NaCl reflection coefficients in proximal tubule apical and basolateral membrane vesicles. Measurement by induced osmosis and solvent drag.

Two independent methods, induced osmosis and solvent drag, were used to determine the reflection coefficients for NaCl (sigma NaCl) in brush border and basolateral membrane vesicles isolated from rabbit proximal tubule. In the induced osmosis method, vesicles loaded with sucrose were subjected to varying inward NaCl gradients in a stopped-flow apparatus. sigma NaCl was determined from the osmolality of the NaCl solution required to cause no initial osmotic water flux as measured by light scattering (null point). By this method sigma NaCl was greater than 0.92 for both apical and basolateral membranes with best estimates of 1.0. sigma NaCl was determined by the solvent drag method using the Cl-sensitive fluorescent indicator, 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ), to detect the drag of Cl into vesicles by inward osmotic water movement caused by an outward osmotic gradient. sigma NaCl was determined by comparing experimental data with theoretical curves generated using the coupled flux equations of Kedem and Katchalsky. By this method we found that sigma NaCl was greater than 0.96 for apical and greater than 0.98 for basolateral membrane vesicles, with best estimates of 1.0 for both membranes. These results demonstrate that sigma NaCl for proximal tubule apical and basolateral membranes are near unity. Taken together with previous results, these data suggest that proximal tubule water channels are long narrow pores that exclude NaCl.     

Maturation of rat proximal tubule chloride permeability

We have previously shown that neonate rabbit tubules have a lower chloride permeability but comparable mannitol permeability compared with adult proximal tubules. The surprising finding of lower chloride permeability in neonate proximals compared with adults impacts net chloride transport in this segment, which reabsorbs 60% of the filtered chloride in adults. However, this maturational difference in chloride permeability may not be applicable to other species. The present in vitro microperfusion study directly examined the chloride and mannitol permeability using in vitro perfused rat proximal tubules during postnatal maturation. Whereas there was no maturational change in mannitol permeability, chloride permeability was 6.3 +/- 1.3 x 10(-5) cm/s in neonate rat proximal convoluted tubule and 16.1 +/- 2.3 x 10(-5) cm/s in adult rat proximal convoluted tubule (P < 0.01). There was also a maturational increase in chloride permeability in the rat proximal straight tubule (5.1 +/- 0.6 x 10(-5) cm/s vs. 9.3 +/- 0.6 x 10(-5) cm/s, P < 0.01). There was no maturational change in bicarbonate-to-chloride permeabilities (P(HCO3)/P(Cl)) in the rat proximal straight tubules (PST) and proximal convoluted tubules (PCT) or in the sodium-to-chloride permeability (P(Na)/P(Cl)) in the proximal straight tubule; however, there was a significant maturational decrease in proximal convoluted tubule P(Na)/P(Cl) with postnatal development (1.31 +/- 0.12 in neonates vs. 0.75 +/- 0.06 in adults, P < 0.001). There was no difference in the transepithelial resistance measured by current injection and cable analysis in the PCT, but there was a maturational decrease in the PST (7.2 +/- 0.8 vs. 4.6 +/- 0.1 ohms x cm2, P < 0.05). These studies demonstrate there are maturational changes in the rat paracellular pathway that impact net NaCl transport during development.     

Na+ Recirculation and Isosmotic Transport

The Na(+) recirculation theory for solute-coupled fluid absorption is an expansion of the local osmosis concept introduced by Curran and analyzed by Diamond & Bossert. Based on studies on small intestine the theory assumes that the observed recirculation of Na(+) serves regulation of the osmolarity of the absorbate. Mathematical modeling reproducing bioelectric and hydrosmotic properties of small intestine and proximal tubule, respectively, predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1 (-/-) mice, and the inverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow. The model reproduces the volume responses of cells and lateral intercellular space (lis) following replacement of luminal NaCl by sucrose as well as the linear dependence of volume absorption on luminal NaCl concentration. Analysis of solvent drag on Na(+) in tight junctions provides explanation for the surprisingly high metabolic efficiency of Na(+) reabsorption. The model predicts and explains low metabolic efficiency in diluted external baths. Hyperosmolarity of lis is governed by the hydraulic permeability of the apical plasma membrane and tight junction with 6-7 mOsm in small intestine and < or = 1 mOsm in proximal tubule. Truly isosmotic transport demands a Na(+) recirculation of 50-70% in small intestine but might be barely measurable in proximal tubule. The model fails to reproduce a certain type of observations: The reduced volume absorption at transepithelial osmotic equilibrium in AQP1 knockout mice, and the stimulated water absorption by gallbladder in diluted external solutions. Thus, it indicates cellular regulation of apical Na(+) uptake, which is not included in the mathematical treatment.     

Water permeabilities and salt reflection coefficients of luminal, basolateral and intracellular membrane vesicles isolated from rabbit kidney proximal tubule

The mechanisms of water transport across the rabbit renal proximal convoluted tubule were approached by measuring osmotic permeabilities and solute reflection coefficients of the brush-border and the basolateral membranes. Plasma and intracellular membrane vesicles were isolated from rabbit renal cortex by centrifugation on a Percoll gradient. Three major turbidity bands were obtained: a fraction of purified basolateral membranes (BLMV), the two others being brush-border (BBMV) and endoplasmic reticulum (ERMV) membrane vesicles. The osmotic permeability (Pf) of the three types of vesicle was measured using stop-flow techniques and their geometry was determined by quasi-elastic light scattering. Pf was equal to 123 +/- 8 microns/s (n = 10) for BBMV, 166 +/- 10 microns/s (n = 10) for BLMV and 156 +/- 9 microns/s (n = 4) for ERMV (T = 26 degrees C). A transcellular water permeability, per unit of apical surface area, of 71 microns/s was calculated considering that the luminal and the basolateral membranes act as two conductances in series. This value is in close agreement, after appropriate normalizations, with previously reported transepithelial water permeabilities obtained using in vitro microperfusion techniques thus supporting the hypothesis of a predominantly transcellular route for water flow across rabbit proximal convoluted tubule. The addition of 0.4 mM HgCl2, a sulfhydryl reagent, decreased Pf about 60% in three types of membrane providing evidence for the existence of proteic pathways. NaCl and KCl reflection coefficients were measured and found to be close to one for plasma and intracellular membranes suggesting that the water channels are not shared by salts.     

Convective paracellular solute flux. A source of ion-ion interaction in the epithelial transport equations

An electrolyte model of an epithelium (a cell and a tight junction in parallel, both in series with a lateral interspace basement membrane) is analyzed using the formalism of nonequilibrium thermodynamics. It is shown that if the parallel structures are heteroporous (i.e., reflection coefficients for two ion species differ between the components), then a cross-term will appear in the overall transport equations of the epithelium. Formally, this cross-term represents an ion-ion interaction. With respect to the rat proximal tubule, data indicating epithelial ionic reflection coefficients less than unity, together with the assumption of no transcellular solvent drag, imply the presence of convective paracellular solute flux. This means that a model applicable to a heteroporous structure must be used to represent the tubule, and, in particular, the cross-terms for ion-ion interaction must also be evaluated in permeability determinations. A series of calculations is presented that permits the estimation of the Na-Cl interaction for rat proximal tubule from available experimental data. One consequence of tubule heteroporosity is that an electrical potential may be substantially less effective than an equivalent concentration gradient in driving reabsorptive ion fluxes.     

Bicarbonate-water interactions in the rat proximal convoluted tubule. An effect of volume flux on active proton secretion

The effect of volume absorption on bicarbonate absorption was examined in the in vivo perfused rat proximal convoluted tubule. Volume absorption was inhibited by isosmotic replacement of luminal NaCl with raffinose. In tubules perfused with 25 mM bicarbonate, as raffinose was increased from 0 to 55 to 63 mM, volume absorption decreased from 2.18 +/- 0.10 to 0.30 +/- 0.18 to -0.66 +/- 0.30 nl/mm X min, respectively, and bicarbonate absorption decreased from 131 +/- 5 to 106 +/- 8 to 91 +/- 13 pmol/mm X min, respectively. This bicarbonate-water interaction could not be attributed to dilutional changes in luminal or peritubular bulk phase bicarbonate concentrations. Inhibition of active proton secretion by acetazolamide abolished the effect of volume flow on bicarbonate absorption, which implies that the bicarbonate reflection coefficient is close to 1 and eliminates the possibility of solvent drag across the tight junction. When the luminal bicarbonate concentration was varied, the magnitude of the bicarbonate-water interaction increased with increasing luminal bicarbonate concentration. The largest interaction occurred at high luminal bicarbonate concentrations, where the rate of proton secretion has been previously shown to be independent of luminal bicarbonate concentration and pH. The results thus suggest that a peritubular and/or cellular compartment exists that limits bicarbonate diffusion, and where pH changes secondary to bicarbonate-water interactions (solute polarization) alter the rate of active proton secretion.     

Rapid development of vasopressin-induced hydroosmosis in kidney collecting tubules measured by a new fluorescence technique.

The pre-steady-state kinetics of the vasopressin-induced increase in collecting tubule osmotic water permeability (Pf) has been measured by a new fluorescence technique. Isolated cortical collecting tubules (CCT) from rabbit kidney were perfused with physiological buffers containing the impermeant fluorophores fluorescein sulfonate (FS) and pyrenetetrasulfonic acid (PTSA). Tubules were subject to a 120 mOsm bath-to-lumen osmotic gradient in the presence and absence of 250 microU/ml vasopressin. The magnitude of transepithelial volume flow was determined from the self-quenching of FS, or from the ratio of PTSA/FS fluorescence, measured at 380 nm excitation and 420 +/- 10 nm (PTSA) and greater than 530 nm (FS) emission wavelengths. Pf was calculated from the magnitude of transepithelial volume flow, lumen and bath osmolarities, lumen perfusion rate, and tubule geometry. The instrument response time for a change in bath osmolality was less than 3 s. At 37 degrees C, CCT Pf was (in units of cm/s x 10(4] 13 +/- 2 (mean +/- SE, 16 tubules) before, and 227 +/- 10 after addition of vasopressin to the bath. CCT Pf began to increase in 23 +/- 3 s after vasopressin addition and was half-maximal after 186 +/- 20 s. At 23 degrees C, Pf was 9 +/- 1 (seven tubules) before, and 189 +/- 12 after vasopressin addition. Pf began to increase in 40 +/- 4 s and was half-maximal after 195 +/- 35 s. After vasopressin removal from the bath, Pf decreased to its baseline value with a half-time of 14 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of the Na+ recirculation theory to ion coupled water transport in low- and high resistance osmoregulatory epithelia

The theory of Na+ recirculation for isosmotic fluid absorption follows logically from Hertz's convection-diffusion equation applied to the exit of water and solutes from the lateral intercellular space. Experimental evidence is discussed indicating Na+ recirculation based upon the following approaches: (i) An isotope tracer method in small intestine. Simultaneous measurement of water flow and ion transport in toad skin epithelium demonstrating, (ii) occasional hyposmotic absorbates, and (iii) reduced fluid absorption in the presence of serosal bumetanide. (iv) Studies of the metabolic cost of net Na+ absorption demonstrating an efficiency that is lower than the 18 Na+ per O2 consumed given by the stoichiometry of the Na+/K+-pump. Mathematical modeling predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1(-/-) mice, the adverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow, and in a non-contradictory way the wide range of metabolic efficiencies from above to below 18 Na+/O2. Certain types of observations are poorly or not at all reproduced by the model. It is discussed that such lack of agreement between model and experiment is due to cellular regulations of ion permeabilities that are not incorporated in the modeling. Clarification of these problems requires further experimental studies.     

Ion exchangers mediating NaCl transport in the renal proximal tubule

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. Studies of isolated brush-border membrane vesicles confirmed the presence of Na+-H+ exchange and identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanisms of uphill Cl- entry. We found that formate and oxalate each stimulate NaCl absorption in microperfused proximal tubules. Stimulation of NaCl absorption by formate was blocked by the Na+-H+-exchange inhibitor EIPA, whereas stimulation by oxalate was blocked by omission of sulfate from the perfusion solutions. These observations were consistent with recycling of formate from lumen to cell by H+-coupled formate transport in parallel with Na+-H+ exchange and recycling of oxalate by oxalate-sulfate exchange in parallel with Na+-sulfate cotransport. Using isoform-specific antibodies, we found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the loop of Henle. The inhibitor sensitivity of Na+-H+ exchange in renal brush-border vesicles and of HCO3- absorption in microperfused tubules suggested that NHE3 is responsible for most, if not all, apical membrane Na+-H+ exchange in the proximal tubule. The role of NHE3 in mediating proximal tubule HCO3- absorption and formate-dependent Cl- absorption was confirmed by studies in NHE3 null mice. Finally, we cloned and functionally expressed CFEX, an anion transporter expressed on the apical surface of proximal tubule cells and capable of mediating Cl(-)-formate exchange.     

Transport of chlorine in the proximal tubule. Its effects on water-electrolyte absorption

Several studies in rat kidney have established that an appreciable fraction of proximal absorption is passive in nature and occurs across the highly conductive paracellular pathway. Passive absorption is generally ascribed to the transepithelial Cl- distribution, luminal Cl- activity (alpha lCl) being higher than plasma Cl- activity (alpha pCl). The inequality alpha lCl greater than alpha pCl generates a transepithelial diffusion potential, lumen positive, which taken together with the chemical potential differences of Cl- and Na+ across the epithelium gives rise to transepithelial electrochemical potential differences for Cl- and Na+ favoring their absorption. The alpha lCl greater than alpha pCl distribution is traditionally ascribed to preferential bicarbonate absorption. We argue that HCO3- absorption alone cannot generate a non equilibrium transepithelial Cl- distribution. Other mechanisms are necessary. Our measurements in amphibian proximal tubule demonstrate that the intracellular Cl- activity, alpha cCl, is higher than the theoretical value predicted for equilibrium. This distribution is the result of two basolateral coupled transport processes (Cl-/HCO3- exchange and Cl-/Na+ cotransport). It contributes to the exit of Cl- from cell to lumen (by passive diffusion and K+/Cl- cotransport), yielding alpha lCl values higher than the theoretical value for equilibrium with regard to plasma. Thus, a small transcellular flux of Cl- (without solvent) proceeds from interstitium to lumen. It compensates the dissipative tendency of a much higher paracellular Cl- absorptive flux (in association with water) on the transepithelial Cl- gradient. The result is a steady-state luminal Cl- distribution above equilibrium, along the major part of the proximal tubule.     

Sodium-sensitive, probenecid-insensitive p-aminohippuric acid uptake in cultured renal proximal tubule cells of the rabbit.

In the intact kidney, renal proximal tubule cells accumulate p-aminohippurate (PAH) via a basolateral, probenecid- and sodium-sensitive transport system. Primary cultures of rabbit proximal tubule cells retain sodium-glucose co-transport in culture, but little is known about PAH transport in this system. Purified proximal tubule cells from a rabbit were grown in culture and assessed for PAH and alpha-methyl-D-glucoside uptake capacities as well as proximal tubule marker enzyme activities. Control PAH uptake on collagen-coated filters (20 +/- 3 pmol/mg protein.min; n = 8) was not significantly different from uptake in the presence of 1 mM probenecid (19 +/- 4 pmol/mg protein.min; n = 8). Uptake from the basal side of the cell was 3.9 +/- 0.7 times greater than that from the apical side. In multi-well plate studies, the uptake was significantly reduced by removing sodium from the medium and stimulated by coating the wells with collagen. Glutarate (10 mM) had no effect on the uptake of PAH. Other differentiated proximal tubule characteristics were retained in culture, including the ability to form domes and to transport glucose by a phlorizin-sensitive system. Phlorizin-sensitive 1 mM alpha-methyl-D-glucoside uptake was 134 +/- 42 pmol/mg protein.min (n = 7; P less than 0.02). The proximal tubule marker enzymes alkaline phosphatase and gamma-glutamyltranspeptidase, increased in activity in the cultures after confluence. It was concluded that whereas some differentiated properties were retained during primary culture of rabbit proximal tubule cells, the PAH transport system was selectively lost or modified from that present in the intact kidney.     

Effect of claudins 6 and 9 on paracellular permeability in MDCK II cells

The neonatal proximal tubule has a lower permeability to chloride, higher resistance, and higher relative sodium-to-chloride permeability (P(Na)/P(Cl)) than the adult tubule, which may be due to maturational changes in the tight junction. Claudins are tight-junction proteins between epithelial cells that determine paracellular permeability characteristics of epithelia. We have previously described the presence of two claudin isoforms, claudins 6 and 9, in the neonatal proximal tubule and subsequent reduction of these claudins during postnatal maturation. The question is whether changes in claudin expression are related to changes in functional characteristics in the neonatal tubule. We transfected claudins 6 and 9 into Madin-Darby canine kidney II (MDCK II) cells and performed electrophysiological studies to determine the resultant changes in physiological characteristics of the cells. Expression of claudins 6 and 9 resulted in an increased transepithelial resistance, decreased chloride permeability, and decreased P(Na)/P(Cl) and P(HCO3)/P(Cl). These findings constitute the first characterization of the permeability characteristics of claudins 6 and 9 in a cell model and may explain why the neonatal proximal tubule has lower permeability to chloride and higher resistance than the adult proximal tubule.     

Chloride reabsorption by renal proximal tubules of necturus

Summary Movement of Cl from the lumen ofNecturus proximal tubule into the cells is mediated and dependent on the presence of luminal Na. Intracellular Cl activity was monitored with ion selective microelectrodes. In Cl Ringer's perfused kidneys, cell Cl activity was 24.5±1.1mm, 2 to 3 times higher than that predicted for passive distribution. When luminal NaCl was partially replaced by mannitol (capillaries perfused with Cl Ringer's) cell Cl decreased showing a sigmoidal dependence on luminal NaCl. Peritubular membrane potential was unaltered. Sulfate Ringer's perfusion of the kidneys washed out all cell Cl but did not alter peritubular membrane potential. Chloride did not enter the cell when the tubule lumen was perfused with 100mm KCl, LiCl, or tetramethylammonium Cl. Luminal perfusion of NaCl caused cell Cl to rise rapidly to the same value as the controls in the Cl Ringer's experiments. Perfusion of the tubule lumen with mixtures of NaCl and Na₂SO₄, while the capillaries contained sulfate Ringer's yielded a sigmoidal dependence of cell Cl on luminal NaCl activity. Chloride movement from the lumen into the proximal tubule cells required approximately equal concentrations of Na and Cl. Current clamp experiments indicated that intracellular chloride activity was insensitive to alterations in liminal membrane potential, suggesting that chloride entry was electrically neutral. The transcellular chloride flux was calculated to constitute about one half of the normal chloride reabsorption rate. We conclude that the cell Cl activity is primarily determined by the NaCl concentration in the tubule lumen and that Cl entry across the luminal membrane is mediated.     

A component of fluid absorption linked to passive ion flows in the superficial pars recta

We studied salt and water absorption in isolated rabbit superficial proximal straight tubules perfused and bathed with solutions providing oppositely directed transepithelial anion gradients similar to those which might obtain in vivo. The perfusing solution contained 138.6 mM Cl- 3.8 mM HCO-3 (pH 6.6) while the bathing solution contained 113.6 mM Cl- and 25 mM HCO-3 (pH 7.4); the system was bubbled with 95% O2-5% CO2. At 37 degrees C, net volume absorption (Jv nl min-1 mm-1) was 0.32 +/- 0.03 (SEM); Ve, the transepithelial voltage (millivolts; lumen to bath), was +3.1 +/- 0.2. At 21 degrees C, Ve rose to +3.7 +/- 0.1 and Jv fell to 0.13 +/- 0.01 (significantly different from zero at P less than 0.001); in the presence of 10(-4)M ouabain at 37 degrees C, Ve rose to +3.8 +/- 0.1 and Jv fell to 0.16 +/- 0.01 (P less than 0.001 with respect to zero). In paired experiments, the ouabain- and temperature-insensitive moieties of Jv and Ve became zero when transepithelial anion concentration gradients were abolished. Titrametric determinations net chloride flux at 21 degrees C or at 37 degrees C with 10(-4) M ouabain showed that chloride was the sole anion in an isotonic absorbate. And, combined electrical and tracer flux data indicated that the tubular epithelium was approximately 18 times more permeable to Cl- than to HCO-3. We interpret these results to indicate that, in these tubules, NaCl absorption depends in part on transepithelial anion concentration gradients similar to those generated in vivo and in vitro by active Na+ absorption associated with absorption to anions other than chloride. A quantitative analysis of passive solute and solvent flows in lateral intercellular spaces indicated that fluid absorption occurred across junctional complexes when the osmolality of the lateral intercellular spaces was equal to or slightly less than that of the perfusing and bathing solutions; the driving force for volume flow under these conditions depended on the fact that sigmaHCO3 exceeded sigmaCl.     

Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex.

Regulation of Cl conductance by protein kinase A may play a role in control of endosomal acidification [Bae, H.-R., & Verkman, A. S. (1990) Nature, 348, 637-639]. To investigate the mechanism of kinase A action, cell-free measurements of Cl transport and membrane protein phosphorylation were carried out in apical endocytic vesicles from rabbit kidney proximal tubule. Cl transport was measured by a stopped-flow quenching assay in endosomes labeled in vivo with the fluorescent Cl indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Phosphorylation was studied in a purified endosomal preparation by SDS-PAGE and autoradiography of membrane proteins labeled by [gamma-32P]ATP. Endosomes had a permeability (PCl) for conductive Cl transport of 3.1 x 10(-8) cm/s at 23 degrees C which was stilbene inhibitable. PCl was increased by 90 +/- 20% by a 10-min preincubation with the catalytic subunit of kinase A (PKA, 10 units/mL) and MgATP (0.5 mM) with anion selectivity Cl greater than I greater than Br. The increase in PCl was blocked by 100 microM N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and was reversed by addition of alkaline phosphatase (AP, 40 units/mL) after incubation with PKA and MgATP; the increase in PCl was not blocked by pretreatment with AP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activated neutrophils increase microvascular permeability in skeletal muscle: role of xanthine oxidase

To determine the role of xanthine oxidase in the microvascular dysfunction produced by activated granulocytes, we examined the effect of xanthine oxidase depletion or inhibition on the increase in microvascular permeability produced by infusion of the neutrophil activator phorbol myristate acetate (PMA). Changes in vascular permeability were assessed by measurement of the solvent drag reflection coefficient for total plasma proteins (sigma) in rat hindquarters subjected to PMA infusion in xanthine oxidase-replete and -depleted animals, in animals pretreated with the xanthine oxidase inhibitor oxypurinol, and in animals depleted of circulating neutrophils by pretreatment with antineutrophil serum (ANS). Xanthine oxidase depletion was accomplished by administration of a tungsten-supplemented (0.7 g/kg diet) molybdenum-deficient diet. In animals fed the tungsten diet, muscle total xanthine dehydrogenase plus xanthine oxidase activity was decreased to less than 10% of control values. Estimates of sigma averaged 0.84 +/- 0.04 in control hindquarters, whereas PMA infusion was associated with a marked increase in microvascular permeability (decrease in sigma to 0.68 +/- 0.03). PMA infusion also caused an increase in the amount of the radical-producing oxidase form of xanthine oxidase (from 3.9 +/- 0.05 to 5.6 +/- 0.4 mU/g wet wt). ANS pretreatment attenuated this permeability increase (sigma = 0.77 +/- 0.04) and diminished the rise in xanthine oxidase activity (4.9 +/- 0.5 mU/g wet wt). Xanthine oxidase depletion with the tungsten diet or pretreatment with oxypurinol had no effect on this neutrophil-mediated microvascular injury (sigma = 0.69 +/- 0.06 and 0.67 +/- 0.03, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Chloride transport in rabbit esophageal epithelial cells

We investigated Cl(-) transport pathways in the apical and basolateral membranes of rabbit esophageal epithelial cells (EEC) using conventional and ion-selective microelectrodes. Intact sections of esophageal epithelium were mounted serosal or luminal side up in a modified Ussing chamber, where transepithelial potential difference and transepithelial resistance could be determined. Microelectrodes were used to measure intracellular Cl(-) activity (a), basolateral or apical membrane potentials (V(mBL) or V(mC)), and the voltage divider ratio. When a basal cell was impaled, V(mBL) was -73 +/- 4.3 mV and a(i)(Cl) was 16.4 +/- 2.1 mM, which were similar in presence or absence of bicarbonate. Removal of serosal Cl(-) caused a transient depolarization of V(mBL) and a decrease in a(i)(Cl) of 6.5 +/- 0.9 mM. The depolarization and the rate of decrease of a(i)(Cl) were inhibited by approximately 60% in the presence of the Cl(-)-channel blocker flufenamate. Serosal bumetanide significantly decreased the rate of change of a(i)(Cl) on removal and readdition of serosal Cl(-). When a luminal cell was impaled, V(mC) was -65 +/- 3.6 mV and a was 16.3 +/- 2.2 mM. Removal of luminal Cl(-) depolarized V(mC) and decreased a by only 2.5 +/- 0.9 mM. Subsequent removal of Cl(-) from the serosal bath decreased a(i)(Cl) in the luminal cell by an additional 6.4 +/- 1.0 mM. A plot of V(mBL) measurements vs. log a(i)(Cl)/log a(o)(Cl) (a(o)(Cl) is the activity of Cl(-) in a luminal or serosal bath) yielded a straight line [slope (S) = 67.8 mV/decade of change in a(i)(Cl)/a(o)(Cl)]. In contrast, V(mC) correlated very poorly with log a/a (S = 18.9 mV/decade of change in a/a). These results indicate that 1) in rabbit EEC, a(i)(Cl) is higher than equilibrium across apical and basolateral membranes, and this process is independent of bicarbonate; 2) the basolateral cell membrane possesses a conductive Cl(-) pathway sensitive to flufenamate; and 3) the apical membrane has limited permeability to Cl(-), which is consistent with the limited capacity for transepithelial Cl(-) transport. Transport of Cl(-) at the basolateral membrane is likely the dominant pathway for regulation of intracellular Cl(-).     

Phalloidin attenuates postischemic neutrophil infiltration and increased microvascular permeability.

The aim of this study was to determine whether phalloidin (1 microM) or antamanide (1 microM), cyclic peptides that stabilize dense peripheral band and stress fiber F-actin in endothelium, would attenuate the increase in microvascular permeability induced by 4 h of ischemia and 30 min of reperfusion (I/R) in the isolated canine gracilis muscle. Changes in microvascular permeability (1 - sigma) were assessed by determining the solvent drag reflection coefficient for total plasma proteins (sigma) in muscles subjected to 4.5 h of continuous perfusion (nonischemic controls), I/R alone, I/R + phalloidin, or I/R + antamanide. Muscle neutrophil content was assessed by determination of myeloperoxidase (MPO) activity in tissue samples obtained at the end of the experiments. Fluorescent detection of nitrobenzoxadiazole-phallicidin in endothelial cell monolayers confirmed that phalloidin enters these cells. I/R was associated with marked increases in microvascular permeability and muscle neutrophil content (1 - sigma = 0.45 +/- 0.07; MPO = 8.9 +/- 0.5 units/g) relative to control (4.5 h continuous perfusion) preparations (1 - sigma = 0.12 +/- 0.03; MPO = 0.5 +/- 0.8 unit/g). These I/R-induced changes were largely prevented by administration of phalloidin (1 - sigma = 0.19 +/- 0.02; MPO = 0.8 +/- 0.4 U/g) or antamanide (1 - sigma = 0.07 +/- 0.11; MPO = 0.9 +/- 0.3 unit/g) at reperfusion. Similar results were obtained when phalloidin was administered before ischemia (1 - sigma = 0.24 +/- 0.04; MPO = 1.2 +/- 1.0 units/g). Although antamanide decreased superoxide production (by approximately 60%) and adherence to plastic (by approximately 75%) by activated neutrophils in vitro, phalloidin failed to alter these aspects of granulocyte function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and partial purification of the thiazide diuretic receptor from rabbit renal cortex

This study was designed to solubilize, characterize and begin to purify the thiazide-sensitive Na/Cl transporter from mammalian kidney. Metolazone, a thiazide-like diuretic drug, binds to receptors in rat renal cortex closely related to the thiazide-sensitive Na/Cl transport pathway of the renal distal tubule. In the current study, [3H]metolazone bound to receptors in rabbit renal cortical microsomes. The portion of [3H]metolazone binding that was inhibited by hydrochlorothiazide reflected binding to a high-affinity class of receptor. The affinity (Kd 2.0 +/- 0.1 nM) and number (Bmax = 0.9 +/- 0.4 pmol/mg protein) of high-affinity receptors in rabbit renal cortical membranes were similar to values reported previously for rat. When proximal and distal tubule fragments were separated by Percoll gradient centrifugation, receptors were restricted to the fraction that contained distal tubules. When compared with cortical homogenates, receptor density was enriched 12-fold by magnesium precipitation and differential centrifugation. The zwitterionic detergent CHAPS solubilized 25-35% of the receptors (at 6 mM). Chloride inhibited and Na stimulated binding of [3H]metolazone to solubilized high-affinity receptors. The receptors could be purified significantly by hydroxyapatite chromatography and size exclusion high performance liquid chromatography (HPLC). The combination of magnesium precipitation and differential centrifugation, hydroxyapatite chromatography, and size exclusion HPLC resulted in a 213-fold enrichment of receptors, compared to renal cortical homogenate. The current results indicate that thiazide receptors from rabbit kidney share characteristics with receptors from rat, and that rabbit receptors can be solubilized in CHAPS and purified significantly by hydroxyapatite chromatography and size exclusion HPLC.     

Basolateral membrane Cl/HCO3 exchange in the rat proximal convoluted tubule. Na-dependent and -independent modes

To examine whether Cl-coupled HCO3 transport mechanisms were present on the basolateral membrane of the mammalian proximal tubule, cell pH was measured in the microperfused rat proximal convoluted tubule using the pH-sensitive, intracellularly trapped fluorescent dye (2',7')- bis(carboxyethyl)-(5,6)-carboxyfluorescein. Increasing the peritubular Cl concentration from 0 to 128.6 meq/liter caused cell pH to decrease from 7.34 +/- 0.04 to 7.21 +/- 0.04 (p less than 0.001). With more acid extracellular fluid (pH 6.62), a similar increase in the peritubular Cl concentration caused cell pH to decrease by a similar amount from 6.97 +/- 0.04 to 6.84 +/- 0.05 (p less than 0.001). This effect was blocked by 1 mM SITS. To examine the Na dependence of Cl/HCO3 exchange, the above studies were repeated in the absence of luminal and peritubular Na. In alkaline Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.57 +/- 0.06 to 7.53 +/- 0.06 (p less than 0.025); in acid Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.21 +/- 0.04 to 7.19 +/- 0.04 (p less than 0.05). The effect of Cl on cell pH was smaller in the absence of luminal and peritubular Na than in its presence. To examine whether the previously described Na/(HCO3)n greater than 1 cotransporter was coupled to or dependent on Cl, the effect of lowering the peritubular Na concentration from 147 to 25 meq/liter was examined in the absence of ambient Cl. Cell pH decreased from 7.28 +/- 0.03 to 7.08 +/- 0.03, a response similar to that observed previously in the presence of Cl. The results demonstrate that Cl/HCO3 (or Cl/OH) exchange is present on the basolateral membrane. Most of Cl/HCO3 exchange is dependent on the presence of Na and may be coupled to it. The previously described Na/(HCO3)n greater than 1 cotransporter is the major basolateral membrane pathway for the coupling of Na and HCO3 and is not coupled to Cl.