Development of optical imaging of intracellular Cl − in neurons in brain slices, that proved the change in the consequence of GABAergic action from Cl − efflux to Cl − influx during development

Abstract

Three different methods have been used to improve a model for fluid secretion in Upper Malpighian Tubules (UMT) of the blood sucking insect Rhodnius prolixus. (I) In the first, UMT double perfusions in 5th instar Rhodnius were used to measure their fluid secretion rate. They were stimulated to secrete with 5-HT. Double perfusions allowed access separately to the basolateral and the apical cell membranes with pharmacological agents known to block different ion transport functions, namely ATPases, cotransporters and/or countertransporters and ion and water channels: ouabain, bafilomycin A1, furosemide, bumetanide, SITS, acetazolamide, amiloride, DPC, BaCl2, pCMBS and DTT. The basic assumption is that changes in water movement reflect changes in ion transport mechanisms. (II) Intracellular Na+ concentrations were measured with a fluorometric method in dissected R. prolixus UMT, under several experimental conditions. (III) ATPase activities were measured in R. prolixus UMT. A tentative model for the function of the UMT cell is presented. We find that (a) at the basolateral cell membrane, fundamental is a Na+–K+–2Cl− cotransporter; of intermediate importance are the Na+–K+-ATPase and a ouabain-insensitive Na+-ATPase, ion channels and Rp-MIP water channels. (b) At the apical cell membrane, most important are a V-H+-ATPase; and a K+ and/or Na+–H+ exchanger.