Past work has shown that adult lobster striated muscle exhibits biochemical, physiological, and pharmacological similarities to both striated and smooth muscle of vertebrates. Furthermore, just as for vertebrate smooth muscle and nervous tissue, the function of lobster striated muscle is modulated by blood borne hormones. Specifically, resting tone, contractility, and relaxation kinetics of lobster muscle are all influenced by amine and peptide hormones synthesized in neurosecretory glands and delivered to muscle via the general circulation. The overall objective of this research proposal is to determine the mechanism whereby these hormones act. In order to achieve this goal, the applicant will investigate hormonal effects on a number of the steps involved in excitation-contraction coupling of the dactyl opener muscle of larval lobsters. Larval muscle is selected because single fibers are readily isolated and investigated with a combination of techniques allowing evaluation of sarcolemmal ion conductances, control of intracellular calcium levels, and monitoring of muscle mechanical activity. The applicant will test two hypotheses proposed to explain the effects of the following hormones on the function of lobster muscle: """"""""...serotonin, octopamine, proctolin, two opioid-like peptides (lobster peptides F1 and F2) and a newly identified peptide which specifically stimulates cGMP metabolism (peptide G1)""""""""; all of these hormones are synthesized by adult lobster neurosecretory glands and released into the circulation. The first hypothesis to be tested is that these hormones act on various steps involved in excitation-contraction coupling. The second hypothesis is that they cause pharmacomechanical coupling; that is, the hormones act on membrane receptors to activate second messenger pathways which """"""""...initiate contraction and modulate muscle tone without activation of excitation-contraction coupling..."""""""". Therefore, the applicant will evaluate the effects of applied second messengers (GTP, cAMP, and IP3) and substances altering their levels within cells (e.g., pertussis toxin, phosphodiesterase inhibitors). In order to test the two proposed hypotheses, the following measurements will be performed before, during, and after exposure to the chemicals noted above: 1. calcium and potassium currents will be recorded with the whole cell patch clamp technique; 2. cytoplasmic concentrations of calcium will be measured with Fura-2 imaging; 3. the sensitivity of contractile proteins to calcium will be measured for muscle fibers permeabilized with Beta-escin and exposed to varying concentrations of extracellular calcium; 4. muscle contractility will be measured from video images of fiber shortening, contractions of single muscle fibers, or absorbance changes at the isobestic point of Fura-2 (260 nm). A distinct advantage of the larval muscle preparation is that measurements 1, 2, and 4 can be performed simultaneously for a single fiber.
