Recently, Na + /K + -ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na + /K + -ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na + /K + -ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na + /H + -exchanger. Altogether the results of the present investigation indicate that apical Na + /K + -ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.Strong luminal alkalinization of up to pH values of 12 has been observed in midgut regions of many larvae of endopterygote insects, including members of the orders Coleoptera, Diptera, Trichoptera and Lepidoptera (for references see Clark, '99). Two tissues have been studied especially intensively with regard to their mechanisms and capacities of alkalinization during the past 20 years: the midgut of the tobacco hornworm (Manduca sexta) and the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti). For the lepidopteran larvae, a transport model has been established in which luminal V-type H + -pumps (V-ATPases) energize luminal, electrogenic K + /2H + -exchangers, resulting in active K + secretion and strong luminal alkalinization (for a review see Wieczorek et al., '99). Unfortunately, this mechanism can so far not be tested with the isolated epithelium, because it loses the capacity for strong luminal alkalinization after isolation, whereas active K + secretion continues in vitro at a high rate (Clark et al., '98). Strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes appears to significantly differ from lepidopterans, including the methodological aspect that it can readily be observed with isolated and perfused midgut preparations (Onken et al., 2008). In larval A. aegypti, the V-ATPase is not localized in the luminal membrane of the anterior midgut, but instead in the basolateral membrane (Zhuang et al., '99). The epithelium of larval yellow fever mosquitoes generates a lumen negative transepithelial voltage (Clark et

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Anterior stomachs of larval mosquitoes maintain luminal pH values of 10–12. Recently, Na+/K+‐ATPase was localized in apical membranes of this tissue in larval Aedes aegypti (Patrick, ML et al. 2006: JEB 209, 4638). We tested the hypothesis that this apical ATPase acts as a primary active Na+/H+‐exchanger, contributing to luminal alkalinization by active H+ absorption. m‐Cresol Purple (mCP, 0.04 %) in the water was used to visualize alkalinization in the anterior stomach of the transparent larvae. In the presence (N = 10) and absence (N = 12) of 5 mM ouabain the anterior stomach was found to be purple, indicating an alkaline content. Isolated anterior stomachs were bathed in mosquito saline and perfused with 100 mM NaCl and mCP. The transepithelial voltage (Vte) was measured and luminal alkalinization was determined by the color change of mCP after luminal perfusion stop. Luminal ouabain (5 mM) did not affect Vte or the alkalinization of the lumen (N = 7). Ouabain‐sensitive ATPase activity was measured in homogenates of single stomachs in the presence (86 ± 3 nmol Pi h−1 gut−1 or 10.6 ± 2.2 ìmol Pi h−1 mg−1, N = 3) and the absence of K+ (0.1 ± 0.1 ìmol Pi h−1 mg−1, N = 3). Omission of K+ abolished the ouabain‐sensitive ATPase activity. The results seem to rule out that apical Na+/K+‐ATPase contributes to luminal alkalinization by generating ATP driven Na+/H+‐exchange across the apical membrane. Supported by NIH (1R01AI063463‐01A2).

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