In view of the large size of principal cells in Malpighian tubules of the yellow fever mosquito Aedes aegypti, we explored the application of two electrode voltage clamp methods (TEVC) that usually are employed to study transport proteins over-expressed in Xenopus oocytes. A single principal cell of the isolated Malpighian tubule was impaled with current and voltage electrodes. In 49 principal cells the intracellular voltage (Vpc) was -86.7 mV, and the input resistance (Rpc) was 388.5 K?. In a subset of 6 principal cells, Ba 2+ (15 mM) hyperpolarized Vpc and increased Rpc from 325.3 K? to 684.5 K? (p<0.001). In the presence of Ba 2+ , leucokinin-VIII (1 µM) increased Vpc to –101.8 mV (p<0.001), and reduced Rpc to 340.2 K? (p<0.002). These data were analyzed on the basis of an electrical equivalent circuit model of transepithelial NaCl and KCl secretion (Pannabecker et al., J. Membr. Biol. 132, 63, 1993). Circuit analysis revealed: basolateral membrane resistance Rbl = 652.0 K?, apical membrane resistance Ra = 340.2 K?, shunt resistance Rsh = 344.3 K?. It follows that the transcellular resistance Rcell = Rbl + Ra is 992.2 K?, that Rcell/Rsh = 3.53, and that the fractional resistance of apical membrane fRa = Ra/Rcell is 0.35. Estimates of Rcell/Rsh and fRa obtained using a different method (cable analysis of in vitro perfused tubules, CA) yield 3.33 and 0.32 respectively, validating the use of TEVC methods in principal cells of Aedes Malpighian tubules. Further comparison of data from TEVC experiments with data obtained using CA in isolated perfused tubules suggests electrical coupling of five to six principal cells. As shown in other abstracts presented at this meeting, TEVC methods allow the functional dissection of transport systems operating at basolateral and apical membranes in intact principal cells in situ. (NSF IBN 9604394)

Index terms: Malpighian tubules, electrolyte and fluid secretion, principal cells, shunt pathway.

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