Amphotericin B enhanced anomalous potential difference response to changes in aqueous K+ in frog cornea.

An increase in aqueous K+ from 0 to 4 mM increased the potential difference (anomalous response of electrogenic (Na+ + K+)-ATPase antiport) by 1.1 mV in Cl(-)-free solutions compared to 6.8 mV in Cl- solutions. With amphotericin B added to the tear solution in Cl(-)-free solutions, the anomalous PD response for the addition of 4 mM K+ to the aqueous solution was about 20 mV, significantly greater than in Cl- solutions. This anomalous response was inhibited by ouabain. These data support the electrogenicity of the (Na+ + K+)-ATPase pump. It is also evident that, for the pump to respond, Na+ should readily enter the cell. This may be accomplished experimentally, either across the basolateral membrane in Cl- solutions or across the apical membrane in Cl(-)-free solutions with amphotericin B present in the tear solution.

Evidence for HCO3- conductance pathways in nutrient membrane of bullfrog antrum.

The effect of changing the nutrient HCO3- concentration on potential difference (PD) and resistance in bullfrog antrum bathing in CI- media was determined. Changes in HCO3- concentration were from 25 mM to several lower concentrations and back to 25 mM. A plot of /delta PD/ versus log [HCO3-] gave a linear relation for changes of HCO3- concentration from 25 down to 3.1 mM and back to 25 mM but deviated to some extent for changes to 1.6 mM. In these experiments, changes from higher to lower HCO3- concentrations gave a less rapid initial PD response than those in the reverse direction. This result eliminated H+ conductance pathways as being predominant. Experiments were done in which in the first part changes were made in nutrient solution from 5 percent CO2 and 25 mM HCO3- to 0.6 percent CO2 and 3 mM HCO3- and in the second part the same changes with a simultaneous changes of secretory solution from 5 percent to 10 percent CO2. The magnitude of PD decrease was greater by 4.5 mV in the second part. This result indicated that HCO3- conductance pathways rather than OH- conductance pathways are predominated . There was no evidence of HCO3-, OH-, and H+ conductance pathways in secretory membranes.

Effect of reduced pH in absence of HCO3(-) on anomalous and normal potential responses in bullfrog antrum.

Effect of changing [K+], [Na+] and [Cl-] in nutrient solution on potential difference (PD) and resistance was studied in bullfrog antrum with and without nutrient HCO3(-) but with 95% O2/5% CO2 in both cases. In both cases, changing from 4 to 40 mM K+ gave about the same initial PD maximum (anomalous response) which was followed by a decrease below control level. Latter effect was much less with zero than with 25 mM HCO3(-). Changing from 102 to 8 mM Na+ gave initial normal PD response about the same in both cases. However, 10 min later the change in PD with zero HCO3(-) was insignificant but with 25 mM HCO3(-) the PD decreased (anomalous response of electrogenic NaCl symport). PD maxima due to K+ and Na+ were largely related to (Na+ + K+)-ATPase pump. Changes in nutrient Cl- from 81 to 8.1 mM gave only a decrease in PD (normal response). Initial PD increases are explained by relative increases in resistance of simple conductance pathways and of parallel pathways of (Na+ + K+)-ATPase pump and Na+/Cl- symport. Removal of HCO3(-) and concurrent reduction of pH modify resistance of these pathways.

Potential difference responses due to K+, Na+ and Cl- changes in bullfrog antrum with and without HCO3-.

The effect of changing [K+], [Na+] and [Cl-] in nutrient solution was studied in bullfrog antrum with and without HCO3- in nutrient. In 25 mM HCO3- (95% O2/5% CO2) and in zero HCO3- (100% O2), nutrient pH was maintained at 7.3. Changing from 4 to 40 mM K+ or from 81 to 8.1 mM Cl- gave a decrease 10 min later in transmucosal PD (nutrient became more negative)--a normal response. These responses were less in zero than in 25 mM HCO3-. A decrease from 102 to 8 mM Na+ decreased PD (anomalous response of electrogenic NaCl symport). This effect was attenuated or eliminated in zero HCO3-. In contrast, change from 4 to 40 mM K+ gave initial anomalous PD response and change from 102 to 8 mM Na+, initial normal PD response with either zero or 25 mM HCO3-. Both responses were associated with (Na+ + K+)-ATPase pump and were greater in zero than in 25 mM HCO3-. Initial PD increases in zero HCO3- are explained as due to increase in the resistance of passive conductance and/or NaCl symport pathways. Thus, removal of HCO3- modifies conductance pathways of nutrient membrane.

Effect of Ba2+ on the K+ conductance pathways in the frog cornea.

Two types of transepithelial potential difference (PD) responses have been observed in the bullfrog, Rana catesbeiana, when the K+ concentration is changed in the aqueous solution. (1) A normal response, that is, a decrease in the positivity of the aqueous solution when the K+ is increased in this solution. (2) An anomalous response, that is, an increase in PD when K+ is increased from 0 to 4 mM in the aqueous solution. In present experiments 2 mM Ba2+ results in a significant decrease in transepithelial PD and an increase in resistance (R), consistent with the well-known effect of Ba2+ on the K+ conductance in other biological membranes. In the presence of Ba2+ compared to its absence the normal PD responses were decreased when K+ was increased from 4 to 20 or to 79 mM in the aqueous solution. Barium enhanced, but not significantly, the anomalous PD response (PD increase) when K+ was increased from 0 to 4 mM. An anomalous PD response (PD decrease) was obtained with Ba2+ when K+ was changed from 4 to 0 mM while in its absence the response was normal (PD increase) or did not change. These findings support the concept that anomalous PD responses as a result of the electrogenic (Na+ + K+)-ATPase may be obtained when the resistance of the simple K+ pathway is increased.

Electrogenicity of the frog gastric mucosa proton pump based on polarization responses in the presence of H(+)-secretion inhibitors.

Recently, we have shown that polarization of an electrogenic H+/K(+)-ATPase pump located in the secretory (luminal) membrane of the frog gastric mucosa is the major factor contributing to the increase in open circuit potential difference (OCPD) induced by voltage clamping. While this transmucosal polarization was not affected by removal of Cl- and Na+ and minimally affected by increasing the K+ concentration to 79 mM in both nutrient and secretory solutions, it was markedly reduced by 10(-3) M famotidine (beta blocker) or 10(-4) M omeprazole (H+/K(+)-ATPase inhibitor) in the nutrient solution. In present experiments, the effects of three other inhibitors of H+ secretion were examined, namely, cimetidine (beta blocker), SCH 28,080 (H+/K(+)-ATPase inhibitor) and SCN- (non-specific inhibitor). While cimetidine and SCH 28,080 markedly reduced the polarization induced by voltage clamp, SCN- affected the polarization to a lesser extent. These data further support the electrogenicity of the frog gastric mucosa proton pump and the lack of a direct effect of SCN- on the pump.

Anomalous potential response and (Na+ + K+)-ATPase in in vitro frog gastric mucosa.

In general, increasing K+ on the nutrient side decreases the transmucosal PD (nutrient becomes more negative) but after bathing the mucosa in zero K+ media for about 30 min, or longer, elevation of K+ on the nutrient side increases the PD, an anomalous effect. In Cl- media, increasing nutrient K+ from zero to 4 mM produces an increase in PD (an anomalous response) of 3.1 and 5.3 mV in 2 and 5 min, respectively. Ouabain (10(-3) M) to the nutrient side abolished the anomalous response as did removal of Na+ (choline for Na+) from bathing media. In SO4(2-) media (SO4(2-) for Cl-), a significant anomalous PD response was observed when K+ on the nutrient side was increased from zero to 1, 2 or 3 mM but not to higher K+ concentrations. In this case, ouabain also abolished the anomalous response. It is postulated, on the basis of the effects of ouabain and the use of choline media, that an electrogenic (Na+ + K+)-ATPase pump is present on the nutrient-facing membrane in which more Na+ than K+ are transported per cycle.

Effect of current direction and K+ on polarization of the frog gastric mucosa proton pump.

When current was sent from serosa (S) to mucosa (M) across the frog stomach, there was a polarization (POL) of the open circuit potential (OCPD). POL was not affected by NaCl-free solutions, but was decreased by inhibitors of the H+ pump. In present experiments, current was sent to clamp the PD (VC) across the mucosa in steps of 20 mV up to 100 mV below the control OCPD, that is, current was sent from M to S. All experiments were performed in NaCl-free solutions. The POL was expressed as a % of the difference between the VC PD and the control OCPD. In 4 mM K+ control solutions, the POL was 11.8%; with 10(-3) M omeprazole (H+/K+ pump inhibitor), 1.1; with 10(-5) M SCH 28080 (H+/K+ pump inhibitor), 3.6; with 10(-3) M famotidine (H2 blocker), 1.6; and with 10(-2) M SCN-, 25.4 (inhibition of H+ sec, but not of the pump); in 79 mM K+ control solutions, 26.2; with 10(-3) M omeprazole, 4.2; with 10(-5) M SCH 28080, 15.9; with 10(-3) M famotidine, 5.6; and with 10(-2) M SCN-, 29.9. POL was higher in high K+ than in low K+ solutions contrary to what was observed in previous experiments with current sent from S to M. Results are explained on the basis of an electrogenic H+/K(+)-ATPase pump which includes a H+ channel, permeable to K+. With high K+ solutions, K+ is driven through the H+ channel onto the antiporter (ATPase) when current is sent from M to S, resulting in a greater POL of the pump.

Diffusion resistance of endothelium and stroma of bullfrog cornea determined by potential response to K+.

Corneas of bullfrog (Rana catesbeiana) were mounted between lucite chambers. A four-electrode system was used and the potential difference (PD) and the electrical resistance were measured. In intact corneas, the PD averaged 25 mV (acqueous side positive) and the electrical resistance 1.5 kQ - cm2. perfusion of the aqueous side with high K+ solutions resulted in a marked decrease in PD and a drop in the electrical resistance. Scraping the epithelium (leaving the stroma plus endothelium) resulted in a drop of the PD to about zero and a decrease in electrical resistance to about 0.1 kQ - CM2 and a very small PD response to a marked elevation of the K+ concentration on the aqueous side. On the basis of the above, it is obvious that the large delta PD in intact corneas, due to elevation of the K+ concentration, must be due to K+ diffusing from the aqueous side across the endothelium and stroma and reaching the epithelium. The duration of the PD response is therefore a measure of the resistance to diffusion of the stroma plus endothelium. A quantitiative analysis shows that under in vitro conditions the resistance of the endothelium plus stroma to the diffusion of ions is very low.

Potential difference responses to secretory K+, Na+ and HCO3- changes in secreting and resting states of frog stomach in Cl(-)-free media.

The effects of changes in secretory concentrations of K+, Na+ and HCO3- on transmucosal potential difference (PD) and resistance in Cl(-)-free (SO4(2-)) solutions were compared for secreting fundus and resting fundus of Rana pipiens. In the resting fundus experiments, histamine was not present in the nutrient solution and cimetidine was primarily used to obtain acid inhibition. Increase of K+ from 4 to 80 mM, decrease of Na+ from 156 to 15.6 mM and decrease of HCO3- from 25 to 5 mM gave, 10 min after the change, in the secreting fundus delta PD values of 39.7, -11.9 and 3.2 mV, respectively. In the resting fundus, 1.5 to 2 h after the addition of cimetidine, the same changes in secretory ion concentration gave delta PD values of 12.2, -5.6 and 1.5 mV, respectively. Replacement of cimetidine with SCN and without histamine yielded a delta PD somewhat lower than that in cimetidine, namely 9 mV for a K+ change from 4 to 80 mM. Subsequent addition of histamine with SCN present gave a delta PD of about 21 mV. The change in PD was attributed to histamine increasing the secretory membrane area, leading to an increase in K+ conductance. Another possibility is that histamine increases the K+ conductance per se.

Microelectrode studies of potential difference responses to changes in stromal K+ in bullfrog cornea.

The effects of changing stromal K+ were studied using microelectrodes in an in vitro preparation of frog cornea. The intracellular potential (V0) responded in two opposite ways under short-circuit conditions: (1) depolarization (normal response) when stromal K+ was increased from 4 to 20 or to 79 mM, about 30 mV per 10-fold K+ concn. change; (2) a hyperpolarization (anomalous response) of 10 mV maximum when stromal K+ was increased from 0 to 4 mM. The increase from 4 to 20 or 79 mM decreased or even reversed the short-circuit current (Isc). The transepithelial conductance (gt) increased when K+ was increased to 79 mM but no change occurred in the apical membrane fractional resistance (fRo). Increase of stromal K+ from 0 to 4 mM increased Isc and minimally changed gt and fRo. Ouabain (10(-3) M) abolished the anomalous responses, that is, the increases in V0 and Isc when stromal K+ was increased from 0 to 4 mM. These results are interpreted in terms of two K+ conductive pathways in the basolateral membrane of the corneal epithelium, a Nernstian conductance and an electrogenic (Na+ + K+)-ATPase pump transporting more Na+ than K+ ions per cycle. The normal or anomalous potential difference responses to changes in stromal K+ appear to depend on the relative resistance of the two pathways at the time stromal K+ is changed.

Potential difference responses to nutrient K+, Cl- and Na+ changes in secreting and resting states of frog stomach.

The effects of changes in nutrient concentrations of K+, Cl- and Na+ on the transmucosal potential difference (PD) and the resistance were compared for secreting fundus and resting fundus of Rana pipiens. Increase of K+ from 4 to 40 mM, decrease of Cl- from 81 to 8.1 mM and decrease of Na+ from 102 to 10 mM gave, 10 min after the change in the secreting fundus, delta PD values of -28.2, -19.8 and -7.5 mV, respectively. In the resting fundus with SCN- inhibition, the same changes in nutrient ion concentration gave delta PD values of -20.1, -17.0 and -10.2 mV, respectively. Changes in Na+ concentration were considered in a set of experiments of high acid secreting stomachs (4 to 6 mu equiv. . h-1 . cm-2). Here, delta PD gave for 10-fold decreases in Na+ concentration in secreting fundus -4.8 mV and in resting fundus with SCN- inhibition -22.6 mV. Omeprazole inhibition gave results quite similar to those with SCN- inhibition. From these results in going from secretion to inhibition, it follows that the increment of K+ conductance if it increased was lower than the increase in NaCl symport conductance since the change in delta PD for K+ decreased and that for Na+ increased. Also HCO3- conductance increased with inhibition. After SCN- inhibition the transmucosal resistance initially increased and later decreased. The decrease can be accounted for by the increase in conductance of the NaCl symport pathway and of the HCO3- pathway.

Potential difference responses to nutrient K+, Na+ and Cl- changes with varying nutrient HCO3- in resting frog stomach.

The effects of changes in nutrient concentrations of K+, Na+ and Cl- on the transmucosal potential difference (PD) and resistance were compared for 25 and 5 mM nutrient HCO3- in resting fundus. With 25 mM HCO3-, increase of K+ from 4 to 40 mM, decrease of Na+ from 100 to 10 mM and decrease of Cl- from 81 to 8.1 mM gave, 10 min after the change, delta PD values of -23.2, -15.1 and -21.3 mV, respectively. With 5 mM HCO3-, the same changes in nutrient ion concentration gave delta PD values of -11.9, -9.4 and -10.0 mV, respectively. From these results, in going from 25 to 5 mM HCO3-, it follows that the resistances of the ionic pathways for K+, Na+ and Cl- increased. The anomalous PD response following the increase in nutrient K+ from 4 to 40 mM with 5 mM nutrient HCO3- gave further evidence that the resistance of the simple K+ conductance pathway increased prior to the increase to 40 mM K+. The fact that 2 mM Ba2+ in the 25 mM HCO3- nutrient gave a smaller increase in resistance, compared to the decrease in nutrient HCO3- from 25 to 5 mM, supported the inference that resistances of ion pathways other than that of the K+ pathway increased.

Normal and anomalous potential responses due to K+ changes in bullfrog antrum.

The effect of changing the K+ concentration in the bathing media was studied in the bullfrog antrum. Usually increasing K+ on the nutrient side in standard Cl- -containing and Cl- -free solutions decreased the transmucosal potential difference (nutrient became more negative) - a normal effect. Similar results were obtained on the secretory side. Moreover, for K+ changes on the nutrient side in Cl- media, a plot of magnitude of delta V vs. log [K+] was linear for [K+] greater than 20 mM with slope of 27 mV per 10-fold change in [K+]. However, after bathing the mucosa in Cl- media with zero K+ for about 20 min, elevating the nutrient [K+] to 4 mM increased the potential difference (V) by 4.8 mV in 5 min and repeating the same sequence increased V by 6.9 mV in 5 min - both anomalous effects. Beyond 20 mM K+ the response was normal. In SO2-4 media, an anomalous potential difference of about 1 mV was obtained for changes from 0 to 3 or 6 mM nutrient K+. Ouabain (1 X 10(-3) M) in the nutrient solution abolished the anomalous response in Cl- and SO2-4 media. The normal response is attributed to passive, conductance pathways and the anomalous response because of the effect of ouabain, to a (Na+ + K+)-ATPase pump on the nutrient-facing membrane in which more Na+ than K+ ions are transported per cycle.

Effect of amphotericin B and Cl- removal on basolateral membrane K+ conductance in frog corneal epithelium.

Increase in stromal K+ concentration from 4 to 79 mM in an in vitro preparation of the frog cornea, in Cl(-)-free solutions, did not change the apical membrane fractional resistance, fR0, or the transepithelial conductance, gt; it depolarized the intracellular potential, V0, by 38 mV and decreased the short-circuit current, Isc by 2.9 microA/cm2. These changes were similar to those observed for the same increase in stromal K+ in control solutions except for the increase in gt in the latter. When stromal K+ was increased with 10(-5) M amphotericin B, AmB, in the tear solution, fR0 increased by 0.27 in control solutions and by 0.08 in Cl(-)-free solutions; respectively, gt increased by 0.40 and by 0.17 mS/cm2; Isc decreased by 12 and by 11 mS/cm2; V0 depolarized by 9 and by 9.5 mV. These results support the concept that: (i) entrance of Cl- into the cell is responsible in part for the bioelectrical changes observed when stromal K+ is increased; and (ii) AmB decreases the partial K+ conductance in the basolateral membrane of the frog cornea epithelium by a decrease in intracellular K+.

Effects of ouabain on frog gastric mucosa in vitro.

The effect of the addition of ouabain to the nutrient solution was determined on resistance, potential difference (p.d.) and H+ secretion rate. In NaCl media, 10(-3) M ouabain decreased significantly the p.d. from 25.6 mV to 16.1 mV in 30 min and to 11.0 mV in 60 min. No significant changes occurred in resistance and H+ secretion rate. In Na2SO4 (Cl(-)-free) media, ouabain produced a biphasic effect on p.d. The p.d. changed from -28.0 mV (nutrient-negative) to a nadir of -37.4 mV in 7 min and then increased to -16.4 mV in 60 min. At the nadir there was no significant change in resistance or H+ secretion rate but at 60 min, unlike Cl- media, resistance increased by 36% and H+ secretion rate decreased by 43%. To decide whether the ouabain-caused decrease in H+ rate in Na2SO4 media was due to an effect on the H+ pump or on resistance of the return pathways, the voltage was clamped at 0 and 40 mV. Clamping the voltage showed that in the case of a marked decrease in the H+ secretion rate, the H+ transport mechanism itself was inhibited (and not the parallel pathway). The decrease in p.d. due to ouabain in Cl- and SO42- media indicates that the (Na+ + K+)-ATPase mechanism may be electrogenic.

Inhibition of acid secretion of fundus of Rana pipiens with a high concentration of potassium on the secretory side.

Inhibition of acid secretion of the frog fundus is generally accompanied by an increase in transmucosal resistance, Rt, and in potential difference, PD (nutrient normally positive). These results are predicted for the intact tissue by an electrogenic proton pump. It has been suggested that the increase in PD with inhibition can also be explained by a neutral proton pump. The latter model postulates a K+ diffusion potential across the secretory (lumen-facing) membrane tending to make the secretory side positive. Upon inhibition, the [K+] in the lumen is assumed to increase, which decreases the diffusion potential, resulting in an increase in the positivity of the nutrient side. To test this theory, we determined the effects of inhibition with a high [K+] on the secretory side. With a high [K+] in the lumina, inhibition would result in only a small change in the ratio of K+ in the cell to that in the lumina, and hence a small change in the diffusion potential. We found, however, that inhibition increased the PD essentially the same as in the controls. With inhibition the resistance also increased with high secretory K+. Elevating the secretory K+ during secretion produced a 44% decrease in Rt indicating a large increase in luminal K+. We conclude that the results are not compatible with the K+ diffusion potential model but are those predicted by the electrogenic concept.

Microelectrode studies of amphotericin B on Na+ and K+ conductance in bullfrog cornea.

Addition of 10(-5) M amphotericin B to the tear solution of an in vitro preparation of the frog cornea increased the transepithelial conductance, gt, and decreased the apical membrane fractional resistance, f(R0), in the presence or absence of tear Na+ and Cl-. In the presence of tear Na+ and Cl-, amphotericin B increased the short-circuit current, Isc, from 3.9 to 8.8 microA.cm-2 and changed the intracellular potential, V0, from -48.5 to -17.9 mV probably due to a higher increase in the Na+ than in the K+ conductance. In the absence of tear Na+ and Cl-, amphotericin B decreased Isc from 5.5 to about 0 microA.cm-2 due to K+ (and possibly Na+) flux from cell to tear and changed V0 from -35.4 to -63.6 mV due to the increase in conductance of both ions. Increase in the tear K+ from 4 to 79 mM (in exchange for choline), in the presence of amphotericin B and absence of tear Na+ and Cl-, decreased f(R0) from 0.09 to 0.06, increased gt from 0.23 to 0.31 mS, increased Isc from 0.63 to 7.3 microA.cm-2, and changed V0 from -65.5 to -17.3 mV due to the change in EK in the presence of a high conductance in the tear membrane. Similar effects were observed with an increase of tear Na+. Results support the concept that the Na+ conductance opened by amphotericin B in the apical membrane is greater than the K+ conductance. Previously observed transepithelial effects of the ionophore may be explained mostly on the basis of its effect on the apical membrane.

Direct current electrical measurement in epithelia: steady-state and transient analysis.

A method has been presented for the determination of resistance of biological tissues in which the PD response to step currents is determined. The delta PD after the dielectric capacitors are charged, divided by the current, gives the resistance, provided the current density is low enough so that the tissue behaves as a linear-bilateral system. In the gastric mucosa the PD continues to increase after the dielectric capacitors are charged and it is shown that this part of the delta PD is due to polarization of EMFs and should not be used in determining the resistance. It has been shown that (1) resistance measurements have enabled us to demonstrate that during acid secretion there is a neutral mechanism(s) for the movement of HCO3- out of and the entrance of Cl- into the oxyntic cells, (2) the transmucosal resistance varies inversely with the rate of acid secretion, and (3) the low resistance of the secreting frog fundus is due to the low resistance of the lumen-tubular cell pathway--the parallel pathways (the TIC or paracellular and surface cell pathways) have high resistances. The results of both the resistance and PD measurements have recently been analyzed with respect to the problem of whether the proton pump is neutral or electrogenic in the intact tissue.

Cell-to-cell communication of osteoblasts.

Osteoblasts were investigated by two methods, electrical conductance and dye injection. Current injection into one cell caused a change in the recorded transmembrane potential of a second cell, indicating high conductance pathways between the two cells. Dyes injected into a single osteoblast were transmitted to numerous surrounding cells.