Dibutyryl cAMP activates bumetanide-sensitive electrolyte transport in Malpighian tubules

Abstract

The effects of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and bumetanide (both 10(-4) M) on transepithelial Na+, K+, Cl-, and fluid secretion and on tubule electrophysiology were studied in isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti. Peritubular DBcAMP significantly increased Na+, Cl-, and fluid secretion but decreased K+ secretion. In DBcAMP-stimulated tubules, bumetanide caused Na+, Cl-, and fluid secretion to return to pre-cAMP control rates and K+ secretion to decrease further. Peritubular bumetanide significantly increased Na+ secretion and decreased K+ secretion so that Cl- and fluid secretion did not change. In bumetanide-treated tubules, the secretagogue effects of DBcAMP are blocked. In isolated Malpighian tubules perfused with symmetrical Ringer solution, DBcAMP significantly hyperpolarized the transepithelial voltage (VT) and depolarized the basolateral membrane voltage (Vbl) with no effect on apical membrane voltage (Va). Total transepithelial resistance (RT) and the fractional resistance of the basolateral membrane (fRbl) significantly decreased. Bumetanide also hyperpolarized VT and depolarized Vbl, however without significantly affecting RT and fRbl. Together these results suggest that, in addition to stimulating electroconductive transport, DBcAMP also activates a nonconductive bumetanide-sensitive transport system in Aedes Malpighian tubules.

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

“…However, the evidence for Na + /K + /2Cl -cotransport is merely pharmacological because this transporter has not been isolated from and/or cloned in any insect Malpighian tubule. In Malpighian tubules of Aedes aegypti, bumetanide was found to significantly inhibit transepithelial K + secretion without affecting transepithelial resistance and the fractional resistance of the basolateral membrane, which is consistent with an effect on a non-conductive transport pathway such as that of an electroneutral transport system (Hegarty et al, 1991). More importantly, the present study illustrates that inhibitors of K + transport significantly increase the transepithelial secretion of Na + and Cl -.…”

“…The electroneutral transport step is identified by the effects of bumetanide, a known blocker of Na + /K + /2Cl -cotransport. In a previous study, bumetanide inhibited 70% of transepithelial K + secretion without significant effects on transepithelial electrical resistance (Hegarty et al, 1991). In the present study, the co-administration of barium and bumetanide completely inhibited transepithelial K + secretion, revealing no other transepithelial K + transport mechanisms.…”

“…Previous attempts to obtain a dose-response curve of the effects of bumetanide were unsuccessful because the effects of bumetanide are cumulative and irreversible. For this reason, we used the bumetanide concentration (0.1·mmol·l -1 ) employed in a previous study (Hegarty et al, 1991).…”

“…Rates of transepithelial fluid secretion were measured as described previously (Hegarty et al, 1991) with the following modifications. With about 80% of the tubule length remaining in a 40·µl droplet of Ringer solution, the open end of the tubule was pulled into the surrounding oil and gently draped around a small steel broach.…”

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

“…This pH drop could then induce the assembly of V 1 and V O complexes and hence increase transepithelial ion secretion. Because cAMP also activates Na + channels and a bumetanide-sensitive Na + /K + /2Cl -co-transporter in the basolateral membrane of Malpighian tubules (Hegarty et al, 1991;Beyenbach, 2003), the competitive status of intracellular Na + for elimination via the apical NHA increases, resulting in natriuresis.…”