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E Kinne-Saffran

Publications and source records attributed to E Kinne-Saffran.

17 recordsLinked to original sources
PubMed1991

Localization and activity of renal carbonic anhydrase (CA) in CA-II deficient mice.

A null allele at the mouse Car 2 locus was induced by ethylnitrosurea; mice homozygous for the new allele lack the carbonic anhydrase (CA)-II isoenzyme. The expression of this genetic lesion was investigated by: (1) using tissue fractionation techniques to determine localization and activity of CA in the kidney, and (2) examining renal response to CA inhibition in CA-II deficient mice (CAD), in normal (N) mice and in heterozygous litter mates (LM). N and LM mice had CA activity in proximal tubule brush border membranes and cytosol. CA activity was also localized to membranes and cytosol of the outer medullary region. CAD mice lacked cytosolic activity but had normal CA activity in all membranes examined. All membrane associated CA had 2-8-fold lower sulfonamide sensitivity than cytosolic CA. These inhibition characteristics suggest that the membrane enzyme is CA-IV. Baseline urinary excretion of Na+, K+, and HCO3- was similar in all groups. Urine pH and Cl- excretion were higher and titratable acid output was lower in CAD mice. Inhibition of CA (methazolamide, 25 mg/kg) led in all groups to equivalent increments of urine pH, urine flow, and HCO3-, Na+, and K+ excretion. Cl- excretion was unchanged. Thus the extent of the genetic deficiency of CA-II mice extends to the kidney cytosol but does not alter membrane localization or levels of CA, probably CA-IV. The similar response to CA inhibition in CAD mice suggests that CA-IV, the membrane bound isoenzyme is the important isoenzyme in proximal tubule HCO3- reabsorption.

Animals↗
PubMed1989

Cadmium inhibition of L-alanine transport into renal brush border membrane vesicles isolated from the winter flounder (Pseudopleuronectes americanus).

Using isolated brush border membrane vesicles from the kidney of the winter flounder (Pseudopleuronectes americanus), we have studied the effect of cadmium on L-alanine transport. Pretreatment of vesicles with 0.1 mM Cd2+ resulted in inhibition of L-alanine uptake in the presence of a NaCl (but not KCl) gradient. Inhibition was due to a specific interaction with the sodium-alanine cotransport system and not a change in the driving forces for alanine transport, since Cd2+ did not affect sodium-dependent D-glucose uptake. The effect of Cd2+ on Na+-alanine cotransport showed mixed-type inhibition which is only partially reversible by EDTA. Cd2+ uptake itself was shown to be time and temperature dependent, resulting in binding to both sides of the membrane. No direct correlation was possible between inhibition of L-alanine transport and the amount of Cd2+ taken up by the membranes. Nevertheless, the striking time dependence of the effect of Cd2+ on sodium-dependent L-alanine uptake and the inability of EDTA to reverse the inhibitory action of Cd2+ suggest that Cd2+ inhibits Na+-alanine cotransport at the cytoplasmic side of the membrane.

Alanine↗
PubMed1989

Renal H+ ATPases.

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Adenosine Triphosphate↗
PubMed1989

A Na/H exchange mechanism in apical membrane vesicles of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) interposed between the vascular system of the choroid and the neural retina performs a variety of functions essential for vision. In order to further elucidate the transport functions of the RPE, apical membranes were isolated from the RPE of the dogfish (Squalus acanthias) by differential precipitation with calcium. Na-K-ATPase, an apical marker enzyme in this tissue, was enriched 15-fold in the final membrane fraction. About 50% of the membranes form right-side-out vesicles in which the membrane has retained its in vivo orientation. Sodium uptake into these vesicles as determined by a rapid filtration method was stimulated 37% by the presence of a proton gradient across the membrane (pHi = 6.1, pHo = 8.1). The stimulation was also observed in membrane vesicles "short-circuited" with valinomycin and K. The pH gradient-dependent sodium uptake but not the uptake in the absence of a pH gradient was completely inhibited by 5 X 10(-4) M amiloride, and 56% inhibition was found at 10(-5) M amiloride. The uptake of 22Na was also strongly decreased in the presence of nonradioactively labelled sodium and lithium; potassium was without effect. pH gradient dependence, amiloride sensitivity, saturability and cation specificity of the sodium flux indicate the presence of a Na/H exchanger in the apical membrane of the retinal pigment epithelium. The presence of the Na/H exchange process might have important implications for the control of pH in the subretinal space, optimum intracellular pH of the RPE and the triggering of other functions of the RPE.

Amiloride↗
PubMed1986

The anion specificity of the sodium-potassium-chloride cotransporter in rabbit kidney outer medulla: studies on medullary plasma membranes.

Plasma membrane vesicles were isolated from rabbit kidney outer medulla and employed in sodium, rubidium, and chloride flux studies. Chloride dependence and bumetanide sensitivity of (part of) the sodium and rubidium flux indicate that this plasma membrane fraction can be used to study the properties of Na-K-2Cl cotransport system present in the luminal membrane of the medullary thick ascending limb. The anion specificity of the cotransporter was investigated by determining the effect of anion replacement on sodium fluxes. When chloride was completely replaced by bromide, iodide, nitrate, or thiocyanate only bromide could effectively substitute for chloride (90% activity), whereas sodium uptake in the presence of iodide, nitrate, and thiocyanate amounted to only 25% of the sodium uptake observed in the presence of chloride. When similar replacement experiments were performed in the presence of 10 mmol/l chloride, bromide could substitute for chloride by 110%, iodide and nitrate by 60%, and thiocyanate by 70%. In the presence of 10 mmol/l bromide iodide, nitrate, and thiocyanate were similarly effective. The effect of nitrate and chloride on sodium flux was additive. Bumetanide-sensitive chloride uptake was inhibited by nitrate, the inhibition was however only partly, amounting to 60%. The results obtained are compatible with the view that the two anion binding sites of the Na-K-2Cl cotransporter can exhibit a different substrate specificity and that the transporter in addition to a 2Cl mode can also operate in a 2Br, Cl-, A- and Br-, A- mode, A- representing iodide, nitrate, or thiocyanate.

Animals↗
PubMed1986

Proton pump activity and Mg-ATPase activity in rat kidney cortex brushborder membranes: effect of 'proton ATPase' inhibitors.

In order to further characterize the ATP driven proton pump present in the luminal membrane of the renal proximal tubule, brushborder membranes were isolated from rat kidney cortex and the effect of various proton ATPase inhibitors on intravesicular ATP hydrolysis in sealed brushborder membrane vesicles and on Mg-ATPase activity in permeabilized brushborder membranes was investigated. The protonophor induced intravesicular ATP hydrolysis (ATP driven proton pump) was inhibited by DCCD and filipin but not by diethylstilbestrol and duramycin. All four compounds decreased Mg-ATPase activity, the two former inhibited the ATPase activity with a lower potency than the proton pump. NEM--up to 10 mM--and orthovanadate did not affect intravesicular ATP hydrolysis nor Mg-ATPase activity. From the relative sensitivity of the proton pump and the Mg-ATPase activity to the inhibitors it is concluded that about 35% of the Mg-ATPase activity found in the brushborder membrane can be attributed to the ATP-driven proton pump. Furthermore, the results obtained with NEM and duramycin suggest that the brushborder membrane proton pump has different properties than the proton pump in clathrin-coated vesicles or endosomes. The results presented above raise the possibility that the brushborder membrane proton pump is predominantly involved in acid secretion by the proximal convoluted tubule whereas the proton pump in clathrin-coated vesicles may be predominantly involved in the endocytosis of larger peptides and proteins.

Animals↗
PubMed1986

Ammonium transport in medullary thick ascending limb of rabbit kidney: involvement of the Na+,K+,Cl(-)-cotransporter.

In order to investigate the question whether ammonium reabsorption in the thick ascending limb of Henle's loop (TALH) proceeds via the Na+,K+,Cl(-)-cotransporter, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and the effect of NH+4 on their transport properties was investigated. It was found that, in the presence of a 78-mmol/liter NaCl gradient, 5 mmol/liter NH+4 inhibited bumetanide-sensitive rubidium flux by 86%; a similar decrease was observed for 5 mmol/liter, K+. Inhibition of bumetanide-sensitive rubidium uptake by NH+4 was competitive and an apparent Ki of 1.9 mmol/liter was found. Bumetanide-sensitive sodium uptake measured in the presence of a 83 mmol/liter KCl gradient was not inhibited by 5 mmol/liter NH+4. A 100-mmol/liter NH4Cl gradient was, however, capable of stimulating bumetanide-sensitive sodium uptake to the same extent as a KCl gradient. These data suggest that NH+4 is accepted by the K+ site of the Na+,K+,Cl-cotransport system and that the transporter can function in a Na+,NH+4,2Cl mode. Since the affinity of the transporter for NH+4 lies in the concentration range found in the TALH lumen in vivo, it is concluded that Na+,NH+4,2Cl-cotransport can contribute to the NH+4 reabsorption in this tubular segment.

Ammonia↗
PubMed1979

Further evidence for the existence of an intrinsic bicarbonate-stimulated Mg2+-ATPase in brush border membranes isolated from rat kidney cortex.

The aim of this study was to provide further evidence for the existence of a nonmitochondrial becarbonate-stimulated Mg2+-ATPase in brush border membranes derived from rat kidney cortex. A plasma membrane fraction rich in brush border microvilli and a mitochondrial fraction were isolated by differential centrifugation. Both fractions contain a Mg2+-ATPase activity which can be stimulated by bicarbonate. The two Mg2+-ATPases are stimulated likewise by chloride, bicarbonate, and sulfite or inhibited by oligomycin and aurovertin, though to different degrees. In contrast to these similarities, only the Mg2+-ATPase activity of the mitochondrial fraction is inhibited by atractyloside, a substance which blocks an adenine nucleotide translocator in the inner mitochondrial membrane. On the other hand, filipin, an antibiotic that complexes with cholesterol in the membranes inhibits exclusively the Mg2+-ATPase of the cholesterol-rich brush border membranes. Furthermore it could be demonstrated by the use of bromotetramisole, an inhibitor of alkaline phosphatase activity, that the Mg2+-ATPase activity in the membrane fraction is not due to the presence of the highly active alkaline phosphatase in these membranes. These results support the assumption that an intrinsic bicarbonate-stimulated Mg2+-ATPase is present in rat kidney brush border membranes.

Adenosine Triphosphatases↗
PubMed1977

The mechanism of action of harmaline on renal solute transport.

The effect of the hallucinogenic drug harmaline was tested on rat kidney proximal tubular solute and water transport, using in vivo micropuncture and electrophysiological techniques as well as in vitro biochemical techniques. During peritubular application harmaline (5 mmol/l) was found to block net tubular volume absorption reversibly (by 85%) through inhibition of active Na+ transport and possibly active HCO-3 transport. The inhibition was accompanied by a rapid strong depolarization of the tubular cell membranes. As a biochemical equivalent harmaline inhibited the Na+-K+-ATPase and the Mg2+-ATPase of peritubular cell membrane fractions as well as the HCO-3-stimulated ATPase of a brush border membrane fraction with similar kinetics. By studying glucose tracer efflux and by measuring cell membrane potential and conductance changes in response to glucose perfusions, no evidence for a direct effect of harmaline on Na+-glucose (or amino acid) cotransport mechanisms in the brush border could be obtained. The data suggest that harmaline does not specifically compete with Na+ for transport sites. Neither are the cotransport systems in the brush border membrane specifically inhibited, nor could the inhibition of the Na+ pump in the peritubular cell membrane simply result from a competition between harmaline and Na+.

Adenosine Triphosphatases↗
PubMed1975

Distribution of parathyroid hormone-stimulated adenylate cyclase in plasma membranes of cells of the kidney cortex.

Free flow electrophoresis was employed to separate renal cortical plasma membranes into luminal (brush border microvilli) and contraluminal (basal-lateral membrane) fractions. During the separation adenylate cyclase activity was found to parallel the activity of Na+-K+-activated ATPase, an enzyme which is present in contraluminal but not in luminal membranes. In the basal-lateral membrane fraction the specific activities of adenylate cyclase and Na+-K+-activated ATPase were 4.4 and 4.6 times greater, respectively, than in the brush border fraction. The adenylate cyclase of the basal-lateral membrane fraction was specifically stimulated by parathyroid hormone which maximally increased enzyme activity eightfold. The biologically active (1-34) peptide fragment of paratyhroid hormone produced a 350% increase in adenylate cyclase activity. In contrast, calcitonin, epinephrine and vasopressin maximally stimulated the enzyme by only 55, 35 and 30%, respectively. These results indicate that adenylate cyclase, specifically stimulated by parathyroid hormone, is distributed preferentially in the contraluminal region of the plasma membrane of renal cortical epithelial cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Distribution of membrane-bound cyclic AMP-dependent protein kinase in plasma membranes of cells of the kidney cortex.

Renal cortical plasms membranes were separated by free flow electrophoresis into luminal (brush border microvilli) and contraluminal (basal-lateral membrane) fractions. These membranes were found to contain an intrinsic, self-phosphorylating system which consists of a cyclic AMP-dependent protein kinase, a phosphorprotein phosphatase and the substrate(s) of these enzymes. The kinase, but not the phosphatase, was stimulated by cyclic AMP; maximal (1.7-fold) stimulation was effected at a cyclic AMP concentration of 0.1 muM. The degree of phosphorylation of the brush borders was six times greater than that of the basal-lateral membranes in the absence of cyclic AMP and 2.3-fold greater in the presence of cyclic AMP. This preferential phosphorylation of the luminal membrane by membrane-associated protein kinase(s) may play a role in the parathyroid hormone-mediated alterations of solute reabsorption in the proximal tubule.

Adenosine Triphosphatases↗
PubMed1975

Sugar transport by renal plasma membrane vesicles. Characterization of the systems in the brush-border microvilli and basal-lateral plasma membranes.

Uptake studies of D-and L-glucose were performed on vesicles derived from brush-border and basal-lateral membranes. The uptake of the sugars into the vesicles was osmotically sensitive and independent of glucose metabolism. In brush-border vesicles D-glucose but not L-glucose transport was Na-+-dependent, wn the presence of an initial Na+gradient. Basal-lateral membranes take up D-glucose faster than L-glucose, but the D-glucose uptake is significantly less sensitive to sodium removal and only moderately inhibited by phlorzin as compared to the prush-border fraction.

Animals↗