I hereby declare that the work presented in this document is based on research carried out by me, and that no part has previously been submitted for a degree in this or any other university

Abstract

Inhibitors of known transport processes, in conjunction with ion substitution were used in biochemical, physiological and microelectrode studies to investigate the mechanisms underlying ion and fluid secretion across the Malpighian tubules of Locusta migratoria. Immunocytochemical localization and cell fractionation methods used in conjunction with biochemical analyses demonstrate the presence of Na(^+)/K(^+)-ATPase activity on the basal cell membranes and HCO(_3)(^-)-stimulated and V-type ATPase activities on apical cell membranes. The biochemical properties of the HCO(_3)(^-)-stimulated and V-type ATPase activities are compared and the results discussed. Specific inhibitors of Na(^+)/K(^+)-ATPase and V-type ATPases inhibited fluid secretion whilst cAMP was found to stimulate fluid secretion. The continued secretion of K(^+) when tubules were bathed in K(^+)-free saline was attributed to the presence of mineral concretions shown in ultrastructural studies to be distributed throughout the cytoplasm. The effects of ouabain, NEM and cAMP on the cationic composition of the "urine" were studied. In control saline, ouabain and NEM increased the level of Na(^+) in the "urine" whereas cAMP caused an increase in the amount of K(^+) secreted. In Rb(^+)-saline, ouabain caused "urine" levels of Na(^+) to increase and levels of Rb(^+) to fall whereas cAMP and NEM had no effect on the cationic composition of the "urine". Ion substitution experiments used in conjunction with intracellular microelectrodes suggest the basal cell membrane contains an inward rectifying K(^+) channel. Blocking the basal inward rectifier with Rb(+) drove the potential of this membrane towards the emf of the apical membrane potential. In the presence of Rb(^+), bafilomycin A(_1), a specific V-type ATPase inhibitor, reduced this potential suggesting the apical membrane potential is produced by a V-type ATPase. The results are discussed and a hypothetical model is proposed to account for the mechanisms of ion and fluid movement across apical and basal cell surfaces.