Effect of ibuprofen and rofecoxib transport parameters in the frog corneal epithelium.

Effects of cyclooxygenase (COX) inhibitors on transport parameters of the frog corneal epithelium were studied. Epithelial cells of the intact cornea were impaled with microelectrodes. Under short-circuit current (I(sc)) conditions, 10(-4) M ibuprofen (IBU) (non-specific COX inhibitor) or 5 x 10(-5) M rofecoxib (COX-2 inhibitor) were added to the tear solution. With ibuprofen, I(sc) decreased by 1.0 from 3.1 microA/cm2; intracellular potential, V(o), depolarized by 14.2 from -56.9 mV; IBU did not affect the transepithelial conductance, g(t), or the apical membrane fractional resistance, fR(o). With rofecoxib, I(sc) decreased by 0.9 from 4.3 microA/cm2; V(o) depolarized by 18 from -62.4 mV; g(t) significantly increased by 0.03 from 0.37 ms/cm2; and fR(o) decreased by 12 from 50. Basolateral membrane K+ and apical membrane Cl- partial conductances were studied by the ion substitution method. Depolarization of V(o) by an increase in stromal K+ from 4 to 79 mM was smaller with IBU (17.5 mV) or rofecoxib (19.2 mV) than without the inhibitors (29.1 and 29.3 mV, respectively). Depolarization of V(o), by a decrease in tear Cl- from 81 to 8.1 mM, was abolished by the COX inhibitors. Decrease in I(sc) and V(o) can be explained by a decrease in the K+ and Cl-? conductances. Experiments with amphotericin B ruled out a major effect of the inhibitors on the Na+/K+ ATPase pump.

Effect of dicyclohexylcarbodiimide (DCCD) on transport parameters in the frog cornea epithelium.

Dicyclohexylcarbodiimide (DCCD) is a carboxyl group modifier and it is an inhibitor of various ATPases. Present experiments, using an in vitro preparation, were designed to study whether DCCD affected the transporters of the bullfrog cornea epithelium, specifically, the Na(+)/K(+) ATPase pump located in the basolateral membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (I(sc)) conditions. Addition of DCCD to a concentration of 10(-4) m to the tear solution gave a marked decrease in I(sc); a marked depolarization of the intracellular potential, V(o); and a significant decrease in the apical membrane fractional resistance, fR(o). There were small and variable although significant changes in the transepithelial conductance, g(t). The effects may be explained by a decrease in the basolateral membrane K(+) conductance, in combination with a partial inhibition of the Na(+)/K(+)-ATPase pump located in the basolateral membrane. There is also evidence for an increase in the apical membrane Cl(-) conductance.

Effect of pentachlorophenol (PCP) on frog cornea epithelium.

Pentachlorophenol (PCP) is a toxic substance that affects many tissues adversely. Present experiments, using an in vitro preparation, were designed to study whether PCP affected the electrophysiological parameters of the bullfrog cornea epithelium, specifically, the Na+/K+ ATPase pump and the K+ conductance located in the basolateral membrane and the Cl- conductance located in the apical membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (Isc) conditions. Addition of PCP to a concentration of 5 x 10-5 M to the tear solution gave a marked decrease in Isc; a marked depolarization of the intracellular potential, Vo; and minimal but significant decreases in the apical membrane fractional resistance, fRo, and in the transepithelial conductance, gt. Isc experiments in Cl--free solutions with amphotericin B in the tear solution confirm results indicating that PCP inhibits the active transepithelial transport mechanism and produces a small increase in the basolateral membrane resistance due to a decrease in the K+ conductance.

Effect of melittin on PD, resistance and short-circuit current in the frog gastric mucosa.

In an in-vitro preparation of gastric mucosae of Rana pipiens, the effect of adding melittin to a concentration of 5x10-6 M in the secretory solution on the transepithelial potential difference (PD), resistance (R) and short-circuit current (Isc) was studied. In 20 min, melittin decreased the PD by 9.3 mV and R by 148 ohm cm2. These changes can be explained by a decrease in the resistance, RP, of the paracellular pathway. To determine whether specific-ion pathways were responsible for the decrease in R, the effect of melittin on the partial conductances of Cl-, K+ and Na+ was also studied using the ion substitution method. Melittin decreased the PD response to changes in nutrient Na+, K+ and Cl- and the PD response to changes in secretory Cl-, but did not affect PD responses to changes in secretory Na+ or K+. Therefore, melittin decreased the nutrient membrane partial conductances of Cl-, K+ and Na+ and secretory membrane partial conductance of Cl-, without affecting the secretory partial conductances of Na+ or K+. Initially, melittin increased Isc in regular and Cl--free but not in Na+-free solutions. There was a delayed decrease in Isc. The results indicate that melittin decreases RP, increases the Na+ conductance of the secretory membrane and inhibits, eventually, the Na+/K+-ATPase pump.

Effect of melittin on the apical membrane Na+ and Cl- conductances of frog cornea epithelium.

The transepithelial conductance increased with 10(-6) M melittin on the tear side of the frog cornea. This effect was attributed to the opening of an apical membrane Na+ conductance. However, the potency of the venom at this concentration (apical membrane fractional resistance (fRo) decreased to near zero) masked this and other effects. With 3 x 10(-7) M on the tear side, the effects were finite (fRo > 0) and reversible. With fRo > 0, the effects of melittin could be readily studied on the transport parameters of Na+, Cl-, and K+. Epithelial cells of intact bullfrog corneas were impaled with microelectrodes using an in vitro preparation. Under short-circuit current (Isc) conditions, 20 min after melittin Isc increased by 3.1 from 8.2 microA/cm2., fRo decreased by 18 from 51%; the intracellular potential, Vo, depolarized by 19.4 from -56.5 mV; and the transepithelial conductance, gv increased by 0.57 from 0.29 mS/cm2. Tenfold decreases in tear Na+ or Cl- concentrations changed the transport parameters consistent with the formation of a Na+ conductance and an increase in the apical membrane Cl- conductance by the venom. These conclusions were further supported by the minimal effect of melittin in Cl(-)-fre and, particularly, in Na(+)-free solutions. Changes in K+ concentration had no effect on transport parameters. These findings indicate that the effect of melittin at this low concentration is upon a Na+ channel protein and not due to nonspecific conductances.

Effect of melittin on ion transport across cell membranes.

Extensive work with melittin has shown that the venom has multiple effects, probably, as a result of its interaction with negatively changed phospholipids. It inhibits well known transport pumps such as the Na(+)-K(+)-ATPase and the H(+)-K(+)-ATPase. Melittin increases the permeability of cell membranes to ions, particularly Na+ and indirectly Ca2+, because of the Na(+)-Ca(2+)-exchange. This effect results in marked morphological and functional changes, particularly in excitable tissues such as cardiac myocytes. In some other tissues, e.g., cornea, not only Na+ but Cl- permeability is also increased by melittin. Similar effects to melittin on H(+)-K(+)-ATPase have been found with the synthetic amphipathic polypeptide Trp-3.

Effect of acetazolamide and melittin on polarization of the frog gastric mucosa proton pump.

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 change in open circuit potential difference (OCPD) induced by voltage clamping. This transmucosal polarization was markedly reduced by H2 blockers famotidine and cimetidine, and by the H+/K+-ATPase inhibitors omeprazole and SCH 28080. SCN-, a nonspecific H+ secretion inhibitor, did not affect the polarization. In the present experiments, the effects of two other inhibitors of H+ secretion were examined, namely, acetazolamide (AA), a carbonic anhydrase inhibitor, and melittin (MEL), an inhibitor of the H+/K+-ATPase enzyme. When AA 10(-3) M or MEL 10(-5) M was added to the nutrient solution, H+ secretion was completely inhibited. While MEL markedly reduced the polarization induced by voltage clamp, AA did not affect the polarization. These data support the concept that MEL directly affects the electrogenic H+/K+-ATPase pump while the inhibition of H+ secretion by AA is by an indirect mechanism. The data further support the electrogenicity of the H+/K+-ATPase.

Effect of melittin on electrophysiological parameters in the frog cornea epithelium.

The effect of melittin on electrophysiological parameters of the bullfrog cornea was studied using an in vitro preparation. Epithelial cells of corneas were impaled with microelectrodes. Experiments were done under short-circuit current (Isc) conditions. Melittin was added in concentrations of 10(-5)M to the stromal or 10(-6)M to the tear solution. The effects of melittin were as follows: (i) stromal side, a decrease in Isc; an increase in the apical membrane fractional resistance, fRo; no change in the transepithelial conductance, gt; and a depolarization of the intracellular potential, Vo; (ii) tear side, an initial (first 10 min) increase and then a decrease in Isc; a decrease in fRo; an initial (first 10 min) increase with subsequent small decrease in gt; and a depolarization of Vo. Changes in tear Na+, but not in tear K+, with melittin present in tear solution, induced changes in some electrical parameters. The effects on the tear side may be explained by opening of nonspecific channels in the apical membrane with some specificity for Na+ channel. The subsequent effects on the tear and the effects on the stromal side may be explained by an inhibition of the primary transport system, that is, of the Na+/K+ -ATPase pump located in the basolateral membrane. In these experiments, there was no evidence of opening of channels in the basolateral membrane.

Effects of nutrient HCO3- and Cl- on potential difference responses to nutrient Na+ changes in secreting and inhibited states of frog stomach.

Previously, it was found that a decrease in nutrient (serosal) Na+ decreased the transmucosal potential difference (PD) in the secreting and inhibited states of fundi of stomachs of Rana pipiens. This PD response, designated as anomalous, was explained by two different models, an electrogenic NaCl symport and an electrogenic NaHCO3 symport. To test these hypotheses, experiments were done at pH approximately 7.4 in which the PD response to a change in nutrient Na+ was compared in 25, 5, and 0 mM nutrient HCO3- solutions. In the H+ secreting state, all HCO3- concentrations gave, for a 10-fold decrease in nutrient Na+, about the same PD response (9-12 mV). The PD response was 8.5, 2.9, and 0 mV higher in the inhibited state for 25, 5, and 0 mM HCO3- , respectively. Experiments with decreasing nutrient Cl- decreased the anomalous PD response to Na+ changes, and the response became insignificant at Cl- concentrations of 20 mM or less in the secreting state. The anomalous PD response to a decrease in Na+ concentration was obtained for all Cl- concentrations (0-79 mM), becoming less with decreasing Cl- in the inhibited state. These results support a NaCl symport to explain the PD response in the secreting state and a NaHCO3 symport to explain the enhancement in the inhibited state.

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.

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.

Effect of inhibitors of gastric secretion on polarization of gastric potential by voltage clamping.

Voltage clamping across the frog gastric mucosa, nutrient positive to secretory, results in an increase in the open-circuit potential difference (OCPD). The increase in OCPD, or polarization, induced by voltage clamping could be caused by a redistribution of ions across the plasma membrane or by a polarization of pump electromotive forces, such as the Na(+)-K(+)-adenosinetriphosphatase (ATPase) located in the nutrient (serosal) membrane or the proton pump located in the secretory (luminal) membrane. The polarization induced by voltage clamping was not affected by removing Cl- and Na+ or by increasing the K+ concentration to 79 mM but was markedly reduced by placing in the nutrient solution 10(-3) M famotidine or 10(-4) M omeprazole. These data suggest that there is a strong contribution of the proton pump to the polarization induced by voltage clamping with some contribution of ion redistribution and/or the Na(+)-K(+)-ATPase. The data support the electrogenicity of the proton pump.

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+.

Further evidence for a simple Cl- conductance pathway in nutrient membrane of frog stomach.

A decrease in nutrient Cl- increases the negativity of the nutrient relative to the secretory side. It seemed possible that Cl- transport could result from a neutral Cl- mechanism in the nutrient membrane coupled to a simple Cl- conductance pathway in the secretory membrane. Experiments in HCO3(-)-free, K(+)-free and Na(+)-free solutions in both bathing media gave for a 10-fold change in nutrient Cl- a PD change of 9.5 mV. Similar experiments with 0.5 mM DIDS in the nutrient solution gave for a 10-fold change in nutrient Cl- a PD change of 7.9 mV. These experiments eliminated a neutral Cl(-)-HCO3- exchanger, a NaCl and a KCl symport. Thus the change in PD could best be explained by a simple Cl- conductance in the nutrient membrane.

Role of polarization of gastric EMFs in evaluation of contemporary HCl models.

The neutral proton pump (NP) model postulates a neutral exchange of K+ for H+ across the secretory membrane and the electrogenic proton pump (EP) model an electrogenic proton pump. Previous evidence is briefly reviewed and polarization of EMFs by transmucosal voltage clamping (VC) is presented. During VC, open circuit potential difference (PD) (VOC) is obtained by breaking the circuit for 2 s (after dielectric capacitors have discharged). The magnitude of polarization in Cl- media is less than in Cl(-)-free media, presumably due to the high conductance of Cl- paths. The magnitude in Cl(-)-free media is from 35 to 50 mV for a VC of 100 mV (nutrient side positive). The Na+/K(+)-ATPase is not essential because with choline sulphate media polarization is typical. With Cl(-)-free media, VOC versus IH (H+ rate) is exponential but (VC-VOC) versus IH is linear. Polarization on the basis of the NP model would be due to changes in K+ diffusion potentials. However, with 80 mM K+ on both sides (Cl(-)-free media) polarization is typical. We conclude that polarization cannot be due to a change in K+ diffusion potentials but to polarization of the EP model. The problem remains of how to incorporate the important finding of the H+/K(+)-ATPase into a model for the intact tissue.

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.

Effect of loop diuretics on bullfrog cornea epithelium.

The effect of loop diuretics on Cl transport was studied on an in vitro preparation of the bullfrog cornea. Bumetanide (10(-4) M) or furosemide (10(-3) M) added to the stromal solution decreased Cl transport measured as the short-circuit current (Isc) to values near zero. Concomitantly, transepithelial conductance (gt) decreased, whereas the intracellular potential (Vo) hyperpolarized and the fractional resistance of the apical membrane (fRo) increased. Substitution of SO4 for Cl in the tear-side solution led to prompt changes in Isc, gt, Vo, and fRo, characteristic of appreciable passive Cl movement across the apical membrane before and after inhibition. Epinephrine (10(-4) M) was similarly effective on apical membrane conductance in inhibited tissues as under control conditions, but the effective electromotive force for transepithelial Cl transport was reduced to approximately 25%. Intracellular Cl activity, measured with ion-selective microelectrodes, decreased so much that the difference in electrochemical Cl potential divided by the Faraday constant (delta mu Cl/F) was close to zero after inhibition of Isc by bumetanide. Apical Cl permeability remained essentially unchanged. Accordingly, loop diuretics inhibit Cl transport in the Cl-secreting cornea epithelium by blocking the Na-Cl symport without secondary apical effects, as believed for other Cl-reabsorbing epithelia.

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.

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.