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

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

Non-selective, calcium-permeable channels in the apical membrane of rabbit renal tubules and in the basolateral membrane of human renal tubules : an exploratory patch clamp study

Includes bibliography.The presence of calcium (Ca) channels has been investigated in the apical membrane of various segments of rabbit renal tubules and in the basolateral membrane of human tubules, using the patch clamp technique. The rabbit tubule segments comprised proximal straight tubules (PST), thick ascending limbs (TAL), distal convoluted tubules (OCT), and cortical collecting ducts (CCD). The human tubule segments could not be identified, but were probably of proximal origin. The luminal surfaces of the individual tubule segments were accessed by perfusing the tubule and inserting the patch pipette through the open end or, more frequently, by tearing open the tubule to allow direct access by a patch electrode. Either Ba (90 mM) or Ca (70 mM) was used in the pipettes. Where possible, channel activity was sought in voltage clamped (30 to -60 m V) excised patches. The data were digitized at l kHz, and filtered (200- 500 Hz) by a six-pole Bessel filter

Characterization of Transepithelial Transport in the Osmoregulation of Freshwater Dipteran Larvae

In the osmoregulatory epithelia of freshwater (FW) animals, transcellular solute transport relies on at least one of the primary ionomotive pumps, Na+/K+-ATPase (NKA) and V-type H+-ATPase (VA), but there are no data on their distribution in FW chironomid larvae. The paracellular solute movement in invertebrate epithelia is controlled by the septate junctions (SJs). However, nothing is known about the SJ components in aquatic insects. In the present set of studies, larvae of the FW chironomid Chironomus riparius and FW mosquito Aedes aegypti were used to examine a role for NKA and VA and SJ proteins, respectively, in the maintenance of salt and water balance in aquatic dipterans. Spatial distribution and activity of NKA and VA along the alimentary canal of C. riparius larvae revealed the importance of the rectum in the ionoregulatory homeostasis. It was found that the rectum absorbed relatively high amounts of K+ into the hemolymph under dilute conditions and decreased K+ absorption in brackish water (BW). This rectal K+ absorption was dependent on the activities of both NKA and VA. Next, genes encoding transmembrane SJ proteins megatrachea, sinuous, kune-kune (Kune), neurexin IV, snakeskin (Ssk), mesh and gliotactin (Gli) were identified in A. aegypti and shown to exhibit tissue specific transcript abundance in larval osmoregulatory epithelia. Ssk and mesh expression was restricted to smooth SJ bearing midgut and Malpighian tubules (MT) whereas Gli was detected in all tissues examined. Kune was confined to SJs in the posterior midgut and rectum and apical membrane domain of the syncytial anal papilla epithelium. Rearing A. aegypti larvae in BW caused an increase in Kune and Gli protein and Ssk and mesh mRNA abundance in the midgut and MT which occurred in conjunction with increased midgut and decreased MT permeability. Paracellular MT permeability was further modulated by leucokinin. When dsRNA was used to reduce Gli abundance in the midgut, paracellular permeability was decreased. Together, this research provides a better understanding of the physiology of transepithelial ion and water transport in aquatic insects and offers significant insight into the role of NKA, VA and SJ proteins in the osmoregulation of FW dipteran larvae