Effects of cyclic 3’, 5’ guanosine monophosphate on larval midgut ecdysone 20-monooxygenase activity of the tobacco hornworm, Manduca sexta

During the last larval stadium of the tobacco hornworm, Manduca sexta, midgut ecdysone 20-monooxygenase undergoes an approximate 50-fold increase in activity, which is temporally coincident with the onset of wandering. In the present study, the effects of the ecdysone agonist and/or second messengers, either alone or in combination with other additives, on midgut ecdysone 20-monooxygenase activity were assessed under in vitro and in vivo conditions. Pre-incubation of M. sexta midgut tissues with second messengers revealed that dibutyryl cGMP elevated midgut monooxygenase activity, whereas dibutyryl cAMP was without effect on the activity compared to untreated controls. Under in vivo conditions, the normal 50-fold increase in midgut monooxygenase activity was diminished by prior injections of the guanylate cyclase inhibitors LY-83583, methylene blue, or ODQ either alone or in combination with RH-5849. The diminution effects of LY-83583, methylene blue, and ODQ were significantly ameliorated if they were co-injected with dibutyryl cGMP, or the guanylate cyclase activator SNAP. Although injections of dibutyryl cGMP alone into competent head- or thorax-ligated pre-wandering stage larvae did not cause an elevation in midgut monooxygenase activity, it was found to synergize when co-injected with a suboptimal dose of RH-5849 and this combination elicited significant increases in enzyme activity. Additionally, radioimmunoassays of midgut cGMP content throughout the M. sexta fifth larval instar revealed that cGMP content increased significantly on day five in concert with the 50-fold increase in monooxygenase activity. Collectively, these data suggest the involvement of a second messenger, cGMP, in the steroid-mediated increase in midgut ecdysone 20-monooxygenase activity.