tubules.     

Immunocytochemical localization of gamma-glutamyl-transferase on isolated renal cortical tubular fragments

The localization of gamma-Glutamyltransferase (gamma-GT, E.C.2.3.2.2) was studied on isolated tubular fragments from rat kidney cortex immunocytochemically. Monospecific antibodies raised in the goat against rat kidney gamma-GT were used. Antigoat immunoglobulin from the rabbit conjugated with ferritin was used for visualisation of the antibody binding sites. The enzyme was found to be localized at the brush border membrane of proximal tubules, the luminal membrane of distal tubules and collecting duct segments. The enzyme could further be localized on the antiluminal or basolateral cell membranes of proximal and distal tubular fragments, whereas no such localization was verified for collecting duct segments. The role of this basolateral gamma-GT localization in context with the kidney's ability to extract over 83% of the renal arterial glutathione (GSH) input during a single passage is discussed.     

Histochemical localization of the soluble arylsulphatase activities in rat brain

Summary The modified method of Goldfischer has been used for the localization of soluble arylsulphatase activities in the rat brain. The highest activity of these enzymes was found in some parts of the drive system and in nervous tracts. The bulk of activity in neurons and glial cells is localized in the lysosomes. Some arylsulphatase activity has been found to be bound to myelin sheaths. We have not been able to ascribe this activity to any defined subcellular structures, by light microscopy.The method of Goldfischer does not permit differentiation of the two soluble arylsulphatases (enzymes: A and B). We suggest however, that these enzymes may have different functions in the brain. Arylsulphatase A (cerebrosidesulphatase) may be primarily connected with the nervous tracts, a hypothesis supported by the character of disorders caused by lack of this enzyme in metachromatic leucodystrophy. Arylsulphatase B, hydrolysing sulphuric esters of catecholamines, may be involved in the function of the drive system.Arylsulphate sulphohydrolase, EC 3. 1.6. 1.     

Succinate dehydrogenase localization in the mitochondria of the renal tubules of cold- and warm-blooded vertebrates

Using electron microscopic histochemical technique, studies have been made on the activity of succinic dehydrogenase in the kidneys of the cod Gadus morrhua and dog. It was shown that chelate granules indicating localization of the enzyme in the mitochondria of nephronal cells, concentrate mainly in two zones -- between the membranes and inside the cristae. This distribution of the enzyme implies the presence of two pools of succinic dehydrogenase in the mitochondria which are utilized at different stages of oxidative phosphorylation. Succinic dehydrogenase content of the cristae is lower in cod than in dog.     

Cystine-glutamate transport interactions in rat renal cortical tubules,brushborder vesicles,and cultured renal tubule cells

Glutamate had no significant effect on the uptake of 0.025 mM cystine by isolated rat renal cortical tubules and brushborder membrane vesicles in contrast to lysine which significantly inhibits cystine transport. Glutamate, however, markedly inhibited cystine uptake by rat renal tubule cells grown in a serum-free, hormonally defined media for 5 days. Lysine also inhibited cystine transport in these cultured renal tubule cells.     

Correlation between cyclic AMP levels and calcium efflux in isolated renal cortical tubules.

Isolated renal cortical tubules from male hamsters were utilized to examine the possible relationship between cyclic AMP (cAMP) and efflux of calcium. Both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) produced dose-related increases in cAMP levels and calcium efflux from isolated tubules. Maximal concentrations of both hormones resulted in changes in cAMP which were 6 fold greater and changes in calcium efflux which were 2 fold greater with PGE1 than with PTH. Effects of sub-maximal amounts of either hormone on both cAMP and calcium efflux were potentiated to tubule incubations resulted in increases in tissue-associated cAMP over the same degree by inclusion of methyl-isobutylxanthine (MIX). Addition of either exogenous cAMP or dibutyryl cAMP (db-cAMP) produced dose-related increases in calcium efflux which occurred more rapidly with db-cAMP than with cAMP. Increasing amounts of cAMP added to the same concentration range resulting in increases in calcium efflux. Addition of 2', 3' cyclic AMP, 5'AMP or db-cyclic GMP had no significant effect on calcium efflux while 3', 5' cyclic CMP significantly reduced this response. The results indicate that cAMP increases efflux of calcium from renal tubules and may play a central role in hormone-dependent transport of this ion.