Unmasking of the Apical Electrogenic H Pump in Isolated Malpighian Tubules <i>(Formica polyctena</i><i>)</i>by the Use of Barium

Abstract

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, Vap, by 16, 17 and 30 mV, respectively, whereas they had virtually no effect on the basolateral membrane potential (Vm). The mild effect on Vap, 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 Vb1 and Vap (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 Vap and Vb1 The reduction in Vap was 67, 67 and 54 mV, respectively. Vb1depolarized 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.