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First published online November 2, 2007
Journal of Experimental Biology 210, 3979-3989 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.006056
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An antidiuretic peptide (Tenmo-ADFb) with kinin-like diuretic activity on Malpighian tubules of the house cricket, Acheta domesticus (L.)

Geoffrey M. Coast1,*, Ronald J. Nachman2 and David A. Schooley3

1 School of Biological and Chemical Sciences, Birkbeck, University of London, Malet Street, London WC1E 7HX, UK
2 US Department of Agriculture, APMRU/SPARC, College Station, TX 77845, USA
3 Biochemistry, University of Nevada, Reno, NV 89557, USA


Figure 1
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  		Fig. 1. Peptides known to reduce primary urine production [Tenmo-ADFa (ADFa), Tenmo-ADFb (ADFb) and Manse-CAP2b (CAP2b)] were tested at 1 µmol l–1 for an effect on cricket tubules, along with their second messenger cyclic GMP [1 mmol l–1 8-bromo cyclic GMP (cGMP)]. The cricket kinin Achdo-KII (1 nmol l–1; AKII) was included in the assay as a positive control. Bars represent mean values for the change in rate of secretion ({Delta} pl mm–1 min–1) following addition of test compounds, and vertical lines represent + 1 s.e.m. for the number of replicates indicated in parentheses. Tenmo-ADFb and 8-bromo cyclic GMP significantly increased (***P<0.001) fluid secretion, and the response to the former was comparable to that of Achdo-KII.

 

Figure 2
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  		Fig. 2. Dose–response curves for the diuretic activity of (A) Tenmo-ADFb and (B) Achdo-KII. Results are expressed as a percentage of the response to a supramaximal concentration (1 nmol l–1) of Achdo-KII. Data points are the means ± 1 s.e.m. of 7–10 replicates. Note the vast difference in potency between Tenmo-ADFb (EC50 1.5 µmol l–1) and Achdo-KII (EC50 10.2 pmol l–1).

 

Figure 3
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  		Fig. 3. Time course for the stimulation of fluid secretion by supramaximal concentrations of Tenmo-ADFb (10 µmol l–1; solid symbols and line) and Achdo-KII (1 nmol l–1; open symbols and dotted line). Results are normalised by being expressed as a percentage of the unstimulated rate of secretion at 45 min. Data points are the means ± 1 s.e.m. of 11–12 replicates. Fluid secretion increases rapidly following addition of the individual peptides (downward arrow) and is not further elevated when they are applied together (upward arrow).

 

Figure 4
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  		Fig. 4. Secreted fluid pH and concentrations of K+, Na+ and Cl– before (open bars) and after (solid bars) the addition of 10 µmol l–1 Tenmo-ADFb (ADFb), 1 nmol l–1 Achdo-KII (AKII) and 1 mmol l–1 8-bromo cyclic GMP (cGMP). Bars represent the means + 1 s.e.m. for the number of replicates shown in parentheses. Asterisks indicate where the change in composition following addition of the secretagogue is significant in a paired t-test; *P<0.05; **P<0.01; ***P<0.001.

 

Figure 5
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  		Fig. 5. Tenmo-ADFb (1 µmol l–1; ADFb) and Achdo-KII (1 nmol l–1; AKII) have no effect on fluid secretion by tubules incubated in low Cl– saline (one-tenth normal [Cl–]). Bars represent the means + 1 s.e.m. of fluid secretion by tubules in normal (open bars) and in low Cl– (solid bars) saline. The number of replicates is shown in parentheses. In the final experimental period, all tubules were moved to normal saline (+ chloride). Note that identical letters indicate mean values that do not differ significantly i.e. P<0.05.

 

Figure 6
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  		Fig. 6. The chloride channel blocker DPC depresses basal secretion and reduces the effect of (A) 10 µmol l–1 Tenmo-ADFb (+ADFb) and (B) 1 nmol l–1 Achdo-KII (+AKII). Bars represent the means + 1 s.e.m. for the number of replicates shown in parentheses. Tubules were held in control saline (open bars) or moved to a saline containing 0.2 mmol l–1 DPC in the first experimental period (+DPC; solid bars). Identical letters indicate significant differences between mean rates of secretion (P<0.01).

 

Figure 7
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  		Fig. 7. (A) Evidence of synergism between 10 µmol l–1 8-bromo cyclic AMP (cAMP) and 0.3 µmol l–1 Tenmo-ADFb (ADFb). Bars represent the means + 1 s.e.m. for the change in rate of secretion ({Delta} pl mm–1 min–1) following addition of the secretagogues individually and together. The number of replicates is shown in parentheses, and identical letters indicate values that differ significantly (P<0.001). The sum of the separate responses to 8-bromo cyclic AMP and Tenmo-ADFb (Sum) is significantly less than when they are tested together (Both). (B) 8-bromo cyclic GMP (1 mmol l–1) increases secretion by tubules that have been maximally stimulated by Tenmo-ADFb (10 µmol l–1) and Achdo-KII (1 nmol l–1; AKII). Bars represent the means + 1 s.e.m. for the change in rate of secretion ({Delta} pl mm–1 min–1) after addition of the peptides alone (open bars) and then together with 8-bromo cyclic GMP (grey bars). Black bars show the diuretic activity of 1 mmol l–1 8-bromo cyclic GMP alone measured in separate groups of tubules. The number of replicates is shown in parentheses, and identical letters indicate values that differ significantly (P<0.001).

 

Figure 8
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  		Fig. 8. Representative recordings showing the change in transepithelial voltage (Vt) following the addition of (A) 10 µmol l–1 Tenmo-ADFb, (B) 1 nmol l–1 Achdo-KII and (C) 1 mmol l–1 8-bromo cyclic GMP. Thick arrows show the time of insertion (downward) and withdrawal (upward) of the microelectrode, and thin arrows show when peptides were added. The horizontal bar in C shows when 8-bromo cyclic GMP was present in the perfusate, and the thin arrow indicates the time of addition of 10 µmol l–1 Tenmo-ADFb.

 

Figure 9
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  		Fig. 9. (A) Representative recording of the transepithelial voltage (Vt) before and after the addition of 0.2 mmol l–1 diphenylamine-2-carboxylate (DPC) to the bathing saline. DPC briefly depolarises Vt but does not inhibit the spontaneous voltage oscillations. Nor does it prevent the reduction of Vt by 10 µmol l–1 Tenmo-ADFb (thin arrow; ADFb). (B) A representative recording of the basal membrane voltage (Vb) showing this is not affected by the addition of either 10 µmol l–1 Tenmo-ADFb or 1 nmol l–1 Achdo-KII (AKII). Thick arrows mark the point of insertion (downward) and withdrawal (upward) of the microelectrode, and horizontal bars show when peptides were present in the perfusate.

 

Figure 10
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  		Fig. 10. A representative recording of spontaneous contractions by a cricket hindgut preparation challenged with first 10 µmol l–1 Tenmo-ADFb (A), then 2 nmol l–1 Achdo-KII (B) and finally with saline alone (C). Bars show when test substances were present in the perfusate.

 




© The Company of Biologists Ltd 2007