Neuropeptide stimulation of the nitric oxide signaling pathway in Drosophila melanogaster Malpighian tubules.

Activation of the nitric oxide (NO) and guanosine 3', 5'-cyclic monophosphate (cGMP) signaling pathway stimulates fluid secretion by the Drosophila melanogaster Malpighian tubule. The neuropeptide cardioacceleratory peptide 2b (CAP2b) has been previously shown to stimulate fluid secretion in this epithelium by elevating intracellular cGMP levels. Therefore, it was of interest to investigate if CAP2b acts through NO in isolated tubules and thus presumably through stimulation of a tubule NO synthase (NOS). We show here by reverse-transcription polymerase chain reaction that Drosophila NOS (dNOS) is expressed in Malpighian tubules. Biochemical assays of NOS activity in whole tubules show that CAP2b significantly stimulates NOS activity. Additionally, fluid secretion and cyclic nucleotide assays show that CAP2b-induced elevation of intracellular cGMP levels and fluid secretion rates are dependent on the activation of a soluble guanylate cyclase. Treatment of tubules with a specific NOS inhibitor abolishes the CAP2b-induced rise in intracellular cGMP levels. These data indicate that CAP2b stimulates NOS and therefore, endogenous NO production, which, in turn, stimulates a soluble guanylate cyclase. This is the first demonstration of stimulation of an endogenous NOS by a defined peptide in Drosophila.

Separate control of anion and cation transport in malpighian tubules of Drosophila Melanogaster.

Microelectrode measurements of basal, apical and transepithelial potentials in the Malpighian tubules of Drosophila melanogaster were obtained under a range of conditions in order to investigate whether each of the three main second messenger systems known to act in the tubules (cyclic AMP, cyclic GMP and Ca2+) acted specifically on either cation or anion transport, or whether they activated both systems. Ion-selective microelectrode determinations of K+ concentration and pH of secreted fluid allowed the role of each signalling system to be analysed further. Stimulation with cyclic nucleotides markedly alters the potential profile across principal cells through the selective activation of an apical electrogenic V-ATPase. By contrast, manipulation of extracellular chloride levels, combined with stimulation with leucokinin, does not affect the potential profile across the principal cells, showing that chloride must pass through another route. The cell-permeant Ca2+ chelator BAPTA-AM was shown to suppress the action of leucokinins (insect peptides that induce rapid fluid secretion), but not those of cyclic AMP, the neuronally derived insect peptide cardioacceleratory peptide 2b (CAP2b) or its intracellular messenger cyclic GMP. This shows that leucokinins act through Ca2+ and not through cyclic nucleotides and that the cyclic nucleotide pathways do not co-activate the intracellular Ca2+ pathway to exert their effects. Taken together, these results show that leucokinin acts through intracellular Ca2+, independently of cyclic AMP or cyclic GMP, to raise the chloride permeability of the epithelium. By contrast, either cyclic AMP or cyclic GMP (upon CAP2b stimulation) acts on the electrogenic cation-transporting apical V-ATPase, with only a negligible effect on anion conductance and without perturbing intracellular [Ca2+]. There is thus a clear functional separation between the control pathways acting on cation and anion transport in the tubules. Given the evidence from D. melanogaster and other species that chloride does not pass through the principal cells, we speculate that these two pathways may also be physically separated within cell subtypes of the tubules.

CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates tubule fluid secretion via cGMP.

A cardioacceleratory peptide, CAP2b, identified originally in the lepidopteran Manduca sexta, stimulates fluid secretion by Malpighian tubules of the dipteran Drosophila melanogaster. High-performance liquid chromatography analyses of adult D. melanogaster reveal the presence of a CAP2b-like peptide, that coelutes with M. sexta CAP2b and synthetic CAP2b and that has CAP2b-like effects on the M. sexta heart. CAP2b accelerates fluid secretion in tubules stimulated by adenosine 3',5'-cyclic monophosphate (cAMP) but has no effect on tubules stimulated by guanosine 3',5'-cyclic monophosphate (cGMP), implying that it acts through the latter pathway. By contrast, the action of leucokinin is additive to both cAMP and cGMP but not to thapsigargin, suggesting that leucokinin acts by the elevation of intracellular calcium. CAP2b stimulation elevates tubule cGMP levels but not those of cAMP. By contrast, leucokinin has no effect on levels of either cyclic nucleotide. Both CAP2b and cGMP increase transepithelial potential difference, suggesting that stimulation of vacuolar-adenosinetriphosphatase action underlies the corresponding increases in fluid secretion. Overall, the results show that a Drosophila CAP2b-related peptide acts to stimulate fluid secretion by Malpighian tubules through the cGMP-signaling pathway.

Amino acid sequence of CAP2b, an insect cardioacceleratory peptide from the tobacco hawkmoth Manduca sexta.

The primary structure of a novel insect neuropeptide, Cardioacceleratory Peptide 2b (CAP2b), from the tobacco hawkmoth Manduca sexta has been established using a combination of mass spectroscopy, Edman degradation microsequencing, amino acid analysis, and biological assays. The sequence of CAP2b, pyroGlu-Leu-Tyr-Ala-Phe-Pro-Arg-Val-amide, has a molecular weight of 974.6 and is blocked at both the amino and carboxyl ends. Examination of several national computer protein data bases failed to reveal other peptides or proteins with any sequence homology to CAP2b indicating that this is likely to be a novel insect neuropeptide. This peptide may be a general activator of insect viscera since it causes an increase in heart rate in Manduca and in Drosophila, and has also been implicated in the regulation of fluid secretion by the Malphigian tubules of Drosophila.