BERNICK, EP*; ONKEN, H; KIRSCHNER, LB; MOFFETT, DF; Washington State University; Washington State University; Washington State University; Washington State University: ATPase activities in Aedes aegypti midgut
Previous electrophysiological studies of isolated, perfused larval Aedes aegypti midgut demonstrated the presence of a lumen negative voltage in the anterior stomach and a lumen positive voltage in the posterior stomach (Clark et al., 1999). These voltages can be stimulated upon exposure of the tissues to 5-HT. ATPases play an active role in powering the transport of ions across epithelia against steep concentration gradients. To quantify the activities of ion-motive ATPases in the midgut of mosquito larvae and to monitor their possible changes by modulators, an ATPase assay was developed and applied to whole midguts from Aedes aegypti larvae. Complete larval midguts (including the ceca, anterior, and posterior midgut) were isolated and homogenized, over ice, in a hand held glass homogenizer (one midgut per 10 µl buffer). Three ATPases have been studied: the Na-K ATPase, the V-ATPase, and the F-ATPase. Their activities were determined through exposure of the homogenate to 5 mM ouabain, 1 µM concanamycin, or 2 mM sodium azide, respectively. The activities found, expressed as mean ± SEM, were: Na-K: 8.87 ± 1.71 nmol Pi gut-1 h-1 (n=9); V: 12.32 ± 3.97 nmol Pi gut-1 h-1 (n=8); F: 34.79 ± 2.28 nmol Pi gut-1 h-1(n=7). First results with 5-HT show that preincubating the midguts in saline containing this drug seems not to affect the ATPase activities of the homogenate. This result does not distinguish between the presence of ATPases sequestered within the tissue versus present at sites where they contribute to transport characteristics. 5-HT may stimulate the transport and/or fusion of ATPase rich vesicles into the cell membrane. Continuing studies will involve assays of different regions of the larval midgut and of additional possible modulators of ATPase activity. This project is supported by NSF (grant #IBN-0091208).
