Slices Of Rat Renal Cortex Research Articles

Effect of type A and B monoamine oxidase selective inhibition by Ro 41–1049 and Ro 19–6327 on dopamine outflow in rat kidney slices

1. The influence of pargyline and of selective inhibitors of type A and B monoamine oxidase (MAO), Ro 41-1049 and Ro 19-6327 respectively, on the outflow of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in slices of rat renal cortex loaded with exogenous L-3,4-dihydroxyphenylalanine (L-DOPA) was examined. Dopamine and DOPAC in the tissues and in the effluent were assayed by means of h.p.l.c. with electrochemical detection. 2. The levels of newly-formed dopamine and DOPAC in the perifusate decreased progressively with time. In control conditions, DOPAC/dopamine ratios in the perifusate were 3 to 5 fold those in the tissue and were found to increase progressively with time. The addition of pargyline (100 microM), produced a marked decrease in the outflow levels of DOPAC (45 to 54% reduction) and significantly increased the levels of dopamine in the effluent (102 to 158% increase); DOPAC/dopamine ratios in the perifusate remained stable throughout the perifusion and were similar to those found in the tissues. The addition of the MAO-A inhibitor Ro 41-1049 to the perifusion fluid also significantly decreased DOPAC outflow (41% to 54% reduction) and increased dopamine outflow (19% to 80% increase). In the presence of Ro 41-1049 DOPAC/dopamine ratios in the perifusate were lower (P < 0.01) than in controls; in contrast with the effect of pargyline, this ratio was found to increase (P < 0.01) throughout the perifusion period. Ro 19-6327 did not reduce the outflow of DOPAC, but significantly increased (by 40-60%) that of dopamine. In the presence of Ro 19-6237, the proportion of DOPAC to dopamine in the perifusate was similar to that of controls and significantly increased throughout the perifusion; however, this increase was less than that observed in the control group.3. When benserazide (50 microM) was added to the perifusion fluid, the levels of both dopamine and DOPAC in the effluent were similar to those observed in the absence of benserazide. However, in the presence of benserazide, DOPAC/dopamine ratios in the perifusate did not increase with time. In conditions of decarboxylase inhibition, the effects of pargyline, Ro 41-1049 and Ro 19-6327 on dopamine and DOPAC outflow were less pronounced than in experiments conducted in the absence of benserazide.4. In conclusion, the results presented here show that the fraction of newly-formed dopamine which leaves the compartment where the synthesis has occurred is a constant source for deamination into DOPAC. The results provide evidence favouring the view that MAO-A is the main form of the enzyme involved in this process; however, the data described here suggest that dopamine would also have access to MAO-B.

Stimulatory effect of ethanol on weak organic acid uptake in rat renal tubules

Ethanol at relatively low concentrations (10–40 mM) significantly stimulated the uphill uptake of a weak organic acid, fluorescein, in the superficial proximal tubules of rat renal cortex slices, but it did not affect the rate of glucose production from lactate or pyruvate in rat renal cortex fragment suspension. In a low Na + medium, ethanol failed to stimulate fluorescein uptake, although under the conditions employed in the present study, the baseline weak organic acid uptake was not dependent on external Na +. The stimulation of fluorescein uptake by ethanol (20 mM) was abolished by an inhibitor of alcohol dehydrogenase (EC 1.1.1.1), pyrazole (1 mM), or an inhibitor of aldehyde dehydrogenase (EC 1.2.1.3), cyanamide (0.3 mM), suggesting that oxidation of ethanol mediated its effect on the uptake. Among gluconeogenesis inhibitors tested, only d-malate (2 mM) abolished the stimulatory effect of ethanol, while the rest either did not affect (quinolinate) or even slightly augmented (α-cyano-4-hydroxycinnamate and phenylpyruvate) it. The effect of ethanol was markedly increased by an inhibitor of the tricarboxylic acid cycle, fluoroacetate. It was concluded that the stimulation by ethanol of weak organic acid uptake in rat renal tubules was mediated by the production of acetate.

Actin cytoskeleton, tubular sodium and the renal synthesis of dopamine

The present study has examined the effect of colchicine and cytochalasin B, two cytoskeleton disrupter compounds, on the formation of dopamine in slices of rat renal cortex loaded with exogenous l-3,4-dihydroxyphenylalanine ( l-DOPA); the deamination of newly formed dopamine into 3,4 dihydroxyphenylacetic acid (DOPAC) was also examined. The accumulation of newly formed dopamine and DOPAC in kidney slices loaded with l-DOPA (10–100 μM) was found to be dependent on the concentration of l-DOPA, being similar in control conditions and in preparations treated with increasing concentrations of colchicine (5, 10 and 50 μM). By contrast, cytochalasin B (5, 10 and 50 μM) was found to produce a concentration-dependent reduction in the formation of dopamine and of its deaminated metabolite DOPAC in kidney slices loaded with l-DOPA (10–100 μM). The inhibitory effect of cytochalasin B on the formation of dopamine was found to be completely abolished in kidney slices pretreated with ouabain (500 μM) or when sodium concentration in the incubation was reduced from 120 to 20 mM. On its own, ouabain (500 μM) was found to reduce the formation of dopamine by 55%; the effect of reducing sodium concentration in the incubation medium to 20 mM was also a significant reduction (53% decrease) in the formation of dopamine. The accumulation of DOPAC did always parallel that of its parent amine. It is concluded that the renal formation of dopamine is dependent on the concentration of sodium in the medium and the integrity of the tubular transport of sodium, namely on the association between actin cytoskeleton and Na +,K +-ATPase, appears to be determinant.

Sodium-dependence and ouabain-sensitivity of the synthesis of dopamine in renal tissues of the rat.

1. The present study has examined the influence of sodium chloride (0-160 mM) and ouabain (100 and 500 microM), an inhibitor of the enzyme Na(+)-K+ ATPase, on the synthesis of dopamine in slices of rat renal cortex loaded with exogenous L-dihydroxyphenylalanine (L-DOPA). The deamination of newly-formed dopamine into 3,4-dihydroxyphenylacetic acid (DOPAC) was also examined. The assay of L-DOPA, dopamine and DOPAC in kidney slices was performed by high performance liquid chromatography (h.p.l.c.) with electrochemical detection. 2. The accumulation of newly-formed dopamine and DOPAC in kidney slices loaded with L-DOPA (50 and 100 microM) was found to be dependent on the concentration of NaCl in the medium. A similar picture could be observed for DOPAC. The fractional rate of accumulation (k; mM NaCl-1) was at 50 and 100 microM L-DOPA, respectively, 0.00305 +/- 0.00036 and 0.00328 +/- 0.00029 for dopamine and 0.00672 +/- 0.00072 and 0.00641 +/- 0.00069 for DOPAC. The sodium-dependent formation of dopamine was completely abolished when the experiments were performed in the absence of oxygen. 3. In experiments performed in the presence of 120 mM NaCl, but not in conditions of low sodium (20 mM NaCl in the medium), ouabain (100 and 500 microM) was found to inhibit the accumulation of newly-formed dopamine and DOPAC (14-57% reduction; P less than 0.05); this effect was more marked at 50 and 100 microM L-DOPA. When the experiments were performed in the absence of oxygen the renal production of dopamine and DOPAC became markedly decreased (respectively, 40% and 77% reduction; P<0.05) and ouabain (100 and 500 microM) was no longer able to reduce the accumulation of both newly-formed dopamine and DOPAC.4. The formation of dopamine and its deamination to DOPAC in kidney homogenates closely depended on the concentration of L-DOPA added to the medium; ouabain (100, 500 and 1000 microM) was found to affect neither the formation of dopamine nor its deamination to DOPAC when homogenates were used instead of kidney slices.5. In conclusion, the results presented here show that the formation of dopamine in kidney slices loaded with L-DOPA is sodium-dependent and ouabain-sensitive suggesting the involvement of a co-transport system of sodium and DOPA into the tubular epithelial cell.

Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates

The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by cAMP-dependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem Chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 n m) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 n m or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological samples.

P2‐, but not P1‐purinoceptors mediate formation of 1,4,5‐inositol trisphosphate and its metabolites via a pertussis toxin‐insensitive pathway in the rat renal cortex

1. The adenosine receptor (P1-purinoceptor) agonists N6-cyclopentyladenosine and N-5'-ethyl-carboxamidoadenosine at concentrations up to 10 mumols 1(-1) affected neither basal, nor noradrenaline- and angiotensin II-stimulated formation of inositol-1-phosphate, inositol-1,4-bisphosphate, and inositol-1,4,5-trisphosphate in slices of rat renal cortex. 2. In contrast, adenine nucleotides (P2-purinoceptor agonists) markedly stimulated inositol phosphate formation. The observed rank order of potency adenosine-5'-O-(2-thiodiphosphate) (EC50 39 mumols 1(-1] greater than adenosine-5'-O-(3-thiotriphosphate) (587) greater than or equal to 5'-adenylylimidodiphosphate (App(NH)p, 899) greater than adenylyl-(beta, gamma-methylene)-diphosphate (4,181) was consistent with the interaction of the compounds with the P2Y-subtype of P2-purinoceptors. AMP and the ADP analogue (alpha, beta-methylene)-adenosine-5'-diphosphate were ineffective. ATP and ADP (less than or equal to 10 mmol 1(-1] did not produce a consistent increase, owing to their hydrolytic degradation in the incubation medium. 3. Whereas the inositol phosphate response to App(NH)p was linear only up to 5 min incubation, the time-dependent stimulation of noradrenaline declined at a slower rate. Following pre-exposure of the renal cortical slices to App(NH)p, renewed addition of App(NH)p caused no further enhancement in the accumulation of inositol phosphates, whilst noradrenaline was still capable of eliciting a response. This suggests that the apparent loss of responsiveness to App(NH)p is not due to substrate depletion or enzymatic inactivation, but most likely attributable to homologous desensitization of the purinoceptor. 4. Pretreatment of the animals with pertussis toxin caused a substantial reduction of functional Gi-protein, as indicated by the lack of [32P]-NAD incorporation in a membrane preparation of the renal cortex. Nevertheless, the increase in inositol phosphate formation induced by noradrenaline, angiotensin II, and App(NH)p was not significantly impaired. 5. We conclude that P2 gamma-purinoceptors are present in the renal cortex; these receptors stimulate formation of inositol phosphates via a pertussis toxin-insensitive pathway and undergo homologous desensitization. On the other hand, our results suggest that renal A,-adenosine receptors do not use stimulation of phosphoinositide breakdown as a transmembrane signalling system.

The volume and ionic composition of cells in incubated slices of rat renal cortex, medulla and papilla

The apparent extracellular space in incubated slices of rat renal cortex, medulla and papilla has been measured using three differently sized marker molecules, mannitol, sucrose and inulin. Cellular volumes have been estimated by following the efflux of 3-O- methyl- d-glucose from equilibrated slices. Sucrose appears to be the most accurate extracellular marker in each of the regions examined, in that the sum of its volume of distribution plus cellular volume approximates most closely to the total slice fluid volume. Inulin has the same volume of distribution as sucrose in cortical slices, but under-penetrates medullary and papillary tissue. Mannitol overestimates the extracellular space in all three regions, although its larger volume of distribution, relative to that of sucrose, was not statistically significant in papillary slices. When cell volume and composition are estimated (a) using sucrose as extracellular marker and (b) making appropriate allowance for the presence of bound tissue electrolytes, it is found that cells in each region have low Na + and high K + concentrations and contents. When papillary slices are incubated in medium of very high osmolality (NaCl plus urea, 2000 mosmol/kg H 2O) there is a moderate (approx. 23%) decrease in cell volume and an increase in cell fluid Na + and Cl − concentrations equal to approx. 50% of the increase in the extracellular concentrations. Cell K + concentrations remain unchanged. The results show that cells in renal slices are able to maintain high K +-to-Na + ratios when incubated in isosmotic (cortex) or moderately hyperosmotic media (medulla and papilla), and suggest that regulation of papillary cell volume following hyperosmotic shock can only partly be ascribed to uptake of extracellular electrolytes.

Uptake of trimethoprim by renal cortex

The purpose of this study was to examine the mechanisms involved in the uptake of the urinary antibacterial drug trimethoprim by incubated slices of rat renal cortex. Concentration-dependent studies of the uptake process demonstrated that a saturable component was involved. The results of inhibitor studies as well as the time-course pattern support the conclusion that at least two processes are involved in the uptake of trimethoprim. These include active transport via the organic cation system, accounting for about 40% of the total uptake, and a second component that continues to operate under conditions of inhibited cellular metabolism. Chromatographic examination of post-incubation bathing medium and slice extracts failed to demonstrate renal cortex metabolism of trimethoprim.

Uptake of inorganic lead in vitro by isolated mitochondria and tissue slices of rat renal cortex

Slices of rat renal cortex were shown to take up Pb 2+ during incubation in vitro; Pb 2+ was also shown to enter mitochondria within the slices. The uptake of Pb 2+ by isolated mitochondria was inhibited by N 3 −, La 3+ and ruthenium red. A steady state of uptake was attained within 60 sec. The concentration dependence of uptake was complex; maximum uptake was attained at 25 μM and inhibition ensued at higher concentrations. A substantial inhibitor-resistant component of Pb 2+ uptake was noted, especially at medium Pb 2+ concentrations greater than 25 μM, and these concentrations also inhibited respiration state 3. The effects on respiration were reduced if the mitochondria had been preincubated with ruthenium red. Slices of renal cortex incubated at 1° in medium with various concentrations of Pb 2+ showed two fractions of uptake, one saturating at 50–100 μM external Pb 2+ and the other at 150–200 μM. Subsequent incubation for 60 min at 25° led to further uptake at all concentrations. Upon isolation of mitochondria from incubated slices, significant amounts of Pb 2+ were detected in the mitochondria within 5 min of addition of Pb 2+ (200 μM), with maximum attained at 30 min. Electron microscopy of slices showed electron-dense particles, apparently of Pb 2+, in the cortical cells but the greatest concentrations was deposited in the basement membranes. The results indicate the importance of the basement membrane in limiting access of Pb 2+ to cortical cells, and of mitochondria in accumulating Pb 2+ once it is in the cells. They also illustrate the importance of interactions between Pb 2+ and Ca 2+.

Characteristics of ionic binding by rat renal tissue in vitro.

A study has been made of Na+ and Cl- binding in metabolically inhibited slices of rat renal cortex and outer medulla incubated in modified Krebs phosphate-bicarbonate Ringer solution. At pH 7.35 in control media (cortex, 147 mmol Na+/l, 105 mmol Cl-/l; outer medulla, 187 mmol Na+/l, 145 mmol Cl-/l) cortical slices bound (mean) 171 nmol Na+ and 56.7 nmol Cl-/mg solute-free dry weight; outer medullary slices bound 188 nmol Na+/mg and negligible amounts of Cl-. In both regions, Na+ was exchangeable on a 1:1 basis for K+ or Li+ in media containing equal concentrations of each cation: Na+ was completely displaced by La3+. In cortical slices in media containing equimolar Cl- and other monovalent anions, binding occurred according to the sequence acetate less than or equal to salicylate less than or equal to Cl- less than SCN-; Cl- was completely displaced by PO4(3-). When medium pH was lowered, Na+ binding was markedly reduced in both regions, whereas Cl- binding increased (and became significant in outer medulla). In NaCl solutions, Na+ binding capacity was saturated at control Na+ concentrations. When [Na+] was progressively reduced (iso-osmolality being maintained by addition of urea), bound Na+ in both regions was nearly linearly related to log medium [Na+]. Raising medium osmolality with urea caused decreased Na+ binding and increased Cl- binding in both regions. Na+ binding in both regions was significantly reduced by pre-treatment with chondroitinase ABC. Binding of both ions was independent of temperature within the range 2-37 degrees C. The possibility is raised that renal ionic binding might influence vectorial ion transport by affecting free ion activity in the region of the transporting cells.

Some effects of monovalent anion replacement on the volume and composition of cells in incubated slices of rat renal cortex

Steady-state cellular water content and cation content and concentration have been studied in slices of rat renal cortex incubated in media in which Cl − was replaced by various monovalent anions at pH 7.35. Anions derived from low molecular weight aliphatic acids caused net uptake of cell water and K +. In the presence of larger anions cell water content fell in a manner related to anionic equivalent weight, but water content in media containing anions from weak aliphatic or alicyclic acids was slightly but consistently higher than that in media containing anions from strong acids. Cell K + content did not decrease in these media. Aromatic anions caused enhanced cell water loss. When external pH adjusted to 6.8 or 7.8 it was found that in media containing anions from weak acids, but not from strong acids, increasing pH was associated with significantly decreased cell water content.

The relationship of 4-aminobutyric acid metabolism to ammoniagenesis in renal cortex

Mitochondrial 4-aminobutyrate aminotransferase in rat kidney can utilize pyruvate as the acceptor for the amino group of 4-aminobutyrate. Renal 4-aminobutyrate aminotransferase activity at saturating equimolar concentration of 4-aminobutyrate and 5 mM pyruvate is 42.8 ± 2.5 μmol/g protein per h (mean ± S.E.M.) or 70% of 4-aminobutyrate aminotransferase activity with equimolar α-ketoglutarate. 4-Aminobutyrate aminotransferase in brain does not transaminate with pyruvate. Since pyruvate is an important mitochondrial metabolite in kidney, net disposal of glutamate via the 4-aminobutyrate pathway is possible. The renal 4-aminobutyrate pathway in the rat has other distinctive features when compared with the pathway in rat brain. Most inhibitors of rat neuronal glutamate decarboxylase were ineffective against the renal form of the enzyme, but 20 mM semicarbazide inhibited the latter form by 80% ( P < 0.001) in vitro and reduced renal 4-aminobutyrate content by 75% ( P < 0.001) in vivo. In the presence of 20 mM semicarbazide, ammoniagenesis by rat renal cortex slices incubated in 1 mM glutamine was inhibited 26% ( P < 0.01). Semicarbazide was proportionately less effective (15% inhibition) when ammoniagenesis was stimulated (+243%) in slices prepared from chronically acidotic animals, and was no deterrant to ammoniagenesis when non-acidotic slices were incubated in supraphysiologic concentrations of 10 mM glutamine. We conclude that whereas integrity of the renal 4-aminobutyrate pathway may contribute to glutamate disposal and thus ammoniagenesis under physiologic conditions, the pathway is a passive participant in the overall process of ammoniagenesis.

Lack of effect of cholinergic agents or endogenous guanosine 3':5'-monophosphate on renin release by slices of rat renal cortex in vitro

Cyclic GMP, in concentrations exceeding 50 μM, and cyclic AMP, in concentrations exceeding 1 μM, significantly increased the release of renin by slices of rat renal cortex. Stimulants of cyclic AMP accumulation, such as epinephrine or norepinephrine, 1 μM, produced a significant increase of renin release but did not alter cyclic GMP levels. In contrast, acetylcholine, carbamylcholine, bethanachol, methachol and NaN 3 increased cyclic GMP accumulation without modifying renin release. These results suggest that exogenous cyclic GMP acts as a weak agonist of cyclic AMP on renin release in renal cortex. The effects observed with exogenous cyclic GMP, however, may not represent a physiological action since various stimulants of endogenous cyclic GMP accumulation did not alter renin secretion.

The effect of diamide on amino acid transport by rat renal cortex slices

Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of α-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4°C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles.

Oxygen Consumptions and Potassium Contents of Slices of Rat Renal Cortex

An oxygen electrode respirometer for determining the oxygen consumption of slices of mammalian renal cortex is described and assessed. Though rat renal cortical slices incubated in potassium-free medium for one hour lost 102 +/- 14 mmol of potassium/kg dry weight, there was only a small, nonsignificant fall in oxygen consumption. In contrast the oxygen consumption of slices incubated in potassium-free medium with 10 mmol.1-1 ouabain was markedly reduced (by 32 +/- 6%), while such slices lost 180 +/- 15 mmol of potassium/kg dry weight. These disproportionate effects on potassium loss and inhibition of oxygen consumption suggest that in renal cortical slices the loss of potassium in low potassium medium is not primarily due to inhibition of the conventional sodium pump.

Alterations in rat renal cortical and medullary guanosine 3′5′-monophosphate accumulation by oxygen- and calcium-dependent and -independent mechanisms: Evidence for a calcium-independent action of oxygen in renal inner medulla

Factors influencing guanosine 3′5′-monophosphate (cGMP) metabolism were examined in slices of rat renal cortex, outer medulla, and inner medulla. In the presence of extracellular Ca 2+ and O 2, a gradation of steady-state cGMP levels was evident among the tissues (inner medulla outer medulla cortex). Carbamylcholine, bradykinin, histamine, and the divalent cation ionophore A23187 significantly increased cGMP in each tissue. The cGMP-stimulating action of these agents was reversibly abolished by exclusion of either Ca 2+ or O 2. The influence of Ca 2+ and O 2 on expression of effects of carbamylcholine and related cGMP agonists was interdependent in each region of the kidney, since both were required for expression of agonist action. By contrast, nitrite, nitroprusside, NH 2OH, and nitrosoguanidine increased cGMP in the presence or absence of Ca 2+ or O 2. Thus, at least two distinct mechanisms for altering cGMP accumulation are operative or expressible in each region of the kidney: one that requires and one that does not require the presence of extracellular Ca 2+ and O 2. Results also suggested a role for transmembrane transport of Ca 2+ in the maintenance of basal cGMP and in the expression of the responses to Ca 2+-dependent agonists in renal cortex, outer and inner medulla. Thus, verapamil, which can block such transport, lowered basal cGMP and abolished these responses while ionophore A23187 enhanced cGMP in cortex and medulla only in the presence of Ca 2+. The interrelationship of Ca 2+ and O 2 in control of basal cGMP levels clearly differed in cortex compared to inner medulla. In cortex, Ca 2+ and/or O 2 deprivation produced quantitatively similar reductions in cGMP. Moreover, expression of the action of O 2 to increase cortical cGMP required Ca 2+. Thus, O 2 effects on cGMP in cortex were closely coupled with or mediated through Ca 2+. By contrast, in inner medulla, O 2 deprivation resulted in more pronounced reduction in basal cGMP than did Ca 2+-deprivation, and O 2 significantly increased inner medullary cGMP in the absence of extracellular Ca 2+. The latter indicates that the action of O 2 to increase cGMP in inner medulla is not totally interdependent with that of Ca 2+. In inner medulla, Ca 2+ or O 2 deprivation had effects on cAMP accumulation that were analogous to those on cGMP, whereas exclusion of extracellular Ca 2+ had no detectable effect on cAMP in cortex. Accordingly, common or related cellular processes may operate in control of accumulation of both cGMP and cAMP in inner medulla.

Selectivity of food colours for different organic acid transport systems in rat renal cortex.

Seventeen food colours were tested as inhibitors of the simultaneous uptake of labelled o-iodohippurate and iodipamide into slices of rat renal cortex. The results have been interpreted in terms of inhibition of two different aniontransport systems: the hippurate of H-system and the liver-like L-system. No clearcut relation was found between inhibition ability and system specificity on the one hand and moleculr weight or structure on the other. However, most disulphonic azo dyes and quinoline yellow show an L-system affinity while tri-and tetra-sulphonic azo dyes, triarylmethanes and erytrosine show very little predilection for either of the two systems.

Effects of replacing medium sodium by choline, caesium, or rubidium, on water and ion contents of renal cortical slices.

1. Renal cortical slices from rat, rabbit, and guinea-pig were incubated in media in which choline, caesium or rubidium replaced sodium.2. Slices of rabbit and guinea-pig renal cortex incubated in oxygenated choline Ringer decreased in volume initially and did not swell over 3 hr at 25 degrees C. There was a steady loss of potassium. Inhibition of metabolism (N(2) + 1 mM iodoacetamide) caused some swelling. Ouabain, 10 mM, in choline Ringer affected neither loss of potassium nor tissue water content.3. Slices of rat renal cortex similarly incubated in choline Ringer swelled over 3 hr at 25 degrees C whether or not metabolism was inhibited; ouabain (15 mM) affected neither tissue potassium loss nor tissue water content.4. Incubation in choline Ringer containing either 0.2 mMp-chloromercuribenzoic acid, or 1 mM ethacrynic acid increased the tissue water content of guinea-pig renal cortical slices.5. Depletion of cellular potassium (by preliminary incubation in oxygenated potassium-free sodium Ringer with 10 mM ouabain at 30 degrees C) resulted in increased tissue water content when rabbit renal cortical slices were subsequently incubated in oxygenated choline Ringer at 25 degrees C for 3 hr.6. There was no evidence of energy-dependent extrusion of water or ions from either equilibrated rat or rabbit renal cortical slices leached at 0.5 degrees C and then reincubated at 25 degrees C in choline Ringer.7. Rat and guinea-pig renal cortical slices leached at 0.5 degrees C and reincubated at 25 degrees C swelled in rubidium Ringer and in caesium Ringer. There was no evidence of energy-dependent water or ion extrusion when metabolism was restored after leaching in either of these media. Metabolizing rat slices but not guinea-pig slices swelled faster than slices whose metabolism was inhibited.8. These results lend no support to the mechano-chemical hypothesis which ascribes cellular volume regulation to a contractile mechanism squeezing isotonic extracellular fluid from the cells. Instead it is suggested that cellular water content in these experiments reflects the balance between the rate of loss of potassium (and chloride) from the cells and the rate of uptake of extracellular cation (and chloride) into the cells - these rates reflecting both the electrochemical potential gradients of the ions and membrane permeability to them. The implications in relation to the hypothesis of ouabain-insensitive cellular volume regulation are discussed.

Heterogeneity of the β-amino-preferring transport system in rat kidney cortex. Differential influence of glutathione oxidation

Taurine, a naturally found β-amino acid, is inert in rat renal cortex slices. Its active accumulation by slices is abolished by anaerobiosis, a strongly acidic media or the removal of Na +. Concentration-dependent uptake studies reveal more than one taurine carrier: the apparent K m value for uptake below 1.1 mM is 0.4 mM and the apparent K m value above 1.1 mM is 14.5 mM. Of all amino acids tested only β-alanine, another β-compound, inhibited uptake. The oxidizing agent diamide was used to lower the concentration of GSH in rat cortex slices. The ability to accumulate taurine by the low K m system was decreased in diamide-treated slices, but not by the high K m system. Diamide was found to greatly augment efflux of taurine taken up from lower concentrations but not from higher concentrations. GSH in the media prevented this diamide-induced inhibition of uptake and enhanced efflux at lower taurine concentrations. A possible mechanism of diamide inhibition of uptake is that intracellular GSH depletion leads to greatly enhanced efflux of taurine, thus preventing uptake.

Non-sterol metabolism of mevalonate [formula omitted]: Artifacts and realities

Commercial [5- 14C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5- 14C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated R-5-phospho-[5- 14C]mevalonate by ion-exchange chromatography. The artifactual 14CO 2 results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the 14CO 2 and [ 14C ]cholesterol produced by rat renal cortex slices incubated with purified [5- 14C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that R[5- 14C]mevalonate is genuinely oxidized to 14CO 2 in vitro , and that purification of substrate before its use is necessary. Production of 14CO 2 and various [ 14C ]lipids from purified [5- 14C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.