Malpighian tubule Na/H exchange during development

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DESCRIPTION (provided by the applicant): Fluid balance in insects is dependent on ion and fluid secretion across Malpighian tubules. The rate of fluid secretion by Malpighian tubules is dependent on the activity of a Na/H exchanger and the generation of the second messenger cAMP by adenylate cyclase. Inhibition of the Na/H exchanger results in a decrease in fluid secretion, a decrease in intracellular pH and a decrease in the extrusion of acid from the Malpighian tubule cells. Inhibition of the cAMP response by blocking the activity of the catalytic subunit of protein kinase A also decreases fluid secretion. These findings of a role for the Na/H exchanger and protein kinase A in regulating fluid secretion has been determined only in adult non-blood fed mosquitoes to date. We have extended these studies to Malpighian tubules from larval, pupal and blood fed female and determined that throughout development and after a blood meal that fluid secretion is dependent on Na/H exchange and cAMP. Additionally, we have cloned and sequenced the cDNA coding for Na/H exchanger from the adult mosquito allowing us to determine the molecular biological and immunological expression of the Na/H exchanger. The goals for the proposed research include a physiological, pharmacological, biochemical and immunological dissection of the role-played by the Na/H exchanger and cAMP during development and after a blood meal. The proposed experiments will investigate the temporal and spatial expression of the Na/H exchanger in individual Malpighian tubules from 1st instar larvae to adults five days after emergence. The role of the Na/H exchanger up to 48 hours after a blood meal will also be determined. The temporal studies will determine the amount of Na/H exchange activity, the effects of NaJH exchanger inhibitors on exchange activity and the amount of mRNA and protein at each developmental stage and after blood feeding. The spatial studies of the Na/H exchanger will immunologically determine the membrane (apical versus basolateral) location of the Na/H exchanger using confocal microscopy. These studies will be repeated in the presence of cAMP and inhibitors of protein kinase A to determine the key role-played by adenylate cyclase in regulating fluid secretion during development and after a blood meal. The results from the experiments will determine the role played by the Na/H exchanger during development and after a blood meal in mosquitoes. This body of knowledge will lead to potential sites for inhibition of fluid secretion during development and blood feeding. The inhibition of fluid secretion will disturb fluid balance in the mosquito and thereby render the mosquito in capable of spreading disease.