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, 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.

Basolateral ion transport mechanisms during fluid secretion byDrosophilaMalpighian tubules: Na+ recycling,Na+:K+:2Cl– cotransport and Cl– conductance

“…Similarly, blockage of K + currents by prior exposure K + -free saline did not block the effect of Ba 2+ on the basolateral membrane potential. This result indicates that the current responsible for the hyperpolarization is not carried by K + , in contrast to proposals for tubules of F. polyctena and A. aegypti Pannabecker et al, 1992;Weltens et al, 1992;Masia et al, 2000). On the other hand, prior exposure to Cl --free saline containing 20·mmol·l -1 K + blocked the hyperpolarization of Vbl in response to Ba 2+ .…”

“…Equivalent circuit analysis shows that increasing basolateral membrane resistance with Ba 2+ will cause the loop current to drive the basolateral membrane potential more negative Pannabecker et al, 1992;Weltens et al, 1992;Masia et al, 2000;Wiehart et al, 2003b). In F. polyctena and A. aegypti tubules it has been proposed that the loop current is carried by inward K + flow through basolateral K + channels Weltens et al, 1992;Masia et al, 2000).…”

“…This finding suggests that Cl -carries the loop current responsible for the hyperpolarization produced by Ba 2+ in D. melanogaster tubules. It is important to point out that Cl -flow from cell to bath would produce a current of the same sign as K + flow from bath to cell, proposed as the basis for the Ba 2+ -induced hyperpolarization of Vbl in tubules of F. polyctena and A. aegypti Pannabecker et al, 1992;Weltens et al, 1992;Masia et al, 2000). Taken together, our results suggest that the effect of Ba 2+ on the basolateral membrane potential in D. melanogaster tubules is the result of an increased influence of the loop current on Vbl caused by the increased membrane resistance.…”

Sulphonylurea sensitivity and enriched expression implicate inward rectifier K+ channels inDrosophila melanogasterrenal function

“…-cotransporter (Ianowski and O'Donnell, 2004) and barium-sensitive potassium channels (Beyenbach and Masia, 2002;Masia et al, 2000;Weltens et al, 1992); their relative importance may vary between insects. Present estimates suggest that, in Drosophila, each contributes approximately equally to the K + flux (Torrie et al, 2004).…”

Comparative physiology of Malpighian tubules: form and function

“…11,41,49 Several studies evidence the presence of a cotransport system and a H + pump in the MTs of Formica polyctena (Hymenopera, Formicidae). [50][51][52][53][54] The presence of active and passive K + uptake mechanisms across the basal membrane was investigated in different bath K + concentrations. The authors proposed a model in which K + channels play a dominant role in high-K + bathing medium, and the results obtained from intracellular and luminal ion-selective measurements have demonstrated that the calculated gradients for Cl 2 and K + are consistent with the transport mechanisms.…”