Importance of basolateral K+ conductance in maintaining Cl- secretion in murine nasal and colonic epithelia

Abstract

1. Epithelia lining the nasal passages and descending colon of wild-type and cystic fibrosis (CF) mice were examined by the short-circuit current technique. Additionally, intracellular Ca2+ ion determinations were made in nasal epithelial cells. Forskolin produced anion secretory currents in wild-type and CF nasal epithelia. It produced similar effects in wild-type colonic epithelia, but not in colonic epithelia from CF mice. 2. After electrogenic Na+ transport was blocked with amiloride and electrogenic Cl- secretion was stimulated with forskolin, the ability of K+ channel blockers to inhibit the forskolin-induced Cl- current was determined. The order of efficiency for nasal epithelium was: Ba2+ > clofilium >>> TEA = azimilide >>> trans-6-cyano-4-(N-ethylsulphonyl-N-methylamino)-3-hydroxy-2, 2-dimethyl-chromane (293B) = charybdotoxin, whereas for the colonic epithelium the order was: Ba2+ = 293B >>> azimilide = TEA >>> clofilium = charybdotoxin. 3. 1-Ethyl-2-benzimdazolinone (1-EBIO) was able to generate large Cl--secretory currents in colonic epithelia which were partially sensitive to charybdotoxin, with the remaining current being inhibited by 293B. In nasal epithelia 1-EBIO produced only a small transient effect on current. 4. Forskolin released intracellular Ca2+ in nasal epithelial cells; this activity was attenuated when more powerful Ca2+-releasing agents were applied first. 5. It is concluded that an action on basolateral cAMP-sensitive K+ channels is an important determinant of the maintained responses to forskolin in nasal and colonic epithelia, in addition to the effects on the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. In CF nasal epithelia the activation of calcium-activated chloride channels (CACs) substitutes for the effect on CFTR. On the basis of the different orders of potency of the blocking agents and the differential response to 1-EBIO it is concluded that the cAMP-sensitive K+ channels are different in the airways and the gut.

Figure 1. Effects of forskolin on anion secretion in nasal epithelia

A, typical SCC traces showing the effects of the sequential addition of amiloride (100 μM, apically), forskolin (10 μM, both sides) and furosemide (1 mM, basolaterally) on nasal epithelia from a wild-type (WT, ±ΔF508; upper trace) and a CF (ΔF508; lower trace) mouse. B and C show the basal, amiloride-sensitive and forskolin-sensitive changes in SCC in 4 groups of nasal epithelia. In B, data for wild-type (consisting of 12 heterozygotes and 6 homozygotes) and CF null mouse tissues are shown, all animals being derived from the same outbred genetic background. In C, comparable data are shown for wild-type (11 heterozygotes and 16 homozygotes) and CF ΔF508 mouse tissues. In both B and C the amiloride-sensitive SCC was significantly greater in the CF tissues (P < 0.0003 and 0.005, respectively; Mann-Whitney U test) while the responses to forskolin in each pair were not different.

Figure 6. SCC traces showing effects of K⁺ channel blockers and 1-EBIO on colonic epithelia

A, lack of effect of clofilium and charybdotoxin and effectiveness of Ba²⁺ and 293B in blocking the action of forskolin on wild-type colons. B, effect of 1-EBIO on wild-type colon and partial inhibition by charybdotoxin and major inhibition by 293B.

Figure 2. Effects of Ba²⁺ ions on the forskolin-sensitive SCC responses in nasal epithelia

The figure shows the SCC increases remaining at steady state 10 min after the addition of the drug and the effect Ba²⁺ (5 mM) had on this value. The 4 groups of tissues are as in Fig. 1. In all instances Ba²⁺ caused a significant reduction in current (Mann-Whitney paired U test). In 30 % of these measurements another agent (either charybdotoxin or 293B) was added before Ba²⁺, but had no effect. Numbers of experiments and statistical significance are shown on the figure.

Figure 3. Effects of clofilium on the responses to forskolin

In A, responses in a wild-type (left) and CF (right) nasal epithelium to 100 μM clofilium (applied basolaterally) are shown. Ten minutes after clofilium was added, Ba²⁺ (5 mM) was added to the same side. Data for 11 wild-type (6 heterozygotes and 5 homozygotes) and 6 CF nasal epithelia are given in B. Mann-Whitney paired U test was used to test for significance. Note, in CF basal epithelia, current was not further reduced by Ba²⁺ after clofilium.

Figure 4. Effects of TEA on the responses to forskolin

In A, responses of a wild-type (left) and a CF (right) nasal epithelium to 30 mM TEA (applied basolaterally) are shown. Ten minutes after TEA was added, Ba²⁺ (5 mM) was added to the same side. Data for 10 wild-type (6 heterozygotes and 4 homozygotes) and 5 CF nasal epithelia are given in B. The forskolin-induced current was significantly inhibited by Ba²⁺, but not TEA (Mann-Whitney paired U test), as indicated on the figure.

Figure 5. Diagrammatic representations of the effects of Ba²⁺ and 293B on responses to forskolin in colonic epithelia

Effects of 5 mM Ba²⁺ (A and B) and 100 μM 293B (C and D) on responses of wild-type (A and C) and CF (B and D) epithelia are shown. Note that in the CF epithelia both Ba²⁺ and 293B produced only transient effects, while the response to forskolin was reversed by furosemide. The effects of Ba²⁺ and 293B on wild-type colonic epithelia were significant (P < 0.0001 and P < 0.001, respectively; Student's t test).

Figure 7. Diagrammatic representations of the effects of 1-EBIO on Cl⁻ secretory responses of wild-type nasal and colonic epithelia

A, the effect of 600 μM 1-EBIO (applied both sides) on nasal epithelia was small and transient. Subsequently, forskolin was able to increase SCC, which was significantly inhibited by furosemide (P < 0.03). B, the current generated by 600 μM 1-EBIO (applied both sides) in the colon was partially sensitive to charybdotoxin (50 nM, P < 0.01) and majorly affected by 293B (100 μM, P < 0.001; Student's t test throughout).

Figure 8. Fura-2 fluorescence in nasal epithelial cells

A, effects of forskolin (10 μM) and TBHQ-ionomycin (12.5 μM and 5 μM, respectively) on [Ca²⁺]_i indicated by the 340 nm/380 nm ratio in fura-2-loaded tissues. B, ratio of the response to TBHQ-ionomycin to that for forskolin depends on the order in which the agents are given. The ratio is significantly greater when TBHQ-ionomycin are given first. C shows examples of 340 nm/380 nm ratios for preparations from wild-type nasal epithelia (top and middle traces) and from a CF preparation (bottom trace).