Serotonin-induced protein phosphorylation in a lobster neuromuscular preparation

Three substances believed to be neurohormones in the lobster--serotonin, octopamine, and proctolin--can modulate the physiology of the dactyl opener muscle of the lobster walking leg. All three act directly on muscle fibers to cause long-lasting contractures. In addition, serotonin enhances the release of transmitter from the excitatory and inhibitory axons that innervate the muscle. We now report that serotonin, applied to intact nerve-muscle preparations, increases the phosphorylation of a soluble 29,000-dalton protein by as much as 20-fold. In two respects the phosphorylation parallels the physiological effects of serotonin: both have a slow time course, and both are caused by low concentrations of serotonin (thresholds of approximately 10(-8) M). The 29,000-dalton protein is present in many lobster tissues, including thoracic ganglia, gills, and several different muscles. This suggests that the phosphorylation participates in some aspect of metabolism common to many types of cells, rather than in an activity unique to nerve or muscle. The function of the phosphorylated protein remains unknown. Several lines of evidence suggest that the phosphorylation is mediated by a cyclic nucleotide, probably cyclic AMP. (1) Adding cyclic AMP or cyclic GMP to nerve-muscle homogenates leads to the phosphorylation. (2) Adding 8-Br-cyclic AMP or 8-Br-cyclic GMP to intact preparations leads to the phosphorylation. (3) In intact tissue, forskolin, an adenylate cyclase activator, and isobutylmethylxanthine, a phosphodiesterase inhibitor, increase cyclic AMP and cyclic GMP and also cause the phosphorylation. (4) Serotonin increases cyclic AMP in intact preparations. (5) RMI 12,330A, which blocks the serotonin-induced cyclic AMP increase, also blocks the phosphorylation. (6) Serotonin has no detectable effect on cyclic GMP. This suggests that cyclic AMP, and not cyclic GMP, is the second messenger that mediates phosphorylation in vivo. In contrast, calcium is not likely to mediate the phosphorylation. Agents that block calcium channels do not block the serotonin-induced phosphorylation, and agents that increase sarcoplasmic calcium do not cause it. Furthermore, calcium does not induce the phosphorylation in a homogenate. One of the other modulators, proctolin, does not increase cyclic AMP or cause the phosphorylation even at concentrations 10(6)-fold greater than the hormone's threshold for producing contractures. The third modulator, octopamine, causes small increases in cyclic AMP and is a weak agonist of the phosphorylation. Neither proctolin nor octopamine alters cyclic GMP levels.