The dependence of electrical transport pathways in Malpighian tubules on ATP

Abstract

Studies of transepithelial transport typically focus on the movement of ions, organic solutes and water across the epithelium, but what drives transport is ultimately metabolism. The latter has not received much attention in studies of epithelial transport. However, metabolism and transepithelial transport must be intimately coupled, especially in actively transporting epithelia, such as Malpighian tubules. Indeed, in Malpighian tubules of the yellow fever mosquito Aedes aegypti, the inhibition of oxidative phosphorylation with dinitrophenol brings transepithelial electrolyte and water secretion immediately to a halt and eliminates voltages across cell membranes and the whole epithelium (Beyenbach, 2001).Since the product of oxidative phosphorylation is ATP, the present study was undertaken to examine the relationship between intracellular ATP concentration ([ATP]i) and electroconductive and electrogenic transport mechanisms in principal cells of Malpighian tubules of the yellow fever mosquito. Principal cells are the sites of Na + and K + secretion from the hemolymph into the tubule lumen (Beyenbach, 1995). The entry of K + into the cell from the hemolymph is electroconductive through K + -channels in the basolateral membrane ( Fig. 1). On the apical side of the cell, extrusion of K + from the cytoplasm into the tubule lumen is thought to be mediated by an nH + /K + exchanger that, in turn, is driven by the electrochemical H + potential generated across the membrane by the activity of the electrogenic V-type H + -ATPase, also located in the apical membrane. To examine the relationship between [ATP]i and electroconductive and electrogenic transport mechanisms in principal cells of the tubule, we measured the effects of metabolic inhibition on [ATP]i in one set of tubules, and the effects on membrane voltage and resistance of principal cells in another set of tubules. We found that a severe reduction in [ATP]i not only inhibited the electrogenic V-type H + -ATPase at the apical membrane but also blocked the K + -conductance of the basolateral membrane at the same time. Simultaneous changes at both apical and basolateral membranes as a function of [ATP]i suggest that ATP integrates the transport steps at these two cell membranes. In the absence of ATP synthesis, the drop of [ATP]i to 10% of control levels effectively isolates the cell

Protein kinase A dependent and independent activation of the V-ATPase in Malpighian tubules ofAedes aegypti.

“…However, when mitochondrial ATP synthesis was inhibited by KCN (Fig.3A), V a , V bl and V t all depolarized towards zero in parallel because of the electrical coupling. Importantly, V a depolarized in the presence of KCN, which is known to reduce intracellular ATP concentrations in A. aegypti Malpighian tubules (Wu and Beyenbach, 2003). Accordingly, the rate of V a depolarization reflected the run-down of the intracellular ATP pool when ATP was no longer produced in the presence of KCN.…”

“…In order to maximize the response to substances that cause an increase in V-ATPase activity, we set out to induce V-ATPase disassembly by reducing the ATP/ADP ratio, as had been demonstrated in vitro for the M. sexta V-ATPase (Huss and Wieczorek, 2007). Therefore, we incubated the Malpighian tubules with the metabolic inhibitor KCN, which reduces intracellular ATP concentration rapidly in a reversible manner (Wu and Beyenbach, 2003). Unexpectedly, in tubules incubated with 1mmoll -1 KCN for 10min the membrane association of V 1 subunits C and D significantly increased by 18 and 23%, respectively (Fig.5A).…”

Mechanisms of K+ transport across basolateral membranes of principal cells in Malpighian tubules of the yellow fever mosquito,Aedes aegypti

“…Measurements of the basolateral membrane voltage (Vbl) and principal cell input resistance (Rpc) were obtained using the methods of TEVC, as described by Masia et al (2000) and Wu and Beyenbach (2003). Again, values of Vbl and Rpc are steady-state values taken between 10·min and 60·min of control and experimental periods.…”

“…Fluid secreted under experimental conditions was collected at the end of the experiment, 25-70·min after introducing the agent of interest to the peritubular Ringer bath. (Beyenbach and Masia, 2002;Masia et al, 2000;Wu and Beyenbach, 2003).…”

“…The large increase in basolateral membrane resistance is expected to decrease transcellular cationic current and, consequently, paracellular current (Fig.·3). Indeed, estimates of transcellular and paracellular currents, or the intraepithelial loop current, show significant reductions in the presence of Ba 2+ (Wu and Beyenbach, 2003). Thus, as loop current decreases, the rate of K + and Na + transport through principal cells decreases and the rate of Cl -transport decreases.…”