NPPB inhibits the basolateral membrane K+ conductance in the isolated bullfrog cornea.

The effects of the Cl- channel blocker, 4-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on active transepithelial Cl- transport were measured in the isolated bullfrog cornea. With a Cl(-)-free Ringers, stromal-side 10(-5) M NPPB elicited a maximum depolarization of the membrane voltage from -72 +/- 6 to -48 +/- 9 mV (n = 6, P less than 0.05) and reduced the magnitude of the depolarization induced by a 10-fold increase in K+ concentration. Subsequent exposure to 10(-4) M ouabain decreased the membrane voltage from -41 +/- 6 mV to -25 +/- 2 mV (n = 6, P less than 0.05). After stimulation with 10(-5) M amphotericin B of a short-circuit current, Isc, largely accounted for by tear to stroma K+ diffusion, this Isc was effectively inhibited by 10(-5) M NPPB on the stromal-side. This decrease reflected a fall in basolateral membrane K+ conductance. In NaCl Ringers, inhibition of the essentially Cl(-)-originated Isc either on the tear- or stromal-sides required instead 10(-4) M NPPB. NPPB depolarized the membrane voltage from -55 +/- 7 to -38 +/- 6 mV (n = 14, P less than 0.05). The direction of the change in the fractional apical membrane resistance (fRo) depended upon its initial value; in those corneas with a lower value it increased whereas if they had a higher fRo, 10(-4) M NPPB consistently caused fRo to fall. However, following exposure to 5 x 10(-3) M Ba2+ and a fall in fRo, NPPB consistently caused fRo to increase significantly from 30 +/- 8 to 53 +/- 4% (n = 5). Therefore, inhibition of active Cl- transport by 10(-4) M NPPB may be associated with declines in: (1) a basolateral membrane K+ conductance that is distinct from a Ba2(+)-sensitive pathway; (2) an apical membrane Cl- conductance. Neither of these effects may be the result of a direct effect of NPPB on a conductance pathway because: (1) the drug was equipotent from either bathing solution; (2) following a one hour washout the Isc had not fully recovered to its control value.

Secretagogues increase apical membrane conductance of isolated bullfrog corneal epithelium pretreated with loop diuretics.

Bullfrog (Rana catesbeiana) corneas were mounted in an Ussing type chamber and impaled with an intracellular microelectrode and the short circuit current was inhibited by pretreatment with the loop diuretics furosemide (0.3 to 1 mM) or bumetanide (10 to 100 microM). Subsequent addition of the secretagogues prostaglandin E2, forskolin, or 3-isobutyl-1-methylxanthine (IBMX) caused the fractional voltage drop of the apical barrier to decrease from 0.72 +/- 0.05 to 0.48 +/- 0.04 and the chloride-dependent conductance to increase by 0.15 +/- 0.03 mS/cm2, but caused only a small, transient increase in short circuit current. The loop diuretics by themselves always greatly reduced the short circuit current but did not consistently reduce conductance or fractional voltage drop of the apical membrane. Because the secretagogues were able to increase the apical membrane conductance of diuretic-inhibited corneas without large effects on the short circuit current, the loop diuretics must have a major effect at a site other than the apical membrane Cl- conductance, presumably at the basolateral membrane. An additional effect of the loop diuretics at the apical membrane is also possible.

Effects of oxytocin on cation content and electrophysiology of frog skin epithelium.

We measured effects of oxytocin on current-voltage (I-V) relations of frog (Rana catesbeiana) skins impaled with an intracellular microelectrode. In both Cl- and Cl(-)-free (SO4(2-) solutions, oxytocin caused an approximate doubling short-circuit current (Isc) and a depolarization of the cell membrane. Increase in apical membrane slope conductance, chord conductance, and permeability after oxytocin correlated with the increase in amiloride-sensitive Isc. Oxytocin also increased basolateral membrane conductance (gb). In Cl-, the shift in the voltage intercept of the apical membrane I-V relation (Ea) implied increased intracellular Na+ activity (a(Na)c) after oxytocin. In isolated frog skin epithelia, a similar increase in intracellular [Na+] after oxytocin was demonstrated by flame photometry. In SO4(2-), changes caused by oxytocin in both Ea and in flame photometrically determined cell [Na+] were minimal. The voltage intercept of the basolateral membrane I-V relations (Eb) was shifted by oxytocin in both Cl- and SO4(2-) solutions. Assuming that the basolateral membrane is selectively permeable to K+, changes in K+ obtained from Eb were in disagreement with those obtained by flame photometry. These results suggest that 1) the increase in a(Na)c caused by oxytocin is not essential to produce either the increase in gb or Isc and 2) ions other than K+ make an important contribution to basolateral membrane conductance.

Insulin action on electrophysiological properties of apical and basolateral membranes of frog skin.

We measured the effects of insulin on the current-voltage (I-V) relations of frog skins impaled with an intracellular microelectrode. The current across the cell membranes was assumed to be equal to the amiloride-inhibitable current. Insulin increased short-circuit current (Isc) approximately 40% from the control value. The increase in Isc was associated with a depolarization of the cell membrane. In addition there was an increase in the value of the parameters that describe the ease of movement of Na+ across the apical membrane, namely, slope conductance (ga), chord conductance (Ga), and permeability (PNa). The values of these parameters show remarkable linear correlations with membrane current both before and after stimulation. Intracellular Na+ activity (acNa) was determined from the I-V relations of the apical membrane. Insulin did not significantly modify acNa. Insulin also increased the value of the basolateral membrane conductance, however, the relationship between this parameter and current was complex. These experiments show that the stimulatory effect of insulin on Isc is associated with an increase in the conductance of both the apical and basolateral membranes.

Role of prostaglandin release in the response of tight epithelia to Ca2+ ionophores.

We have studied the effects of the Ca2+ ionophores A23187 and ionomycin on ion transport across amphibian skin and urinary bladder. Both A23187 and ionomycin stimulated transepithelial Na+ transport across the skin. Ionomycin also markedly increased the conductance of an amiloride-insensitive pathway. Both ionophores markedly stimulated the release of prostaglandin E2 (PGE2) into the solution bathing the serosal surface of the skin. Addition of indomethacin to the serosal bathing solution of the skin blocked both the stimulation of short-circuit current (Isc) and the release of prostaglandin caused by the ionophores. Acetylsalicylic acid also blocked the ionomycin-induced stimulation of Isc. These results suggest that the stimulation of Na+ transport caused by Ca2+ ionophores is mediated by the release of a product of the cyclooxygenase pathway, very likely PGE2. Ca2+ ionophores also stimulated the release of PGE2 in urinary bladders; however, they generally depressed Isc. Since the effect on Isc caused by the addition of exogenous PGE2 was different in urinary bladders than in skins, we suggest that at least part of the difference in the action of ionophores is due to the difference in the sensitivity of these epithelia to PGE2. Our results suggest that the heterogeneity of effects that Ca2+ ionophores cause in the physiological parameters of tight epithelia are not always the direct result of increased cytoplasmic Ca2+ but that they may be mediated by other tissue responses triggered by the addition of the ionophores.

Current-voltage relations of the apical and basolateral membranes of the frog skin.

We determined the current-voltage (I-V) relations of the apical and basolateral barriers of frog skins by impaling the cells with an intracellular microelectrode and assuming that the current across the cellular pathway was equal to the amiloride-inhibitable current. We found that: (a) The responses in transepithelial current and intracellular potential to square pulses of transepithelial potential (VT) varied markedly with time. (b) As a consequence of these transient responses, the basolateral I-V relation was markedly dependent on the time of sampling after the beginning of each pulse. The apical I-V plot was much less sensitive to the time of sampling within the pulse. (c) The I-V data for the apical barrier approximated the I-V relations calculated from the Goldman constant field equation over a relatively wide range of membrane potentials (+/- 100 mV). (d) A sudden reduction in apical bath [Na+] resulted in an increase in apical permeability and a shift in the apical barrier zero-current potential (Ea) toward less positive values. The shift in Ea was equivalent to a change of 45 mV for a 10-fold change in apical [Na+]. (e) The transient responses of the skin to square VT pulses were described by the sum of two exponentials with time constants of 114 and 1,563 ms, which are compatible with the time constants that would be produced by an RC circuit with capacitances of 65 and 1,718 microF. The larger capacitance is too large to identify it comfortably with a true dielectric membrane capacitance.

Basolateral membrane responses to transport modifiers in the frog skin epithelium.

Application of transepithelial square voltage pulses to the frog skin leads to responses in the transepithelial current and intracellular potential which include transient components. Determinations at 600 ms allow for meaningful estimates of basolateral membrane responses to transport modifiers. Oxytocin produced a large and sustained increase in the amiloride-inhibitable short circuit current (Im) which was accompanied by a large increase of both apical and basolateral membrane conductance (ga and gb, respectively). While Im and ga increased nearly simultaneously, gb started to increase several minutes after the increase in the two other parameters. Insulin also increased Im, ga and gb. As with oxytocin, the increases in Im and ga often preceded the changes in gb. Ouabain reduced Im and ga. The effects on gb were more complex, since sometimes the inhibition of Im was first accompanied by an increase followed by a decrease while in other instances only minor changes in conductance could be observed. The currently available information regarding the control of cytoplasmic [Ca2+] and the effects of Ca2+ on cell membrane properties are used to construct a model in which changes in cytoplasmic [Ca2+] account for the observed behavior of the basolateral membrane.

Chloride transport inhibition by piretanide and MK-196 in bullfrog corneal epithelium.

Two new diuretic agents, piretanide and MK-196, inhibited short-circuit current (SCC) across the isolated frog corneal epithelium. The effect is explained as an inhibition of active Cl transport. A definite decrease in SCC and an increase in electrical resistance was observed with both diuretics in concentrations as low as 10(-6) M. Piretanide, at 10(-4) M, reduced the SCC by 90%, reduced th unidirectional forward Cl flux from 0.60 to 0.28 mueq x h-1 x cm-2, and increased the resistance by 60%. There was no effect on the Cl backflux. At 10(-4) M, MK-196 reduced the SCC by 83% and increased the resistance by 72%, from 1.68 to 2.91 k omega x cm2. Replacement of Cl by SO4 in the bathing solutions resulted in a larger increase in resistance, from 1.68 to 3.80 k omega x cm2. The diuretics had no effect on active Na transport across the corneal epithelium. After the permeability of the apical side was increased by amphotericin B, the drugs could not inhibit the Cl-originated SCC. These results suggest that piretanide and MK-196 selectively inhibit active Cl transport in the cornea by blocking Cl permeability of the apical side of the epithelial cells.

Metabolic requirements for anaerobic active Cl and Na transport in the bullfrog cornea.

In the bullfrog cornea, the relationships between the rates of aerobic and anaerobic glycolysis and active Cl and Na transport were studied. In NaCl Ringer (glucose-free), the short-circuit current (SCC) declined much more slowly under aerobic than under anaerobic conditions. The aerobic lactate effluxes in glucose-free and glucose-rich NaCl Ringer were 0.08 and 0.23 micromol/h.cm2, respectively. The transition to anoxia caused these values to increase significantly and was accompanied by depletion of endogenous glycogen in glucose-free Ringer. In Na2SO4 Ringer, amphotericin B (10(-5) M) stimulation of the aerobic SCC was not dependent on the presence of glucose but under anoxia, SCC stimulation required glucose. In Na2SO4 (glucose-rich) Ringer, amphotericin B stimulated the aerobic lactate efflux from 0.26 to 0.36 mumol/h.cm2 and anoxia increased it to 0.55 micromol/h.cm2. In NaCl Ringer, the addition of either 0.5 mM adenosine or 1 mM ATP with 26 mM glucose restored the anaerobic-inhibited SCC and lactate efflux of glucose-depleted corneas. The results show that the reactions of glycolysis are a sufficient energy source for supporting active Na and Cl transport.

An inexpensive, high output voltage, voltage clamp for epithelial membranes.

A circuit is described that can produce a high output voltage to automatically short-circuit an epithelial tissue mounted in an Ussing chamber. Because of its high output voltage it can short-circuit preparations requiring over 500 muA even in conjunction with high-resistance agar bridges. The circuit, which is easy to build, uses an inexpensive, low-voltage, integrated circuit operational amplifier, which is electrically isolated from the high-voltage part of the circuit by an optical isolator. The device can also be modified for use as a high impedance preamplifier for monitoring the spontaneous membrane potential.

Selective effects of bumetanide on chloride transport in bullfrog cornea.

Frog corneas were mounted in a modified Ussing chamber and short-circuit current (SCC) and unidirectional Cl fluxes were measured. Bumetanide, a loop diuretic, at concentrations as low as 10(-7) M, reduced the SCC 29%. At 10(-5) M, bumetanide reduced the SCC 96% and increased transcorneal electrical resistance 20-51%. The forward Cl flux declined from 0.71 +/- 0.04 to 0.20 +/- 0.03 mueq/h.cm2 (n, 7), while, in separate experiments, the backward Cl flux did not change significantly (from 0.22 +/- 0.03 to 0.23 +/- 0.04; n, 7). When corneas were mounted in Cl-free Ringer and the net Na transport was stimulated with amphotericin B, 10(-5) M bumetanide had no effect on the SCC. In separate experiments the effect of 10(-5) M bumetanide on the O2 consumption was measured in a stirrer bath assembly. Bumetanide decreased the O2 consumption from 352 +/- 14 to 297 +/- 19 microliter/h.cm2 (significantly different from sham-treated controls). This decrease was similar to that obtained with furosemide or when Cl was removed from the bathing medium. We infer from these results that bumetanide is a selective inhibitor of active Cl transport in the bullfrog cornea.

Prevention of 5-fluorouracil-caused growth inhibition in Sordaria fimicola.

Growth (dry weight accumulation) of Sordaria fimicola in standing liquid culture (sucrose-nitrate-salts-vitamins) is inhibited by the presence of 5 muM 5-fluorouracil in the medium. This inhibition is completely prevented by uracil, deoxyuridine, and 5-bromouracil, partly prevented (40 to 90% of growth observed without 5-fluorouracil) by uridine, thymidine, and 5-bromodeoxyuridine, and slightly prevented by trifluorothymine, cytosine, cytidine, deoxycytidine, and 5-methylcytosine (all at 0.5 to 1 mM). Thymidine and thymine riboside were without any apparent effect. Growth is also inhibited by 0.2 mM 6-azauracil, and this inhibition was completely prevented by uracil and uridine, partly prevented by deoxyuridine, 5-bromouracil, cytidine, and 5-methylcytosine, and slightly prevented by thymine, thymidine, 5-bromodeoxyuridine, cytosine, and deoxycytidine. The data suggest that the observed inhibition of growth by 5-fluorouracil is due to inhibition of both ribonucleic acid and deoxyribonucleic acid synthesis. The data also allow inferences concerning pyrimidine interconversions in S. fimicola; i.e., thymine can be anabolized to thymidylic acid without first being demethylated, although demethylation appears to occur also.

Selective effects of purine and pyrimidine analogues and of respiratory inhibitors on perithecial development and branching in sordaria.

The initiation of perithecia in the homothallic ascomycete Sordaria fimicola was completely suppressed, without seriously inhibiting vegetative growth, by growing the fungus on an agar medium containing one of the following additions: 1) 1 mum 5-fluorouracil, 2) 10 to 100 mum 6-azauracil, 8-azaguanine or 8-azaadenine, 3) 50 to 500 mum cyanide or azide, 4) 5% (w/v) casein hydrolysate. In contrast to the selective activity of the analogues of 3 RNA bases, whose inhibition could be reversed by the appropriate normal bases only, none of the analogues of thymine were active, neither were the thio-derivatives of RNA bases. Other inhibitors of RNA and protein synthesis, like actinomycin D, puromycin and cycloheximide, were also without selective activity, although the last of these inhibited perithecial maturation at 0.1 mum concentration but not initiation. Amino acid analogues were inactive, as were the metabolic inhibitors thiourea, 2,4-dinitrophenol and fluoride. The compounds which inhibited the formation of perithecia also lowered the branching frequency of leading hyphae, but not their linear growth rates. Consequently, the branch densities were diminished in their presence. Hypotheses to account for these findings are discussed in terms of inhibition of growth in general, of the synthesis of some specific messenger RNAs, and of RNA-mediated transport across membranes, the last of which seeming the most fruitful for further work.