Douglas C. Eaton

Basolateral membrane potential of a tight epithelium: ionic diffusion and electrogenic pumps.

SummaryThe contribution of specific ions to the conductance and potential of the basolateral membrane of the rabbit urinary bladder has been studied with both conventional and ion-specific microelectrode techniques. In addition, the possibility of an electrogenic active transport process located at the basolateral membrane was studied using the polyene antibiotic nystatin. The effect of ion-specific microelectrode impalement damage on intracellular ion activities was examined and a criterion set for acceptance or rejection of intracellular activity measurements. Using this criterion, we found (K+)=72mm and (Cl−)=15.8mm. Cl− but not K+ was in electrochemical equilibrium across the basolateral membrane. The selective permeability of the basolateral membrane was measured using microelectrodes, and the data analyzed using the Goldman, Hodgkin-Katz equation. The sodium to potassium permeability ratio (PNa/PK) was 0.044, and the chloride to potassium permeability ratio (PCl/PK) was 1.17. Since K+ was not in electrochemical equilibrium, intracellular (K+) is maintained by active metabolic processes, and the basolateral membrane potential is a diffusion potential with K+ and Cl− the most permeable ions. After depolarizing the basolateral membrane with high serosal potassium bathing solutions and eliminating the apical membrane as a rate limiting step for ion movement using the polyene antibiotic nystatin, we found that the addition of equal aliquots of NaCl to both solutions caused the basolateral membrane potential to hyperpolarize by up to 20 mV (cell interior negative). This popential was reduced by 80% within 3 min of the addition of ouabain to the serosal solution. This hyperpolarization most probably represents a ouabain sensitive active transport process sensitive to intracellular Na+. An equivalent electrical circuit for Na+ transport across rabbit urinary bladder is derived, tested, and compared to previous results. This circuit is also used to predict the effects that microelectrode impalement damage will have on individual membrane potentials as well as time-dependent phenomena; e.g., effect of amiloride on apical and basolateral membrane potentials.

Single-Channel Recordings from Two Types of Amiloride-Sensitive Epithelial Na+ Channels

We report here the first evidence in intact epithelial cells of unit conductance events from amiloride-sensitive Na+ channels. The events were observed when patch-clamp recordings were made from the apical surface of cultured epithelial kidney cells (A6). Two types of channels were observed: one with a high selectivity to Na+ and one with relatively low selectivity. The characteristics of the low-selectivity channel are as follows: single-channel conductance ranged between 7 and 10 pS (mean = 8.4 +/- 1.3), the current-voltage (I-V) relationship displayed little if any nonlinearity over a range of +/- 80 mV (with respect to the patch pipette) and the channel Na+/K+ selectivity was approximately 3-4:1. Amiloride, a cationic blocker of the channel, reduced channel mean open time and increased channel mean closed times as the voltage of the cell interior was made more negative. Amiloride induced channel flickering at increased negative potentials (intracellular potential with respect to the patch) but did not alter the single-channel conductance or the I-V relationship from that observed in control patches. The characteristics of the high-selectivity channel are: a single-channel conductance of 1-3 pS (mean = 2.8 +/- 1.2), the current-voltage relationship is markedly nonlinear with a Na+/K+ selectivity greater than 20:1. The mean open and closed times for the two types of channels are quite different, the high-selectivity channel being open only about 10% of the time while the low-selectivity channel is open about 30% of the time.

Effects of nystatin on membrane conductance and internal ion activities inAplysia neurons

SummaryTwo methods were used to study effects of the antibiotic, nystatin, on giant neurons ofAplysia. In the first method the effects of various concentrations of nystatin on the current-voltage relationship were evaluated at a fixed time after exposure to the antibiotic using a two-microelectrode voltage clamp. Nystatin increased membrane conductance in a dose-dependent manner. The dose-response relation was very steep, with little or no effect below 15 mg/liter and an effect too large to measure at concentrations greater than 30 mg/liter. Upon return to antibiotic-free solution, membrane conductance returned to pre-treatment levels within 30 minutes. The second type of experiment involved use of ion-specific microelectrodes to measure changes of intracellular univalent ion activities which attended the nystatin-induced permeability. meability was also increased. Nystatin may therefore be used to selectively rearrange the internal ionic milieu to study the effect of such a change on membrane tranpsort or electrical properties.

Current-voltage relationship of the basolateral membrane of a tight epithelium

The polyene antibiotic nystatin is used to reduce selectively to zero the apical membrane resistance of the rabbit descending colon, allowing the measurement of the current-voltage curve of the basolateral membrane. The I--V relationship is described by the Goldman-Hodgkin-Katz equations allowing calculation of PNa/PK, PCl/PK and PK for the basolateral membrane. Cs+ is found to block inward current (serosa to mucosa) in a manner similar to that found in excitable membranes.

Ionic permeabilities of anAplysia giant neuron

SummaryIn a giant neuron ofAplysia californica, permeabilities and conductances obtained by measuring net fluxes of Na+, K+ and Cl− with ion-specific microelectrodes were compared with those obtained by measuring transmembrane current and potential changes when the three ions were varied in the external solution. Net fluxes were measured with ion-specific microelectrodes, after blocking metabolic processes, thus allowing movement of ions down their electrochemical gradients. Permeabilities and conductances obtained from the “chemical” measurements (i.e., ion-specific electrodes) were generally comparable to the values obtained from “electrical” measurements. Where discrepancies occurred, they could be explained by showing that some of the assumptions necessary to use the “electrical” method were not quantitatively true in this system. The absolute magnitudes of the permeabilities are significantly less than those found in many axonal preparations. There is also a relatively highPNa/PK ratio. The selectivity of the membraneagainst ions such as Tris+ and MeSO3− is not good, Tris+ being nearly as permeable as Na+ and MeSO3− about one-half as permeable as Cl−. These properties may be characteristic of somal membranes.

Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder

SummaryThe apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na+ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal22Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na+ flux was a linear function of mucosal Na+ concentration. Ouabain-sensitive Na+ flux and ouabain-sensitive, Na+-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na+ with the basolateral transport process is assumed to involve the interaction of some number of Na+ ions,n, with a maximal flux,Mmax, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value forMmax of 287.8pm cm−2 sec−1 with an intracellular Na concentration of 2.0mm Na+ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na+-induced current, we calculate a Na+ to K+ coupling ratio of 1.40±0.07 for the transport process.

Acid pH and weak acids induce Na−Cl contransport in the rabbit urinary bladder

SummaryWe have described a coupled Na−Cl entry step at the apical membrane of a tight epithelium, the rabbit urinary bladder. Mucosal pH values, more acid than 4.6, stimulate a 20 to 40-fold increase in mucosal-to-serosal Na+ and Cl− flux. The flux increase is almost completely blocked by low concentrations of bumetanide. The transepithelial movement of Na+ and Cl− is normally electroneutral; however, when weak acids (such as acetate) are present in the mucosal solution, the acid-induced increase in flux is accompanied by a large increase in short-circuit current. Besides blockage by bumetanide, both the increase in flux and short-circuit current are blocked by: (1) Na+-free solutions on the mucosa; (2) Cl−-free solutions on the mucosa; (3) phosphodiesterase inhibitors; (4) ouabain in the serosal solution; (5) K+-free solutions on the serosa; and (6) HCO3−-free solutions on the serosa. The increase in the fluxes and the short-circuit current is unaffected by: (1) amiloride application in the mucosal solution; (2) mucosally applied stilbene derivatives which block Cl−/HCO3− exchange (SITS); and (3) Cl−-free solutions applied to the serosa. We interpret these results to imply a coupled Na−Cl uptake step at the apical membrane which is stimulated by intracellular acetate (or pH). The uptake step leads to a movement of Na+ and Cl− across the basolateral membrane, which is mediated by the Na+, K+-ATPase and a Na/Cl/HCO3− exchange mechanism. Our results demonstrate that “tight” epithelia may, under appropriate circumstances, demonstrate mechanisms of ion movement which are similar to “leaky” epithelia.

Amiloride-inhibited Na+ uptake into toad bladder microsomes is Na+-H+ exchange

Amiloride-inhibited Na+ transport into toad urinary bladder microsomes is sensitive to a pH gradient across the vesicular membrane. The magnitude of the gradient was measured directly with acridine orange. Also Na+ could stimulate amiloride-sensitive proton efflux from the microsomes. These results indicated that the transport process was Na+-H+ exchange.

Sulfhydryl reagents affect Na+ uptake into toad bladder membrane vesicles

SummaryThe effect of sulfhydryl reagents on the Na+ permeability mechanisms of toad urinary bladder vesicles was examined. The reagents 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), iodosobenzoate, and ethylenimine were able to decrease amiloride-inhibited sodium uptake into vesicles when used at low concentrations. When used at higher concentrations these reagents were able to induce large increases in vesicle Na+ permeability that were not sensitive to amiloride. The reagentp-chloro-mercuribenzene sulfonate was able to induce such leaks even at low concentrations. The reagent N-ethylmaleimide was incapable of substantially affecting vesicle Na+ transport in any way. All of the effects observed could be reversed by removing the reagents from the solution surrounding the vesicles. Our results help explain the varied actions of sulfhydryl reagents on intact epithelial tissue.

Tea blocks potassium current in squid axon

Abstract 1. 1. Internal perfusion of squid axon with an infusion of tea (orange pekoe and pekoe) caused an irreversible loss of voltage-activated potassium conductance ( g K ) in squid axon. 2. 2. Internal application of tannic acid similarly reduced g K , while theophylline had little effect on potassium currents. 3. 3. Both tea and tannic acid reduced sodium inactivation.