Antagonistic control of fluid secretion by the Malpighian tubules of Tenebrio molitor: effects of diuretic and antidiuretic peptides and their second messengers
U. I. M. Wiehart1,
S. W. Nicolson1,*,
R. A. Eigenheer2 and
D. A. Schooley2
1 Department of Zoology, University of Cape Town, Rondebosch 7701, South Africa and
2 Department of Biochemistry, University of Nevada, Reno, NV 89503, USA
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Fig. 1. Dose–response curves for the effect of Tenebrio molitor diuretic peptides Tenmo-DH37 (filled squares) and Tenmo-DH47 (open circles) on fluid secretion rates. Results are expressed as a percentage of the maximal response obtained in the presence of 8-bromo-cyclic AMP (0.1 mmol l–1). Data points are the mean of 6–12 determinations for Tenmo-DH37 and 6–7 determinations for Tenmo-DH47; vertical lines represent ± 1 S.E.M. The EC50 values are 0.12 nmol l–1 for Tenmo-DH37 and 26 nmol l–1 for Tenmo-DH47 (r2 values for the curve fits were 0.95 and 0.96, respectively).
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Fig. 2. Lack of synergism between Tenmo-DH37 (DH37) and Tenmo-DH47 (DH47). Diuretic peptides were tested at 0.2 nmol l–1 and 40 nmol l–1, approximately 1.5 times their respective EC50 values, after which the Ringer’s solution was changed to one containing both peptides (at these same concentrations). No change in secretion rates was observed. Data are presented as means + 1 S.E.M. (N=8).
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Fig. 3. Dose–response curve for the effect of cyclic AMP on fluid secretion by Tenebrio molitor Malpighian tubules. Data are presented as means ± S.E.M. of 7–12 tubules. The EC50 was 350 µmol l–1 (r2=0.999 for the curve fit). Note that data for the two highest cyclic AMP concentrations tested are shown in the graph, but were excluded from the non-linear regression analysis because they indicate some receptor desensitisation at high levels, and this cannot be accommodated by the algorithm used.
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Fig. 4. Comparison of secretory response to cyclic AMP and Tenmo-DH37 (DH37). No significant differences in the response time or maximum rates of secretion were observed when Malpighian tubules of Tenebrio molitor were stimulated with 1 mmol l–1 cyclic AMP (broken line) or 100 nmol l–1 Tenmo-DH37 (solid line). Each point shows the mean ± S.E.M. for 8–12 tubules. The horizontal filled bar indicates the period during which tubules were exposed to either cyclic AMP or Tenmo-DH37.
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Fig. 5. Dose–response curve for the effect of cyclic GMP on fluid secretion. Data are presented as means ± 1 S.E.M., N=5–11. The EC50 value determined was 490 µmol l–1 (r2=0.999, 95 % confidence intervals 99.7 µmol l–1 to 2.4 mmol l–1). Note that data for the highest cyclic GMP concentration have been excluded from the non-linear regression analysis because they indicate some receptor desensitisation at high levels, and this cannot be accommodated by the algorithm used.
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Fig. 6. Antagonistic effect of cyclic GMP on Malpighian tubules of Tenebrio molitor already stimulated with cyclic AMP. Each point shows the mean ± S.E.M. for 8–10 tubules. Responses are shown for 0.5 mmol l–1 cyclic AMP in combination with 0.5 mmol l–1 cyclic GMP (broken line), 0.5 mmol l–1 cyclic AMP with 1 mmol l–1 cyclic GMP (dashed line) and 0.25 mmol l–1 cyclic AMP with 1 mmol l–1 cyclic GMP (solid line). The horizontal dotted bar indicates the period during which tubules were exposed to cyclic AMP, and the horizontal filled bar indicates the period of exposure to cyclic GMP.
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Fig. 7. Antagonism between Tenebrio molitor peptides controlling Malpighian tubule secretion and between their second messengers. Effect of adding Tenmo-ADF (ADF) to tubules already stimulated with Tenmo-DH37 (DH37) (both at concentrations of 100 nmol l–1) compared with the effect of adding cyclic GMP to tubules already stimulated with cyclic AMP (both at concentrations of 0.5 mmol l–1). Secretion rates were measured after 45 min of each treatment. Values are means + 1 S.E.M. for eight tubules (peptides) or seven tubules (second messengers). C, control rates.
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Fig. 8. Dose–response curve for the effect of CAP2b on fluid secretion by Tenebrio molitor tubules. Data are presented as means ±1 S.E.M. for 8–9 tubules. The EC50 value determined was 85 nmol l–1 (r2=0.79, 95 % confidence intervals 4.3 nmol l–1 to 1.7 µmol l–1).
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Fig. 9. Antagonism between CAP2b and Tenmo-DH37 (DH37). Inhibition of fluid secretion by 1 µmol l–1 CAP2b and subsequent slow stimulation by Tenmo-DH37 (10 nmol l–1). Data are presented as means ±1 S.E.M. for five tubules. The horizontal filled bar indicates the period of exposure to CAP2b, and the horizontal dotted bar indicates the period of exposure to Tenmo-DH37.
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Fig. 10. Effects of stimulants on secretion rates of Tenebrio molitor larval (filled columns) and adult (open columns) Malpighian tubules. Tubules were stimulated with 100 nmol l–1 Tenmo-DH37 (DH37) and 1 mmol l–1 cyclic AMP. Data are presented as means + 1 S.E.M. (N=9–10). C, control rates.
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© The Company of Biologists Ltd 2002
