Journal of Neuroscience, Vol 4, 611-626, Copyright © 1984 by Society for Neuroscience
Serotonin-induced protein phosphorylation in a lobster neuromuscular preparation
MF Goy, TL Schwarz and EA Kravitz
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.
