norpA and itpr mutants reveal roles for phospholipase C and inositol (1, 4, 5)-trisphosphate receptor in Drosophila melanogaster renal function

Abstract

Mutants of norpA, encoding phospholipase Cß (PLCß), and itpr, encoding inositol (1,4,5)-trisphosphate receptor (IP₃R), both attenuate response to diuretic peptides of Drosophila melanogaster renal (Malpighian) tubules. Intact tubules from norpA mutants severely reduced diuresis stimulated by the principal cell- and stellate cell-specific neuropeptides, CAP_2b and Drosophila leucokinin (Drosokinin), respectively, suggesting a role for PLCß in both these cell types. Measurement of IP₃ production in wild-type tubules and in Drosokinin-receptor-transfected S2 cells stimulated with CAP_2b and Drosokinin, respectively, confirmed that both neuropeptides elevate IP₃ levels. In itpr hypomorphs, basal IP₃ levels are lower, although CAP_2b-stimulated IP₃ levels are not significantly reduced compared with wild type. However, CAP_2b-stimulated fluid transport is significantly reduced in itpr alleles. Rescue of the itpr^90B.0allele with wild-type itpr restores CAP_2b-stimulated fluid transport levels to wild type. Drosokinin-stimulated fluid transport is also reduced in homozygous and heteroallelic itpr mutants. Measurements of cytosolic calcium levels in intact tubules of wild-type and itpr mutants using targeted expression of the calcium reporter, aequorin, show that mutations in itpr attenuated both CAP_2b- and Drosokinin-stimulated calcium responses. The reductions in calcium signals are associated with corresponding reductions in fluid transport rates. Thus, we describe a role for norpA and itpr in renal epithelia and show that both CAP_2b and Drosokinin are PLCß-dependent, IP₃-mobilising neuropeptides in Drosophila. IP₃R contributes to the calcium signalling cascades initiated by these peptides in both principal and stellate cells.