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First published online August 18, 2005
Journal of Experimental Biology 208, 3281-3291 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01760

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Mosquito natriuretic peptide identified as a calcitonin-like diuretic hormone in Anopheles gambiae (Giles)

Geoffrey M. Coast^1,*, Christopher S. Garside¹, Simon G. Webster², Kathleen M. Schegg³ and David A. Schooley³

¹ Department of Biology, Birkbeck (University of London), London WC1E 7HX, UK
² School of Biological Sciences, University of North Wales, Gwynedd LL57 2UW, UK
³ Biochemistry Department, University of Nevada, Reno, NV 89557, USA

View larger version (14K): [in a new window]   Fig. 1. Exogenous 8-bromo-cyclic AMP stimulates secretion of Na+-rich urine by An. gambiae Malpighian tubules. Fluid secretion and urine Na+ and K+ concentrations were initially measured over a 30 min control period and then at 10–15 min intervals after the addition of 1 mmol l–1 8-bromo-cyclic AMP. Data points show the means ± S.E.M. for five tubules. (A) Fluid secretion (solid line) and the tubule fluid [Na+]:[K+] ratio (broken line) increase after the addition of 8-bromo-cyclic AMP. (B) This reflects the selective stimulation of transepithelial Na+ transport (solid line) compared with K+ transport (broken line). Arrows show the time of addition of 8-bromo-cyclic AMP.

View larger version (10K): [in a new window]   Fig. 2. Representative recordings of (A) transepithelial voltage (Vt) and (B) principal cell basolateral membrane voltage (Vb) in Malpighian tubules challenged with 100 µmol l–1 8-bromo-cyclic AMP. Exogenous cyclic AMP hyperpolarises Vt and depolarises Vb to a similar extent. Horizontal bars indicate when 8-bromo-cyclic AMP was present in the bath.

View larger version (16K): [in a new window]   Fig. 3. Anoga-DH44 has diuretic activity but does not selectively stimulate Na+ transport. Fluid secretion and urine Na+ and K+ concentrations were initially measured over a 30 min control period and then at 10–15 min intervals after the addition of 1 µmol l–1 Anoga-DH44 alone (first arrow) and then in combination with 1 mmol l–1 8-bromo-cyclic AMP (second arrow). Data points show the means ± S.E.M. for five tubules. (A) Fluid secretion (solid line) is increased 3-fold by Anoga-DH44, whereas the urine [Na+]:[K+] ratio (broken line) fell slightly. The addition of 8-bromo-cyclic AMP to the same batch of tubules further accelerates fluid secretion and increases the [Na+]:[K+] ratio. (B) Both Na+ (solid line) and K+ (broken line) transport are increased by Anoga-DH44, whereas 8-bromo-cyclic AMP selectively stimulates the secretion of Na+.

View larger version (16K): [in a new window]   Fig. 4. Anoga-DH31 stimulates diuresis and natriuresis, mimicking the actions of exogenous cAMP. Fluid secretion and urine Na+ and K+ concentrations were initially measured over a 30 min control period and then at 10–15 min intervals after the addition of 1 µmol l–1 Anoga-DH31 alone (first arrow) and then in combination with 1 mmol l–1 8-bromo-cyclic AMP (second arrow). Data points show the means ± S.E.M. for five tubules. (A) Anoga-DH31 stimulates fluid secretion (solid line) and increases the urine [Na+]:[K+] ratio (broken line) to the same extent as 8-bromo-cyclic AMP. (B) Transepithelial Na+ transport (solid line) is selectively stimulated by Anoga-DH31, which has relatively little effect on K+ transport (broken line).

View larger version (17K): [in a new window]   Fig. 5. Sequential addition of Anoga-DH44 and Anoga-DH31 to the same batch of tubules demonstrates that only the latter has pronounced natriuretic activity. Fluid secretion and urine Na+ and K+ concentrations were measured over 30 min intervals under control conditions and after the addition of 1 µmol l–1 Anoga-DH44 alone and in combination with 1 µmol l–1 Anoga-DH31. Bars represent the means + S.E.M. for five tubules. Anoga-DH44 stimulates fluid secretion (open bars) without affecting the [Na+]:[K+] ratio (solid bars) of the secreted urine, which increases dramatically after the addition of Anoga-DH31 along with a further acceleration of urine flow. Identical letters indicate values that do not differ significantly (P>0.05).

View larger version (11K): [in a new window]   Fig. 6. Representative recordings of the K+ concentration (blue line) of urine secreted by tubules challenged with either (A) 1 µmol l–1 Anoga-DH44 or (B) 1 µmol l–1 Anoga-DH31. The concentration of Na+ (red line) in the secreted fluid was calculated assuming the sum of Na+ and K+ concentrations was 200 mmol l–1. Arrows show the time of addition of the diuretic peptides.

View larger version (9K): [in a new window]   Fig. 7. Representative recordings of principal cell basolateral membrane voltage (Vb) in tubules stimulated with either (A) 100 nmol l–1 Anoga-DH44 or (B) 100 nmol l–1 Anoga-DH31. The response to Anoga-DH44 is triphasic, commencing with a transient hyperpolarisation (Phase 1) followed by the depolarisation (Phase 2) and repolarisation (Phase 3) of Vb before the peptide is washed off. Horizontal bars indicate when the peptides were present in the bath.

View larger version (13K): [in a new window]   Fig. 8. Representative recording of the basolateral membrane voltage (Vb) in a principal cell challenged sequentially with 100 nmol l–1 Anoga-DH31, 100 nmol l–1 Anoga-DH44 and 100 µmol l–1 8-bromo-cyclic AMP. Note that only Anoga-DH44 gives a triphasic response beginning with a brief hyperpolarisation. Horizontal bars indicate when peptides or cyclic AMP analogue were present in the bath. Arrows show when the principal cell was impaled (downward arrow) and the microelectrode withdrawn (upward arrow).

View larger version (17K): [in a new window]   Fig. 9. Anoga-DH44 does not selectively stimulate Na+ transport after phosphodiesterase activity is inhibited by IBMX. Fluid secretion and urine Na+ and K+ concentrations were first measured under control conditions (30 min) and then in the presence of 100 µmol l–1 IBMX (50 min). Subsequently, the tubules were challenged with 1 µmol l–1 Anoga-DH44 alone (40 min) and then in combination with 1 µmol l–1 Anoga-DH31 (40 min) in the continued presence of IBMX. Fluid secretion and ion concentrations were measured at the end of each collection period. Bars represent the means + S.E.M. for eight tubules. Fluid secretion (open bars) increases in the presence of IBMX, but the urine [Na+]:[K+] ratio (solid bars) is unchanged. Anoga-DH44 promotes a further increase in secretion but has no effect on the [Na+]:[K+] ratio, whereas both parameters increase dramatically after the addition of Anoga-DH31. Identical letters indicate values that do not differ significantly (P>0.05).

View larger version (13K): [in a new window]   Fig. 10. Representative recording of the basolateral membrane voltage (Vb) in a principal cell challenged separately and together with 100 nmol l–1 Anoga-DH31 and 100 nmol l–1 Musdo-K. Anoga-DH31 depolarises and Musdo-K hyperpolarises Vb, but in combination they produce a triphasic response mimicking that obtained with Anoga-DH44 (cf. Fig. 7A). Horizontal bars indicate when the peptides were present in the bath. Arrows show when the principal cell was impaled (downward arrow) and the microelectrode withdrawn (upward arrow).

View larger version (16K): [in a new window]   Fig. 11. Anoga-DH31 has natriuretic activity in tubules previously challenged with Musdo-K. Fluid secretion and urine Na+ and K+ concentrations were measured over 30 min intervals under control conditions and after the addition of 1 µmol l–1 Musdo-K alone and then in combination with 1 µmol l–1 Anoga-DH31. Bars represent the means + S.E.M. for eight tubules. Fluid secretion (open bars) is stimulated 4-fold by Musdo-K, but the urine [Na+]:[K+] ratio (solid bars) is unchanged. Both parameters are increased >6-fold after the addition of Anoga-DH31. Identical letters indicate values that do not differ significantly (P>0.05).