The protective nature of Chironomus luridus larval tubes against copper sulfate.

The objective of this study was to determine whether the tubes in which Chironomus larvae dwell protect them against chemical toxicants. A laboratory culture of an Israeli benthic midge, Chironomus luridus, was exposed to copper sulfate. Two conditions were tested in bioassay experiments: larvae within silt tubes and larvae without tubes. The non toxic, anionic, fluorescent dye, fluorescein, was used to examine the effect of sub-lethal copper sulfate concentrations on the permeability of cuticular, gill and gut epithelia of the chironomids. Increased cell permeability, which is the cause of cell damage, was reflected by an increase in fluorescence intensity. Following exposure to copper sulfate, higher fluorescence was found in different body compartments: midgut, hindgut, tracheal gills, fat body and muscles, and the Malpighian tubules. The effect was significantly higher in tube-free larvae when compared to silt tube dwelling larvae. We conclude that in addition to its other functions in feeding, respiration, and anti-predation shelter, the Chironomus luridus tube protects its inhabitant from toxins such as copper sulfate.

The influence of the lipid bilayer phase state on the p-aminohippurate (PAH) transport and the activity of the alkaline phosphatase in brush-border membrane vesicles from normal and mutant rats.

The kinetic parameters of p-aminohippurate transport and activity of the alkaline phosphatase were studied using brush-border membrane vesicles isolated from the kidney cortex of normal and mutant (strain of Campbell) rats. p-Aminohippurate (PAH) transport of both normal and mutant animals was carried out by the mechanism of facilitated diffusion. The apparent Michaelis constant at 36 degrees C was equal to 7 mM, the maximal rate of PAH transport was 15 nmol/min per mg protein and the constant of inhibition by probenecid was 0.5 mM for normal rats and, respectively, 29 mM, 62 nmol/min per mg protein and 1.4 mM for mutant rats. The Arrhenius plot for the PAH transport and activity of the alkaline phosphatase showed the breakpoints at 28-30 degrees C for normal rats and at 36-38 degrees C for the Campbell strain rats. The thermotropic phase transitions detected by the EPR method with 5-doxylstearate as a probe were recorded at 21-30 degrees C and 30-35 degrees C for normal and mutant rats, respectively. Therefore, characteristic features of the PAH carrier and alkaline phosphatase activity in normal and Campbell strain rats are determined by the difference in the phase state of their membrane lipid bilayers. We suppose that mutation in the Campbell strain gives rise to a membrane pleiotropic effect which enables us to understand the mechanism of genetic control of the lipid structure and membrane fluidity.

[Physicochemical mechanisms of organic acid transport in the apical membrane vesicles of the cells of proximal kidney tubules in rats. II. Kinetic parameters of transport and phase state of the lipid bilayer in mutant Campbell strain rats]. / Fiziko-khimicheskie mekhanizmy transporta organicheskikh kislot v vezikulakh apikal'noi membrany kletok proksimal'nykh kanal'tsev pochki krys. II. Kineticheskie parametry transporta i fazovoe sostoianie lipidnogo bisloia u mutantnykh krys linii Kempbell.

The kinetics of 14C-para-aminohippuric acid (PAH) transport in the vesicles and the influence of the temperature on the initial rate of this transport were studied using a purified fraction of the apical membrane isolated from the kidney cortex of the Campbell strain rats with an autosomic recessive gene. The transport was brought about owing to the facilitate diffusion mechanism. At 36 degrees C the apparent Michaelis constant was equal to 29 mM, the maximum rate--62 nmol/min on 1 mg of protein, the inhibition constant for the PAH-transport by probenecid--1.5 mM. The temperature dependence of the initial rate of PAH-transport in vesicles and that of the rate of substrate splitting by alkaline phosphatase show the break point on the Arrhenius plot at 36 degrees C-38 degrees C. The analysis of electron magnetic resonance reveals the thermotropic transition at temperatures near 30 degrees-35 degrees C. Therefore the affinity of the carrier to its substrates in vesicles of the Campbell strain rats is strongly reduced and the lipid layer is more viscous than in the normal rats. We decide therefore that the mutation taking place in the Campbell strain leads to pleotropic membrane reconstructions in different organs (eye, kidney). The discovery of such a mutation is of considerable biological interest and promotes bases for development of the membrane biochemical genetics.

[Physico-chemical mechanisms of organic acid transport in the apical membrane cells of the proximal kidney tubules in rats. I. Kinetic transport parameters and effect of the lipid phasic state on transport]. / Fiziko-khimicheskie mekhanizmy transporta organicheskikh kislot v vezikulakh apikal'noi membrany kletok proksimal'nykh kanal'tsev pochki krys. I. Kineticheskie parametry transporta i vliianie fazovogo sostoianiia na transport.

The kinetics of the transport of 3H-para-aminohippuric acid (PAH) and the influence of the temperature on the initial rate of transport were studied on the vesicles of a purified fraction of the apical membrane isolated from cells of kidney proximal tubules. The PAH transport is accomplished owing to the facilitate diffusion mechanism. The apparent Michaelis constant at 36 degrees C was equal to 7.0 + 1.0 mM, the maximum rate was 15 nmol/min on 1 mg of protein, the inhibition constant for the PAH transport by probenecid being 0.5 mM. At 22 degrees C the apparent Michaelis constant was drastically increased. When the temperature dependence of the initial rate of PAH transport into vesicles was replotted in the form of the Arrhenius plot, there was a turning-point of the line at 28-30 degrees C. The same turning-point is shown on the Arrhenius plot for temperature dependence of alkaline phosphatase activity (a marker enzyme for the apical membrane). The electron paramagnetic resonance spectra analysis of 5-doxylstearate-labeled apical membrane preparation reveals a thermotropic transition near 21-29 degrees C. It is concluded that the function of the carrier and the activity of alkaline phosphatase depend on the phasic state of membrane lipids; the normal function of membrane proteins is possible under the liquid-crystalline state of the lipid bilayer.

[Effect of a NaCl gradient on the transport of para-aminohippuric acid into the vesicles of the basolateral membrane of the kidney cortex]. / Vliianie gradienta NaCl na transport paraaminogippurovoi kisloty v vezikuly bazolateral'noi membrany kory pochki.

Basolateral membrane vesicles were isolated from the rat kidney cortex by a modified method of cation precipitation. Different steps of preparation were analysed using the marker enzymes: Na+,K+-ATPase (for basolateral membrane), alkaline phosphatase (for apical membrane), glucose-6-phosphatase (for membranes of endoplasmic reticulum) and succinate dehydrogenase (for mitochondria). The basolateral membrane was purified by a 8-9-fold treatment with Na+,K+-ATPase, while other membrane contaminations were as low as 2% (as compared to homogenate). The transport of 3H-p-aminohippurate (3H-PAH) by basolateral membrane vesicles was measured under different experimental conditions. The 3H-PAH uptake was found to be Na-gradient dependent. The initial rate of 3H-PAH uptake in the presence of NaCl gradient (500 pM/mg X min) was higher than without the gradient (88 pM/mg X min). It is concluded that the PAH transfer across the basolateral membrane may be energized by the Na+ chemical gradient.

[Specific binding of organic acids by a fraction of the basolateral membrane of the rat kidney cortex isolated by osmotic shock]. / Spetsificheskoe sviazyvanie organicheskikh kislot fraktsiei bazolateral'noi membrany kory pochki krysy, poluchennoi metodom osmoticheskogo shoka.

Non-vesiculated membrane fragments of the basolateral membrane of the rat kidney cortex were isolated by the osmotic shock method and fractionated by means of differentional centrifugation. Formation and purity of membrane fragments were tested morphologically (contact luminescent, phase-contrast and electron microscopy) and biochemically (determination of the activity of marker enzymes--Na+, K+-dependent ATPase and alkaline phosphatase). The activities of Na+, K+-ATPase and alkaline phosphatase in the purified fraction of the basolateral membrane were 21 and 0.2%, respectively, of those in the kidney cortex homogenate. The binding of 14C-hyppuric and 14C-uric acids with basolateral membrane fragments was studied by means of filtration through the millipore filters. The existence of competitive inhibition and substrate saturation of the binding testify to the presence of organic acid carrier in the basolateral membrane. The affinity of the carrier to hyppurate in membrane preparations was proved to be the same as in the intact proximal tubules (the apparent Michaelis constant is equal to 0.7 mM). The equilibrium constant (Kf) for the carrier-hyppurate complex does not exceed 10 M-1. That means that the complex of the carrier with hyppurate is not strong.

[Effect of functional loading on the operation of the active transport system for organic acids in the proximal kidney tubules of rats after unilateral nephrectomy and in the early postnatal period]. / Vliianie funktsional'noi nagruzki na rabotu sistemy aktivnogo transporta organicheskikh kislot v proksimal'nykh kanal'tsakh pochki krys posle odnostoronnei nefréktomii i v rannem postnatal'nom periode.

The influence of a prolonged introduction of exogenic organic acid penicillin (that is functional loading) on the level of accumulation of an anionic dye (fluorescein) in renal proxima tubules was studied after unilateral nephrectomy and early postnatal period. Injection of penicillin 2 days after unilateral nephrectomy slowly increased Na-independent and strongly increased Na-dependent component of active fluorescein transport in renal proximal tubules of randombred, but strongly decreased both Na-independent and Na-dependent transport in renal tubules of the Campbell rats. When newborn random-bred, Wistar and Campbell rats were pretreated with penicillin, we obtained a slow increase in Na-independent and a strong increase in Na-dependent component of fluorescein transport in renal tubules of random-bred and Wistar rats, but a significant reduction in both Na-independent and Na-dependent transport. It is concluded that the ability for adaptive (or substrate) stimulation of active transport of organic anion in renal proximal tubules is controlled genetically. Adaptive stimulation of organic acid transport in renal tubules referred to in literature as "carried induction", was accomplished apparently by the increase in driving force of the active transport, that is evidently the level of electrochemical Na+-gradient.

[Work of the active transport system of organic acids in the kidney proximal tubules of rats with compensatory hypertrophy]. / Rabota sistemy aktivnogo transporta organicheskikh kislot v proksimal'nykh kanal'tsakh pochki krys pri kompensatornoi gipertrofii.

The active transport of organic acids in proximal renal tubules of the Campbell, Wistar and random-bred rats was studied by contact microfluorometry with anion dye fluorescein as a marker, on 1, 2, 3, 5, 10, 30 and 90 days after a unilateral nephrectomy. Sham-operated rats were used as a control. Transport was investigated under certain conditions when fluorescein was transported by Na-dependent (at 30 degrees C) or Na-independent (at 20 degrees C) systems. It is shown that fluorescein accumulation in renal tubules of operated random-bred rats at 30 degrees C is (if compared to the corresponding control) 81, 105 and 75% on 1, 2 and 3-10 days, resp., and about 90% on 30 and 90 days. Fluorescein accumulation at 20 degrees C in all investigated rats was about 90% of the corresponding control. A specific poison of Na+, K+-ATPase = ouabain inhibited fluorescein accumulation on day 1 after sham-operation by 70% to control, but there was no inhibition on day 1 after the operation (unilateral nephrectomy). P-aminohippuric acid in both media inhibited fluorescein accumulation in renal tubules of sham-operated rats better than in operated rats. It is concluded that either the amount of the carried of transport system for organic acids decreased in membranes of renal tubules of operated rats, or the affinity of carrier to organic acids is aggravated.

[Cadmium stimulation of Na-independent organic acid transport in the kidney tubules]. / Stimulirovanie kadmiem Na-nezavisimogo transporta organicheskoi kisloty v pochechnykh kanal'tsakh.

The influence of Cd++ (as well as of Hg++ and Cu++) on the uptake of an organic acid (fluorescein) in superficial proximal tubules of the surviving rat kidney was studied at 20 degrees C, when the active transport of fluorescein does not depend on the external Na. In contrast to mercury and copper, cadmium stimulated the uptake of fluorescein from the beginning of incubation. The minimal effective concentration of Cd++ was 5 X 10(-6)M, the relative effect of Cd++ on the uptake being the same within the concentration range from 5 X 10(-6) to 10(-3) M. A 60 minutes pre-incubation with Cd++ at 20 degrees C resulted in a significant increase in the stimulatory effect of acetate on the fluorescein transport. The stimulation of the fluorescein transport by cadmium was prevented by ouabain or by omissing Na from the incubating medium, although neither ouabain nor the absence of Na affected the transport of fluorescein under these conditions. It is supposed that the stimulation by Cd++ of the fluorescein transport may result from the activated oxidation of NAD-linked substrates due to acceleration of the active transepithelial transport of Na ions.

[The system of active transport of organic acids in the proximal tubules of rats with hereditary retinal degeneration (Campbell strain)]. / Sostoianie sistemy aktivnogo transporta organicheskikh kislot v proksimal'nykh kanal'tsakh krys s nasledstvennoi degeneratsiei setchatki (liniia Kempbell).

Active transport of organic acids (ATOA) in proximal renal tubules of Campbell, Wistar and random-bred rats was studied by contact microfluorometry with anion fluorescein as a marker. Beginning with the 60th day of life this transport in Campbell rats was characterized by a lower equilibrium uptake of fluorescein by the tubules, by altered sensitivity to AMP and adenosine, and by no response to unilateral nephrectomy. It is assumed that ATOA in Campbell rats plays a certain role in the pathogenesis of retinitis pigmentosa.

[Effect of artificial NaC1 and KC1 gradients on the active transport of organic acids in energy-depleted kidney proximal tubules. II. The KC1 gradient]. / Vliianie iskusstvennykh gradientov NaC1 i KC1 na aktivnyi transport organicheskikh kislot v deénergizovannykh proksimal'nykh kanal'tsakh pochki. II. Gradient KC1.

The stimulatory effect of KCL in bath medium on fluorescein uptake in superficial proximal tubules in the absence of Na in bath medium was observed on Na- and energy-depleted slices of the rat kidney outer cortex. In the absence of K gradient between bath medium and tubular cells, the fluorescein uptake increased with raising the intracellular K concentration. The efflux of fluorescein from the tubules was inversely proportional to K concentration in the cells. Visual control showed that at high K concentrations fluorescein was accumulated mainly in the tubular walls (i. e. in the cytoplasm of tubular cells). It is suggested that the raise of K concentration in cells results in the intracellular binding of organic acid.

[Effect of artificial NaCl and KCl gradients on the active transport of organic acids in energy-depleted proximal kidney tubules. I. The NaCl gradient]. / Vliianie iskusstvennykh gradientov NaCl i KCl na aktivnyi transport organicheskikh kislot v deénergizovannykh proksimal'nykh kanal'tsakh pochki. I. Gradient NaCl.

The present work has been undertaken to obtain a direct evidence of the involvement of chemical gradient of Na+ in organic acid transport in the renal tubules. Superficial tubules of rat and turtle kidneys were depleted of Na and K during cold preincubation to be then incubated in the presence of ouabain under anoxic conditions at 30 degrees C. Uptake of an organic acid, fluorescein, was detected in proximal tubules of the essential surface of kidneys by means of contact microfluorometry. The creation of artificial gradient of NaCl (medium to cells) stimulated the uphill uptake of fluorescein in the tubules. The dissipation of Na gradient was accompanied with the efflux of fluorescein from tubules. The presence in the medium of harmaline, an inhibitor of Na-dependent transport systems, as well as the replacement of Cl- for SO2- or SCN- resulted in the disappearance of the stimulatory effect of Na gradient on the fluorescein transport. It is concluded that the fluorescein transfer across the baso-lateral membrane of tubular cells may be energized by Na chemical gradient, and this process significantly depends on the anionic environment.

[Inorganic and organic anion interrelations in the active transport system for organic acids in the proximal kidney tubules]. / Issledovanie vzaimootnoshenii neorganicheskikh i organicheskikh anionov v rabote sistemy aktivnogo transporta organicheskikh kislot v proksimal'nykh kanal'tsakh pochki.

A mechanism of inhibitory action of inorganic anions (SCN- and SO 2/4-) on the transport of an organic acid (fluorescein) into proximal tubules was investigated in slices of the rat's kidney outer cortex. The peritubular S14CN- is shown to be accumulated in the cortical tissue by means of simple diffusion, this accumulation not depending on the presence of fluorescein in the medium. The peritubular 35SO 2/4- is accumulated in the tissue against the gradient of its electrochemical potential. It is very likely that the peritubular sulphate penetrates via intercellular contacts into the tubular lumen to be then actively reabsorbed by the Na+-dependent transport system in the luminal membrane. The sulphate accumulation is not affected by fluorescein added to the medium. It is concluded that the inhibition of organic acid transport after replacement of Cl- in the medium by SCN- or SO 2/4- may not be considered as a competition for a common carrier between organic and inorganic anions.

[Active transport of organic acids in the proximal tubules of the surviving rat kidney normally and under various influences. II. The effect of exogenous cyclic 3',5'-AMP and of substances that alter its intracellular concentration]. / Aktivnyi transport organicheskikh kislot v proksimal'nykh kanal'tsakh perezhivaiushchei pochki krysy v norme i pri nekotorykh vozdeistviiakh. II. Vliianie ékzogennogo tsiklicheskogo 3',5'-AMF i veshchestv, izmeniaiushchikh ego vnutrikletochnuiu kontsentratsiiu.

Exogenous cyclic 3',5'-AMP (cAMP) and substances known to increase the intracellular concentration of this nucleotide (isoproterenol, theophylline, noradrenaline, lactate) were shown to inhibit the transport of fluorescein (a weak organic acid) into the rat renal proximal tubules at 20 degrees C. Carbacholine decreasing intracellular cAMP concentration stimulated the transport. Propranolol, a beta-adrenergic blockator, diminished significantly the inhibitory effect of noradrenaline on the transport. Lactate and carbacholine when added simultaneously, neutralize their action. The inhibitory action of intracellular cAMP on the transport is supposed to be a result of the diminition of a pool of endogenous weak organic acids which may take part in the exchange of diffusion with the marker anion across basal plasma membrane.

On the active transport of organic acids (fluorescein) in the choroid plexus of the rabbit.

The kinetics of active transport of an organic acid (fluorescein) through the membranes of the choroid plexus from the lateral ventricules of the brain of rabbit was studied both morphologically and functionally. It was shown that fluorescein is actively translocated through the apical and basal membrane of the epithelium and is accumulated in blood capillaries at a concentration exceeding one order of magnitude that in the incubation medium. The kinetic curves displaying saturation and the demonstration of inhibition by other acids shows that a specific carrier is involved in the transfer across the membrane. The active transport of fluorescein at 20 degrees C was found to be sodium independent. Total exclusion of sodium from the incubation medium does not change the Michaelis constant (Km) and maximal velocity (V). The active transport depends on the operation of (Na+ + K+)-ATPase as energy source but obviously no specific complexes with the participation of sodium are involved.

[Active transport of organic acids in the proximal tubules of a surviving rat kidney normally and under certain actions. I. The effect of temperature, aeration conditions and Na ions]. / Aktivnyi transport organicheskikh kislot v proksimal'nykh kanal'tsakh perezhivaiushchei pochki krysy v norme i pri nekotorykh vozdeistviiakh. I. Vliianie temperatury, uslovii aératsii i ionov Na.

The transport of a marker organic acid (fluorescein) in the intact proximal tubules was studied with the aid of contact microfluorimetry of the surface of surviving rat kidneys. The kinetics of transport at 20 and 37 degrees obeys to the Michaelis-Menten equation. The increase of oxygen content in the gas phase, from 21 to 100%, results in raising V max by 1.7 times, with an apparent Km being unchanged. With the 100% oxygen content taken as a gas phase, the fluorescein transport rate is maximal at 37--40 degrees, the temperature raising from 20 to 37 degrees results in decreasing Km by 4.5 times and in increasing V max by 45%. Both Na-free medium and the addition of strophantin K inhibit fluorescein uptake at temperature higher than 25 degrees only. At 37 degrees the omission of Na+ from the bath medium inhibits the fluorescein transport via Km augmentation with Vmax being unchanged. Thus, active transport of fluorescein is Na+-dependent in physiological range of temperature and the motive force of the transport is an electrochemical Na+-gradient created by means of Na+, K+-ATPase operation.