An intact globe technique for electrophysiological studies on cornea.

An intact globe method was developed to determine the characteristics of the cornea of the bullfrog, Rana catesbeiana. With this method the anterior chamber could be perfused and the transcorneal potential difference (PD) and electrical resistance determined. It was found for the endothelium plus stroma (epithelium scraped) that the PD was essentially zero and the electrical resistance was only a small fraction of that of the intact cornea. Elevation of K+ or decrease in Cl- concentration in the anterior chamber produced in intact corneas a large and rapid change in PD while with the epithelium scraped (stroma and endothelium intact) these elevations produced a negligible change in PD. It is concluded that ions can rapidly move across the endothelium and stroma of the cornea.     

Histochemical study of alkali-burned rabbit anterior eye segment in which severe lesions were prevented by aprotinin treatment

Summary Activities of different enzymes (acid glycosidases, phosphatases, Na⁺–K⁺-dependent ATPase, proteases, dehydrogenases) and acid glycosaminoglycans were studied by histochemical methods in sections of rabbit anterior eye segments after experimental alkali burn and treatment with aprotinin, an inhibitor of plasmin and other serine proteinases. Solutions of sodium hydroxide (0.25–1.0M) were applied on corneas using 12-mm-diameter plastic tube for 15–60 s. After wiping with cotton and rinsing with tap water, aprotinin solutions were applied in saline (in experimental animals) and saline (in control animals) dropwise in 12-h intervals for a month. Within the first two weeks aprotinin was used at a concentration of 5000 IU/ml. During the subsequent two weeks the aprotinin concentration was reduced to 2500 IU/ml.Striking differences in enzyme activities and in the healing between treated and untreated eyes were found. Without aprotinin, ulcers developed in most corneas within 3 weeks and plasmin was regularly demonstrated in tears and in the aqueous. When aprotinin treatment was started within 24 h after the burn, the number of enzymatically active inflammatory cells was significantly lower, not only in the cornea itself but also in the whole anterior eye segment. With aprotinin treatment no ulcerations and no plasmin in tears and the aqueous were observed and the corneas healed within a month. The healing process started from the zone of enzymatically activated corneal cells in the unburned zone at the corneal periphery. In the regenerating epithelium and endothelium high activities of Na⁺–K⁺-dependent ATPase, -glutamyltransferase, lactate and succinate dehydrogenases appeared very soon. Keratocytes displayed high activities of all enzymes studied. The restoration of corneal transparency depended on concentration of alkali used and parallelled the regeneration of the stroma and normalization of corneal hydration. Our results demonstrate that aprotinin is a potent therapeutic agent in the treatment of experimentally induced corneal ulcers, presumably due to its inhibitory action on plasmin and other serine proteases present in the alkali-burned anterior eye segment.     

Changes in membrane potential, membrane resistance, and intracellular H, K, Na, and Cl activities during the progesterone-induced maturation of urodele amphibian oocytes

Changes in intracellular activities of H⁺, K⁺, Na⁺, and Cl⁻ ions were recorded with ion-selective microelectrodes during progesterone-induced maturation of full-grown oocytes of the urodele amphibians Ambystoma mexicanum and Pleurodeles waltlii. The membrane potential (E_m) and electrical resistance (R_m) were also determined. During the first hours after initiation of maturation, the oocytes slowly depolarized and R_m gradually increased. By the end of maturation of Pleurodeles oocytes E_m had stabilized at about −10 mV and R_m had increased from 410 to 1760 kΩ. The same initial pattern was observed for Ambystoma, but in most oocytes a rapid transition occurred at about the time of germinal vesicle breakdown (GVBD): E_m spontaneously shifted from about −15 to about +30 mV; simultaneously R_m dropped from 1230 down to 100 kΩ (i.e., less than the initial 270 kΩ resistance). The internal K⁺ activity did not show any important variation during maturation of Ambystoma and Pleurodeles oocytes. Na⁺ activity increased slightly at the onset of GVBD in Ambystoma; a further marked increase of Na⁺, accompanied by an increase in Cl⁻ activity, was observed as soon as E_m shifted to a positive value. In Pleurodeles sodium activity was also more elevated in matured than in immature oocytes. The average pH of Ambystoma immature oocytes was 7.48 ± 0.05 (external pH 7.5). A transient alkalinization to 7.64 ± 0.04 took place during the first 4–6 hr postprogesterone. Cytoplasmic pH was restored to 7.50 ± 0.07 between 10 and 12 hr postprogesterone, before the onset of GVBD and the shift of E_m. The difference between the measured oocyte pH and the calculated equilibrium pH decreases during the course of maturation, due partly to the depolarization of E_m.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

Disturbances in the rabbit cornea after short-term and long-term wear of hydrogel contact lenses

Summary The influence of soft contact lenses (SCL) with low (37%, L) and high (65%, H) water content on rabbit corneas was investigated. The lenses were worn continuously for 1, 2, 4, 7, 10, 14, 21 or 28 days. The changes in corneal transparency, hydration and enzyme activities were studied. A slight change in corneal transparency due to higher hydration caused by a decreased activity of Na⁺–K⁺-dependent adenosine triphosphatase (Na⁺–K⁺-ATPase) in the corneal endothelium is followed by a decrease in the activity of -glutamyl transferase (GGT). Slight morphological disturbances appear within 4 days in animals wearing SCL (L). SCL (H) produce similar changes one week later. Subsequently, the corneal epithelium becomes thinner and changes in the size of corneal endothelial cells are obvious. Disturbances of enzyme activities in cells of all corneal layers are present. In the epithelium highly increased activities of acid glycosidases, acid phosphatase, and dipeptidyl peptidase I and II, in keratocytes decreased activities of alkaline phosphatase and GGT, and in the endothelium decreased activity of Na⁺–K⁺-ATPase and GGT were found. These changes are more severe after SCL (L). In this case, inflammatory cells displaying high activities of lysosomal hydrolases appear in the anterior part of the stroma during the 3rd and 4th weeks and local degradation of glycosaminoglycans and proteins takes place. In contrast, after SCL (H) a remarkable thinning of the corneas was observed during extended wear, accompanied by decreased stainability of stromal glycosaminoglycans and highly decreased enzyme activities in keratocytes. The histochemical methods proved very useful in the assessment of tesions caused by a continuous wear of SCL.     

Effects of external cations and respiratory inhibitors on electrical potential of the xylem exudate of excised corn roots

Glass capillary microelectrodes were used to study the electrical potential difference (PD) between the xylem exudate of excised corn roots, Zea mays L. Golden Bantam hybrid, and the external solution. A survey of the effects of various ions on the PD was made. With 1 mm single salt solutions, the PD was between 25 and 50 mv, exudate negative. The PD responded to concentration differences in single salt solutions of K⁺, Na⁺, and Ca²⁺ in a manner suggestive of cation selectivity and cation diffusion potentials. With Ca²⁺ present, the PD was insensitive to concentration changes of other cations. Substitution of NO₃⁻ for Cl⁻ in K⁺ solutions increased the PD by 2 to 5 mv, although in general the PD showed little response to anion concentration changes. The PD was partially abolished by cyanide. The remaining fraction of the PD was sensitive to concentration changes in external K⁺, and we postulate that the PD is the result of both a diffusion potential and an electrogenic pump.     

Modulation of red cell K transport by membrane lipids

Alterations in the state of the membrane lipids affect human red cell K⁺ transport. Depletion of membrane cholesterol by 29–34% significantly inhibited both total K⁺ influx and ouabain-sensitive K⁺ influx. Addition of the hydrophobic anesthetic, chlorpromazine, in concentration from 2 · 10⁻⁵ to 2 · 10⁻⁴ M increased both total K⁺ influx and ouabain-sensitive K⁺ influx. In each case the effect on both processes was almost identical which indicates a linkage between K⁺ “pump” and “leak”. Further, these results demonstrate that red cell K⁺ transport can be modulated by local conditions in the micro-environment of the transport system.     

Seasonal differences in activity of perch (Perca flavescens) gill Na/K ATPase

We measured Na⁺/K⁺ ATPase activity in homogenates of gill tissue prepared from field caught, winter and summer acclimatized yellow perch, Perca flavescens. Water temperatures were 2–4°C in winter and 19–22°C in summer. Na⁺/K⁺ ATPase activity was measured at 8, 17, 25, and 37°C. V_max values for winter fish increased from 0.48±0.07 μmol P mg⁻¹ protein h⁻¹ at 8°C to 7.21±0.79 μmol P mg⁻¹ protein h⁻¹ at 37°C. In summer fish it ranged from 0.46±0.08 (8°C) to 3.86±0.50 (37°C) μmol P mg⁻¹ protein h⁻¹. The K_m for ATP and for Na⁺ at 8°C was ≈1.6 and 10 mM, respectively and did not vary significantly with assay temperature in homogenates from summer fish. The activation energy for Na⁺/K⁺ ATPase from summer fish was 10 309 (μmol P mg⁻¹ h⁻¹) K⁻¹. In winter fish, the K_m for ATP and Na⁺ increased from 0.59±0.08 mM and 9.56±1.18 mM at 8°C to 1.49±0.11 and 17.88±2.64 mM at 17°C. The K_m values for ATP and Na did not vary from 17 to 37°C. A single activation energy could not be calculated for Na/K ATPase from winter fish. The observed differences in enzyme activities and affinities could be due to seasonal changes in membrane lipids, differences in the amount of enzyme, or changes in isozyme expression.     

Local osmotic coupling to the active trans-endothelial bicarbonate flux in the rabbit cornea

The present experiments investigate HCO₃⁻, Cl⁻ and fluid fluxes across partially destromalised corneas. Although there is no net flux of Cl⁻, there is a net flux of HCO₃⁻ across the endothelium from stromal side to aqueous side which is accompanied by a flux of water in the same direction. Bulk phase osmosis cannot account for the initiation of the flux of fluid. Local osmotic coupling between ions and water is postulated to occur in the preparation. The exudate is hypertonic to the bathing Ringer solution.     

Effects of tryptamine on active sodium and chloride transport in the isolated bullfrog cornea

The effects of the serotonin analogue, tryptamine, on the active transepithelial transport of Na⁺ and Cl⁻ in the in vitro bullfrog cornea were studied. Tryptamine, 1 mM, inhibited both the short-circuit current (I_sc) and potential difference (PD) of corneas transporting either Na⁺ alone or both Na⁺ and Cl⁻. The electrical resistance, R, increased in all cases. Both unidirectional Na⁺ and Cl⁻ fluxes were decreased by tryptamine and these changes accounted for the inhibitory effects on the I_sc. The effects of tryptamine were considered along with with those of 2 mM theophylline and 0.1 mM ouabain. Tryptamine inhibited the I_sc and both undirectional Cl⁻ fluxes which were previously stimulated by theophylline. Theophyline addition, after tryptamine preincubation, increased the Cl⁻ undirectional fluxes but did not restore the inhibited I_sc. The inhibitory effects of tryptamine on active Na⁺ and Cl⁻ transport were different from those of ouabain. While both drugs inhibited the forward Na⁺ and Cl⁻ fluxes, their backfluxes decreased with tryptamine and increased with ouabain. The addition to the bathing solution of tryptamine after ouabain preincubation reduced the ouabain-increased backward Cl⁻ flux and further increased the electrical resistance. These results are analyzed in terms of an electrical model from which it appears that tryptamine's mechanism of action was to decrease cellular permeability to the transepithelial movement of Na⁺ and Cl⁻.     

Effect of taurine on the isolated retinal pigment epithelium of the frog: Electrophysiologic evidence for stimulation of an apical,electrogenic Na+−K+ pump

Summary The apical surface of the retinal pigment epithelium (RPE) faces the neural retina whereas its basal surface faces the choroid. Taurine, which is necessary for normal vision, is released from the retina following light exposure and is actively transported from retina to choroid by the RPE. In these experiments, we have studied the effects of taurine on the electrical properties of the isolated RPE of the bullfrog, with a particular focus on the effects of taurine on the apical Na⁺–K⁺ pump.Acute exposure of the apical, but not basal, membrane of the RPE to taurine decreased the normally apical positive transepithelial potential (TEP). This TEP decrease was generated by a depolarization of the RPE apical membrane and did not occur when the apical bath contained sodium-free medium. With continued taurine exposure, the initial TEP decrease was sometimes followed by a recovery of the TEP toward baseline. This recovery was abolished by strophanthidin or ouabain, indicating involvement of the apical Na⁺–K⁺ pump.To further explore the effects of taurine on the Na⁺–K⁺ pump, barium was used to block apical K⁺ conductance and unmask a stimulation of the pump that is produced by increasing apical [K⁺] ₀ . Under these conditions, increasing [K⁺] ₀ hyperpolarized the apical membrane and increased TEP. Taurine reversibly doubled these responses, but did not change total epithelial resistance or the ratio of apical-to-basal membrane resistance, and ouabain abolished these responses.Collectively, these findings indicate the presence of an electrogenic Na⁺/taurine cotransport mechanism in the apical membrane of the bullfrog RPE. They also provide direct evidence that taurine produces a sodium-dependent increase in electrogenic pumping by the apical Na⁺–K⁺ pump.     

Transient outwardly rectifying potassium channel in the rabbit corneal endothelium

Summary Ionic currents from freshly dissociated rabbit corneal endothelial cells were examined using patch-clamp technology and a perforated patch technique. Whole-cell current recordings revealed a transient outward K⁺-selective current that was blockable in a dose-dependent manner by 4-aminopyridine (4-AP) and quinidine. This current is similar to the A-type current present in many excitable cells and is the first reported instance of such a current in any epithelial cell type. In addition to the transient current, an outwardly rectifying nonselective cation current was also observed. This current is also blocked by quinidine.To examine the possible role of these currents in the stromal volume regulatory function of the endothelium, corneas were perfused under a specular microscope with a glutathionebicarbonate Ringer's solution (GBR) or GBR plus either 1 mM quinidine or 10 mM 4-AP. For quinidine perfusions, control corneas swelled at a rate of 6 m/hr, while quinidine-perfused corneas swelled at a rate of 48 m/hr. For 4-AP perfusions, control corneas deswelled at a rate of –2 m/hr, while 4-AP perfused corneas swelled at a rate of 24 m/hr. One possible mechanism of the stromal swelling induced by these K⁺ channel blockers may be the result of loss of the K⁺ recycling pathway necessary for proper Na⁺/K⁺ ATPase function.We would like to thank Dr. William Bourne for the use of his specular microscopy corneal perfusion apparatus and Helen Hendrickson for her technical assistance. This work was supported by NIH grants EY06206, EY03282, EY06005, and an unrestricted award from Research to Prevent Blindness.     

The transepithelial potential difference in the gills of the fiddler crab,Uca tangeri: Influence of some inhibitors

Summary Euryhaline Crustacea living in dilute media, counterbalance the salt loss by active absorption of NaCl across the gill epithelium. To investigate the mechanisms involved in salt absorption, transeptithelial potential difference (PD_te) was measured in isolated, perfused gills of the fiddler crab,Uca tangeri. The influence of some specific inhibitors of epithelial ion transport on the PD_te was tested.With symmetrical conditions on both sides of the epithelium, the posterior gills ofUca tangeri showed a spontaneous PD_te of +5 to +10 mV, that is an active transport potential which was positive on the bath side as referred to the hemolymph side. This potential decreased considerably after application of KCN or 2,4-dinitrophenol (DNP) to the perfusion saline.Omission of K⁺ from the perfusion saline or addition of ouabain led to a reversible drop of the PD_te, suggesting that the absorption of Na⁺ and also of Cl^– is driven by the (Na⁺+K⁺)ATPase located in the basolateral membrane of the epithelial cells.Perfusion of the hemolymph space with saline containing diphenylamine-2-carboxylate (DPC) or the loop diuretic furosemide resulted in a decrease of the PD_te.After application of amiloride to the bath saline the PD_te increased. Half-maximum response to amiloride was reached at a concentration of about 10^–5 mol·l^–1. This suggests that one of the Na⁺ pathways across the apical membrane may consist of Na⁺ channels.Abbreviations PD _te transepithelial potential difference - DPC diphenylamine-2-carboxylate - R _ps resistance of perfusate shunt - R _te transepithelial resistance - R _in input resistance - DNP 2,4-dinitrophenol Parts of this study have been reported at the 1st Congress of Comparative Physiology and Biochemistry, Liège 1984, and at the Vth European Colloquium on Renal Physiology, Frankfurt, 1985     

Effects of ouabain on chloride movements across the seawater eel intestine

Summary Simultaneous measurements of transepithelial potential difference (PD) and net water flux were made in the stripped intestine of seawater eels, and the effects of ouabain on these two parameters were examined in normal Ringer solution or under a chloride concentration gradient. Ouabain reduced the serosa-negative PD and the net water flux in normal Ringer solution with a linear relationship between the PD and the net water flux. Removal of K⁺ from the Ringer solution on both serosal and mucosal sides also reduced the PD and the net water flux to approximately zero. On the other hand, blocking the Na⁺–K⁺ pump by ouabain, K⁺-free or Na⁺-free Ringer solution increased the diffusion potential for Cl^–. Inhibition of Cl^– transport and increment in Cl^– permeability by ouabain occurred almost simultaneously. It is likely, therefore, that Cl^– transport as well as Cl^– permeability is dependent on Na⁺–K⁺ pump activity. A possible mechanism of dependence of Cl^– transport on the Na⁺–K⁺ pump is discussed in relation to the increment in Cl^– permeability.     

Intracellular K+ activities and cell membrane potentials in a K+-transporting epithelium,the midgut of tobacco hornoworm (Manduca sexta)

Summary Transbasal electrical potential (V _b) and intraepithelial potassium chemical activity ((K⁺) _i ) were measured in isolated midgut epithelium of tobacco hornworm (Manduca sexta) using double-barrelled glass microelectrodes. Values ofV _b ranging from +8 to –48 mV (relative to blood side) were recorded. For all sites, (K⁺) _i is within a few millivolts of electrochemical equilibrium with the blood side bathing solution. Sites more negative than –20 mV show relatively high sensitivity ofV _b to changes in blood side K⁺ concentration: 43% of these sites can be marked successfully with iontophoresed Lucifer yellow CH dye and shown to represent epithelial cells of all three types present in the midgut. In about half of successful marks, dye-coupling of several adjacent cells is seen. Low potential sites — those withV _b less negative than –20 mV —typically do not show high sensitivity ofVb to changes of external K⁺, but rather (K⁺) _i rapidly approaches the K⁺ activity of blood side bathing solution. These sites can seldom be marked with Lucifer yellow (4% success). The mean (K⁺) _i of the high potential sites is 95±29 (sd)mm under standard conditions, a value which is in accord with published values for the whole tissue.     

Membrane transport parameters in frog corneal epithelium measured using impedance analysis techniques

Summary Active Cl^– transport in bullfrog corneal epithelium was studied using transepithelial impendance analysis methods, and direct-current (DC) measurements of membrane voltages and resistance ratios. The technique allows the estimation of the apical and basolateral membrane conductances, and the paracellular conductance, and does not rely on the use of membrane conductance-altering agents to obtain these measurements as was requisite in earlier DC equivalent-circuit analysis studies. In addition, the analysis results in estimates of the apical and basolateral membrane capacitances, and allows resolution of the paracellular conductance into properties of the tight junctions and lateral spaces. Membrane capacitances (proportional to areas) were used to estimate the specific conductances of the apical and basolateral membranes, as well as to evaluate coupling between the cell layers. We confirm results obtained from earlier studies: (1) apical membrane conductance is proportional to the rate of active Cl^– transport and is, highly Cl^– selective; (2) intracellular Cl^– activity is above electrochemical equilibrium, thereby providing a net driving force for apical membrane Cl^– exit; (3) the paracellular conductance is comparable to the transcellular conductance. We also found that: (1) the paracellular conductance is composed of the series combination of the junctional conductance and a nonnegligible lateral space resistance; (2) a small K⁺ conductance reported in the apical membrane may result from Cl^– channels possessing a finite permeability to K⁺; (3) the basolateral membrane areas is 36 times greater than the apical membrane area which is consistent with the notion of electrical coupling between the five to six cell layers of the epithelium; (4) the specific conductance of the basolateral membrane is many times lower than that of the apical membrane; (5) the net transport of Cl^– is modulated primarily by changes in the conductance of the apical membrane and not by changes in the net electrochemical gradient resulting from opposite changes in the electrical and chemical gradients; (6) the conductance of the basolateral membrane does not change with transport which implies that the net driving force for K⁺ exit increases with transport, possibly due to an increase in the intracellular K⁺ activity.     

The comparison of vanadyl(IV) and insulin-induced hyperpolarization of the mammalian muscle cell

Extracellularly applied vanadyl(IV) hyperpolarized the membrane potential of mouse diaphragm muscle from about −74.0 mV up to −81.7 mV. The hyperpolarizing effect of 10⁻⁴ mol·l⁻¹ vanadyl(IV) is comparable with hyperpolarization induced by 100 mU·ml⁻¹ insulin. Both compounds increased the intracellular K⁺ concentration, the hyperpolarizing effect of vanadyl(IV) and insulin is blocked by ouabain and is unaffected by removal of K⁺ from the external medium. Triggering of the release of intracellular K⁺ associated with cellular proteins is proposed as the mechanism of vanadyl(IV) and insulin-induced hyperpolarization.     

Microsomal (Na + K)-activated ATPase from frog skin epithelium. Cation activations and some effects of inhibitors

A method is described for the extraction of microsomal ouabain-sensitive (Na⁺ + K⁺)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na⁺ + K⁺)-stimulated activity in the range of 30–40 nmol · mg⁻¹ · min⁻¹ at 26 °C. This portion, which is also ouabain sensitive, is about half of the total activity in media containing Mg²⁺, Na⁺ and K⁺. These preparations also contain Mg²⁺-dependent or Ca²⁺-dependent activities which are not additive and which are not significantly affected by ouabain, Na⁺, K⁺ or Li⁺.The activations of the ouabain-sensitive ATPase activity by Mg²⁺, Na⁺, and K⁺ are similar to those described in other tissues. It is found that Li⁺ does not substitute for Na⁺ as an activator but in high concentrations does produce partial activation in the presence of Na⁺ with no K⁺. These results are pertinent to the reported observations of ouabain-sensitive Li⁺ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li⁺ on the sodium pump.     

Chloride movement across the basolateral membrane of proximal tubule cells

Summary Electrophysiologic and tracer experiments have shown that Cl^– entersNecturus proximal tubule cells from the tubule lumen by a process coupled to the flow of Na⁺, and that Cl^– entry is electrically silent. The mechanism of Cl^– exit from the cell across the basolateral membrane has not been directly studied. To evaluate the importance of the movement of Cl^– ions across the basolateral membrane, the relative conductance of Cl^– to K⁺ was determined by a new method. Single-barrel ion-selective microelectrodes were used to measure intracellular Cl^– and K⁺ as a function of basolateral membrane PD as it varied normally from tubule to tubule. Basolateral membrane Cl^– conductance was about 10% of K⁺ conductance by this method. A second approach was to voltage clamp the basolateral PD to 20 mV above and below the spontaneous PD, while sensing intracellular Cl^– activity with the second barrel of a double-barrel microelectrode. An axial wire electrode in the tubule lumen was used to pass current across the tubular wall and thereby vary the basolateral membrane PD. Cell Cl^– activity was virtually unaffected by the PD changes. We conclude that Cl^– leavesNecturus proximal tubule cells by a neutral mechanism, possibly coupled to the efflux of Na⁺ or K⁺.     

Complexation of lithium(I), sodium(I), silver(I) and thallium(I) by 4,7,13,16-tetraoxa-1,10-diazabicyclo[8.5.5]eicosane and 4, 7, 13-trioxa-1,10-diazabicyclo[8.5.5]eicosane in trimethyl phosphate. A potentiometric titration and Li and Na nuclear magnetic resonance study

Complexation of M⁺=Li⁺, Na⁺, Ag⁺ and TI⁺ by the cryptands 4, 7, 13, 18-tetraoxa-l, 10-diazabicyclo[8.5.5]eicosane (C211) and 4,7,13-trioxa-1,10-diazabicyclo[8.5.5]eicosane (C21C₅) to form the cryptates [M.C211]⁺ and [M.C21C₅]⁺ has been studied in trimethyl phosphate by potentiometric titration and ⁷Li and ²³Na NMR spectroscopy. For [M.C211]⁺ the logarithm of the apparent stability constants, log K (dm³ mol^-1)=6.98±0.05, 5.38±0.05, 9.82±0.02 and 3.95±0.02 for M⁺ =Li⁺, Na⁺, Ag⁺ and TI⁺, respectively; and for [M.C21C₅]⁺ log K (dm³ mol^-1)=2.40±0.10, 1.90±0.05, 6.04±0.02 and 2.42±0.10 for M⁺=Li⁺, Na⁺, Ag⁺ and Tl⁺, respectively. The decomplexation kinetic parameters for [Na.C211]⁺ are: k_d (298.2 K)=6.924±0.50 s^-l, ΔH_d≠=62.2±0.9 kJ mol^-1, and ΔS_d≠= -20.3±2.7 J K^-1 mol^-1; and those for [Li.C21C₅]⁺ are: k_d (298.2 K)=23.3±0.4 s^-1, ΔH_d≠ =61.2±1.1 kJ mol^-1, and ΔS_d≠= -13.6±3.6 J K^-1 mol^-1. Metal ion exchange on [Li.C211]⁺ is in the very slow extreme of the NMR timescale up to 390 K and k_d « 4 s^-1 at 298.2 K, while in contrast exchange on [Na.C21C₅]⁺ is in the fast extreme of the NMR timescale at 298.2 K (k_d≈ 10⁴ s^-1). These data are compared with those obtained in other solvents.