Cyclic 3′, 5′ guanosine monophosphate and 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 (E20M) activity increases 50-fold coincident with wandering onset. In our study, the effects of the ecdysone agonist RH-5849 and second messengers, dibutyryl cyclic 3′, 5′ adenosine monophosphate (dibutyryl cAMP) and dibutyryl cyclic 3′, 5′ guanosine monophosphate (dibutyryl cGMP), either alone or combined with other additives, on midgut E20M were assessed in vitro and in vivo. Pre-incubation of midguts with dibutyryl cAMP was ineffective, but dibutyryl cGMP elevated E20M activity. In vivo, the 50-fold increase in E20M activity was diminished by injections of guanylate cyclase inhibitors alone or combined with RH-5849. Guanylate cyclase inhibitor effects were significantly diminished when co-injected with dibutyryl cGMP, or a guanylate cyclase activator. Although dibutyryl cGMP injections into head- or thorax-ligated pre-wandering larvae did not elevate midgut E20M activity, they did synergize when co-injected with a suboptimal dose of RH-5849, thereby eliciting significant activity increases. Radioimmunoassays of midgut cGMP content throughout the M. sexta fifth larval instar revealed that levels of cGMP significantly increased between days four and five consistent with the 50-fold E20M activity increase. These data indicate cGMP involvement in the ecdysone-mediated increase in midgut E20M activity.