We have developed a model neuromuscular preparation for studying interactions of conventional neurotransmitters and peptide neuromodulators that are co-released by motoneurons. The neuromuscular system, which controls slow flexion of one hemisegment in the crayfish abdomen, consists of 6 identified motoneurons and approximately 40 muscle fibers. We have demonstrated that in addition to the conventional neurotransmitter L-glutamate, three of the motoneurons also contain the peptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH). Proctolin is released by the motoneurons in response to neural stimulation. Perfused proctolin does not induce tension in resting muscles at any concentration, but it amplifies depolarization-induced tension in the muscle fibers by as much as 400% at low concentrations (10-9 M). Proctolin amplifies tension without depolarizing the muscle or changing synaptic potentials. The contribution of neurally-release proctolin to the magnitude of tension generated in the muscle is large in our isolated preparation and appears most significant when tension is sustained. Our primary goal will be to investigate the biochemical basis of the peptide effect. Preliminary results indicate multiple actions of the peptide, involving the increase of membrane-bound voltage- dependent Ca2+ currents as well as modulation of alter steps of the excitation-contraction coupling pathway. we propose to elucidate the biochemical pathway using convergent techniques that allow the concomitant measurement of tension, membrane conductance, internal calcium concentration, membrane currents and membrane channel kinetics during controlled voltage steps coupled with applications of proctolin or step increases in cytosolic concentrations of intracellular messengers such as inositol 1,4,5-triphosphate, cyclic AMP, cyclic GMP and calcium. We will continue to relate our findings to the role of peptide neuromodulation in normal muscle function. Since biochemical processes underlying tension production are conserved among organisms, our results should provide general insights into the mechanisms and functions of peptide co- transmission in neuromuscular systems, which in turn may provide new approaches to investigations of neuromuscular diseases.
