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Mechanism and regulation of the electrogenic alkali cation pumpin KCl secreting epithelia

Objective


Cellular transport mechanisms of potassium chloride (KC1) in both basal and apical membranes in Malpighian tubules of the ant have been studied using, secretion rate measurements combined with different electrophysiological and microfluorescence techniques.

From the analysis of the electrical equivalent model and the finding of a high apical over basolateral membrane resistance it could be explained why changes in the basolateral electromotive forces were reflected on the apical membrane. The bath potassium concentration can regulate fluid secretion by decreasing the height of the electrical and concentration steps to be overcome by potassium transport across the apical barrier.

From the effect of barium, monoiodoacetic acid and the inhibitors of V-type proton adenosine triphosphatase (ATPase), bafilomycin A1 and N-ethyl maleimide, on fluid secretion rate and electrophysiological parameters it was concluded that a proton pump of the V-type is present in the apical membrane of the Malpighian tubules of Formica, which may act as the prime mover for potassium transport.

From luminal and intracellular potassium and pH measurements in the presence of different bath potassium concentrations, luminal and basolateral electrochemical potassium and pH gradients were estimated. It was clearly demonstrated that the bath potassium determines the amount of cell potassium available for apical extrusion and that the electrochemical gradient of potassium across the basal membrane is in favour of a net entry through a conductive channel. The proton concentration gradient, built up across the apical membrane in different bath concentrations and in the presence of barium and dinitrophenol, indicated that the electrogenic V-type hydrogen pump can indeed be the prime mover in the active potassium transport. An apical electroneutral potassium hydrogen antiporter could explain the potassium extrusion in the lumen.

The effort of diuretic extracts from ants on electrophysiological parameters was determined and the intracellular second messenger mechanism was investigated. It was found that cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP) may acta as second messengers fro the endogenous factors found in extracts from different body parts of Formica. Moreover intracellular calcium seems to play an important role in the hormonal control of fluid secretion.
Structural, molecular and physiological features of the electrogenic alkali cation pump and the mechanisms of its regulation will be studied in Malpighian tubules of Formica, producing a KC1 rich fluid as it's primary urine. Different and complementary approaches will be used : morphology at the light microscopical and ultrastructural level in relation to different physiological conditions ; immunocytochemistry with monoclonal antibodies against the various subunits of the pump ; analysis of the nucleotide sequences of these various subunits ; in situ hybridisation ; transport and electrophysiological studies on the intact isolated tubule ; measurements of single channel currents ; fluorescence measurements of intracellular ion activities on intact segments of perfused tubules ; extraction of diuretic factors. The collaboration and the specific contribution of each participating laboratory is essential to characterize the mechanisms involved in the KCI secretion.

Coordinator

Limburgs Universitaire Centrum
Address
Universitaire Campus
3590 Diepenbeek
Belgium

Participants (3)

Albert-Ludwigs-Universität Freiburg
Germany
Address
Hermann-herder-straße 7
79104 Freiburg
Ludwig-Maximilians-Universitdt München
Germany
Address
Luisenstrasse
80333 München 2
Universidad Pública de Navarra
Spain
Address
273,Apartado
31080 Pamplona