Immunological, biochemical and physiological analyses of cardioacceleratory peptide 2 (CAP₂) activity in the embryo of the tobacco hawkmoth Manduca sexta

The cells in the embryonic CNS of the tobacco hawkmoth, Manduca sexta, that synthesize a cardioacceleratory peptide 2 (CAP₂)-like antigen were identified using immunohistochemical techniques. Two distinct neurosecretory cell types were present in the abdominal ventral nerve cord (VNC) that contain CAP2-like immunoreactivity during late embryogenesis: a pair of large (diameter range 15-20 μm) cells lying along the posterior, dorsal midline of abdominal ganglia A4-A8, and a bilateral set of four smaller (diameter range 6-11 μm) neurons which lie at the base of each ventral root in abdominal ganglia A2-A8. CAP2-like accumulation appeared to follow independent patterns in the two cell types. CAP₂-like immunoreactivity began at 60% of embryo development (DT) in the medial cells, accumulated steadily throughout embryogenesis, and dropped markedly during hatching. Lateral cells synthesized the CAP₂-like antigen later in development (70 % DT) and showed a sharp drop in antigen levels between 75 % and 80 % of embryonic development. Extracts from developing M. sexta embryos were found to contain a cardioactive factor capable of accelerating the contraction frequency of the pharate adult moth heart in a fashion similar to CAP₂. Immunoprecipitation with a monoclonal antibody that specifically recognizes the two endogenous Manduca cardioacceleratory peptides and purification using high pressure liquid chromatography identified this factor as cardioacceleratory peptide 2 (CAP₂). Using an in vitro heart bioassay, the levels of this cardioactive neuropeptide were traced during the development of the M. sexta embryo. As with the immunohistochemical results, two periods during embryogenesis were identified in which the level of CAP₂ dropped markedly: between 75 % and 80 % development, and at hatching. Embryo bioassays of CAP₂ activity were used to identify possible target tissues for physiological activity during these two putative release times. CAP₂ was found to accelerate contraction frequency in the embryonic heart and hindgut of Manduca in a dose-dependent fashion. Of these two possible targets, the hindgut proved to be more sensitive to CAP₂, having a lower response threshold and a longer duration of response to a given concentration of the exogenously applied peptide. Based on these immunocytochemical, pharmacological and biochemical results, and on a previously published detailed analysis of Manduca embryogenesis, we conclude that CAP₂ is probably released from a specific set of identified neurosecretory cells in the abdominal VNC to modulate embryonic gut activity at 75-80 % of embryo development during ingestion of the extraembryonic yolk.