Transepithelial Response Research Articles

Swelling-Activated Cl−Channels Support Cl−Secretion by Bovine Ciliary Epithelium

To determine whether swelling-activated Cl- currents (ICl,swell) observed in isolated nonpigmented ciliary epithelial (NPE) cells contribute to Cl- secretion across the ciliary epithelium. Ion transport across intact bovine ciliary epithelium was monitored electrically. Native isolated bovine NPE cells were harvested enzymatically. Cell volume changes were measured by calcein-fluorescence quenching. Bilateral reduction in osmolality transiently increased short-circuit current (Isc), averaging 60% to 70%. Bilateral pretreatment with 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), a Cl- channel blocker, reduced Isc stimulation by approximately 60%, suggesting that transcellular ICl,swell largely mediates the increased current. The hypotonically-triggered Isc stimulation was also inhibited by phloretin, a blocker of swelling-activated Cl- channels and by flufenamic acid, a blocker of Cl- and nonselective cation channels. Cyclamate substitution for bath Cl- reduced the baseline Isc and the increase in hypotonically-triggered Isc. In that case, addition of either NPPB or flufenamic acid did not produce further inhibition. The transepithelial responses were correlated with regulatory volume responses of freshly harvested NPE cells. Hypotonicity elicited a regulatory volume decrease (RVD) over a period comparable to that of the hypotonicity-triggered increase in Isc. The RVD was also inhibited by Cl--channel blockers and by Cl- substitution. ICl,swell of NPE cells is functionally expressed in intact ciliary epithelium and is oriented to subserve aqueous humor formation. NPE cell volume can be measured with calcein-fluorescence quenching. ICl,swell may be stimulated by increased stromal fluid uptake and delivery to the NPE cells, facilitating Cl- secretion and increasing fluid release into the posterior chamber.

Microelectrode measurements of the effects of basolateral adenosine in polarized human intestinal epithelial cells in culture.

Activation of the basolateral receptor for adenosine in HT-29cl.19A cells, by 100 microM adenosine, increased the equivalent short-circuit current (DeltaIsc= 24+/-2 microA/cm2), depolarized the intracellular potential (DeltaVa= 26+/-2 mV) and decreased the fractional apical membrane resistance (DeltafRa=-0.48). The changes in all parameters reached their peak values simultaneously. This suggests that the primary action of the adenosine-activated pathway is on only one membrane. Bumetanide inhibited the transepithelial response and repolarized the cell potential. After preincubation with 100 microM forskolin, application of 300 microM adenosine caused a significant further change in Va, Isc, the transepithelial potential (Vt) and fRa. Together with the results from ion-replacement studies, the observations indicate that adenosine activates channels other than the cystic fibrosis transmembrane conductance regulator (CFTR). The rank order of potencies of adenosine and adenosine analogues implies that the receptor is of the A2 subtype. Preincubation with 4-bromophenacyl bromide (4-BPB) inhibited the effect of an adenosine analogue by 50%, indicating that activation of phospholipase A2 may be involved in the adenosine-induced response.

Sugar-induced transepithelial responses across the tongue epithelium of the frog Rana catesbeiana

Abstract Changes in transepithelial potential difference were induced across the dorsal tongue epithelium of frogs ( Rana catesbeiana ) by stimulation with various sugars. These were observed as a positive phase followed by a negative phase in the submucosa. The characteristics of the positive phase differed in many respects from those of the negative phase, and its profile resembled the activity of the gustatory nerve. The response was composed of a positive component superimposed on and masked by a negative component. In conclusion, the tissue absorbed sugar coupled with sodium and induced a positive component, and this may be related to gustatory transduction in frogs.

Activation of ion transport by combined effects of ionomycin, forskolin and phorbol ester on cultured HT-29cl.19A human colonocytes

The differentiated clone 19A of the HT-29 human colon carcinoma cell line was used as a model to study the intracellular electrophysiological effects of interaction of the cAMP, the protein kinase C (PKC) and the Ca2+ pathways. (a) A synergistic effect between ionomycin and forskolin was observed. From intracellular responses it was concluded that the synergistic effect is caused by activation of an apical Cl- conductance by protein kinase A and a basolateral K+ conductance by Ca2+. (b) A transient synergistic effect of ionomycin and the phorbol ester phorbol dibutyrate (PDB) was found. The decrease of the response appeared to be due to PKC-dependent inactivation of the basolateral K+ conductance. The synergism is caused by PKC-dependent increase of the apical Cl- conductance and Ca(2+)-dependent increase of the basolateral K+ conductance. (c) The effects of carbachol and PDB were not fully additive presumably because of their convergence on PKC activation. (d) Forskolin and PDB, when added in this order, had a less than additive effect. Results of cell-attached patch-clamp studies, presented in the accompanying paper, showed a synergistic effect of forskolin and PDB on non-rectifying small-conductance Cl- channels. Assuming that these channels are involved in the transepithelial responses it is suggested that forskolin and PDB induce a modulatory, synergistic increase of the apical Cl- conductance when both pathways are activated simultaneously. (e) The HT-29cl.19A cells differ from T84 cells in that the latter did n ot respond with an increase of the short-circuit current to addition of phorbol ester. this may be due to a very low expression of PKA alpha.

Regulation of apical Cl- conductance and basolateral K+ conductances by phorbol esters in HT-29cl.19A cells.

The effect of phorbol esters [4 beta-phorbol 12,13-dibutyrate (PDB) and phorbol 12-myristate 13-acetate (PMA)] on potential differences and resistances was studied with the conventional microelectrode technique applied to confluent filter-grown monolayers of the human colon carcinoma cell line HT-29cl.19A. Phorbol esters (PDB or PMA from 10(-7) to 10(-6) M) evoked 1) a transient increase in the transepithelial potential difference (peak value 3.5 +/- 0.5 mV), 2) a depolarization of the cell potential by 23 +/- 2 mV at the peak of the transepithelial potential change and a continued decrease during the decline of the transepithelial potential, and 3) a decrease of the fractional resistance of the apical membrane consisting of two phases, a relative rapid one (time constant 1.2 +/- 0.2 min) and a much slower further decrease during the second phase (time constant 34 +/- 1 min). Ion replacements and electrical circuit analyses indicate that PDB activates an apical Cl- conductance and slowly inhibits the basal K+ conductance of the basolateral membrane. PDB reduced the transepithelial response to forskolin due to inhibition of the basal K+ conductance. The Ca2+ ionophore ionomycin accelerated that effect of PDB. Staurosporine inhibited the effects of PDB, suggesting that the PDB effects are mediated via activation of a protein kinase C.

Comparison of the basolateral K+ conductance of the epithelial cells in frog gastric fundus mucosa

Frog stomach transepithelial potential difference (Vt) and resistance (Rt) as well as the voltage divider ratio (VDR) and serosal membrane potential (VS) of surface epithelial (SEC) and oxyntic (OC) cells were recorded at rest and during stimulation with histamine. Serosal membrane K+ permeability was tested by sudden elevation of serosal K+ concentration from 4 to 13 mmol l-1. Stimulation decreased both Vt and Rt and increased VDR of the OC (from 9.4 +/- SD 3.0 to 14.4 +/- 4.1, n = 10, P less than 0.001), while VS remained virtually unchanged (-66.3 +/- 4.5 mV, n = 10); in SEC, however, VDR as well as VS increased, the latter from -67.3 +/- 5.9 to -75.7 +/- 7.3 mV, n = 9, P less than 0.001. Elevation of serosal K+ reversibly diminished Vt and Vs in both cell types. The transepithelial response to K+ increased after stimulation. However, the cell potential response delta Vs,K increased only in the SEC (from +16.0 +/- 2.9 to +18.5 +/- 2.6 mV, n = 9, P less than 0.001) but not significantly in the OC. We conclude that in frog stomach both OC and SEC are stimulated by histamine: the SEC respond with a hyperpolarization, which reflects an increase in their basolateral K+ conductance; the OC do not respond with a hyperpolarization, possibly because histamine increases the basolateral membrane K+ conductance as well as other ion conductances which have not yet been identified.

Origin of the fast oscillation in the electroretinogram of the macaque

To investigate the origin of the fast oscillation, a phenomenon in the electroretinogram evoked with stimulus frequencies of about 8 mHz (a period time of about 2 min), we recorded responses from retina and pigment epithelium in the macaque. Micropipettes were placed in the subretinal space and in the vitreous close to the retina; the reference electrode was in the orbit behind the eye. Thus, simultaneous recordings were obtained of the trans-epithelial, the trans-retinal and the trans-tissue (vitreal) potential. At 10 mHz the trans-retinal and the trans-epithelial responses are of about equal magnitude but of opposite phase, resulting in a small and rather variable vitreal potential. The origin of the fast oscillation evoked with repetitive stimuli lies in subtle differences between retinal and pigment epithelial potentials, in which a pigment epithelial event plays an important role. For single stimuli lasting 60 s again the trans-epithelial and trans-retinal responses were of equal magnitude and opposite polarity. The epithelial responses were found to return more quickly towards the baseline than the retinal responses. In vitreal recordings this causes a trough between the c-wave and the light peak which is referred to as the “trough” fast oscillation. Most of the “trough” fast oscillation is caused by a pigment epithelial event. In view of the complexity of the fast oscillation evoked with repetitive stimuli it might be difficult to relate pathology to specific neuro-epithelial structures.

Cellular and transepithelial responses of goldfish intestinal epithelium to chloride substitutions

In goldfish intestine chloride was substituted by large inorganic anions (gluconate or glucuronate) either mucosally, serosally or bilaterally. Changes in intracellular activities of chloride (a i Cl−), sodium (a i Na+) and potassium (a i K+), pHi, relative volume, membrane and transepithelial potentials, transepithelial resistance and voltage divider ratio were measured. Control values were:a i Cl−=35 meq/liter, a i Na+=11 meq/liter and a i K+=95 meq/liter. During bilateral substitution the latter two did not change while a i Cl− dropped to virtually zero.

Sodium-dependent sugar and amino acid transport in isolated goldfish intestinal epithelium: electrophysiological evidence against direct interactions at the carrier level.

The effects of mucosal application of monosaccharides and amino acids on transepithelial and membrane potentials in isolated goldfish intestinal epithelium were investigated. Isosmotic replacement of mucosal mannitol by sugars or L-amino acids resulted in a rapid depolarization of the mucosal membrane potential psi mc followed by a slow repolarization. Phlorizin inhibited the responses to sugar but not those to amino acids. D-Amino acids did not induce any electrical response in the epithelium. Dose-response curves for L-amino acids showed simple saturation. Simultaneous application of L-amino acid and glucose induced transepithelial responses of about 80% of the sum of the separate responses to the application of amino acid or glucose alone. Simultaneous application of different amino acids in saturating concentrations did not increase the magnitude of the electrical responses. From the measured changes in potentials we calculated the change in electromotive force across the mucosal (delta Em) and serosal (delta Es) membrane. The change in Em induced by combined application of alanine and glucose was 90% of the sum of the calculated values induced by glucose and alanine alone. The response of Es to both substrates was accelerated with respect to that of separate substrates alone. We conclude that by application of glucose in addition to alanine the influx of sodium is increased, thereby stimulating the basolaterally located electrogenic Na+/K+-pump. There are no indications for direct interaction of sugars and amino acids at the mucosal membrane of the intestinal epithelial cell.