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In a previous study [Lohrmann et al: Pflügers Arch Eur J Physiol 1995;429:517-530] we have shown that chromanol K⁺ channel blockers inhibit Cl^– secretion in rabbit colon. Their effect was easily demonstrable after stimulation by hormones acting through increases of cytosolic cAMP. The present study was undertaken to test in more detail the mechanism of action of one of these compounds (293 B). Two types of studies were performed: Ussing chamber experiments in rabbit distal colon and whole cell patch clamp studies in the isolated in vitro perfused rat colonic crypts. Carbachol (CCH, 100 µmol/l) enhanced Cl^– secretion, quantified as equivalent short circuit current (I_sc), in rabbit colon significantly more than did prosta-glandin E₂ (PGE₂). In whole cell patch clamp studies in rat colonic crypt cells from the base, CCH hyperpolarized the membrane voltage (V_m) and enhanced whole cell conductance (G_m). In agreement with previous impalement studies, 10 µmol/l 293 B depolarized V_m in forskolin-treated rat colonic crypt base cells even further and reduced G_m. In Ussing chamber experiments in rabbit colon, 293 B abolished the I_sc induced by PGE2. CCH, in the continued presence of 293 B, still induced a large I_sc. These data indicate that 293 B specifically inhibits the forskolin- but not the CCH-induced Cl^– secretion, and supports our previous conclusion that this class of substances inhibits a cAMP-regulated K⁺ conductance.

In the present study evidence is given for the presence of an electrogenic, vacuolar type ATPase (V type ATPase) in the apical membrane of malpighian tubules of Formica. Barium (6 mM), the metabolic inhibitor monoiodo-acetic acid (MIA; 5 10^-4M) and two inhibitors of V type proton ATPases, i.e. bafilomycin A1 (Baf-A1; 5·10^-6M) and N-ethylmaleimide (NEM; 5 10^–4M), all inhibited fluid secretion significantly (p < 0.05). This is in agreement with the hypothesis that K enters passively via K channels in the basolateral membrane and that a V type ATPase is involved in the active transport step at the apical membrane. Also MIA, NEM and Baf-A1 slightly depolarized the apical membrane potential, V_ap, by 16, 17 and 30 mV, respectively, whereas they had virtually no effect on the basolateral membrane potential (V_m). The mild effect on V_ap, in contrast with the pronounced effect on fluid secretion, can be explained by the high apical over basolateral membrane resistance: the voltage divider ratio, VDR, was 47 ± 9 (n = 6). As a consequence the basolateral membrane will impose its value on the other barriers. VDR was decreased to 1.4 ± 0.2 (n = 19) by Ba. Ba also caused a strong and reversible hyperpolarization of both V_b1 and V_ap (from – 16 ± 1 to – 84 ± 4 (n = 8) and from -51 ± 4 to-96 ± 6 mV, respectively). As expected MIA, NEM and Baf-Al now had a much more pronounced depolarizing effect, i.e. they drastically reduced the Ba-induced hyperpolarization of both V_ap and V_b1 The reduction in V_ap was 67, 67 and 54 mV, respectively. V_b1depolarized from -73 ± 4to-15 ± 4mV (n = 7), from-72 ± 10 to 13 ± 2mV(n≈5)and from – 78 ± 3 to – 30 ± 5 mV (n = 12) in the presence of Ba and MIA, NEM or Baf-A1, respectively. From cable analysis and total transepithelial resistance in the absence and presence of barium it was also possible to make an estimate of the resistances across the different barriers: total basolateral resistance = 10 Ω·cm², total apical resistance = 475 Ω·cm², total shunt resistance = 228 Ω·cm². It was concluded that in malpighian tubules of Formica an H pump of the V type is present in the apical membrane. As suggested in other epithelia this pump can be the prime mover in active K transport: the proton concentration gradient built up across the apical membrane can drive a K/H exchanger.

In this study we examined by impalement techniques properties of the macroscopic K+ conductances in the luminal and basolateral membrane of principal cells from isolated perfused cortical collecting ducts (CCD) of the rat. Both membranes possess a dominating K+ conductance. Compared to their behaviour with K+, both membranes appear much less permeable to NH+4 and Rb+, and the K+ conductances of both membranes are inhibited by these cations. In light of these findings, it is very unlikely that significant amounts of NH+4, which is secreted in the CCD, cross the principal cells as NH+4. Several inhibitors with known effects on K+ channels in patch-clamp studies have been examined. Tetraethylammonium, which inhibits the excised K+ channels of these cells, has no effect on the macroscopic K+ conductances of either membrane. Verapamil, which inhibits the K+ channels in the luminal membrane, acts predominantly on the basolateral membrane K+ conductance in the intact tubule. Therefore, some of the macroscopic properties of the K+ conductances are distinct from those of single channels thus far observed in patch-clamp studies.