Mode of Action of aDrosophilaFMRFamide in Inducing Muscle Contraction

Abstract

Drosophila melanogaster is a model system for examining the mechanisms of action of neuropeptides. DPKQDFMRFamide was previously shown to induce contractions in Drosophila body wall muscle fibres in a Ca 2+ -dependent manner. The present study examined the possible involvement of a G-protein-coupled receptor and second messengers in mediating this myotropic effect after removal of the central nervous system. DPKQDFMRFamide-induced contractions were reduced by 70% and 90%, respectively, in larvae with reduced expression of the Drosophila Fmrf receptor (FR) either ubiquitously or specifically in muscle tissue, compared with the response in control larvae in which expression was not manipulated. No such effect occurred in larvae with reduced expression of this gene only in neurons. The myogenic effects of DPKQDFMRFamide do not appear to be mediated through either of the two Drosophila myosuppressin receptors (DmsR-1 and DmsR-2). DPKQDFMRFamide-induced contractions were not reduced in Ala1 transgenic flies lacking activity of calcium/calmodulin-dependent protein kinase (CamKII), and were not affected by the CaMKII inhibitor KN-93. Peptide-induced contractions in the mutants of the phospholipase C-β (PLCβ) gene (norpA larvae) and in IP 3 receptor mutants were similar to contractions elicited in control larvae. The peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. Peptide-induced contractions were not potentiated by 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and were not antagonized by inhibitors of cAMP-dependent or cGMPdependent protein kinases. Additionally, exogenous application of arachidonic acid failed to induce myogenic contractions. Thus, DPKQDFMRFamide induces contractions via a G-protein coupled FMRFamide receptor in muscle cells but does not appear to act via cAMP, cGMP, IP 3 , PLC, CaMKII or arachidonic acid.

Cell-selective modulation of theDrosophilaneuromuscular system by a neuropeptide

“…We investigated the most abundant peptide encoded in the Drosophila dFMRF gene, DPKQDFMRFa, which has been isolated and purified from Drosophila tissue and is thought to be released as a neurohormone (Nambu et al 1998;Nichols et al 1999;White et al 1986). Previous work showed that this peptide can increase transmitter release from motor neurons in a cellspecific manner (Dunn and Mercier 2005;Klose et al 2010) and that it acts directly on muscle cells to elicit slow contractions (Clark et al 2008;Milakovic et al 2014). We now present evidence that DPKQDFMRFa alters input resistance preferentially in some muscle cells and elicits stronger contractions in these cells.…”

“…Following dissection, the anterior dissection pin was replaced with the Grass FT03 Force Displacement Transducer (Grass Instrument, Quincy, MA) as described previously (Clark et al 2008;Milakovic et al 2014). Contractions were amplified using a MOD CP122A amplifier (Grass Telefactor, West Warwick, RI), digitized using DATAQ data acquisition (Model DI-145; DATAQ Instruments, Akron, OH), and viewed using WinDaq software (DATAQ Instruments).…”

“…Specific details for RT-quantitative PCR (qPCR) are reported elsewhere (Milakovic et al 2014). Briefly, total RNA was isolated using Norgen's Total RNA Purification Kit (St. Catharines, Ontario, Canada), 500 ng of total RNA were reverse-transcribed with iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA), and SYBR Green qPCR SuperMix (Invitrogen) was added to cDNA and primers.…”

Octopamine and tyramine regulate the activity of reproductive visceral muscles in the adult female blood-feeding bug, Rhodnius prolixus

“…Proctolin was also shown to reduce octopamine-induced cAMP levels in oviducts, suggesting that the components of octopamine and proctolin signalling pathways interact to modulate oviduct contraction (Nykamp and Lange, 2000). The interaction of RhoprFIRFa and octopamine is not known, although it is not likely that octopamine interacts with the RhoprFIRFa signalling pathway according to a recent study in Drosophila (Milakovic et al, 2014). Milakovic et al (2014) found that FMRFamide-induced muscle contraction is independent of the well-known intracellular players such as calmodulin kinase II, IP 3 , cAMP, etc.…”

“…The interaction of RhoprFIRFa and octopamine is not known, although it is not likely that octopamine interacts with the RhoprFIRFa signalling pathway according to a recent study in Drosophila (Milakovic et al, 2014). Milakovic et al (2014) found that FMRFamide-induced muscle contraction is independent of the well-known intracellular players such as calmodulin kinase II, IP 3 , cAMP, etc. ; however, we do not know the pathway used by RhoprFIRFa in this preparation.…”