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1. Frog proximal tubular cells were fused into giant cells. We measured membrane potential (Vm), its changes (delta Vm), and current-induced voltage changes (delta psi) in single cells, during control and experimental states. Each cell served as its own control. 2. In the presence of a physiological Ringer solution, the transference number for potassium (tK) was 0.50. Barium (3 mM) reduced membrane conductance (Gm) by 50%; low-Cl- solutions and low-Na+ solutions also diminished Gm, by 52 and 30%, respectively. The association of barium and low-NaCl solutions decreased Gm to approximately 38% of control, indicating that the impermeant substitute of a physiological ion may interact with other pathways; alternatively, blockade of steady-state conductances may activate physiologically silent processes. 3. In an attempt to enhance the contribution of the partial K+ conductance (GK) to Gm, fused cells were exposed to low-Cl- solutions, containing in addition 0.1 mM-methazolamide, to inhibit the rheogenic Na(+)-HCO3-symport, and 1 microM-amiloride, to block Na+ conductance (GNa). tK went up to 0.83. 4. The high tK preparation was challenged with barium (3 mM) or quinine (Quin, 1 mM). These blockers produced large depolarizations (approximately 60 mV), however, although Gm decreased along early- and mid-depolarization, Gm plateaued and eventually it increased with larger and larger depolarization. 5. Depolarization-associated increase in Gm reflects activation of other conductances. These are Na+, cationic, and K+ conductance(s) poorly sensitive to quinine or barium. In the presence of Ba(2+)- or Quin-induced depolarization, injection of depolarizing current produces delayed increase in conductance. 6. Depolarization-induced activation of cationic conductance (Gcat) and GNa results in enlargement of the K+ electrochemical potential difference, to about 70 mV; this difference allows recycling of K+ ions outwards, since a GK is still detected and may contribute up to 38% of the total conductance.

Because proton secretion and K+ reabsorption in the late distal tubule of amphibians are active, we evaluated whether these processes could be mediated by an H(+)-K(+)-ATPase similar to the gastric H(+)-K+ pump and to the K(+)-ATPase previously described in the terminal segments of the mammalian nephron. K(+)-stimulated ATPase activity was detected in microdissected segments of frog and Necturus nephron: its activity was high in the late distal and collecting tubules, whereas it was undetectable in the proximal convoluted tubule and early distal tubule. In frog collecting tubule, K(+)-ATPase had a high affinity for K+ (Km approximately 0.30 mM), was inhibited by vanadate, omeprazole, and the imidazopyridine Sch 28080, and was insensitive to ouabain. Furthermore, in vivo administration of Sch 28080 to anesthetized Necturus induced a significant rise of the steadystate intratubular pH in the late distal tubule, demonstrating that this drug inhibited tubular fluid acidification. It is suggested that K(+)-ATPase present in the terminal segments of amphibian nephron is similar to the gastric H(+)-K+ pump and is involved in urinary acidification.

To see whether the Na/H antiporter plays a role in digitalis cardiotoxicity, we investigated the influence of modulators of Na/H exchange on the toxic effects of ouabain in isolated, paced (0.4 Hz) rat left atria. Ouabain (1 mmol/l) caused a transient positive inotropic effect followed by toxic events, including a complete loss of developed force and a gradual increase in resting force. In the presence of hexamethyleneamiloride (3 and 10 mumol/l), an inhibitor of Na/H exchange, ouabain (1 mmol/l) caused a sustained positive inotropic effect without toxicity. By contrast, phenylephrine (100 mumol/l), an alpha-adrenoceptor agonist reported to stimulate the antiporter, hastened the development of ouabain's toxicity. Neither ouabain, at a subtoxic concentration (650 mumol/l), nor phenylephrine (100 mumol/l) affected diastolic force, but in their combined presence, a substantial contracture developed and twitch contractions disappeared. Phenylephrine (30 or 100 mumol/l) or adrenaline (30 mumol/l), in the presence of a beta-adrenoceptor antagonist, increased the intracellular pH by up to 0.15 pH unit, as measured using ion-selective microelectrodes in quiescent preparations. This effect on pHi was prevented by hexamethyleneamiloride (10 mumol/l). Consistent with phenylephrine's ability to stimulate Na+ influx via the Na/H antiporter, phenylephrine (100 mumol/l) increased intracellular Na+ activity by about 3 mmol/l in ouabain (650 mumol/l)-treated atria. These findings indicate that modulators of Na/H exchange affect the cardiotoxicity of digitalis glycosides and imply that the stimulation of myocardial alpha-adrenoceptors may aggravate digitalis toxicity.

This study was carried out to assess whether the amphibian distal tubule possesses a basolateral Na/(HCO3)n greater than 1 cotransport. The experiments were performed in the kidney of Necturus maculosus in vivo, by means of double-barreled selective microelectrodes. Basolateral membrane potential (Vm), intracellular pH (pHi), intracellular sodium activity (alpha Nai) and intracellular chloride activity (alpha Cli), were recorded during selected disturbances of peritubular fluid composition. The following results were obtained. (a) A sudden decrease of [HCO3]o leads to Vm depolarization, intracellular acidification and decrease of alpha Nai. (b) A rapid fall of [Na]o elicits Vm depolarization and decreases pHi: these patterns are not substantially altered in the presence of millimolar amiloride concentrations or in the nominal absence of peritubular Cl. (c) An acute decrease of [Na]o does not alter alpha Cli. (d) In the functional absence of CO2/HCO3 buffer (nominally CO2-free solution plus methazolamide), the reduction of [Na]o has no effect on Vm and/or pHi. We conclude that the distal tubule basolateral cell membrane is endowed with an electrogenic chloride-independent Na/base carrier, mediating Na and base efflux. The blockade of this carrier by carbonic anhydrase inhibitor indicates that the cotransported base is HCO3 or a related species.

A review of the literature on elastomeric impression materials, is presented in this paper. The article mentions the composition and the most important properties of the elastomeric impression materials used in dental practice. The clinical significance of these materials, physical and mechanical properties are also emphasized. In addition some new elastomeric impression materials with improved properties and a new (experimental) light-cured impression material, are mentioned. Another part of this article is the biocompatibility of these materials. In the end the great significance of handling is outlined.

The properties of the Na-K pump were studied by electrophysiological techniques in the proximal tubule of Necturus kidney. Peritubular capillaries were perfused for a short time with different artificial solutions, then ouabain (50 microM-1 mM) was added to each perfusate and its effects on cell membrane potential were assessed. Addition of ouabain, 1 mM, in a physiologic Ringer's solution (K concentration = 3 mM) failed to produce immediate depolarization. The introduction of the cardiac glycoside (50 microM) in solutions in which external K+ concentration was lowered to 0.75 or 0.3 mM resulted in depolarization by 4.1 +/- 0.8 and 9.5 +/- 1.4 mV, respectively. Furthermore, ouabain brought about an increase of membrane input resistance when added in a solution of lowered K+ concentration, but not in the presence of physiologic K+ concentrations. Readmission of K+ into a K-free perfusate flowing through the renal capillaries for more than 15 min resulted in hyperpolarization, 7.2 + 2.0 mV. These and other observations suggest that the Na-K pump operates in electrogenic fashion when external K+ concentration is lowered. Yet, part of the depolarizing response to ouabain under these conditions is mediated via ouabain-elicited changes of cell membrane permeabilities. The stoichiometry of the Na-K pump at physiologic external K concentrations cannot be assessed from the present experiments.

We studied the effects of millimolar Ba2+ concentrations on the properties of the basolateral membrane of the proximal tubule in Necturus kidney. Ba2+ was added in the peritubular perfusate by means of capillary microperfusion experiments. Basolateral membrane p.d. and conductance were continuously monitored in single tubules (their lumen was filled with oil) during reversible application of Ba2+ in peritubular fluid. The effect was membrane depolarization by 14mV and a decrease of membrane conductance to 63% of control values. This association strongly suggests that the main effect of Ba2+ is a decrease of the partial conductance to K+ at the basolateral membrane. However, in other experiments, performed also in oil-filled tubules, Ba2+ was shown to decrease intracellular K+ activity, recorded continuously by means of double-barreled (selective vs non selective) K+ microelectrodes. This and other, indirect evidence support the hypothesis that Ba2+ elicits also a small increase of the partial conductance to sodium at the basolateral membrane.

The effects of 0.5 mM 4-acetamido-4'-isothiocyano-stilbene-2,2' disulfonic acid on the electrical properties of the peritubular membrane were studied in the proximal tubule of the perfused Necturus kidney. The addition of stilbene isothiocyanate disulfonic acid in peritubular perfusate resulted in an average 4.5 mV hyperpolarization with no detectable changes of peritubular membrane input conductance. The depolarization elicited by high-K media was enhanced by 18% in the presence of stilbene isothiocyanate disulfonic acid, an observation indicating that the inhibitor increased the contribution of potassium to membrane potential, presumably by decreasing anionic permeabilities. The hyperpolarizing effect of stilbene isothiocyanate disulfonic acid was abolished when peritubular bicarbonate was removed from the medium and isoosmotically replaced by chloride. These data suggest that (a) intracellular bicarbonate activity is higher than that predicted from passive distribution, (b) stilbene isothiocyanate disulfonic acid decreases P HCO3, thus hyperpolarizing the membrane, (c) chloride distribution appears to be passive when bicarbonate is removed from the peritubular perfusate. The state of Cl distribution when extracellular bicarbonate is at physiologic concentration cannot be assessed from the present data.

Double-barreled, selective microelectrodes filled with liquid ion exchanger were used to determine proximal tubule cell pH (pHcell), luminal pH (pHlum), and peritubular capillary blood pH (pHbl.pt) in Necturus kidney in vivo. The average pHbl.pt of 16 animals was 7.64 +/- 0.3; pHcell was 7.36 +/- 0.02 (n = 50), and pHlum was 7.50 +/- 0.05 (n = 16). Because of the variability in pHbl.pt from one animal to another, we studied the blood/cell/lumen pH differences. We sequentially measured with a single microelectrode pHcell and pHlum, and then pHbl.pt in an adjacent peritubular capillary. In 25 such paired determinations, the average pHbl.pt - pHcell difference was 0.28 +/- 0.03, cell acid, and the pHbl.pt - pHlum difference was 0.14 +/- 0.02, lumen acid. The pHcell in this series was significantly more acid than the pHlum (by 0.14 +/- 0.02), but in a few instances the pH gradient across the apical cell membrane was inversed. All measurements were performed in the initial portion of the proximal tubule. We conclude that 1) proximal cell pH is acid with regard to peritubular blood pH, 2) the proximal tubule of Necturus kidney is capable of establishing a small transepithelial pH difference (lumen acid), and 3) pHcell is generally more acid then pHlum.

The effects of D-glucose addition to a glucose-free luminal perfusate were investigated in the proximal tubule of Necturus kidney, by electrophysiological techniques. The main findings are: (1) In the presence of sodium, D-glucose produces 10.5 mV +/- 1.1 (S.E.) depolarization. (2) Phlorizin reduces the magnitude of this response to 2.1 +/- 0.1 mV. (3) The glucose-evoked depolarization, delta VG, does not alter the intracellular K+ activity nor is it affected by peritubular addition of ouabain. (4) Isosmotic reduction of Na+ concentration in luminal perfusate from 95 to 2 mmol/l (choline or Li+ substituting for Na+) does not change the magnitude of delta VG; complete removal of sodium from the lumen lowers the value of delta VG (3.2 +/- 0.2 mV) but the response is not abolished. This observation suggests that the D-glucose carrier of renal tubules in Necturus is poorly specific with regard to the cotransported cation species.

Single proximal convoluted tubules (PCT) of Necturus kidney were impaled with three microelectrodes in the sequence M1, M2, M3. M1 was used for injecting short DC current pulses, M2 for recording peritubular membrane potential, V, and M3 for injecting longer DC current steps and thereby shifting V to a new baseline potential, V'. We define the p.d. changes at M2 due to M1-induced pulses as delta V and delta V' (for baselines V and V', respectively). Our objective was to test whether delta V' was equal to delta V. The main finding is that when V depolarized by 10 to 80 mV delta V'/delta V remained close to 1.00. Care was taken to ensure that this apparent stability of the pulse ratio was not due to opposite changes of apical and basolateral membrane conductances (g(A) and g(B) respectively), to changes of the sum g(A)+g(B) compensated for by changes of the cell-to-cell junctional conductance, g(j), or to a distortion of the delta V'/delta V ratio as a function of interelectrode distance, masking voltage-dependent changes of cell membrane conductances. Hyperpolarization of V produced gradual electrical uncoupling between cells as V' became increasingly negative, by a mechanism yet to be determined.

The significance of the "leaky" tight junction might be understood better if cells of the epithelial monolayer possessed mechanisms to regulate molecular flow through the junction. To test this possibility, Necturus gallbladder, a representative leaky epithelium, was studied before, during, and after mucosal exposure to plant cytokinins and two other microfilament-active drugs, cytochalasin B and phalloidin. Concomitant with morphological changes in microfilaments, cytokinins induced rapid reversible increases in transepithelial resistance and potential difference (PD) and decreases in NaCl dilution potentials, with no change in the ratio of relative cell membrane resistances. Cytochalasin B (0.2-1.2 microM) and phalloidin (0.6-12.7 microM) caused similar changes in transepithelial resistance and PD. When the intramembranous structure of tight junctions was studied by freeze fracture, peak cytokinin-induced increments in transepithelial resistance were associated with more disorder in the strand meshwork resulting in a small increase in tight junction depth, but there was no evidence of de novo strand assembly. These studies suggest that permeability of the tight junction of Necturus gallbladder is subject to rapid reversible modulation, possibly under cytoskeletal control.

Transepithelial potential difference (p.d.) was measured in the proximal tubule of Necturus kidney in vivo, by means of microelectrodes filled either with a 3M KClion or with a Ringer's solution for amphibians. The average transepithelial p.d., measured with KCl-tips, was: -1.4 +/- 2.4 mV (early convolutions), -0.1 +/- 2.0 mV (middle convolutions) and +0.1 +/- 2.4 mV (straight segment). The corresponding values obtained with Ringer's-filled microelectrodes were -2.3 +/- 1.8 mV, -1.3 +/- 1.1 mV and +0.1 +/- 1.2 mV, respectively. Tip localization into the lumen was ascertained by luminal injection of either oil (KCl electrode measurements) or artificial solutions which produced a measurable shift of transepithelial p.d. (determinations obtained with Ringer's-tips). Transepithelial p.d. in split-drops (mean reabsorptive half time 27.1 +/- 2.5 min) was -1.8 +/- 1.1 mV. The magnitude of transepithelial p.d. is discussed with respect to an equivalent electrical circuit; it is shown that high transepithelial p.d.'s are inconsistent with the known values of relative conductances of cell membranes in series and shunt pathway, respectively.

To assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl- activity (alpha iCl) was studied in the proximal tubule of Necturus kidney. Paired measurements of cell membrane PD (VBL) and Cl-selective electrode PD (VBLCl) were performed in single tubules, during reversible shifts of peritubular or luminal fluid composition. Steady-state alpha iCl was estimated at 14.6 +/- 0.6 mmol/liter, a figure substantially higher than that predicted for passive distribution. To determine the site of the uphill Cl- transport into the cell, an inhibitor of anion transport (SITS) was added to the perfusion fluid. Introduction of SITS in peritubular perfusate decreased alpha iCl, whereas addition of the drug in luminal fluid slightly increased alpha iCl; both results are consistent with basolateral membrane uphill Cl- transport from interstitium to the cell. TMA+ for Na+ substitutions in either luminal or peritubular perfusate had no effect on alpha iCl. Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO3- outflux), resulted in an increase of alpha iCl, presumably related to enhanced Cl- cell influx: we infer that Cl- is exchanged against HCO3- at the basolateral membrane. The following mechanism is suggested to account for the rise in luminal Cl- concentration above equilibrium values: intracellular CO2 hydration gives rise to cell HCO3- concentrations above equilibrium. The passive exit of HCO3- at the basolateral membrane energizes an uphill entry of Cl- into the cell. The resulting increase of alpha iCl, above equilibrium, generates downhill Cl- diffusion from cell to lumen. As a result, luminal Cl- concentration also increases.

Unlabelled: K+-selective liquid ion-exchanger double-barreled microelectrodes were used to measure intracellular K+ activity (alpha Ki) of proximal tubular cells in Necturus kidney. SEveral methodological problems inherent in the construction and in vivo application of such microelectrodes were considered, in particular the identification of leaky impalements and the correct assessment of alpha Ki during reversible perfusion of peritubular capillaries from control to a test solution. Peritubular potassium concentration (CpK) could be altered by means of double-barreled micropipettes containing a physiologic and a high or low K solution. The control alpha Ki value averaged 57.9 +/- 8.5 mM (mean +/- SD, n = 38) and the basolateral membrane potential was -70.4 +/- 5.8 mV. Thus, the K+ equilibrium potential across the basolateral membrane (EK) was -81.1 +/- 3.4 mV. Increasing CpK from 3.0 to 30.0 mM raised alpha Ki by 7.0 +/- 1.5 mM, and lowering CpK to 0.3 mM decreased alpha Ki by 9.2 +/- 2.9 mM., Conclusions: 1) the steady-state K+ distribution across the basolateral membrane indicates that potassium is actively pumped into the cell. 2) The relative stability of alpha Ki in the face of large CpK variations underscores the effectiveness of cell homeostasis; passive mechanisms appear to contribute to this end.

This study was undertaken to determine the passive electrophysiological properties of the diffusive barriers of the late distal tubule (LDT) in Necturus. The transepithelial resistance (RT) determined by cable analysis was 1,130 omega.cm2, which puts the LDT in the class of "tight" epithelia. Using two different methods, we did not find significant cell-to-cell electrical coupling. The fractional apical resistance was 0.93, and it did not vary with distance from the current-injecting electrode. Relative permeabilities of K+, Na+, and Cl- during peritubular ion concentration changes were assessed by circuit analysis. The conclusions are as follows. The basolateral cell membrane is highly permeable to K+; its apparent K+ transference number is 0.78. Basolateral chloride transference was very small. Sodium removal from peritubular fluid produced depolarization, suggesting carrier-mediated electrogenic Na+ transport. The high fractional resistance of the apical cell membrane prevented assessment of apical transference numbers. However, Cl- removal from luminal fluid produced cell hyperpolarization; the underlying mechanism has not been established with certainty. The paracellular pathway does not discriminate between Na+, Cl-, and some of their substitutes; it is poorly permeable to gluconate and prefers K+ to Na+.

1. Double-barrelled (selective vs. conventional) microelectrodes were used to assess the steady-state activities (a) of the ions Cl-, K+, Na+ and H+ in peritubular blood capillaries (abld) and in cell (acell) and lumen (alum) of the late distal tubule (l.d.t.) of Necturus. 2. a(cell)cl, a(lum)cl and a(bld)cl were 5.5 +/- 0.3, 11.8 +/- 1.0 and 70.5 +/- 0.1 mM, respectively. They were used to compute the chemical potentials for Cl- across the three diffusive barriers of the tissue. Basolateral and apical membrane potentials were -74.3 +/- 1.1 and -60.1 +/- 2.0 mV, respectively (cell negative); the lumen was thus negative with respect to blood, by 13.6 +/- 1.5 mV. The electrochemical potential difference (e.p.d.) for Cl- of 42 mV across the apical membrane opposes Cl- absorption, implying active apical Cl- uptake, since Cl- is known to be absorbed in the l.d.t. Basolateral Cl- exit is favoured by an e.p.d. of 10 mV. 3. a(cell)K, a(lum)K and a(bld)K were 65.8 +/- 0.8, 2.5 +/- 0.1 and 2.5 +/- 0.1 mm, respectively. The electrochemical distribution of K+ indicates that K+ absorption, if present, proceeds against an adverse apical e.p.d. of 18 mV. Basolateral K+ distribution is close to its electrochemical equilibrium, suggesting high K+ permeability at this membrane. 4. a(cell)Na was 9.0 +/- 0.4 mM, a(bld)Na 71.0 +/- 0.3 mM, and a(lum)Na was approximated at about 9 mM. Diffusive Na+ entry from lumen to cell is favoured by an e.p.d. close to 65 mV. Basolateral Na+ exit must be active, since it proceeds against an e.p.d. of 130 mV. 5. Cell, luminal and blood pH were 7.14 +/- 0.03, 6.52 +/- 0.08 and 7.37 +/- 0.04, respectively. The luminal electrochemical potential of H+ is higher than that of cell (by 91 mV) and blood (by 34 mV) indicating that proton secretion into the lumen must be active. 6. The e.p.d. of each ion across the epithelium opposes, by its orientation, the established direction of net transepithelial ion transport, suggesting that the shunt pathway may serve only for back-diffusion.

The electrical properties of the proximal tubule of the in vivo Necturus kidney were investigated by injecting current (as rectangular waves) into the lumen or into the epithelium of single tubules and by studying the resulting changes of transepithelial (VL) and/or cell membrane potential (VC) at various distances from the source. In some experiments paired measurements of VL and VC were performed at two abscissas x and x'. The luminal length constant of about 1,030 micrometer was shown to provide a good estimate of the transepithelial resistance, specific resistance (RTE = 420 omega.cm2) and/or per unit length (rTE = 1.3 x 10(4) omega.cm). The apparent intraepithelial length constant was subject to distortions arising from concomitant current spread in the lumen. The resistances of luminal membrane (rL), basolateral membrane (rB), and shunt pathway (rS) were estimated by two independent methods at 3.5 x 10(4), 1.2 x 10(4), and 1.7 x 10(4) omega.cm, respectively. The corresponding specific resistances were close to 1,200, 600, and 600 omega.cm2. There are two main conclusions of this study. (a) The resistances of cell membranes and shunt pathway are of the same order of magnitude. The figure of the shunt resistance is at variance with the notion that the proximal tubule of Necturus is a leaky epithelium. (b) A rigorous assessment of the conductive properties of concentric cylindrical double cables (such as renal tubules) requires that electrical interactions arising from one cable to another be taken into account. Appropriate equations were developed to deal with this problem.

The effects of peritubular salicylate for chloride substitution were studied in the isolated perfused Necturus kidney. This substitution resulted in changes of cell membrane p.d., varying from tubule to tubule; the withdrawal of the test-anion invariably produced a steep and prolonged depolarization. Exposure of the tissue to salicylate brought about, in addition, electrical uncoupling of junctional membranes, which was not related to concomitant changes of membrane p.d.

The in vivo effects of the diuretic muzolimine were studied in the late distal tubule of the amphibian Necturus. Conventional and ion-selective microelectrodes were used to determine basolateral membrane potential, intracellular Cl- activity and luminal activities of Cl- and K+. Muzolimine depolarized basolateral membrane potential by about 30 mV in 1 min, in a reversible fashion. We attributed this depolarization to blockade of a K+ conductance, because the effects of muzolimine and barium on the highly K-selective basolateral membrane were not additive. In addition, muzolimine elicited a reversible increase of intracellular Cl- activity from 7.5 +/- 0.5 to 14.5 +/- 2.6 mM (concomitant to the basolateral membrane potential depolarization) and of luminal activities of Cl- from 12.4 +/- 1.5 to 22.3 +/- 2.5 mM, within approximately 1 min; both disturbances relaxed toward control values after withdrawal of the diuretic. That muzolimine increases Cl- activity in both the cell and the lumen of the late distal tubule (Cl- is accumulated in these compartments), indicates that retention of Cl- results from hindrance of the basolateral exit step rather than of apical Cl- uptake. Inasmuch as muzolimine failed to increase the luminal activity of K+, Cl- is believed to accumulate in the lumen as NaCl, not KCl.

Published values of transepithelial potential differences (VTE) in the proximal tubule of Necturus vary from approximately 0 to approximately -15 mV. In view of this disparity, we reinvestigated VTE. Our measured VTE was on the average -1.0 mV in early convolutions and +0.6 mV in terminal proximal segments. In the course of this study, we considered five distinct causes of artifacts. 1) Tip and pre-tip potentials: their occurrence was minimized by using Ringer's filled microelectrodes. 2) Interstitial tip localizations: the position of the tip was ascertained by the shift in potential, resulting in response to peritubular perfusion with gluconate solutions. 3) Leaky impalements: VTE responses to gluconate, input resistance determinations and the presence of positive VTE's rule out the leak hypothesis. 4) Zeroline shifts between pre- and postimpalement stages, and 5) Spontaneous VTE drifts in the positive direction, due to gradual passage of the tip from cell to lumen, or in the negative direction, resulting from tip contact with the lower cellular layer. All five causes of artifacts may be involved in the controversy regarding past VTE estimates.

1. A study has been made of the effects of anionic substitutions on the electrical potential difference (p.d.) and conductance characteristics of the antiluminal (peritubular) membrane of the proximal tubule of Necturus kidney. The tubular lumina were filled with oil in order to minimize potential and conductance contributions from luminal membrane and from paracellular shunt pathway.2. Isosmotic substitutions, [A](o) for [Cl](o), produced the following average changes in membrane p.d. (mV): F(-) +1.7, BrO(3) (-) +0.1, Br(-) -4.5, ClO(3) (-) -5.2, I(-) -7.9, NO(3) (-) -12.1, ClO(4) (-) -17.8, SCN(-) -25.3.3. The amplitude of the depolarization caused by increase in K concentration (K-depolarization) in the peritubular perfusate was found to increase during perfusion of the tissue with ClO(4) (-) (by 78%), SCN(-) (45%), I(-) (23%), NO(3) (-) (20%), Br(-) (16%); it decreased with F(-) (by 17%).4. Comparison of membrane p.d. at peak K-depolarization in the control state (during KCl perfusion) with that obtained in the experimental state (during KA perfusion) was found to be more reliable than determination of bi-ionic potentials as a qualitative estimate of the permeabilities of the various anions (P(A)) relative to that of chloride (P(Cl)).5. Study of both peak K-depolarization p.d. and bi-ionic potentials yielded the following sequence for halide anion permeabilities: P(F) > P(Cl) > P(Br) > P(I). The peritubular membrane was found to be substantially more permeable to NO(3) (-), ClO(4) (-) and SCN(-) than to Cl(-).6. The sequence of membrane conductances during anionic substitutions was Cl(-) approximately BrO(3) (-) < Br(-)

The development of liquid-ion-exchanger microelectrodes displaying tip diameters less than or equal to 1 micron has permitted direct measurement of the transmembrane chemical and electrochemical gradients of several permeant ion species in cells of small size. We have used Cl- and H+ resins to study the intracellular Cl- activity (alpha iCl) and cell pH (pHi) in the proximal tubule of Necturus kidney. These determinations were performed in association with perfusion of peritubular capillaries by several artificial solutions, in order to assess the dependence of alpha iCl and pHi on the composition of physiologic plasma constituents and selected inhibitors. The main findings are: Intracellular chloride activity, alpha iCl, is higher than the theoretical value predicted from electrochemical equilibrium. Peritubular application of SITS resulted in a decrease of alpha iCl and increase of pHi; these observations are taken to indicate that Cl- uptake is achieved across the basolateral membrane in exchange for HCO-3 by a mechanism sensitive to SITS. Na+ removal from peritubular fluid elicited a small reduction of alpha iCl, suggesting the presence of carrier-mediated Cl--Na+ cotransport from interstitium to cell, contributing to the rise of alpha iCl above equilibrium. In conclusion, two carrier-mediated processes (Cl-/HCO-3 exchange and Cl--Na+ symport) located at the basolateral membrane of the proximal tubule may account for the establishment of alpha iCl values above equilibrium, at steady state. The physiologic role of these carriers is discussed in relation to proximal electrolyte absorption.

In the perfused Necturus kidney, the ratio of transepithelial sodium-to-chloride permeabilities (PNa/PCl) was estimated from dilution potentials at 1:7. The ratio of transepithelial permeabilities PHCO3/cl was estimated from bi-ionic potentials at 1:4 and that of Plact/PCl at 1:12. The permeability sequence, therefore, is PCl greater than PHCO3 greater than than PNa or PnaCl greater than PNaHCO3. The latter is similar to the situation in the rat. However, because of the low Na permeability, passive NaCl absorption by solvent drag is probably less important in Necturus than in the rat. The measurement of transepithelial input conductance during replacement of extracellular chloride by either bicarbonate or lactate shows that the former reduces the transmural conductance to 31% of control values as compared with 40% for the latter. Such discrepancies between permeability and conductance sequences are consistent either with an interference of the test anion with chloride permeation or with the presence of positive fixed changes within the shunt pathway.

Cell membrane potential, Vm, was monitored in rabbit hypertrophic cartilage metatarsals, amphibian proximal tubule and muscle cells during application of 1,25-dihydroxy vitamin D-3, 25-hydroxy vitamin D-3 or cholesterol (10(-10) M). 1,25-Dihydroxy vitamin D-3 elicited quick variations of Vm (in less than 1 min) in proximal tubular cells (whether injected in the lumen or in peritubular capillaries) and in cartilage. The precursor 25-hydroxy vitamin D-3 and cholesterol produced a small shift of Vm in proximal tubule only when applied from the luminal side, but this change was significantly smaller than that observed with 1,25-dihydroxy vitamin D-3. Muscle cells were unresponsive to both metabolites and cholesterol. It is concluded that rapid effects of 1,25-dihydroxy vitamin D-3 on Vm, in target cells, are specific, most likely due to permeability changes and not related to nuclear protein synthesis; they may contribute to early modulation of cell function.

The transepithelial potential difference (VTE) and transepithelial resistance (RTE) were determined along the length of the distal tubule of the amphibian Triturus alpestris. The site of impalements was determined at the end of each experiment by latex injection and microdissection. Two segments, differing by their distribution at the surface of the kidney, by their respective diameters and by their electrical properties could be identified: the early distal tubule (EDT) and the late distal tubule (LDT). VTE was invariably positive in the EDT; it increased from approximately + 5 mV to approximately + 22 mV within the first 30% of this segment and remained roughly constant distally to this site. RTE was estimated at 57 omega X cm2 in the EDT. The LDT exhibited essentially negative VTE figures with the exception of its very initial portion; the peak negativity at the end of the LDT was -35 mV. RTE was assessed from 8 measurements performed in the first 30% of the LDT; the tentative transepithelial resistance was 530 omega X cm2. Provisional evidence suggests that the transepithelial resistance of the terminal portion of the LDT may be substantially larger than the resistance measured in other parts of this segment.

Glaphenine, a nonsteroid analgesic compound, administered by gastric gavage in rats (800 mg/kg), induced nonoliguric reversible acute renal failure (ARF). Intratubular deposits were found in medullary collecting ducts. Intratubular hydrostatic pressure (Pt) increased from 11.7 +/- 0.7 to 30.0 +/- 0.9 mmHg. Renal failure was almost completely prevented by concomitant high water and solute diuresis, achieved by furosemide infusion in Wistar rats and by high salt intake in Brattleboro rats with diabetes insipidus. In the latter protected animals, Pt was only slightly elevated (17.0 +/- 0.5 mmHg). Urinary excretion of prostaglandin E2 (PGE2) dropped dramatically after glaphenine administration in Wistar rats; the fall was slight in Brattleboro rats in which PGE2 excretion was normally low. We conclude that tubular obstruction plays a prominent role in glaphenine-induced ARF in the rat. High water and solute diuresis prevented tubular obstruction. Reduced renal PGE2 synthesis is probably not involved in the pathophysiology of this ARF model, inasmuch as Brattleboro rats on a high salt intake were protected despite low basal urinary excretion of PGE2.

Double-barreled microelectrodes selective to either potassium or sodium were used to determine the transepithelial potential difference (VTE) and the intraluminal activity of potassium (alpha LuK) or sodium (alpha LuNa) in the early distal tubule (EDT) of Triturus waltlii in vivo; luminal activities were compared with the corresponding plasma ion activities, alpha PtK and alpha PtNa. The transepithelial equilibrium potentials for potassium (EK(TE] and sodium (ENa(TE] were computed from the respective transmural chemical distributions: they were used to assess the transepithelial electrochemical potential differences [(V-EK)TE and (V-ENa)TE]. By dividing the raw data into three groups of 30% total tubular length (0-30, 31-60, 61-90%), the following results were obtained. 1) VTE increases from +15 to +20 mV (lumen positive) between the first and second portion of the EDT but remains constant thereafter. 2) The alpha LuK/alpha PtK ratio decreases steadily along the EDT from 1.92 to 1.66 and then to 1.32. 3) The values of alpha LuNa/alpha PtNa in the same three subdivisions are 0.79, 0.44, and 0.45. 4) The (V-EK)TE difference is largely positive along the whole EDT: +32, +33, and +27 mV. 5) The (V-ENa)TE difference declines from +9 mV (first portion) to values statistically not different from zero in the last two thirds of the EDT.(ABSTRACT TRUNCATED AT 250 WORDS)

Eleven different secosteroids or steroids (10(-10) to 10(-8) M) were acutely and reversibly introduced in solutions delivered to the lumen of single proximal tubules of the amphibian Necturus kidney while recording basolateral cell membrane potential Vm. Seven of these molecules (1,25(OH)2D3, 25(OH)D3, 24,25(OH)2D3, 5,6-trans-25(OH)D3, 19-diol-cholesterol, estradiol and testosterone) resulted in changes of Vm (delta Vm) occurring in a few seconds, the largest delta Vm being observed with 1,25(OH)2D3, +6.5 +/- 0.75 mV (n = 19); these seven (seco)steroids, but not the four inactive sterols (vitamin D3, cholesterol, 1 alpha D3 and aldosterone) possess a hydroxyl group on at least one carbon of the C17 to C25 lateral chain of the sterol ring. The delta Vm effect was present in Na+-free or Cl-free media, but it was abolished in HCO3-free media. Depolarization of cell membrane potential by addition of glucose, 11 mM, in luminal perfusion fluid abolished the 1,25(OH)2D3-evoked delta Vm effect, suggesting dependence of the latter on the absolute value of membrane potential. Barium, a blocking agent of K+ conductances, suppressed the 1,25(OH)2D3-evoked delta Vm effect, even when the proper effects of barium of cell membrane potential were canceled by current clamp. Pretreatment with quinine, a putative blocker of Ca2+-dependent K+ channels also abolished the 1,25(OH)2D3-evoked depolarization. Such observations are consistent with the presence of Ca2+-dependent K+ channels at the apical cell membrane of the proximal tubule, these channels being inactivated by 1,25(OH)2D3 and probably by other (seco)steroids.

The effect of foreign anions on transepithelial potential difference and transepithelial input conductance was studied in the isolated perfused Necturus kidney. Two microelectrodes (recording and current-injecting) were inserted into the lumen of single proximal tubules and the peritubular perfusate was shifted reversibly for 30-60 sec from a physiologic Ringer's solution to a test solution in which chloride was replaced isosmotically by a foreign anion. The permeability sequence, obtained by potential measurements, was: lactate less than glutamate less than gluconate less than pyruvate less than benzene sulfonate less than or equal to acetate less than or equal to F less than propionate less than BrO3 less than formate less than ClO3 less than Cl than ClO4 less than I less than or equal to Br less than NO3 less than SCN. Transepithelial conductance decreased when the tissue was perfused with anions less permeable than chloride but the conductance sequence was different from the permeability sequence. Such discrepancies were more pronounced during perfusion with hyperpolarizing anions; ClO4 and I- (both more permeable than chloride) produced an important decrease in transepithelial conductance, followed by incomplete reversibility when the perfusion was shifted again to chloride Ringer's. The results are best explained by the presence of weak positive fixed charges, governing anion permeation, at the shunt pathway of the proximal tubule. An analysis of the data allows tentative estimates of shape and size of the sites.

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.