Peter S. Reinach

Transepithelial electrical measurements on the isolated rabbit iris-ciliary body

Transmural electrical properties of the isolated rabbit iris-ciliary body (I-CB) were measured in Ussing-Zerahn-type chambers. Control p.d. across the preparation was -1.2 +/- 0.1 mV, with the ciliary process (aqueous)-side consistently negative with respect to the ciliary body (blood)-side and the short-circuit current (SCC) was 7.9 +/- 0.6 microA cm-2. Bilateral bathing solution substitutions demonstrated absolute requirements for the presence of Na+, K+ and HCO3-for the maintenance of the p.d. Addition of 5 X 10(-5) M ouabain to the aqueous-side chamber increased the p.d. and SCC initially, with a subsequent decline to zero. Only a declining phase was observed when ouabain was added to the blood-side. Ouabain inhibited oxygen consumption by 28% in Tyrodes solution. Respiratory rate was also approximately 28% lower in Na+-free and K+-free Tyrodes solution and ouabain had no additional inhibitory effect in either of these two solutions. Thus the biphasic effect of ouabain on the electrical parameters cannot be explained by a toxic effect but rather as a selective inhibition of the Na+-, K+-pump. Our results indicate that Na+-, K+-ATPase activity and the presence of HCO3- are required for active ion transport in this preparation.

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

SummaryActive 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.

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.

Basolateral Membrane K Permselectivity and Regulation in Bullfrog Cornea Epithelium

SummaryIn the isolated bullfrog cornea epithelium, under short-circuit conditions the regulation of the K permeability of the basolateral membrane was studied with conventional and K-selective microelectrodes in Cl-free Ringers In Cl-free Ringers, the transcellular current is less than 1 μA/cm2, allowing estimation of the basolateral membrane electromotive force from measurements of the membrane voltage (Vsc). The apparent basolateral membrane K conductance was determined from measurements of the effects of single ion substitutions of K for Na on theVsc. An increase of K from 2.5 to 25mm on the stromal side depolarized the membrane voltage by 29 mV, whereas additional increases to 56 and 100mm resulted in depolarizations consistent with a Nernstian prediction. In the range between 25 and 56mm K, these decreases in membrane voltage were smaller after either decreasing the stromal-side pH from 8.1 to 7.2 or substitution of sulfate with gluconate. In contrast, preincubation with 0.1mm oubain did not change the membrane voltage depolarizations over any of the K ranges between 2.5 and 100mm. Equivalent circuit analysis, based on the effects of nystatin on the electrical parameters, was, used to validate the changes in the apparent basolateral, membrane K conductance following increases in [K], substitution of SO4 with gluconate and Na:K pump inhibition. An increase in the [K] to 120mm decreased the basolateral membrane resistance nearly three-fold, whereas gluconate substitution resulted in a 2.5-fold increase of the basolateral membrane resistance. This resistance increased an additional 2-fold after exposure to 5mm Ba. However, exposure to 0.1mm oubain had no significant effect on this resistance. Therefore, there is an agreement between the results of circuit analysis and the magnitude of membrane voltage depolarization resulting from increases in [K], gluconate substitution and pump inhibition with ouabain. Na:K pump inhibition with ouabain caused the K activity to decline slightly after 30 min from 98±7 to 83±8mm, which is consistent with a small basolateral membrane Na conductance. The estimated K permeability ranged from 3.7×10−7 to 1.1×10−6 cm/sec. The less than Nernstian predicted decline of the membrane voltage between 2.5 and 25mm K and the small basolateral membrane Na conductance suggest that the basolateral membrane is also permeable to another unknown ion.

Implications of an anomalous intracellular electrical response in bullfrog corneal epithelium

SummaryThe ionic dependencies of the transepithelial and intracellular electrical parameters were measured in the isolated frog cornea. In NaCl Ringers the intracellular potential differenceVsc measured under short-circuit conditions depolarized by nearly the same amount after either increasing the stromal-side KCl concentration from 2.5 to 25mm or exposure to 2mm BaCl2 (K+ channel blocker). With Ba2+ the depolarization of theVsc by 25mm K+ was reduced to one-quarter of the control change. If the Cl-permselective apical membrane resistanceRo remained unchanged, the relative basolateral membrane resistanceRi, which includes the lateral intercellular space, increased at the most by less than twofold after Ba2+. These effects in conjunction with the depolarization of theVsc by 62 mV after increasing the stromal-side K+ from 2.5 to 100mm in Cl-free Ringers as well as the increase of the apparent ratio of membrane resistances (a=Ro/Ri) from 13 to 32 are all indicative of an appreciable basolateral membrane K+ conductance. This ratio decreased significantly after exposure to either 25mm K+ or Ba2+. The decline ofRo/Ri with 25mm K+ appears to be anomalous since this decrease is not consistent with just an increase of basolateral membrane conductance by 25mm K+, but rather perhaps a larger decrease ofRo thanRiAlso an increase of lateral space resistance may offset the effect of decreasingRi with 25mm K+. In contrast,Ro/Ri did transiently increase during voltage clamping of the apical membrane potential differenceVo and exposure to 25mm K+ on the stromal side. This increase and subsequent decrease ofRo/Ri supports the idea that increases in stromal K+ concentration may produce secondary membrane resistance changes. These effects onRo/Ri show that the presence of asymmetric ionic conductance properties in the apical and basolateral membranes can limit the interpretative value of this parameter. The complete substitution of Na+ withn-methyl-glucamine in Cl-free Ringers on the stromal side hyperpolarized theVsc by 6 mV whereas 10−4m ouabain depolarized theVsc by 7 mV. Thus the basolateral membrane contains K+, Na+ and perhaps Cl− pathways in parallel with the Na/K pump component.

Roles of cyclic AMP and Ca in epithelial ion transport across corneal epithelium: A review

The messenger roles of cyclic AMP and the calcium ion in stimulus-secretion coupling are considered in the frog and bovine corneal epithelium, respectively. In the frog cornea, epinephrine stimulates net C1 transport by increasing cyclic AMP content. This stimulation is associated with a larger apical membrane C1 conductance and basolateral membrane ionic conductance. The response of the apical membrane conductance is thought to result from an increase in cyclic AMP content whereas the basolateral membrane ionic conductance increase is unrelated based on measurements of the effects of the calcium channel antagonist, diltiazem, and the beta agonist, isoproterenol, on the electrical parameters and cyclic AMP content. The basolateral membrane is essentially K permselective since the K channel blocker, Ba, depolarized the intracellular potential difference and increased the basolateral membrane resistance. Diltiazem had even larger effects on these parameters suggesting that this compound is a more effective inhibitor of K channel activity than barium. In broken cell preparations of bovine corneal epithelium, a high affinity form of Ca + Mg activated ATPase is present (Km = .06 microM for Ca) and is essentially of plasma membrane origin. This ATPase activation is at a Ca activity similar to the expected intracellular value and suggests that this activity is the enzymatic basis for net Ca transport.

Identification of calmodulin-sensitive Ca2+-transporting ATPase in the plasma membrane of bovine corneal epithelial cell

ATP-dependent Ca2+ uptake was characterized in a plasma membrane enriched fraction obtained from the bovine corneal epithelium. This uptake essentially represented intravesicular accumulation because 72% of the Ca2+ content was releasable following exposure to 10(-6) M A23187. The substrate and Ca2+ requirements for maximal transport activity were similar to those described in the red blood cell because: (1) exogenous calmodulin (3 microM) significantly decreased the apparent Km for Ca2+ to 0.31 microM and increased the rate of Ca2+ uptake; (2) a hydroxylamine labile Ca(2+)-dependent phosphoenzyme intermediate was identified with an apparent molecular size of 140 kDa; (3) Ca(2+)-dependent binding of 125I-labelled calmodulin to this protein was demonstrated which could be antagonized with a calmodulin antagonist, trifluoperazine. These results show that the plasma membrane contains an ATP-dependent Ca2+ transporter. However, its relationship to a previously described high affinity form of Ca(2+)-stimulated Mg(2+)-dependent ATPase is not apparent because their [Mg2+] requirements to elicit maximal activity differed by two orders of magnitude.

Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists.

SummaryIn the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazems inhibitory efficacy. This antagonist depolarized the intracellular potential differenceVm from −54 to −18 mV (tear: reference) and the voltage divider ratioFR0 decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 105m amphotericin B in Cl-free Ringers did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringers (SCC=0), 1 ×103m diltiazem depolarized theVm from −78 to −9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×103m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×103m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.

Calcium Does Not Act as a Second Messenger for Adrenergic and Cholinergic Agonists in Corneal Epithelial Cells

The role of changes in intracellular [Ca2+]i as a second messenger in response to either adrenergic or cholinergic agonists was determined in isolated bovine corneal epithelial cells. [Ca2+]i was measured in suspensions of cells loaded with either of the fluorescent indicators quin2 or indo-1, as well as in single cells loaded with fura-2. Fluorescence from the cell suspensions was measured in a spectrofluorometer while single cell fluorescence was measured using a modified fluorescence microscope with a photon counting photometer. Cells were loaded with these dyes by incubation in Ringers (pH 8.1) containing 2-50 microM of the acetoxymethyl ester of the indicator. Fluorescence was measured before and after exposure to either, one of the adrenergic agonists isoproterenol, phenylephrine or epinephrine, or the cholinergic agonist carbachol. The resting [Ca2+]i level from the quin2 experiments was 115 nM +/- 41 nM (SEM) (n = 23) whereas with fura-2 it was 71 +/- 10 nM (n = 30). In no case did we see any change in [Ca2+]i within 15 min after addition of any agonist but we were able to observe increased calcium when 0.5 microM ionomycin was added to either the same or untreated cells. The disparity in the resting levels determined by the two methods may result from various calibration problems. Our results indicate that changes in [Ca2+]i have no second messenger role in response to these agonists.

Changes in cellular membrane and paracellular conductances by amphotericin B in the epithelium of the bullfrog cornea

In the isolated bullfrog cornea, measurements of DC electrical parameters in conjunction with AC impedance and ultrastructural analyses were used to determine the effects of 10(-5) M amphotericin B on epithelial cellular membrane and paracellular conductances. In NaCl Ringers, amphotericin B elicited a 3.5-fold increase in the specific apical membrane conductance (Ga/Ca); where Ga and Ca are the apical membrane conductance and capacitance, respectively. The basolateral membrane conductance (Gb) and the basolateral membrane capacitance (Cb) fell by 57% and 50%, respectively. In the paracellular pathway, the tight junctional complex (Gj) was unchanged whereas the lateral intercellular space resistance (Rp) decreased by 55%. The declines in Gb and Cb were suggestive of cell volume shrinkage because these changes were consistent with a previously described decline in intracellular K+ content and reduction in exposed basolateral membrane area to current flow. Ultrastructural analysis validated that amphotericin B caused cell volume shrinkage because there was: (1) increased folding of the basolateral membrane and waviness of the basal aspects- of the plasma membrane; (2) dilatation of the lateral intercellular spaces. This agreement suggests that intracellular activity decreased following exposure to amphotericin B which resulted in cell volume shrinkage and an impairment of Cl- uptake across the basolateral membrane.