Role of ATP in regulating and integrating transport transport across principal cells of malpighian tubules

K. W. Beyenbach

Dept. of Biomedical Sciences, Cornell University, VRT 8014, Ithaca, NY 14853, USA

 

Using X-ray analysis, we showed that cAMP causes diuresis by stimulating transepithelial secretion of NaCl and not KCl in Malpighian tubules of the yellow fever mosquito Aedes aegypti (Williams & Beyenbach, J. Comp. Physiol., B 154, 301, 1984). Cyclic AMP was later identified as second messenger of mosquito natriuretic peptide and other CRF-like diuretic peptides. Cyclic AMP increases basolateral membrane Na + conductance in principal cells, thereby increasing Na + entry and stimulating active transport into the lumen (Sawyer & Beyenbach, Am. J. Physiol. 248, R339, 1985). Using electrophysiological methods, we found that transepithelial Cl - secretion is passive, mediated outside principal cells, and driven by the transepithelial lumen-positive voltage (Hayes et al., Life Sciences 44, 1259, 1989). Cl - passing through the shunt serves as counterion for active Na + and K + transport through principal cells. Leucokinin stimulates secretion of both NaCl and KCl by increasing shunt Cl - conductance (Pannabecker et al., J. Membr. Biol. 132, 63, 1993). Though the site of the shunt, stellate cells or paracellular junctions, is controversial, there is agreement on separate active (Na + , K + ) and passive (Cl - ) transport pathways, controlled respectively by CRF-like and leucokinin-like diuretic peptides (O'Donnell et al., J. exp. Biol. 199, 173, 1996; Beyenbach, J. Insect Physiol. 41, 197, 1995). Using the methods of two electrode voltage clamp in a single principal cell, we now have learned that apical and basolateral membrane voltages are electrically coupled via the shunt. Thus, the voltage generated by the vacuolar H + -ATPase at the apical membrane determines the electrical driving force for Na + and K + entry at the basolateral membrane, coupling entry and exit steps. Inhibition of metabolism by dinitrophenol (DNP) reversibly increases basolateral membrane resistance almost 8-fold (652 K? to 5038 K?) and increases apical membrane resistance nearly 20-fold (340 K? to 6718 K?). Hence, conductive pathways shut down at both membranes, allowing transport to spring back again upon DNP washout. The effects of DNP can be duplicated (reversibly) by cyanide (CN - ) and fluoride (F - ). DNP collapses the mitochondrial proton gradient, CN - inhibits cytochrome oxidase, and F - inhibits glycolysis, but all inhibit ATP synthesis. Effects shared in common by DNP, CN - and F - suggest a role of ATP in regulating and integrating diverse conductive pathways (pumps, carriers and channels) at basolateral and apical membranes. The on/off effect of DNP with switch-like speed points to high turnover rates of ATP. (NSF IBN 9604394)

Index terms: CRF-like peptides, leucokinin, cAMP, ATP, principal cells, shunt.

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