Age-related alterations in cultured human fibroblast membrane structure and function

Membrane enzyme activities, lipid composition, and fluorescence probe characteristics in isolated plasma membranes, microsomes and mitochondria of cultured human fibroblasts were used to determine if structural alterations occurred as a function of donor age. The cells were sex matched and allowed to undergo approximately 8 population doublings under identical culture conditions. Plasma membrane (Na⁺,K⁺)-ATPase, microsomal NADPH cytochrome c reductase, and mitochondrial succinate cytochrome c activities showed variation as a function of increasing donor age but these changes were not statistically significant. At the same time the cholesterol/phospholipid molar ratio was unaltered in plasma membranes, decreased 50% in microsomes, and unchanged in mitochondria with increasing donor age. The phosphatidylcholine/phosphatidylethanolamine ratio increased in all three membrane fractions with increasing age of the fibroblast donor. The ratio of unsaturated/saturated fatty acids decreased in the phospholipids of microsomes but not of plasma membranes or mitochondria. The structural properties of the membranes were determined with two different fluorescence probe molecules, trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene. These probe molecules indicated that the fluorescence lifetime and/or fluorescence polarization of the trans-parinaric acid probe decreased in microsomes, mitochondria, and in the plasma membrane, such that the limiting anisotropy, indicative of restrictions to probe motions, was significantly lower (high fluidity) with increasing subject age in plasma membranes, microsomes and mitochondria. The trans-parinaric acid fluorescence lifetime displayed two components in plasma membranes, microsomes, and mitochondria, a finding consistent with the co-existence of fluid and solid membrane lipid areas in the cultured human fibroblast subcellular membranes. The trans-parinaric acid partitioned preferentially into solid membrane     

Isolation of axolemma-enriched fractions from bovine central nervous system

Axolemmal fractions are isolated by discontinuous gradient centrifugation of purified myelinated axons which have been osmotically shocked to yield axons, myelin and axolemma. The axolemmal fractions are enriched in surface membrane marker enzymes such as Na⁺,K⁺-activated ATPase, 5′-nucleotidase and acetylcholinesterase. Mixing experiments with labeled microsomes and mitochondria show that these contaminants are effectively screened out by the preparation procedure. The fractions contain significant amounts of 2′,3′-cyclic nucleotide-3′-phosphohydrolase and are enriched in higher molecular weight proteins. These membrane preparations may be useful in analytical studies of mammalian axolemmal composition.     

The localization of the Na+−K+-ATPase in the cells of rat kidney cortex

Summary Plasma membrane fractions of rat kidney cortex were subdivided by centrifugation on a continuous and a discontinuous sucrose gradient and by carrier free continuous electrophoresis. In the different fractions the activity of alkaline phosphatase and aminopeptidase, enzymes which are present in the brushborder membrane, as well as Mg⁺⁺-ATPase, Na⁺–K⁺-ATPase, 5-nucleotidase, acid phosphatase and glucose-6-phosphatase were determined.The distribution of alkaline phosphatase, aminopeptidase and 5-nucleotidase is identical, indicating the localization of these enzymes in the brushborder membrane. Na⁺–K⁺-ATPase does not show an identical distribution with any of the enzymes studied.Using carrier free continuous electrophoresis fractions can be obtained which are enriched in alkaline phosphatase by a factor of 15 when compared to the cortex homogenate, whereas the specific activity of Na⁺–K⁺-ATPase is reduced to one third of the starting material. On the other hand fractions can be isolated in which the specific activity of Na⁺–K⁺-ATPase is 16 times higher than in the homogenate. No enrichment of alkaline phosphatase occurs in these fractions.It is therefore concluded that the Na⁺–K⁺-ATPase is not present in the brushborder membrane nor in the lysosomes or endoplasmatic reticulum. The most probable localization of the Na⁺–K⁺-ATPase are the basal infoldings of the plasma membranes of the cells.A preliminary report has been published by Kinneet al. [28, 29].Major part of this work was done by J. E. Schmitz for his degree of M. D.     

Modification of K+ channel properties induced by fatty acids in neuroblastoma cells

The effects of fatty acids on voltage-dependent potassium (K⁺) channels in neuroblastoma cells were studied using the whole-cell current recording technique. At a concentration of 5 M, unsaturated and medium chain length (C₁₀–C₁₄) saturated fatty acids accelerated the apparent inactivation of the K⁺ current. This effect was reversed by albumin. In the absence of exogenous fatty acids, albumin slowed the inactivation of the K⁺ current. The acceleration of the K⁺ current inactivation induced by unsaturated fatty acids was associated with an increase in the sensitivity of K⁺ channels to 4-amino-pyridine. It is concluded that kinetic and pharmacological properties of K⁺ channels are, in part, controlled by membrane fatty acids which, in this way, should contribute to an apparent diversity of K⁺ channels and the modulation of cell excitability.     

Sorghum stem extract modulates Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase,ecto-5′-nucleotidase,and acetylcholinesterase activities

Sorghum stem (Sorghum bicolor) has been in use in traditional medicine systems for the management of neurodegenerative conditions. However, there is dearth of information on the scientific basis for its use in the treatment of such conditions. This study sought to assess the antioxidant activity and effects of phenolic extract from sorghum stem (Sorghum bicolor) on some cholinergic (acetylcholinesterase (AChE)) and purinergic (Na⁺/K⁺-ATPase and ecto-5′-nucleotidase) enzymes associated with neurological conditions. Phenolic-rich extract was prepared using methanol: 1 N HCl (1:1, v/v) mixture and characterized using high-performance liquid chromatography-diode array detector (HPLC-DAD). In vitro tests were used to investigate the effects of the phenolic extract on AChE, Na⁺/K⁺-ATPase, and ecto-5′-nucleotidase activities. Furthermore, the hydroxyl (OH) radical scavenging and Fe²⁺-chelating abilities of the extract were investigated. HPLC-DAD analysis revealed the presence of some phenolic acids such as caffeic acid (120.58 mg/g), ferulic acid (76.45 mg/g), gallic acid (17.48 mg/g), chlorogenic acid (16.25 mg/g), and flavonoids such as kaempferol (15.98 mg/g), rutin (51.07 mg/g), quercetin (263.16 mg/g), and quercitrin (89.21 mg/g) in the phenolic extract. The extract significantly inhibited AChE and ecto-5′-nucleotidase activities in a dose dependent manner with IC₅₀?=?24.88 μg/ml and IC₅₀?=?37.49 μg/ml, respectively, and increased Na⁺/K⁺-ATPase activity in a dose dependent manner. Furthermore, the phenolic extract also scavenged OH radicals and was able to chelate Fe²⁺ in a dose dependent manner with IC₅₀?=?54.27 μg/ml and 18.47 μg/ml, respectively. This study revealed the antioxidant and modulatory effects of phenolic extracts from sorghum stem on some cholinergic and purinergic enzymes.     

A comparative study of the lipids of plasma membranes of normal cells and those infected and transformed by rous sarcoma virus

Comparative studies of the phospholipids of Rous sarcoma virus (RSV)-transformed chicken embryo cells, uninfected normal cells, and cells infected but not transformed by the transformation-defective mutant of RSV td105, were done. Phospholipids of whole cells as well as those of isolated plasma membranes were analyzed by thin-layer and gas-liquid chromatography. The plasma membrane preparations used were significantly enriched in 5′-nucleotidase enzyme activity over that of whole cell homogenates. Our results indicated that transformation by RSV and infection by td105 do not significantly alter the molar ratios of the various phospholipids compared to uninfected normal cells. However, comparison of the fatty acyl chains of total phospholipid as well as isolated specific phospholipids revealed that the phospholipids of transformed cells have higher percentages of unsaturated fatty acyl chains than those of uninfected normal cells; phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SP) have higher percentages of unsaturated fatty acyl chains, while phosphatidylcholine (PC) and phosphatidylserine (PS) remained unchanged. The percentages of unsaturated fatty acyl chains of PE and PI of td 105-infected cells increased, while that of PC and PS decreased resulting in no overall change. These results are consistent with the concept that altered fluidity of membranes of transformed cells is due to the selection of phospholipids with specific fatty acyl chains by new or modified proteins incorporated into the membranes of transformed and/or virus-producing cells.     

Enhancement of theShaker B Δ6–46 current by fatty acids depends on the activation of the lipoxygenase metabolic pathway

Voltage-gated K⁺ channels are modulated by extracellular free unsaturated fatty acids. Both increases and attenuations of K⁺ channel activities have been observed. We studied the effect of cis-unsaturated fatty acids onShaker B Δ6–46 and the endogenous outward rectifier K⁺ channels expressed in COS cell line, using the whole cell recording technique. For both K⁺ channels, exogenously applied unsaturated fatty acids dramatically increased the outward K⁺ currents. This enhancement was not mediated by cylooxygenase or epoxygenase (P450) enzymes. However, nordihydroguaiaretic acid, a lipoxygenase metabolic pathway blocker, did prevent the arachidonic acid-inducedShaker current enhancement.     

Plasma cell membranes of the rat kidney

Summary A fraction enriched with plasma cell membranes (PMF) was isolated from rat kidney homogenate by differential centrifugation. Before NaJ treatment electron micrographs of the preparation showed a membraneous fraction with only a small contamination of mitochondria. After treatment with NaJ the residual PMF exhibited a low microsomal glucose 6-phosphatase activity. Marker enzymes of other subcellular fractions were not detected. The NaJ extracted PMF revealed a high specific activity of ATPase, 91% of which was stimulated by Na⁺ and K⁺. The MgNaK-ATPase was characterized by Michaelis Menten kinetics. In contrast, Hill coefficients (n) of NaK-ATPase for the activation by Na⁺, K⁺ and Mg-ATP were greater than one. Experiments with various nucleotide tri-, di- and monophosphates revealed a high substrate specificity of the NaK-ATPase. The pH optimum was in the range of 7.2. SH-reagents and ouabain depressed the Na⁺ and K⁺ stimulated enzyme activity. PMF isolated from rat kidneys exhibited an acylphosphatase and a nitrophenylphosphatase activity, both of which were stimulated by K⁺. Furthermore 5-nucleotidase and leucine aminopeptidase activities were present in the fraction.—Thus, NaK-ATPase of the PMF revealed the typical properties of the NaK-ATPase demonstrated in the microsomal preparations, which has been referred to by Skou (1965) as the enzymatic basis of active cation transport.

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Abbreviations PMF Plasma Membrane Enriched Fraction - DOC Na-Deoxycholate - NaK-ATPase Activation of basal Mg-ATPase by Na⁺ and K⁺ (=MgNaK-ATPase (–) Mg-ATPase) Supported by grants of the Deutsche Forschungsgemeinschaft.Supported by the Senate of West-Berlin. Present address: Instituto Di Patologia Speciale, Medica e Metodologia Clinica, Policlinico, Napoli.Other abbreviations have the usual connotation     

Evidence that the depolarization of glial cells by inhibitory amino acids is caused by an efflux of K+ from neurones

Summary The action of inhibitory amino acid transmitters GABA, glycine, -alanine and taurine has been studied on the membrane potential of cultured astrocytes and on the extracellular K⁺-concentration ([K⁺]₀) using K⁺-sensitive microelectrodes. All four amino acids caused a depolarization of glial cells and an increase of [K⁺]₀. The effects produced by GABA were usually more pronounced than those caused by the other amino acids. Simultaneous recordings of the action of GABA and glycine on the glial membrane potential and on [K⁺]₀ usually revealed a good correlation in time course, but often there were differences between the amplitudes of glial depolarizations and the values calculated from the [K⁺]₀ increase. 4-Aminopyridine, which blocks K⁺-conductance of excitable membranes, reversibly abolished both the glial depolarization and the [K⁺]₀ increase produced by GABA and glycine. From these results it is concluded that unlike neurones, glial cells do not have receptors for these amino acid transmitters and that their action on glial cells is caused by the efflux of K⁺ from activated neurones.     

Phospholipase A Activity in the Hemolysis of Sheep and Human Erythrocytes by Rickettsia prowazeki

Incubation of Rickettsia prowazeki with sheep or human erythrocytes resulted in lysis of the erythrocytes and formation of free fatty acids and lysophosphatides. Inhibitors of lysis were also invariably inhibitors of this phospholipase A activity. The target for this activity was the glycerophospholipids of the erythrocyte and not those of the rickettsia. Rickettsial phosphatidylethanolamine labeled with ³²PO₄ or [³H]acetate remained intact during lysis, and the composition of the free fatty acids released resembled that of the erythrocyte species used and not the rickettsiae. The products of hydrolysis remained associated with the sedimentable material in the reaction mixture under the usual conditions but partitioned into the supernatant fluid when bovine serum albumin was present. Initially, the time course of phospholipase A activity and lysis was identical, but the release of free fatty acids continued for a short time after the release of hemoglobin was complete. Both the inner and outer leaflets of the erythrocyte membrane were accessible to this rickettsial phospholipase A activity since both phosphatidylcholine and phosphatidylethanolamine were substrates in human erythrocytes. The questions of whether rickettsiae possess their own phospholipase A or activate a latent erythrocyte enzyme and what the role of the energy requirement is in these processes remain unanswered.     

The adsorption and degradation of foot-and-mouth disease virus by isolated BHK-21 cell plasma membranes

Foot-and-mouth disease virus (FMDV) was examined for its ability to adsorb specifically to plasma membranes isolated from BHK-21 cells. The membranes were prepared by the polyethylene glycol-dextran method, and characterized by increases in specific activity of ouabain-sensitive Na⁺K⁺-ATPase and 5′-nucleotidase, and by enrichment in ³H-fucose over unfractionated homogenates. The membranes adsorbed purified radiolabeled FMDV type A₁₂119 with kinetics characteristic of intact cells. Plasma membranes prepared from cells pretreated with trypsin were unable to adsorb virus. The adsorption of labeled FMDV was inhibited by unlabeled virus. Treatment of virus with trypsin, which cleaves capsid protein 3, greatly reduced its ability to adsorb to both plasma membranes and intact cells. After adsorption of virus to membranes at 4°, subsequent incubation at 37° under physiological conditions resulted in a rapid elution of bound virus in an unmodified form which reached approximately 80% by 1 hr. Incubation of the membrane-virus complex at 33° under low-salt conditions degraded the virus particles to intact and fragmented viral RNA and 12 S protein subunits. Membrane-induced viral degradation did not occur at 4° but was observed within 5 min after shifting to 33°. Thus, isolated plasma membranes from BHK-21 cells retain receptors for FMDV possessing uncleaved capsid protein 3. In addition, the eclipse and uncoating of FMDV in intact cells probably occurs at the plasma membrane, and in confirmation of previously reported results, the postadsorptive degradation, unlike that of other picornaviruses, occurs in a single step without the production of intermediate subviral particles.     

Depolarization of cultured astrocytes by glutamate and aspartate

The excitatory transmitter substances glutamate and aspartate are known to have a depolarizing action on cultured CNS neurones, the depolarization being associated with an increase in membrane conductance. When the effects of these amino acids (at a concentration of 10^?4 M) were studied on the membrane potential and resistance of cultured glial cells, they also caused a depolarization of many astrocytes but without producing significant changes in membrane resistance. The majority of glial cells depolarized by glutamate and aspartate were lying in the vicinity of neurones in the dense zone of the cultures, whereas isolated astrocytes in the outgrowth zone were usually not affected by the amino acids. 4-Aminopyridine (5 mM), a substance known to block K⁺-conductance in various excitable membranes, reversibly reduced or abolished the depolarization caused by glutamate and aspartate on glial cells, but had no or only a small effect on the depolarization of neurones caused by these amino acids.These results suggest that the depolarization of glial cells by glutamate and aspartate is caused by an increase in the concentration of extracellular K⁺ which is released from neighbouring neurones during their activation by the amino acids.     

Age-related changes in 5′-nucleotidase and alkaline phosphatase activities in mouse thymocytes

Enzymatic activities of thymocytes isolated from Swiss albino mice were studied at various ages from immediately post weaning until 100 weeks of age, approaching the life expectancy of these animals. Between 5 and 10 weeks of age, the activities of lactate dehydrogenase and alkaline phosphatase decreased to a level that was maintained throughout the remainder of the aging profile. Neutral β-glycerophosphatase (pH 7.5) activity in a thymus membrane preparation was similar in all age groups. The activity of membrane-bound 5′-nucleotidase, that is, AMP-hydrolyzing activity inhibited by 100 μM α,β-methyleneadenosine 5′-diphosphate, progressively increased as a function of age, indicating thymocyte population changes occurring very late in life.In thymocytes of the oldest mice examined (100 weeks of age), 5′-nucleotidase specific activity was approximately ten-fold greater than the activity found in 5-week-old mice. Thus, membrane-bound 5′-nucleotidase activity in thymocytes increased markedly as a function of age in Swiss albino mice; yet several other enzymatic activities, including alkaline phosphatase, remained relatively unchanged in mature mice.     

Biochemical characteristic of non-streptomycin-producing mutants of Streptomyces griseus. II. Lipids and fatty acid composition of vegetative mycelia

Five non-streptomycin-producing non-aerial-mycelium-forming mutants (Str^-Amy^-) of Streptomyces griseus obtained either by spontaneous degeneration or during continuous cultivation of the high-producing aerial-mycelium-forming parent strain HP (Str⁺Amy⁺) were checked with regard to the composition of mycelial lipid material. All the Str^-Amy^-derivatives differed from their ancestor strain HP by an increased ration of 12-methyltetradecanoic acid (aC15:0) to isopalmitic acid (iC16:0) during growth on a chemically defined medium lacking branched-chain amino acids. This finding attests alterations in the availability of precursors for the biosynthesis of methyl- branched fatty acids. The qualitative composition of phospholipids and other polar lipids in one mutant group was found to be similar to the progenitor strain but, additionally, both a yellow pigment and a neutral lipid component were produced in excess. A second type of mutant differed by its incapability to form ornithinolipids even under phosphate limitation. Changes of phospholipid composition were demonstrated in the course of fermentation. Formation of ornithinolipid was suppressed by an excess of inorganic phosphate in the medium, while the portions of phosphatidylethanolamine and cardiolipin increased strongly. Furthermore, the formation of ornithinolipids was influenced by nitrogen sources. These results suggest that the composition of membrane of S. griseus varies in dependence upon the composition of the medium and the age of the mycelium.     

A voltage-sensitive transient potassium current in mouse pancreatic acinar cells

Cellular and luminal pH of isolated ant Malpighian tubules were measured in different bath K⁺ concentrations using double-barrelled pH microelectrodes. The electrochemical gradient for H⁺ across the basolateral and the apical cell membranes was estimated. In control Ringer (51 mmol/l K⁺) cell and luminal pH were alkaline with respect to the basolateral solution: 7.77 and 7.36, respectively, versus 7.25. On lowering basolateral K⁺ concentration to 5 mmol/l or increasing it to 113 mmol/l, luminal pH and to a lesser extent cell pH followed: luminal pH changed to 7.14 and 7.43 and cell pH to 7.69 and 7.82, respectively. In all conditions a cell inward electrochemical gradient for protons across both membranes was observed. Increasing basolateral K⁺ concentration, which was positively correlated with secretion rate, decreased the cell inwardly directed apical proton gradient; moreover, the apical membrane potential difference decreased as well, from –93 mV in 5 mmol/l K⁺ to –65 mV in 113 mmol/l K⁺. Therefore the turnover rate of the electrogenic active proton pump at the apical membrane is facilitated in a high basolateral K⁺ concentration. The calculated electromotive force of this pump is –159 mV. Comparing the proton with the K⁺ electrochemical gradient, taken from another study in the same experimental conditions, we find that the apical proton electrochemical gradient can drive K⁺ extrusion into the lumen for each value of secretion rate.     

Transient changes in the size of the extracellular space in the sensorimotor cortex of cats in relation to stimulus-induced changes in potassium concentration

Summary The time course of local changes of the extracellular space (ES) was investigated by measuring concentration changes of repeatedly injected tetramethylammonium (TMA⁺) and choline (Ch⁺) ions for which cell membranes are largely impermeable. After stimulus-induced extracellular [K⁺] elevations the [TMA⁺] and [Ch⁺] signals recorded with nominally K⁺-selective liquid ion-exchanger microelectrodes increased by up to 100%, thus indicating a reduction of the ES down to one half of its initial size. The shrinkage was maximal at sites where the K⁺ release into the ES was also largest. At very superficial and deep layers, however, considerable increases in extracellular K⁺ concentration were not accompanied by significant reductions in the ES. These findings can be explained as a consequence of K⁺ movement through spatially extended cell structures. Calculations based on a model combining the spatial buffer mechanism of Kuffler and Nicholls (1966) to osmolarity changes caused by selective K⁺ transport through primarily K⁺ permeable membranes support this concept.Following stimulation additional iontophoretically induced [K⁺]_o rises were reduced in amplitude by up to 35%, even at sites where maximal decreases of the ES were observed. This emphasizes the importance of active uptake for K⁺ clearance out of the ES.This investigation was supported by DFG grants Lu 158/10 and He 1128/1     

Properties of the potassium conductances of principal cells of rat cortical collecting ducts

In this study we examined by impalement techniques properties of the macroscopic K⁺ conductances in the luminal and basolateral membrane of principal cells from isolated perfused cortical collecting ducts (CCD) of the rat. Both membranes possess a dominating K⁺ conductance. Compared to their behaviour with K⁺, both membranes appear much less permeable to NH ₄ ⁺ and Rb⁺, and the K⁺ conductances of both membranes are inhibited by these cations. In light of these findings, it is very unlikely that significant amounts of NH ₄ ⁺ , which is secreted in the CCD, cross the principal cells as NH ₄ ⁺ . Several inhibitors with known effects on K⁺ channels in patch-clamp studies have been examined. Tetraethylammonium, which inhibits the excised K⁺ channels of these cells, has no effect on the macroscopic K⁺ conductances of either membrane. Verapamil, which inhibits the K⁺ channels in the luminal membrane, acts predominantly on the basolateral membrane K⁺ conductance in the intact tubule. Therefore, some of the macroscopic properties of the K⁺ conductances are distinct from those of single channels thus far observed in patchclamp studies.Supported by DFG Schl 277/2-1 and Gr 480/10     

Evidence for contribution of Ca2+ storage sites on unitary K+ channel currents in inside-out membrane of rabbit portal vein

While making use of the inside-out membrane patch, we examined the effects of caffeine and heparin on unitary currents of the large conductance Ca²⁺ -dependent K⁺ (maxi-K⁺) channel in the rabbit portal vein. About half of the inside-out membranes we used contained a functional Ca²⁺ -store site which facilitated modification of the maxi-K⁺ channel.When high-K⁺ solution containing 0.05mM EGTA was superfused in the bath, simultaneous openings of more than 20 maxi-K⁺ channels were observed in 39 of 83 patch membranes, and multi-channel opening appeared periodically or continuously at the holding potential of – 10mV. Most channel activities of these patch membranes were inhibited by caffeine or heparin, and some heparin-insensitive channel activities were inhibited by caffeine. The remaining patch membranes (44 out of 83) showed low activity of the maxi-K⁺ channel, and neither caffeine nor heparin modified channel activity.Therefore, in our experimental set-up, half the number of excised patch membranes contained a Ca²⁺ store site. Most Ca²⁺ store sites have inositol 1,4,5-trisphosphate (InsP₃)-activated Ca²⁺ release (IACR) and caffeine-activated Ca²⁺ release (CACR) channels and few lack the IACR channel. The mechanisms of activation of the maxi-K⁺ channel in relation to release of Ca²⁺ from the store sites can be examined in detail using the approaches we have described.     

Electrochemical potentials of potassium in proximal renal tubule of rat

Summary By means of double-barreled K⁺ selective liquid ion-exchange microelectrodes, the electrical potential differences across individual cell membranes were determined simultaneously with the K⁺ concentration in single cellular elements of the proximal tubular epithelium of the rat. Proximal tubular fluid [K⁺] and plasma [K⁺] were also determined electrometrically. Thin cortical slices of the rat kidney analyzed by flame photometry yielded a mean [K⁺] of 136.3±4.2 mM per kg cell water. This electrometric study yielded a mean intracellular [K⁺] of 54.4±2.5 mM, a value which is about 1/3 of the total K⁺ content of proximal tubule cells. The electrometric mean proximal tubule fluid (second half) [K⁺] was 3.7±0.1 mM while plasma [K⁺] was 4.3±0.1 mM, yielding a fluid/plasma concentration ratio of 0.85±0.02. The calculated K⁺ equilibrium potentials (E _K )across the two individual from their respective measured membrane electrical PDs. This signifies that K⁺ exhibits an electrochemical equilibrium distribution across the luminal and peritubular cell boundaries of the proximal tubular epithelium. Thus it is no longer necessary to postulate the presence of an active K⁺ pump in either the luminal or peritubular cell membranes.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.