The permeability of the human erythrocyte to sodium and potassium

Measurements have been made on the permeability of the human erythrocyte to Na and K in vitro, using radioactive tracers to observe the system in the steady state. The average inward K flux is 1.67 m.eq./liter cells hour, and the apparent activation energy is 12,300 ± 1300 calories/mol. The inward K flux is independent of the external K concentration in the range of concentrations studied (4 to 16 m.eq. K/liter plasma). Rb appears to compete with K for transport into the cell, whereas Na and Li do not. The average inward Na flux is 3.08 ± 0.57 m.eq. Na/liter cells hour, and the apparent activation energies are 20,200 ± 2700 calories/mol for inward transport, and 14,900 ± 3,400 calories/mol for outward transport. The inward Na flux is dependent on the external Na concentration, but not in a linear fashion. Li appears to compete with Na for inward transport, whereas K and Rb do not. An approximate maximum estimate shows that the energy required for cation transport is only 8.8 calories/mol liter cells hour of the 110 calories/mol liter cells hour available from the consumption of glucose. A working hypothesis for the transport of Na and K is presented.     

Involvement of Reactive Oxygen Species in a Feed-forward Mechanism of Na/K-ATPase-mediated Signaling Transduction

Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-l-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial ²²Na⁺ transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, ouabain stimulated direct carbonylation of two amino acid residues in the actuator domain of the Na/K-ATPase α1 subunit. Taken together, the data indicate that carbonylation modification of the Na/K-ATPase α1 subunit is involved in a feed-forward mechanism of regulation of ouabain-mediated renal proximal tubule Na/K-ATPase signal transduction and subsequent sodium transport.     

THE THREE-DIMENSIONAL STRUCTURE OF THE BASAL BODY FROM THE RHESUS MONKEY OVIDUCT

The structure of the oviduct basal body has been reconstructed from serial, oblique, and tangential sections This composite information has been used to construct a three-dimensional scale model of the organelle The walls are composed of nine equally spaced sets of three tubules, which run from base to apex pitched to the left at a 10°–15° angle to the longitudinal axis. The transverse axis of each triplet set at its basal end intersects a tangent to the lumenal circumference of the basal body at a 40° angle (triplet angle). As the triplet set transverses from base to apex, it twists toward the lumen on the longitudinal axis of the inner A tubule; therefore, the triplet angle is 10° at the basal body-cilium junction. Strands of fibrous material extend from the basal end of each triplet to form a striated rootlet. A pyramidal basal foot projects at right angles from the midregion of the basal body. In the apex, a 175 mµ long trapezoidal sheet is attached to each triplet set. The smaller of the two parallel sides is attached to all three tubules while the longitudinal edge (one of the equidistant anti-parallel sides) is attached to the C tubule. The sheet faces counterclockwise (apex to base view) and gradually unfolds from base to apex; the outside corner merges with the cell membrane.     

THE ACCUMULATION OF ELECTROLYTES : IV. INTERNAL VERSUS EXTERNAL CONCENTRATIONS OF POTASSIUM

Lowering the potassium in the sea water from 0.011 M to 0.006 M caused an exit of potassium from cells of Valonia macrophysa. Sodium continued to penetrate and the ratio K ÷ Na fell off. The cells ceased to grow but there was no evidence of injury. Increasing the external potassium brought about an increase of the internal concentration of potassium, of halide, of total cations, and of the ratio K ÷ Na inside. These phenomena are to be expected on theoretical grounds.     

Serum and Urinary Folate in Liver Disease

During the active phase of viral hepatitis urinary folate loss was found to be 8·0 to 48·3 (mean 31·1) μg./day, compared with a normal urinary folate excretion of 0·1 to 18·0 (mean 9·5) μg./day. In cirrhosis and cardiac failure with congestive hepatomegaly the corresponding values were 25·8 to 55·0 (mean 35·7) μg./day and 2·5 to 61·6 (mean 26·9) μg./day, respectively. Urinary folate loss may be a significant factor in the aetiology of folate deficiency of chronic liver disease, particularly when dietary intake is poor.After prolonged dialysis in Visking casing urinary folate was almost totally dialysable, but an appreciable fraction of serum folate was not, even after 72 hours. The dialysable (free) folate fraction of serum and urine disappeared maximally during the first six hours'' dialysis, and was virtually cleared after 24 hours'' dialysis; clearance curves in normal individuals and in liver disease were comparable. The non-dialysable serum folate fraction was of similar magnitude in all subjects studied, in spite of marked variation in total folate, and probably represented protein-bound folate.     

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium

Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.     

Cat Heart Muscle in Vitro : I. Cell volumes and intracellular concentrations in papillary muscle

Methods have been developed for the simultaneous determination of total water, inulin space, and K and Na content in muscles of 0.5 to 10 mg. wet weight. These methods have been used to define steady state conditions with respect to intracellular K concentration in papillary muscles from cat hearts perfused and contracting isometrically at 27–28°C. and at 37–38°C. Cell volumes and intracellular ionic concentrations have been followed as a function of the external K concentration and compared with values predicted on the basis of electroneutrality and osmotic equilibrium.     

Active ion transport in the renal proximal tubule. II. Ionic dependence of the Na pump

The dependence of Na pump activity on intracellular and extracellular Na+ and K+ was investigated using a suspension of rabbit cortical tubules that contained mostly (86%) proximal tubules. The ouabain- sensitive rate of respiration (QO2) was used to measure the Na pump activity of intact tubules, and the Na,K-ATPase hydrolytic activity was measured using lysed proximal tubule membranes. The dependence (K0.5) of the Na pump on intracellular Na+ was affected by the relative intracellular concentration of K+, ranging from approximately 10 to 15 mM at low K+ and increasing to approximately 30 mM as the intracellular K+ was increased. The Na pump had a K0.5 for extracellular K+ of 1.3 mM in the presence of saturating concentrations of intracellular Na+. Measurements of the Na,K-ATPase activity under comparable conditions rendered similar values for the K0.5 of Na+ and K+. The Na pump activity in the intact tubules saturated as a function of extracellular Na at approximately 80 mM Na, with a K0.5 of 30 mM. Since Na pump activity under these conditions could be further stimulated by increasing Na+ entry with the cationophore nystatin, these values pertain to the Na+ entry step and not to the Na+ dependence of the intracellular Na+ site. When tubules were exposed to different extracellular K+ concentrations and the intracellular Na+ concentration was subsaturating, the Na pump had an apparent K0.5 of 0.4 mM for extracellular K. Under normal physiological conditions, the Na pump is unsaturated with respect to intracellular Na+, and indirect analysis suggests that the proximal cell may have an intracellular Na+ concentration of approximately 35 mM.     

Axonal transport of [3H]serotonin in an identified neuron of Aplysia californica

The distribution of (Na+ + K+) ATPase over the plasma membranes of the proximal convoluted tubule from canine renal cortex has been determined. Ultrathin frozen sections of this tissue were stained with rabbit antibodies to this enzyme and ferritin-conjugated goat antirabbit gamma-globulin. It is demonstrated that high concentrations of this enzyme uniformly line the intercellular spaces of this epithelium. The consequences of this observation are discussed in terms of the low resistant tight junctions of these tubules and the isotonic fluid transport which they support. Furthermore, antibodies to (Na+ + K+) ATPase recognize an antigen on the luminal surfaces of the tubules within the brush border. It is proposed that the enzyme is present in this region of the plasma membrane as well, although at much lower concentration. To further substantiate this conclusion, a brush border fraction has been purified from rabbit kidney and been shown to contain significant (Na+ + K+) ATPase. These results contradict earlier conclusions about the location of (Na+ + K+) ATPase in this tissue.     

THE AXOSTYLE OF SACCINOBACULUS : II. Motion of the Microtubule Bundle and a Structural Comparison of Straight and Bent Axostyles

Undulations of the flagellate Saccinobaculus result from motility in its axostyle, a bundle consisting of thousands of cross-bridged microtubules. In its resting state, the axostyle is a helix of large pitch and slowly varying radius. The active state as seen by light microscopy involves first a bending of the anterior end of the axostyle to a radius of about 8 µm with a circular arc ranging from 60° to 180°, and then the propagation of this bend without damping to the posterior end of the organism at speeds up to 100 µm/s. The cross section of an unbent axostyle is crescent shaped. This crescent flattens as the bend arrives and reappears as the bend passes by. Intertubule bridges impart to the axostyle tubules an axial periodicity of about 150 Å which can serve as a marker for the investigation of tubule sliding or contraction associated with bend formation. Optical diffraction measurements on electron micrographs of the bend demonstrate that the axostyle tubules slide over one another and that the tubules on the inside of a bend usually contract, sometimes by as much as 25%. Possible relationships between the contraction and sliding of the tubules are discussed.     

Intracellular ion activities in Necturus proximal tubule

Ion-sensitive microelectrodes were used to measure the intracellular activities of Na, K, and Cl in proximal tubules of the perfused Necturus kidney. Cell Cl was 2-3 times higher than the value predicted for passive distribution during perfusion with normal Ringer; intracellular Na was far below the level for passive distribution. Cell Na and Cl fell to very low values when the lumen was NaCl-free. Cl entry into the tubule cell from the lumen required luminal Na. Na entered the cell across the luminal membrane both by diffusion and by coupled movement with Cl.     

Morphometric parameters of nutria kidney structures in postnatal ontogeny

Data on the morphometric parameters of the renal corpuscle, renal tubules, and collecting ducts of male and female nutrias in postnatal ontogenesis were obtained. It was found that the area of the renal corpuscle, glomerulus, the cavity and lumen of the capsule, and the proximal tubule diameter in the right and left kidney of female and male nutrias in the first year of life increase. The distal tubule diameter also increases; however, the dynamics of its changes becomes sinuous after 4.5 months. The collecting duct diameter varies depending on gender, age, and renal topography. The nuclear-cytoplasmic ratio in the cells of proximal and distal tubules and collecting ducts changes in a sinuous manner and depends on the gender and age of nutrias. The minimum mean value of the nuclear-cytoplasmic ratio was found in the proximal tubule cells in the left kidney of 12-month-old female nutrias (0.162 ± 0.002), and the maximum value was found in the distal tubule cells in the left kidney of newborn male nutrias (0.435 ± 0.007).     

Na+,K+ pump and Na+-coupled ion carriers in isolated mammalian kidney epithelial cells: regulation by protein kinase C.

This review updates our current knowledge on the regulation of Na+/H+ exchanger, Na+,K+,Cl- cotransporter, Na+,Pi cotransporter, and Na+,K+ pump in isolated epithelial cells from mammalian kidney by protein kinase C (PKC). In cells derived from different tubule segments, an activator of PKC, 4beta-phorbol 12-myristate 13-acetate (PMA), inhibits apical Na+/H+ exchanger (NHE3), Na+,Pi cotransport, and basolateral Na+,K+ cotransport (NKCCl) and augments Na+,K+ pump. In PMA-treated proximal tubules, activation of Na+,K+ pump probably plays a major role in increased reabsorption of salt and osmotically obliged water. In Madin-Darby canine kidney (MDCK) cells, which are highly abundant with intercalated cells from the collecting duct, PMA completely blocks Na+,K+,Cl- cotransport and decreases the activity of Na+,Pi cotransport by 30-40%. In these cells, agonists of P2 purinoceptors inhibit Na+,K+,Cl- and Na+,Pi cotransport by 50-70% via a PKC-independent pathway. In contrast with MDCK cells, in epithelial cells derived from proximal and distal tubules of the rabbit kidney, Na+,K+,Cl- cotransport is inhibited by PMA but is insensitive to P2 receptor activation. In proximal tubules, PKC-induced inhibition of NHE3 and Na+,Pi cotransporter can be triggered by parathyroid hormone. Both PKC and cAMP signaling contribute to dopaminergic inhibition of NHE3 and Na+,K+ pump. The receptors triggering PKC-mediated activation of Na+,K+ pump remain unknown. Recent data suggest that the PKC signaling system is involved in abnormalities of dopaminergic regulation of renal ion transport in hypertension and in the development of diabetic complications. The physiological and pathophysiological implications of PKC-independent regulation of renal ion transporters by P2 purinoceptors has not yet been examined.     

Potential Role of Pyrophosphate:Fructose 6-Phosphate Phosphotransferase in Carbohydrate Metabolism of Cold Stored Tubers of Solanum tuberosum cv Bintje

To gain a better understanding of the mechanism of cold induced sweetening, sugar accumulation in potato, Solanum tuberosum cv Bintje, was compared to the maximum activity of inorganic pyrophosphate (PPi):fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) and the concentration of two regulatory metabolites. Mature tubers accumulated reducing sugars and sucrose at an almost linear rate of 13.4 and 5.2 micromole per day per gram dry weight at 2°C and 4.5 and 1.3 micromole per day per gram dry weight, respectively, at 4°C. During storage at 8°C sugar accumulation was nil. Sugar accumulation was preceded by a lag phase of about 4 days. The accumulation of reducing sugars persisted for at least 4 weeks, whereas sucrose accumulation declined after 2 weeks of storage. The ratio of glucose:fructose changed concomitantly with sugar increase from 65:35 to equimolarity. The maximum activity of PPi:fructose 6-phosphate 1-phosphotransferase was 2.51 and 2.25 units per gram dry weight during storage at 2 and 8°C, respectively. The temperature coefficient of this enzyme from potatoes kept at 2 or 8°C was 2.12 and 2.48, respectively. The endogenous concentration of fructose 2,6-biphosphate increased from 0.15 to 1 nanomole per gram dry weight during storage at 2 and 4°C but remained the same throughout storage at 8°C. After exposure to 2°C an initial increase in the concentration of PPi was observed from 4.0 to 5.6 nanomoles per gram dry weight. Pyrophosphate concentration did not change during storage at 4°C but decreased slightly at 8°C. All observed changes became annulled after transfer of cold stored tubers to 18°C. These data strongly indicate that PPi:fructose 6-phosphate 1-phosphotransferase can be fully operational in cold stored potato tubers and the lack of increase in PPi concentration supports the functioning of this enzyme during sugar accumulation.     

A cytophotometric analysis of the activity of oxidative enzymes and Na, K-ATPASE in vertebrate nephrons at different levels of sodium transport in the kidney.]

Using quantitative cytochemistry, activities of Na, K-ATPase, succinate dehydrogenase (SDH) and alpha-keto-glutarate dehydrogenase (alpha-KDH) was investigated in cells of renal tubules at different levels of sodium reabsorption in the kidney. The activity of these enzymes in mammals and birds renal tubule cells was found to be higher than in the cells of corresponding renal tubules of cold-blooded vertebrates. This corresponds to the increased total amount of reabsorbed sodium in the kidney of warm-blooded animals. The summer frogs, as compared to the winter ones, exhibit higher activities of SDH and Na,K-ATPase in the proximal tubule cells where changes in sodium reabsorption are also noted. In the kidney of marine teleosts, a negative correlation between U/PNa and the activity of SDH and Na,K-ATPase in the cells of proximal and distal tubule was observed. Aldosterone was found to stimulate sodium reabsorption and to activate Na,K-ATPase.SDH and alpha-KDH mainly in the distal convoluted tubule. Furosemide was observed to inhibit sodium reabsorption and to reduce SDH and Na,K-ATPase activities in cells of the proximal tubule and Henle's loop. In the kidney of adrenalectomized rats, both sodium reabsorption and activities of Na,K-ATPase, SDH, alpha-KDH decreased in all the segments of the nephron. The data obtained suggest that changes in sodium reabsorption may be coupled with those in the activities of the investigated enzymes.     

Active ion transport in the renal proximal tubule. III. The ATP dependence of the Na pump

The dependence of the Na pump activity of intact renal tubules on the ATP concentration was investigated using a suspension of rabbit cortical tubules. Rotenone (an inhibitor of mitochondrial oxidative phosphorylation) was used in graded fashion to alter the cellular ATP, and the Na pump activity was measured when the pump was stimulated by adding KCl to tubules suspended in a K+-free medium. The K+ uptake into the tubule was measured using an extracellular K+ electrode, and the oxygen consumption (QO2) was measured using a Clark-type oxygen electrode. The Na pump activity was found to have a linear, nonsaturating dependence on the ATP concentration. However, the Na,K- ATPase hydrolytic activity (assayed biochemically) of lysed proximal tubule membranes demonstrated saturation and had a K0.5 value of 0.4 mM ATP. Presumably, unknown cytosolic factors present in the intact renal cell but not normally present in the biochemical assay accounted for the differences between the two measurements. The data suggest that an alteration in the intracellular ATP will result in a proportional change in active ion transport activity. Moreover, additional findings also suggest that the basal (non-transport-related) QO2 may be redirected to support the proximal Na pump activity when transport activity is stressed. Thus, basal respiration is not invariant under all conditions, and ion transport activity appears to be maintained foremost among cellular ATP-dependent processes.     

Calcium Efflux from Internally Dialyzed Squid Giant Axons

Calcium efflux has been studied in squid giant axons under conditions in which the internal composition was controlled by means of a dialysis perfusion technique. The mean calcium efflux from axons dialyzed with 0.3 µM calcium and 5 mM ATP was 0.26 pmol/cm²·s at 22°C. The curve relating the Ca efflux with the internal Ca concentration had a slope of about one for [Ca]_i lower than 0.3µM and a slope smaller than one for higher concentrations. Under the above conditions replacement of [Na]_o and [Ca]_o by Tris and Mg causes an 80% fall in the calcium efflux. When the axons were dialyzed with a medium free of ATP and containing 2 mM cyanide plus 5µg/ml oligomycin, analysis of the perfusion effluent gave values of 1–4 µM ATP. Under this low ATP condition, replacement of external sodium and calcium causes the same drop in the calcium efflux. The same effect was observed at higher [Ca]_i, (80 µM). These results suggest that the Na-Ca exchange component of the calcium efflux is apparently not dependent on the amounts of ATP in the axoplasm. Axons previously depleted of ATP show a significant transient drop in the calcium efflux when ATP is added to the dialysis medium. This effect probably represents the sequestering of calcium by the mitochondrial system. The consumption of calcium by the mitochondria of the axoplasm in dialyzed axons was determined to be of the order of 6.0 x 10^-7 mol Ca⁺⁺/mg of protein with an initial rate of 2.6 x 10^-8 mol Ca⁺⁺/min·mg of protein. Axons dialyzed with 2 mM cyanide after 8–10-min delays show a rise in the calcium efflux in the presence of "normal" amounts of exogenous ATP. This effect seems to indicate that cyanide, per se, can release calcium ions from internal sources.     

The Integrin β1 Subunit Regulates Paracellular Permeability of Kidney Proximal Tubule Cells

Epithelial cells lining the gastrointestinal tract and kidney have different abilities to facilitate paracellular and transcellular transport of water and solutes. In the kidney, the proximal tubule allows both transcellular and paracellular transport, while the collecting duct primarily facilitates transcellular transport. The claudins and E-cadherin are major structural and functional components regulating paracellular transport. In this study we present the novel finding that the transmembrane matrix receptors, integrins, play a role in regulating paracellular transport of renal proximal tubule cells. Deleting the integrin β1 subunit in these cells converts them from a “loose” epithelium, characterized by low expression of E-cadherin and claudin-7 and high expression of claudin-2, to a “tight” epithelium with increased E-cadherin and claudin-7 expression and decreased claudin-2 expression. This effect is mediated by the integrin β1 cytoplasmic tail and does not entail β1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the β1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin β1 tail plays a key role in regulating the composition and function of tight and adherens junctions that define paracellular transport properties of terminally differentiated renal proximal tubule epithelial cells.     

中华鳖肾脏的组织化学特性研究

利用光镜组织化学反应对中华鳖肾单位的结构和组织化学特性进行了详细的观察和分析。结果表明,中华鳖肾脏为分叶形的实质器官,肾小叶由被膜和实质组成,实质无髓质和皮质之分,但可以区分为外侧区和内侧区。外侧区嗜酸性,主要分布有近端小管和集合管。内侧区呈弱嗜酸性,肾小体、颈段、中间段和远端小管主要分布在内侧区。肾小球PAS反应呈阳性,但其琥珀酸脱氢酶(SDH)弱阳性,碱性磷酸酶(ALPase)、Na+/K+-ATPase和阿利新兰(AB)反应为阴性。足细胞酸性磷酸酶(ACPase)反应呈阳性。近端小管刷状缘嗜伊红,PAS反应以及ALPase、ACPase和Na+/K+-ATPase酶反应呈阳性,而SDH弱阳性。中间段、远端小管、集合管弱嗜酸性,SDH阳性。中间段Na+/K+-ATPase弱阳性。远端小管细胞侧面呈PAS阳性,腔面显示AB阳性。集合管胞质含有许多ACPase阳性颗粒,腔面呈PAS强阳性,AB阳性。甲苯胺兰(TB)染色可见集合管腔面有阳性颗粒,肾小管上皮含有亮、暗两种细胞。上述组化反应和分布结果表明,鳖的肾小管细胞类型较多,近端小管在原尿的重吸收中起主要作用,远端小管和集合管具有分泌黏液作用。中华鳖肾单位的结构与组化特性不仅与哺乳类和鸟类有一定差异,也与其他爬行动物不完全相同。