James A. Schafer

Sodium transport by rat cortical collecting tubule. Effects of vasopressin and desoxycorticosterone.

We have used rat cortical collecting tubules perfused in vitro to study the effects of antidiuretic hormone (ADH) and desoxycorticosterone (DOCA) on the unidirectional fluxes of sodium. We found that in the basal state, lumen-to-bath flux (Jlb) and bath-to-lumen flux (Jbl) of 22Na were approximately equal, 39.5 +/- 3.9 and 41.8 +/- 11.0 pmol X min-1 X min-1, respectively, resulting in no net flux. Addition of 100 microU/ml ADH to the bath produced a stable increase in Jlb to 58.3 +/- 4.7 pmol X min-1 X mm-1. Pretreatment of the animal with DOCA for 4 to 7 d (20 mg/kg per d) increased baseline Jlb to 81.6 +/- 8.7 pmol X min-1 X mm-1. Addition of ADH to a tubule from a DOCA-pretreated rat caused an increase in Jlb to 144.1 +/- 12.0 pmol X min-1 X mm-1 X Neither hormone had an effect on Jbl X Thus ADH produced a greater absolute and fractional increase in Jlb when the animal was pretreated with DOCA, and the ADH-induced increase over baseline was greater than the DOCA-induced increase. Both the ADH-and DOCA-induced stimulation of Jlb were completely abolished by 10(-5) M luminal amiloride, suggesting that the route of sodium transport stimulated by both hormones involves apical sodium channels. However, ADH and DOCA have very different time courses of action; ADH acted within minutes, while aldosterone and DOCA are known to require 90-180 min. The facilitating action of ADH on DOCA-induced stimulation of sodium transport may be important for maximal sodium reabsorption and for the ability to achieve a maximally concentrated urine.

Electrophysiological studies in principal cells of rat cortical collecting tubules ADH increases the apical membrane Na+-conductance

The mechanism of ion transport across principal cells of rat cortical collecting tubules (CCT) and its regulation by vasopressin (ADH) has been studied in the isolated perfused tubule. To amplify the response to ADH rats were treated with 5 mg I. M. desoxycorticosterone 4–9 days prior to the experiments. Addition of 2·10−10 mol·1−1 ADH increased the transepithelial voltage from −5.1 ±0.7 mV to −16.1±1.4 mV (n=37) and decreased the transepithelial resistance from 51±4 Ω cm2 to 39±2 Ω cm2 (n=33). Optical and functional differentiation of impalements of principal and intercalated cells was made and only data of principal cells are presented. ADH depolarized the apical membrane from 79±1 mV to 66±2 mV (n=26) and decreased the fractional resistance of the apical membrane from 0.76±0.04 to 0.70±0.04 (n=13). These ADH effects were prevented by 10−5 or 10−4 mol·1−1 luminal amiloride which hyperpolarized the apical membrane when added in the presence or absence of ADH. Apical and basolateral membranes were dominated by large K+ conductances and addition of 3 mmol·1−1 barium to bath or lumen perfusates increased transepithelial resistance almost two-fold, whereas luminal amiloride increased the transepithelial resistance only by 26–35%. Ouabain (0.5 mmol·1−1, bath) depolarized the basolateral membrane and decreased its K+ conductance. These effects were prevented by the simultaneous presence of apical amiloride suggesting that the only route of Na+ entry into the principal cells occurred via the amiloride sensitive Na+ conductance. We conclude that ADH stimulates Na+ reabsorption and K+ secretion in the rat CCT primarily by increasing the Na+ conductance in the apical cell membrane.

cAMP Increases Density of ENaC Subunits in the Apical Membrane of MDCK Cells in Direct Proportion to Amiloride-sensitive Na+ Transport

Antidiuretic hormone and/or cAMP increase Na+ transport in the rat renal collecting duct and similar epithelia, including Madin-Darby canine kidney (MDCK) cell monolayers grown in culture. This study was undertaken to determine if that increment in Na+ transport could be explained quantitatively by an increased density of ENaC Na+ channels in the apical membrane. MDCK cells with no endogenous ENaC expression were retrovirally transfected with rat α-, β-, and γENaC subunits, each of which were labeled with the FLAG epitope in their extracellular loop as described previously (Firsov, D., L. Schild, I. Gautschi, A.-M. Mérillat, E. Schneeberger, and B.C. Rossier. 1996. Proc. Natl. Acad. Sci. USA. 93:15370–15375). The density of ENaC subunits was quantified by specific binding of 125I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4–8 nM. Transepithelial Na+ transport was measured as the amiloride-sensitive short-circuit current (AS-I sc) across MDCK cells grown on permeable supports. Specific M2 binding was positively correlated with AS-I sc measured in the same experiments. Stimulation with cAMP (20 μM 8-p-chlorothio-cAMP plus 200 μM IBMX) significantly increased AS-I sc from 11.2 ± 1.3 to 18.1 ± 1.3 μA/cm2. M2 binding (at 1.7 nM M2) increased in direct proportion to AS-I sc from 0.62 ± 0.13 to 1.16 ± 0.18 fmol/cm2. Based on the concentration dependence of M2 binding, the quantity of Na+ channels per unit of AS-I sc was calculated to be the same in the presence and absence of cAMP, 0.23 ± 0.04 and 0.21 ±0.05 fmol/μA, respectively. These values would be consistent with a single channel conductance of ∼5 pS (typically reported for ENaC channels) only if the open probability is <0.02, i.e., less than one-tenth of the typical value. We interpret the proportional increases in binding and AS-I sc to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the I sc increase produced by cAMP.

Cellular constraints to diffusion: The effect of antidiuretic hormone on water flows in isolated mammalian collecting tubules

These experiments were intended to evaluate the effects of antidiuretic hormone (ADH) on dissipative water transport in cortical collecting tubules isolated from rabbit kidney. In the absence of ADH, the osmotic (P(f), cm sec(-1)) and diffusional (P(DW) cm sec(-1)) water permeability coefficients were, respectively, 6+/-6 and 4.7+/-1.3 (SD). When ADH was added to the bathing solutions, P(f) and P(DW) rose to, respectively, 186+/-38 and 14.2+/-1.6 (SD). In the absence of ADH, the tubular cells were flat and the lateral intercellular spaces were closed when the perfusing and bathing solutions were, respectively, hypotonic and isotonic; in the presence of ADH, the cells swelled and the intercellular spaces dilated. These data suggest that ADH increased the water permeability of the luminal membranes of the tubules. It was possible that the ADH-dependent P(f)/P(DW) ratio was referable to the resistance of the epithelial cell layer (exclusive of luminal membranes) to water diffusion (R(DW), sec cm(-1)). Such a possibility required that R(DW) be approximately 650, i.e., approximately 25-fold greater than in an equivalent thickness of water. To test this view, it was assumed that R(Di) values for lipophilic solutes in lipid bilayer membranes and in luminal membranes were comparable. In lipid bilayer membranes, R(Di) was substantially less than 90 sec cm(-1) for pyridine, n-butanol, and 5-hydroxyindole. In renal tubules, R(Di) for these solutes ranged from 795 to 2480 with and without ADH. It was assumed that, in the tubules, R(Di) was referable to cellular constraints to diffusion; for these solutes, the latter were 12-25 times greater than in water. Accordingly, it is possible that the ADH-dependent P(f)/P(DW) ratio was also due to cellular constraints to diffusion.

The effect of reversal of Na+ and K+ electrochemical potential gradients on the active transport of amino acids in Ehrlich ascites tumor cells

1. 1. The net uptake of α-aminoisobutyric acid (AIB) in Ehrlich ascites tumor cells has been studied under a variety of transmembrane concentration gradients of Na+, K+ and AIB itself. 2. 2. Before the transport measurements, the cells were prepared in such a way as to abolish or minimize exchange of extracellular AIB with endogenous amino acids, although the incubation with AIB was shown to have no significant effect on the intracellular concentrations of endogenous amino acids, as determined by an amino acid autonanalyzer. 3. 3. The results are in agreement with the ion gradient hypothesis, in that the uptake of AIB rises in proportion to the increase of the parallel electrochemical potential gradient of Na+ and the antiparallel electrochemical potential gradient of K+. 4. 4. Reversal of the net AIB flux was observed only after very drastic inversion of the above-mentioned gradients, i.e. if the driving forces were on the order of — 4000 joules·mole−1 in opposition to the inward movement of AIB. 5. 5. We have concluded that an additional driving force of at least 4000 joules·mole−1 (approximately 950 cal·mole−1) must be present in addition to the electrolyte gradients. This unexplained driving force may be provided by direct coupling of amino acid transport to cellular metabolism, although alternative explanations such as an unequal distribution of alkali metal ions in the cellular space, e.g. between the nucleus and cytoplasm, cannot be completely excluded at this time. 6. 6. In the presence of 0.5 mM ouabain, the deficit in driving force is reduced to 2240 joules·mole−1, but it is not abolished.

A simplified method for isolation of large numbers of defined nephron segments

We describe a simplified method for the isolation of large numbers of nephron segments from rat and rabbit kidneys. In contrast to most previous protocols, the kidneys are not perfused. After removal from the animal, the kidney is sliced and torn in pieces that are subsequently digested in culture medium containing 0.5 mg/ml of collagenase at 37°C. If the preparation is agitated only very gently and infrequently, then the tissue gradually falls apart into a suspension containing long nephron fragments, often consisting of multiple connected segments. These are easily sorted into homogeneous segment populations that can be used for enzyme assays, protein extraction for immunoblotting, and RNA extraction for reverse transcription-polymerase chain reaction, all of which have been done successfully in our laboratory. For comparison, we have also examined cortical collecting tubule segments and cells prepared by the more rigorous protocol described previously (E. Schlatter, U. Fröbe, and R. Greger. Pflügers Arch. 421: 381-387, 1992). Even after the isolation of single cells in a Ca2+-free medium, the cells maintain their normal architecture and a distinct separation of apical and basolateral membranes.

Principal cells of cortical collecting ducts of the rat are not a route of transepithelial Cl− transport

The rat cortical collecting duct (CCD) exhibits high rates of NaCl reabsorption when stimulated by mineralocorticoid and antidiuretic hormone (ADH). The present study was undertaken to determine if there is significant transcellular Cl− movement across the principal cells of the rat CCD. CCDs were dissected from kidneys of rats that had been injected with deoxycorticosterone (5 mg, i.m.) 2–9 days prior to the experiment. The ducts were perfused in vitro with identical perfusing and bathing solutions, except that 200 pmol.l−1 ADH was added to the bathing solutions. The basolateral membrane voltage (PDbl) of principal cells was −77±1 mV and the luminal membrane voltage (PD1) was −68±1 mV (mean ± SEM, n=124). Separate impalements with single-barrelled Cl−-selective microelectrodes gave an apparent intracellular Cl− activity of principal cells of 17±2 mmol.l−1. Transepithelial PD and PDbl were unaffected by luminal furosemide, hydrochlorothiazide (HCT), 4-acetamido-4-isothiocyanostilbene2,2-disulphonic acid, (SITS), or the Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB); bath addition of SITS or the Cl− channel blocker diphenylamino-2-carboxylic acid; or replacement of bath HCO3−by Cl−. The intracellular Cl− activity (acellCl) also remained unchanged with the addition of HCT, SITS or the Cl− channel blockers to either the perfusing or bathing solutions, or with replacement of the bathing solution HCO3−. With Cl− replacement in both solutions, acellCldecreased to 9 mmol.l−1, but not until after 4–6 min, indicating a very low rate of Cl− transport in these cells, even under conditions of maximal stimulation of NaCl reabsorption by mineralocorticoid plus ADH. The remaining acellClcould be attributed to interference with the Cl− selective electrodes by other cytosolic anions. We conclude that acellClof principal cells in the rat CCD is not far above passive equilibrium, and that these cells do not contribute significantly to transepithelial Cl− reabsorption, which must occur by alternative routes such as the paracellular pathway, and/or through intercalated cells.

The effect of antidiuretic hormone on solute flows in mammalian collecting tubules

These experiments were intended to evaluate the antidiuretic hormone (ADH)-dependent reflection coefficients of urea, sucrose, and NaCl in cortical and outer medullary collecting tubules isolated from mammalian kidney. In one group of experiments, the ADH-dependent osmotic water flows, when the perfusing solutions contained hypotonic NaCl solutions, were indistinguishable from control observations when either urea or sucrose replaced, in part, NaCl in isotonic bathing solutions (cortical collecting tubules). Similarly, both in cortical and outer medullary collecting tubules exposed to ADH, there was zero net osmotic volume flow when a portion of the NaCl in the bathing and/or perfusing solutions was replaced by either sucrose or urea, so long as the perfusing and bathing solutions were isosmolal. Taken together, these observations suggest that the ADH-dependent reflection coefficients of NaCl, urea, and sucrose, in these tubules, were identical. Since the effective hydrodynamic radii of urea and sucrose are, respectively, 1.8 and 5.2 A, it is likely that sigma(i), for urea, sucrose, and NaCl, was unity. In support of this, the diffusion permeability coefficient (P(Di) cm sec(-1)) of urea was indistinguishable from zero. Since the limiting sites for urea penetration were the luminal interfaces of the tubules, these data are consistent with the view that ADH increases diffusional water flow across such interfaces.

Transport ofl-cystine in isolated perfused proximal straight tubules

Unidirectional fluxes ofl-35S-cystine and intracellular35S activity were measured in isolated perfused segments of rabbit proximal straight tubule. The absorptive (lumen-to-both) flux ofl-35S-cysteine showed a tendency toward saturation within the concentration limits imposed by the low solubility of cystine (0.3 mmol·l−1). In contrast, for the bath-to-lumen fluxes, there was a linear relation between the bathing solution concentration ofl-35S-cystine and the rate of35S appearance in the lumen. Nonlinear fitting of both sets of unidirectional flux data gave a maximal cystine transport rate (Jmax) of 1.45±0.27 (SEM) pmol min−1 mm−1, a Michaelis constant (Km) of 0.20±0.07 mmol·l−1, and an apparent permeability coefficient of 0.27±0.11 pmol min−1 mm−1 (mmol·l−1)−1 (approximately 0.06 μm/s). The35S concentration in the cell exceeded that in the lumen by almost 60-fold during the lumen-to-bath flux, and exceeded the bathing solution concentration by 4.7-fold during the bath-to-lumen flux. Thus cystine was accumulated by the cells across either membrane, but over 77% of the intracellular activity was in the form of cysteine. Although the presence of luminall-lysine or cycloleucine inhibited the absorptive flux of cystine, neither amino acid affected the bath-to-lumen flux.

Evidence for activation of an active electrogenic proton pump in Ehrlich ascites tumor cells during glycolysis

SummaryThe addition of glucose to a suspension of Ehrlich ascites tumor cells results in rapid acidification of the extracellular medium due to lactic acid production. The nature of the H+ efflux mechanism has been studied by measuring the time course of the acidification, the rate of proton efflux, the direction and relative magnitude of the H+ concentration gradient, and the voltage across the membrane. Using the pH-sensitive dye acridine orange, we have established that after addition of 10mm glucose an outward-directed H+ concentration gradient develops. As the rate of glycolysis slows, the continued extrusion of H+ reverses the direction of the H+ concentration gradient. Changes in absorbance of the voltagesensitive dye diethyloxadicarbocyanine iodide (DOCC), and changes in the distribution of the lipid permeant cation tetraphenyl phosphonium, showed a dramatic and persistent hyperpolarization of the membrane voltage after glucose addition. The hyperpolarization was prevented by the protonophore tetrachlorosalicylanalide (TCS) and by valinomycin, but not by the neutral-exchange ionophore nigericin. Inhibitors of lactate efflux were found to reduce the rate of acidification after glucose addition but they had no effect on the magnitude of the resulting hyperpolarization. On the basis of these and other data we suggest that an active electrogenic pump mechanism for H+ efflux may be activated by glucose and that this mechanism operates independently of the lactate carrier system.