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First published online July 2, 2004
Journal of Experimental Biology 207, 2877-2888 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01102

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The mechanism of action of the antidiuretic peptide Tenmo ADFa in Malpighian tubules of Aedes aegypti

Richard C. Massaro¹, Lenora W. Lee¹, Ankit B. Patel¹, Daniel S. Wu¹, Ming-Jiun Yu¹, Brett N. Scott¹, David A. Schooley², Kathleen M. Schegg² and Klaus W. Beyenbach^1,*

¹ Department of Biomedical Sciences, VRT 8004, Cornell University, Ithaca, NY 14853, USA
² Department of Biochemistry, University of Nevada, Reno, NV 89557, USA

^* Author for correspondence (e-mail: KWB1{at}cornell.edu)

Accepted 19 May 2004

The mechanism of action of Tenebrio molitor antidiuretic factor `a' (Tenmo ADFa) was explored in isolated Malpighian tubules of Aedes aegypti. In the Ramsay assay of fluid secretion, Tenmo ADFa (10^–9 mol l^–1) significantly inhibited the rate of fluid secretion from 0.94 nl min^–1 to 0.44 nl min^–1 without significant effects on the concentrations of Na⁺, K⁺ and Cl^– in secreted fluid. In isolated perfused tubules, Tenmo ADFa had no effect on the transepithelial voltage (V_t) and resistance (R_t). In principal cells of the tubule, Tenmo ADFa had no effect on the basolateral membrane voltage (V_bl) and the input resistance of principal cells (R_pc). Tenmo ADFa significantly increased the intracellular concentration of cyclic guanosine monophosphate (cGMP) from 2.9 µmol l^–1 (control) to 7.4 µmol l^–1. A peritubular [cGMP] of 20 µmol l^–1 duplicated the antidiuretic effects of Tenmo ADFa without inducing electrophysiological effects. In contrast, 500 µmol l^–1 cGMP significantly depolarized V_bl, hyperpolarized V_t, and reduced R_t and R_pc, without increasing antidiuretic potency beyond that of 20 µmol l^–1 cGMP. A plot of peritubular cGMP concentration vs V_bl revealed a steep dose–response between 300 µmol l^–1 and 700 µmol l^–1 with an EC₅₀ of 468 µmol l^–1. These observations suggest a receptor- and cGMP-mediated mechanism of action of Tenmo ADFa. Tenmo ADFa and physiological concentrations of cGMP (<20 µmol l^–1) reduce the rate of isosmotic fluid secretion by quenching electroneutral transport systems. The inhibition reveals that as much as 50% of the normal secretory solute and water flux can stem from electrically silent mechanisms in this highly electrogenic epithelium.

Key words: antidiuresis, isosmotic fluid secretion, cGMP, inhibition of electroneutral transport, Na/H exchange, Na/K/2Cl cotransport, Tenebrio molitor, antidiuretic factor `a' (Tenmo ADFa), Malpighian tubule, Aedes aegypti

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