Inhibitory effect of valproate on weak organic acid uptake in rat renal proximal tubules.

The effects of an antiepileptic drug, valproic acid (VPA), on transport mechanisms involved in renal excretion of anionic xenobiotics were investigated on rat renal proximal tubules in vitro. It was found that VPA (0.1-1 mM) dose dependently inhibited the baseline uptake of a marker organic anion, fluorescein, in the tubules. The inhibition could not be exclusively accounted for by competition between VPA and fluorescein. Taking into account a proposed relationship between the weak organic anion uptake and ammoniagenesis, the influence of VPA (0.5 mM) on the effects of glutamine and glutamate (both at 5 mM) on fluorescein uptake and ammonia production were examined. Glutamine stimulated ammonia production by the tubules, with the glutamine-induced ammoniagenesis being further augmented by VPA, while glutamate failed to affect the basal ammoniagenesis. Both glutamine (5 mM) and glutamate (5 mM) slightly inhibited fluorescein uptake, with the inhibitory effects not modified by VPA. Thus, there was no coincidence in the effects of VPA on organic anion uptake and renal ammoniagenesis. At the same time, the inhibitory effect of VPA (0.5 mM) on fluorescein uptake was largely overcome by addition of pyruvate (5 mM) to the incubation medium. In addition, VPA strongly inhibited glucose production from pyruvate. A known modulator of pyruvate metabolism, dichloroacetic acid (DCA, 1 mM), also inhibited fluorescein uptake, although its inhibitory effect was less pronounced than that of VPA. Both inhibitors failed to alter the tissue content of alpha-ketoglutarate or lactate but did slightly augment the pyruvate level. The inhibitory effects of VPA and DCA on the baseline fluorescein uptake were not additive, suggesting their similar intracellular targeting. It is assumed that the inhibitory effect of VPA on baseline fluorescein uptake in rat renal proximal tubules in vitro may be associated with its action on pyruvate metabolism.

Weak organic acid uptake in rat renal tubules in vitro: stimulation by pent-4-enoic acid.

A hypoglycemic agent, pent-4-enoic acid (4-PA; 0.1-1.0 mM), stimulated baseline uptake of a weak organic anion, fluorescein, in superficial proximal tubules of rat kidney and inhibited the rate of glucose production from pyruvate (but not lactate or endogenous substrates) by rat renal cortex fragment suspension. The stimulation of the fluorescein uptake was not observed in a low Na+ medium. Maleate (0.1-1.0 mM) and Cd2+ (0.1 mM), known similarly to 4-PA to induce the renal Fanconi syndrome, also stimulated the fluorescein uptake in the Na-dependent manner. Both 4-PA and Cd2+ and maleate elevated intracellular content of alpha-ketoglutarate and increased ammonia formation from endogenous substrates in the suspension of the rat renal cortex fragments. The stimulatory effects of 4-PA, maleate and Cd2+ on the fluorescein uptake were markedly attenuated by LiCl (5 mM), suggesting that the Na-coupled re-uptake of alpha-ketoglutarate is involved in energization of the fluorescein uptake in the exchange for the cytoplasmic dicarboxylate.

Effects of metabolic substrates and inhibitors on weak organic anion transport in rat renal proximal tubules.

Stimulatory effects of intermediates of the tricarboxylic acid cycle on renal uptake of a weak organic anion, fluorescein, were studied with the aid of the method of contact microfluorimetry of individual convoluted proximal tubules ascending to the surface of the rat renal cortex slices. The study was undertaken for verifying the hypothesis that energization of renal excretion of anionic exenobiotics is mediated through their transport across the basolateral membrane in exchange for cytoplasmic alpha-ketoglutarate serving as a counter-anion. Effects of inhibitors of the tricarboxylic acid cycle such as fluoroacetate, malonate and 5-methoxyindole-2-carboxylate on the fluorescein uptake and renal gluconeogenesis in the presence of the metabolic substrates were investigated in order to outline metabolic pathways that could be responsible for elevation of the cytoplasmic alpha-ketoglutarate. Obtained data evidence that the stimulatory effects of the tricarboxylic acid cycle intermediates on the transport process under study depend on the metabolic state of the mitochondria and involve an activation of certain reactions but not the cycle as a whole. It has been suggested that an elevation of the cytoplasmic alpha-ketoglutarate resulting from this activation can be conditioned by export of isocitrate from the mitochondria with its subsequent transformation into alpha-ketoglutarate in the cytoplasm in the isocitrate dehydrogenase reaction.

The effect of frusemide on oxytocin-induced contractions of the rat myometrium.

Oxytocin-induced contractions of isolated strips of oestradiol-treated rat myometrium were found to be affected by exposure to the diuretic frusemide. At a concentration of 20 microM. frusemide transiently increased the force of contraction over a period of approximately 10 min. After this time there was a progressive fall in contractile force. At a higher concentration of 200 microM, only the progressive fall in force was seen until contractions were completely abolished. Frusemide has been reported to increase the activity of cAMP-phosphodiesterase in tissue extracts from oestradiol-treated rat myometrium. Therefore, the changes in contraction due to exposure to frusemide may be a reflection of the changes in intracellular cAMP resulting from a stimulation of cAMP-phosphodiesterase activity. In support of this idea, addition of dibutyryl cAMP was found to partially restore contractions after frusemide treatment. These data suggest that frusemide may be a useful tool in the manipulation of tissue cAMP levels in order to determine the different roles of cAMP in the oestradiol-treated rat myometrium.

Stimulation of weak organic acid uptake in rat renal tubules by cadmium and nystatin.

The uphill uptake of a weak organic acid, fluorescein, in superficial proximal tubules of the rat kidney was stimulated by CdCl2 (0.1 mM) or nystatin (20 microM) in the absence of metabolic substrates in the incubation medium. The stimulation could be observed during the initial period of incubation (up to 30 min) only and was prevented completely by ouabain (0.1 mM), fluoroacetate (1 mM), malonate (10 mM), alpha-cyano-4-hydroxycinnamate (0.1 mM), phenylpyruvate (1 mM), D-malate (2 mM) or phenazine methosulfate (20 microM). In the renal cortex fragment suspension, both Cd2+ and nystatin increased the ouabain-sensitive, basal oxygen consumption and inhibited the rate of glucose production from pyruvate, but not from lactate. In the presence of lactate (0.5-5 mM) in the incubation medium, Cd2+ and nystatin rather inhibited fluorescein uptake, while externally added pyruvate did not influence their stimulatory effects. Taken together, these data suggest that both activation of the tricarboxylic acid cycle and export of reducing equivalents from the mitochondria to the cytosol are necessary for the stimulatory effects of Cd2+ and nystatin on the weak organic acid uptake to develop.

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 (alpha-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.

[The mechanisms of the effect of furosemide on the water permeability of the frog bladder and on oxytocin-stimulated contractions of the rat uterus]. / Mekhanizmy vliianiia furosemida na vodnuiu pronitsaemost' mochevogo puzyria liagushki i stimulirovannye oksitotsinom sokrashcheniia matki krysy.

Furosemide increased the hydrosmotic water flow in the frog urinary bladder and promoted the ADH-like effect of inhibitors of phosphodiesterase cAMP, potentiated hydrosmotic effects of theophylline and serosal osmotic hypertonicity but failed to change the effect of pituitrin. Fur reversibly suppressed oxytocin-induced contractions in the rat myometrium, inhibited the activity of the frog urinary bladder PDE cAMP, whereas the activity of the enzyme from the rat medulla and myometrium was activated by saluretic. Incubation of the myometrium strips in Fur resulted in a decrease in the cAMP content of the tissue. The cAMP seems to play an important role both in the myometrium smooth muscle relaxation and in the oxytocin-activated contractions.

Effects of inhibitors of gluconeogenesis on weak organic acid uptake in rat renal tubules.

Using inhibitors of gluconeogenesis (phenylpyruvate, alpha-cyano-4-hydroxycinnamate, quinolinate, D-malate, aminooxyacetate), we analysed mechanisms by which the gluconeogenic substrates, lactate and pyruvate, as well as a short-chain fatty acid, acetate, stimulate the uptake of a weak organic acid, fluorescein, in the rat kidney. We have shown that these inhibitors modified both the rate of glucose production from lactate and pyruvate in the renal cortex fragment suspension and the stimulatory effects of the metabolic substrates on fluorescein uptake in superficial proximal tubules in the renal cortex slices. The peculiarities of the effects of lactate and pyruvate on the uptake were correlated with the partial divergence of the pathways of gluconeogenesis from these precursors. The linkage of the weak organic acid uptake with gluconeogenesis is interpreted in terms of the hypothesis that the uptake is controlled by the cytoplasmic pyridine nucleotide redox potential, which is maintained with the participation of certain processes involved in glucose synthesis.

[Acetylcholinesterase and the ADH-dependent transport of water in the amphibian bladder]. / Atsetilkholinésteraza i ADG-zavisimyi transport vody v mochevom puzyre amfibii.

It was found that acetylcholine (ACh) at the concentration of 10(-3) M inhibited ADH-stimulated water transport through the wall of amphibian urinary bladder. This effect was suggested to be caused by an interaction of ACh with acetylcholinesterase (AChE) rather than by a stimulation of the M- or N-cholinoreceptor. The inhibitory action of ACh was completely suppressed in the presence of various AChE inhibitors (physostigmine, proserine, armine, Gd-42, acridine-iodmethylate), while an inhibitor of butyrylcholinesterase (BuChE), AD-4, failed to affect it. In accord with this observation the activity of AChE (but not of BuChE) was demonstrated in the urinary bladder epithelium. Since, in addition to the hydrosmotic effects of pituitrine, 8-arginine-vasopressin or oxytocin, ACh blocked also effects of forskolin or cyclic AMP, one may conclude that it acts at some post-cyclic AMP production stage. AChE-dependent inhibition of the ADH-stimulated water transport decreased significantly when the serosal pH was raising from 7.2 to 8.0, but was augmented by serosal acidification (pH 6.8), whereas such pH alterations did not affect the activity of the epithelium AChE. The effect of ACh under consideration was suppressed by adding amiloride (10(-4) M) to the serosal solution. Similarly, the ACh effect was blocked by an inhibitor of Ca-dependent K+ channels, 4-aminopyrdine, which in addition prevented the inhibition of the ADH-stimulated water transport by the serosal acidification. It was noteworthy that some other K+ channel blockers (Ba2+, Cs+, tetraethylammonium, apamine, quinine) did not affect either the water transport or the antipituitrine effect of ACh. In conclusion, we suggest that the inhibitory action of ACh on the ADH-stimulated water transport in the urinary bladder is mediated through the intracellular acidification resulting from ACh interaction with AChE. It is unlikely that the acidification is merely a consequence of the ACh hydrolysis, rather the ACh-AChE interaction induces directly an increase in the proton conductivity of the basolateral membrane of the urinary bladder epithelium.

[Membrane phosphodiesterase of cyclic nucleotides in the chemosensory structures of fishes and amphibians]. / Membrannaia fosfodiésteraza tsiklicheskikh nukleotidov v khemosensornykh strukturakh u ryb i amfibii.

Comparison of the activities of phosphodiesterase in membrane and cytosol preparations obtained from the olfactory mucosa of the frog and taste epithelium of the catfish revealed high content of the membrane-bound enzyme and high resistance of phosphodiesterase from the olfactory mucosa to phenylacetic acid (10(-9)-10(-4) M).

[The presence of calmodulin in chemosensory structures, its purification and content]. / O nalichii kal'modulina v khemosensornykh strukturakh, ego ochistka, soderzhanie.

Chemosensory tissues, bovine olfactory epithelium and barbel of dwarf sheat-fish (Ictalurus nebulosus) rich in gustatory buds were shown to contain calmodulin. The fraction of thermostable proteins which activate brain phosphodiesterase of cyclic nucleotides was purified to homogeneity by stepwise ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-300. Some properties of calmodulin from chemosensory tissues (e. g., content, molecular weight, electrophoretic mobility, degree of activation of phosphodiesterase) are similar to those of brain calmodulin.

[Interaction of leucine with the plasma membranes of catfish taste buds]. / Vzaimodeistvie leitsina s plazmaticheskimi membranami vkusovykh lukovits karlikovogo somika.

The binding of L-[3H]leucine by the plasma membranes from the catfish Ictalurus nebulosus taste organ was studied. The two types of specific binding centers for amino acid with high (KD = 2,5 X 10(-10) M) and low (KD = 1,04 X 10(-9) M) affinities were found. Concentrations of high and low affinity centers were 11,85 nmol/mg and 26 nmol/mg respectively. The structural rearrangement of the surface layer of the chemoreceptor membrane was revealed by spin label technique using 5-doxylstearic acid at leucine concentrations 10(-4)-10(-9) M.

[Properties of cyclic nucleotide phosphodiesterase from lingual taste papillae]. / Svoistva fosfodiesterazy tsiklicheskikh nukleotidov vkusovykh sosochkov iazyka.

Phosphodiesterase activity is estimated in extracts and partially purified preparations from functionally different parts of bovine tongue. The enzyme activity varied from 4.0 to 10.4 nmole/mg of protein/min. Properties of phosphodiesterase from circumvallate papillae are studied, the pH optimum being 8.0--8.5, Km for cAMP--1.5.10(-4) M and for cGMP--6.5.10(-5) M. The enzyme activity did not change after the treatment with trypsin, protamine sulphate (0.01--1.0%), heparin (0.01--1.0) and taste agents: L-leucine (from 1.10(-2) M to 1.10(-5) M), quinine (from 4.10(-3) M to 4.10(-8) M) and D-glucose (from 1.10(-1) M to 1.10(-4) M). The protein inhibitor of the enzyme, isolated from retina external rod-cell segments considerably suppressed phosphodiesterase activity, and the protein activator from brain tissue stimulated it insignificantly. Thermostable protein modulators, which inhibit or activate (depending on experimental conditions) phosphodiesterase activity, are isolated from circumvallate papillae.