Non-neuronal acetylcholine-induced nitric oxide signaling promotes fluid transport in Malpighian tubules of Mythimna separata.

Non-neuronal acetylcholine (ACh) has been shown to elevate intracellular calcium levels in the Malpighian tubules (MTs) of Mythimna separata. However, the physiological functions and mechanisms of this ACh-mediated signaling in MTs remain largely unknown. In this study, pharmacological analyses suggest that muscarinic receptors trigger ACh-induces Ca2+ signal via the phospholipase C (PLC) and inositol trisphosphate receptor (IP3R) pathway in M. separata MTs. This Ca2+ signal activates a Ca2+-dependent nitric oxide synthase (NOS1), which is specifically localized within the principal cells. NADPH diaphorase and pharmacological analyses confirmed that ACh stimulates NOS activity and elevates intracellular NO levels. This effect was inhibited by the NOS inhibitor L-NAME and by inhibitors of the mAChR-PLC-IP3R cascade. The resulting NO production led to elevated intracellular cGMP levels. Ramsay assay revealed that ACh significantly stimulated fluid secretion, and this effect was suppressed by inhibiting any component of the mAChR-PLC-IP3R-NOS-cGMP signaling pathway. These findings indicate that non-neuronal ACh triggers NO release in the principal cells of M. separata MTs primarily through PLC-IP3R pathway, and that this NO signal in turn promotes fluid transport of the tubules. This study provides new evidence for a functional link between non-neuronal ACh and intracellular NO production in insects.