NEUROPEPTIDE STIMULATION OF THE NITRIC-OXIDE SIGNALING PATHWAY IN DROSOPHILA-MELANOGASTER MALPIGHIAN TUBULES

Activation of the nitric oxide (NO) and guanosine 3',5'-cyclic monopho sphate (cGMP) signaling pathway stimulates fluid secretion by the Dros ophila melanogaster Malpighian tubule. The neuropeptide cardioaccelera tory peptide 2b (CAP(2h)) has been previously shown to stimulate fluid secretion in this epithelium by elevating intracellular cGMP levels. Therefore, it was of interest to investigate if CAP(2b) acts through N O in isolated tubules and thus presumably through stimulation of a tub ule NO synthase (NOS). We show here by reverse-transcription polymeras e chain reaction that Drosophila NOS (dNOS) is expressed in Malpighian tubules. Biochemical assays of NOS activity in whole tubules show tha t CAP(2b) significantly stimulates NOS activity. Additionally, fluid s ecretion and cyclic nucleotide assays show that CAP(2b)-induced elevat ion of intracellular cGMP levels and fluid secretion rates are depende nt on the activation of a soluble guanylate cyclase. Treatment of tubu les with a specific NOS inhibitor abolishes the CAP(2b)-induced rise i n intracellular cGMP levels. These data indicate that CAP(2b) stimulat es NOS and, therefore, endogenous NO production, which, in turn, stimu lates a soluble guanylate cyclase. This is the first demonstration of stimulation of an endogenous NOS by a defined peptide in Drosophila.