Kinetic analysis of mechanisms of Na+-dependent transport of organic acids in the proximal tubules of surviving frog kidney. I. Transport of fluorescein and uranin in a sodium-free environment]

By direct microfluorimetry, the kinetic characteristics of active transport system for organic acid were studied. A removal of sodium from the incubation medium is shown to affect the affinity between a carrier and acids (KS), and the rate of translocation of the transporting complex (which includes a carrier and a substrate) across the plasma membrane. The data obtained are in good accordance with the concept of Na+-dependent transport.     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions II. Relationship between counter-flows of fluorescein and sodium ion across cell layer

With the aid of a direct microfluorimetric method a dependence of organic onion (fluorescein) transport into proximal tubules of surviving frog kidney on Na⁺-flow in the opposite direction was studied. It was shown that the complete removal of Na⁺ from the tubules lumen resulted in inhibition of fluorescein transport of about 30%. After a specific inhibitor of sodium channels, amiloride (10^-3M) having been introduced into lumen of the tubules, the fluorescein transport was inhibited to the same extent. Amiloride affects only when Na⁺ is present in the tubular lumen. S present in the tubular lumen. Strophantin K (5 · 10^?5 M), a specific inhibitor of (Na⁺, K⁺)-ATPase, reduced fluorescein transport about twice. Substances increasing the 3′,5′-AMP level in cells (theophylline, NaF) and exogenous 3′,5′-AMP inhibited fluorescein transport while substance that decreased the 3′,5′-AMP level intracellularly (carbachol) stimulated it. For realization of these effects Na⁺ should be present in proximal tubules lumen.Thus, the various effects on the Na⁺ flow from lumen of the tubules to medium at the level of both the basal and apical membranes alter the rate of organic acid active transport from medium to lumen as a result of changes in the maximum rate of transport ($\text{V}$) with unchanged $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. It is suggested that the system of Na⁺ extrusion from proximal tubules produces peritubular membrane-side (near the membrane) gradient of Na⁺ concentration which may be higher than the summary Na⁺ gradient between the medium and the cytoplasm. The magnitude of this gradient affects the maximal rate value of Na⁺-dependent organic acid transport. So, there is a double dependence of the active transport system on Na⁺, and the stages where Na⁺ is needed are: (1) the formation of a carrier-substrate-Na⁺ complex and (2) the production of substantial membrane-side Na⁺ gradient at the expense of Na⁺ extrusion from the tubules.     

Inhibition by cyclic AMP and dibutyryl cyclic AMP of transport of organic acids in kidney cortex.

1. Cyclic adenosine 3',5'-monophosphate and N-6-2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate decrease the initial entry rate and the steady-state uptake of p-aminohippurate and uric acid by rabbit kidney cortex slices. 2. N-6-2'-O-Dibutyryl adenosine 3'-5'-monophosphate inhibits the tubular transport of p-aminohippurate competitively. 3. Isoproterenol, known to increase cyclic nucleotide concentration of the cortical tubules by activation of adenyl cyclase, decreases p-aminohippurate transport. Antidiuretic hormone which is known to stimulate only medullary adenyl cyclase has no effect on p-amino-hippurate uptake by cortical slices. 4. Theophylline, which inhibits cyclic nucleotide phosphodiesterase and, therefore, enhances the cellular accumulation of endogenous cyclic nucleotide, depresses p-aminohippurate transport.     

Mutants of Serratia marcescens lacking cyclic nucleotide phosphodiesterase activity and requiring cyclic 3',5'-AMP for the utilization of various carbohydrates.

Adenine requiring mutants of Serratia marcescens SM-6-F'lac+ have been found to grow well in minimal-glucose medium solely supplemented with cAMP. From one of these ade strains double mutants (called ade cpd) were isolated which could no longer utilize cAMP but which still grew on 5'AMP. Dialyzed cell extracts (soluble fraction) of the double mutants, assayed for cAMP phosphodiesterase, were unable to hydrolyze cAMP whereas cell extracts of the parental strains yielded 5'AMP at a rate of 1.6-2.0 mumoles min-1 mg-1 protein. The loss of the phosphodiesterase activity in S. marcescens cpd W 1181 did not cause an accumulation of large amounts of cAMP as was found for the diesterase-negative mutant AB257pc-1 of Escherichia coli. The induced synthesis of beta-galactosidase in mutant cpd W 1181 showed about the same sensitivity to transient and permanent catabolite (glucose) repression as the corresponding cpd+ strain. Starting from S. marcescens cpd W 1182 three independent double mutants (called cpd cya) were isolated which required exogenous cAMP for utilizing various carbohydrates as carbon source, for motility and for the formation of extracellular lipase and the red pigment prodigiosine. The intracellular concentration of cAMP in these mutants, grown in nutrient broth, was 40-60% of that of the parental strain which is about 4 x 10(-4) M. However, the adenylate cyclase in cell extracts of the mutants W 1237 and W 1270 was like that of the corresponding cya+ strain (about 2 x 10(-2) mumoles min-1 mg-1 protein).     

Regulation of pyruvate dehydrogenase by Ca2+, 3'5' cyclic AMP and adrenalin in isolated rat kidney tubules.

Renal pyruvate dehydrogenasea activity was measured in suspensions of kidney cortex tubules from fed rats after incubation with various concentrations of Ca2+, 3'5' cyclic AMP or adrenalin. At certain concentrations these agents all decreased pyruvate dehydrogenasea activity and increased glucose formation from pyruvate. There appeared to be some interdependence between the effects of Ca2+ and 3,5, cyclic AMP on pyruvate dehydrogenasea activity. Effects of adrenalin on pyruvate dehydrogenasea activity were no longer observed when palmitate was included in incubations. The possibility that regulation of pyruvate dehydrogenasea may indirectly contribute to the control of gluconeogenesis by influencing pyruvate metabolism is briefly discussed.     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions I. Influence of sodium ions in bath medium on the uptake and run out of fluorescein and uranin

With the aid of direct microfluorimetric determination of marker organic anions (fluorescein and uranin) accumulated in the proximal tubules the influence of Na⁺ in the bath medium on the active transport of these anions was studied. Kinetic analysis of the rate dependence of organic acid active transport into tubules on their concentration in the bath medium with a constant Na⁺ concentration permitted to define values of apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}$ for uranin and fluorescein transport in the medium with different Na⁺ content. It was shown that a decrease of Na⁺ concentration in the medium increases $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and lowers the $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio with uncharged $\text{V}$. By varying the Na⁺ concentration in the medium with a constant concentration of the marker anion the and values for fluorescein and uranin transport were determined. A value for fluorescein in twice as much that for uranin. The $\text{1/K}{}_{\mathrm{<mtext>m</mtext>}}$ value for uranin transport is a linear function of Na⁺ concentration, while for fluorescein transport it is a quadratic one. Therefore it is concluded that two Na⁺ from the medium participate in active transfer of one fluorescein anion whereas only one Na⁺ from the medium is required for active transfer of one uranin anion. The run out of fluorescein from tubules preloaded with this acid is sharply reinforced by the Na⁺ omission from the medium. Thus, active transport of organic acids in proximal tubules of frog kidney is Na⁺-dependent, and Na⁺ from the medium is likely to participate directly in formation of a transport complex. When Na⁺ is absent in the medium a carrier fulfils a facilitated diffusion only.     

Elaboration of chloramphenicol acetyltransferase by cells of E. coli K-12 under conditions altering the intracellular concentration of cyclic adenosine-3',5'-monophosphate]

The influence of cAMP, ACTH and glucose on the stimulation of chloramphenicol acetyl transferase synthesis in the cells of E. coli CSH-2/R222 and E. coli WZ-78/R222. (cya855) was examined. Glucose proved to decrease the enzyme synthesis in E. coli CSH-2/R222, causing catabolite repression; 5 mM of cAMP and 1000 mug/ml ACTH overcame the latter. The enzyme synthesis in E. coli WZ-78/R222 was insensitive to the catabolite repression; as to ACTH - it failed to cause stimulation ofchloramphenicol acetyl transferase synthesis in E. coli WZ-78/R222.     

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

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.     

Dissociation of parathyroid hormone and cyclic-3', 5'AMP effects on Na-Pi uptake by cells isolated from proximal straight tubules of rat kidney.

Studies in respiratory alkalotic or short-term phosphate deprived rats raised the possibility that in straight portion of proximal tubules (PST) cAMP might be not a mediator of PTH in inhibition of phosphate reabsorption. The present experiments directly compared the sensitivity of Na-dependent phosphate [32P] (Na-Pi) uptake to PTH or cAMP by PCT or PST cells freshly prepared from outer cortex and outer stripe of outer medulla of rat kidney. The purity of the cells was examined by activity of enzymes specific for PST i.e. glutamine synthetase, gamma-glutamyl transpeptidase and creatine kinase, a marker enzyme for medullary thick ascending limb (MTAL) and distal convoluted tubule. Similar inhibition of Na-Pi uptake by 1-34 bPTH by PST and PCT cells was observed: -33.0 and -30.0% (ns), respectively. In contrast, dibutyryl cAMP decreased Na-Pi uptake only by PCT but not by PST cells: -31.0 and -3.6% (p<0.02), respectively. The 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, resulted in slight stimulation of Na-Pi uptake by PST but strong inhibition by PCT cells: 7.8 vs -26.0% (p<0.001). In contrast to PCT in PST cells cAMP seems to play a minor role as a mediator of inhibition of Na-Pi uptake by PTH.     

Cardiotropic effects of luliberin. Effects of luliberin on the activities of phosphorylase A and ornithine decarboxylase and concentration of 3', 5'-AMP]

The level of luliberin, the luteinizing hormone-releasing hormone (LH-RH), and the possibility of the hormonal control of metabolic and biosynthetic processes in the heart were studied. It was shown that the rat heart contains a factor, which is immunochemically and chromatographically related to LH-RH (19--46 picograms per organ). Intraperitoneal injection of LH-RH increases the activities of phosphorylase A and ornithine decarboxylase and the concentration of 3',5'-AMP and potentiates the stimulating effect of adrenaline on ornithine decarboxylase.     

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

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.     

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

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.     

Interaction of "aza" and "deaza" analogs of adenosine cyclic 3', 5'-phosphate with some enzymes of adenosine cyclic 3', 5'-phosphate metabolism: evidence that the lone pair electrons of N-3 are involved in the binding of adenosine cyclic 3', 5'-phosphate to type II adenosine cyclic 3', 5'-phosphate-dependent protein kinase.

Five hetercyclic analogs of adenosine cyclic 3',5'-phosphate (cyclic AMP) were examined for their ability (1) to stimulate type II cyclic AMP-dependent kinases from bovine brain, bovine heart, and rat liver; (2) to serve as substrates for "high Km" (Km for cyclic AMP = 0.13-0.43 mM) cyclic nucleotide phosphodiesterases from bovine heart, rabbit kidney, and rat liver; and (3) to inhibit the hydrolysis of cyclic AMP catalyzed by "low Km" (Km for cAMP = 0.32-1.5 muM) cyclic nucleotide phosphodiesterases from bovine brain, bovine heart, dog heart, rabbit liver, rat brain and rat liver. The analogs all had a purine ring system which had been modified by replacement of a ring carbon with nitrogen or vice versa to yield 2-aza-cAMP (7-amino-4-beta-D-ribofuranosylimidazo [4,5-d] -v-triazine cyclic 3',5'-phosphate); 8-aza-cAMP (7-amino-3-beta-D-ribofuranosyl-v-triazolo-[4,5-d]-pyrimidine cyclic 3',5'-phosphate); 1 deaza-cAMP (7-amino-3-beta-D-ribofuranosylimidazo [4,5-b[pyridine cyclic 3',5'-phosphate); 3-deaza-cAMP (4-amino-1-beta-D-ribofuranosylimidazo[4,5-c]pyridine cyclic 3',5'-phosphate) and 7-deaza-cAMP (7-amino-4-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidine cyclic 3',5'-phosphate).