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SUMMARY The mechanism of primary urine production and the activity of a diuretic kinin, Achdo-KII, were investigated in Malpighian tubules of Acheta domesticus by measuring intracellular Na+, K+ and Cl− activities, basolateral membrane voltage (Vb), fluid secretion and transepithelial ion transport. Calculated electrochemical gradients for K+ and Cl− across the basolateral membrane show they are actively transported into principal cells, and basolateral Ba2+-sensitive K+ channels do not contribute to net transepithelial K+ transport and fluid secretion. A basolateral Cl− conductance was revealed after the blockade of K+ channels with Ba2+, and a current carried by the passive outward movement of Cl− accounts for the hyperpolarization of Vb in response to Ba2+. Ion uptake via Na+/K+/2Cl− cotransport, driven by the inwardly directed Na+ electrochemical gradient, is thermodynamically feasible, and is consistent with the actions of bumetanide, which reduces fluid secretion and both Na+ and K+ transport. The Na+ gradient is maintained by its extrusion across the apical membrane and by a basolateral ouabain-sensitive Na+/K+-ATPase. Achdo-KII has no significant effect on the intracellular ion activities or Vb. Electrochemical gradients across the apical membrane were estimated from previously published values for the levels of Na+, K+ and Cl− in the secreted fluid. The electrochemical gradient for Cl− favours passive movement into the lumen, but falls towards zero after stimulation by Achdo-KII. This coincides with a twofold increase in Cl− transport, which is attributed to the opening of an apical Cl− conductance, which depolarises the apical membrane voltage.

SUMMARYThe pioneering work of Simon Maddrell established that the rapid postprandial diuresis of the haematophagous insect Rhodnius prolixusis controlled by a diuretic hormone and demonstrated the role of the Malpighian tubules in meeting the volumic, osmotic and ionic challenges posed by an enormous blood meal. A number of diuretic and antidiuretic hormones that control secretion of primary urine by Malpighian tubules have now been identified, but little is known of the interplay between these hormones and those that regulate transport processes in the hindgut. This review therefore focuses on the control of ionic homeostasis in Rhodnius and mosquitoes, because primary urine is voided virtually unchanged during the rapid diuresis that follows a blood meal. At such times, the hindgut has a negligible impact on the volume and composition of the final urine, and neurohormones acting on the Malpighian tubules have a dominant role in the control of ionic homeostasis.

SUMMARY Acheta domesticus is reported to have an antidiuretic hormone that reduces Malpighian tubule secretion. Identified peptides known to work in this way (Tenmo-ADFa and ADFb, and Manse-CAP2b) were tested as candidates for the unidentified hormone, along with their second messenger,cyclic GMP. Only Tenmo-ADFb was active, but was diuretic, as was 8-bromo cyclic GMP. The activity of Tenmo-ADFb is comparable to that of the cricket kinin neuropeptide, Achdo-KII, but it is much less potent. Its activity was unaffected by deleting either the six N-terminal residues or the C-terminal phenylalanine. At high concentrations, tubule secretion is doubled by Tenmo-ADFb and Achdo-KII, but their actions are non-additive, suggesting they have a similar mode of action. Both stimulate a non-selective KCl and NaCl diuresis, which is consistent with the opening of a transepithelial Cl–conductance. In support of this, the diuretic response to Tenmo-ADFb and Achdo-KII is prevented by a ten-fold reduction in bathing fluid chloride concentration, and both peptides cause the lumen-positive transepithelial voltage to collapse. The Cl– conductance pathway appears likely to be transcellular, because the Cl– channel blocker DPC reduces both basal and peptide-stimulated rates of secretion. The effects of 8-bromo cyclic GMP on transepithelial voltage and composition of the secreted fluid are markedly different from those of Tenmo-ADFb. This is the first report of the antidiuretic factor Tenmo-ADFb stimulating tubule secretion. Although the actions of Tenmo-ADFb are indistinguishable from those of Achdo-KII, it is unlikely to act at a kinin receptor, because the core sequence (residues 7–12) lacks the Phe and Trp residues that are critical for kinin activity.

SUMMARYMosquito natriuretic peptide (MNP), an uncharacterised peptide from the yellow fever mosquito, Aedes aegypti, acts via cyclic AMP to stimulate secretion of Na+-rich urine by opening a Na+ conductance in the basolateral membrane of Malpighian tubule principal cells. Corticotropin releasing factor (CRF)-related peptides and calcitonin (CT)-like diuretic peptides use cyclic AMP as a second messenger and were therefore considered likely candidates for MNP. BLAST searches of the genome of the malaria mosquito Anopheles gambiae, gave sequences for the CRF-related peptide Anoga-DH44 and the CT-like peptide Anoga-DH31, which were synthesised and tested for effects on Malpighian tubules from An. gambiae and Ae. aegypti,together with 8-bromo-cyclic AMP.The cyclic AMP analogue stimulated secretion of Na+-rich urine by An. gambiae Malpighian tubules, reproducing the response to MNP in Ae. aegypti. It also depolarised the principal cell basolateral membrane voltage (Vb) while hyperpolarising the transepithelial voltage (Vt) to a similar extent.Anoga-DH44 and Anoga-DH31 stimulated production of cyclic AMP, but not cyclic GMP, by Malpighian tubules of An. gambiae. Both peptides had diuretic activity, but only Anoga-DH31 had natriuretic activity and stimulated fluid secretion to the same extent as 8-bromo-cyclic AMP. Likewise, Anoga-DH31 reproduced the effects of cyclic AMP on tubule electrophysiology, whereas Anoga-DH44initially hyperpolarised Vb and depolarised Vt, which is the opposite of the effect of Anoga-DH31.Anoga-DH44 and Anoga-DH31 were also tested for effects on fluid secretion and ion transport by Ae. aegypti tubules. As in An. gambiae, the CRF-related peptide Anoga-DH44 had a non-specific effect on the transport of Na+ and K+,whereas the CT-like peptide Anoga-DH31 specifically stimulated transepithelial Na+ transport.We conclude that the CT-like peptide Anoga-DH31 is the previously uncharacterised mosquito natriuretic peptide.

Previously, a corticotropin releasing factor (CRF)-like diuretic peptide (Man-duca-DH) has been isolated from Manduca sexta and shown to stimulate fluid excretion in vivo in post-eclosion Pieris rapae adults and in pre-wandering postfeeding Manduca sexta larvae. However, Manduca-DH was reported to have no effect on Malpighian tubules in vitro. Manduca-DH and [Nle2,11]-Manduca-DH were synthesized in Texas and assayed in London on isolated Malpighian tubules of Acheta domesticus. Man-duca-DH stimulated fluid secretion by about 60% of the maximum response achievable with extracts of corpora cardiaca and increased the production of cyclic AMP. In combination with 10−4moll−1 3-isobutyl-l-methyl xanthine (IBMX), Manduca-DH stimulated maximal secretion. A number of CRF-related peptides also stimulated fluid secretion and cyclic AMP production in cricket tubules, and the CRF antagonist α-helical-CRF[9–41] blocked the stimulation of fluid secretion by Manduca-DH. [Nle2,11]-Manduca-DH was more active than Manduca-DH in both assays, suggesting that methionine residues in the natural peptide may become oxidized. Taken in conjunction with previous in vivo studies, the present findings suggest that a Manduca-DH-like diuretic peptide is the hormone controlling post-eclosion diuresis in butterflies, and Manduca-DH was shown to stimulate both fluid secretion and cyclic AMP production in Malpighian tubules from 1–12 h posteclosion Pieris rapae adults. The function of the peptide in Manduca sexta is discussed.