A major part of the present understanding of the molecular basis of signal transduction has been gained from in vitro studies using classical biochemical methods. In this study, we used 31P NMR spectroscopy to investigate the response of live M2R mouse
melanoma cells to stimulation by
melanocyte-stimulating hormone (
MSH;
melanotropin). In the presence of
3-isobutyl-1-methylxanthine and a synergistic dose of
forskolin (1.67 microM),
MSH induced a transient (approximately 60-min) rise in the cellular concentration of 3',5'-cyclic
adenosine monophosphate (cAMP), which coincided in time with an equivalent decrease (approximately 40%) in
ATP. However, no detectable change in
phosphocreatine concentration was observed. Concomitantly,
MSH induced a striking and unexpected increase in the concentration of three phosphomonoester (PME) metabolites (approximately 2-fold increase in total PME signal area); one signal has been assigned to
phosphoethanolamine. The levels of the PMEs remained high for 2-4 hr and declined slowly (approximately 10 hr) to basal level, following perfusion with fresh culture medium. The increase in PME was also observed after stimulation with
MSH alone. In contrast, stimulation with a high dose of
forskolin (50 microM) and isobutylmethylxanthine (0.2 mM), although effective in stimulating the production of cAMP, did not induce the PME response. Evaluation of the cells' energetics indicated that the enhanced production of
phosphoethanolamine is probably not due to
ethanolamine phosphorylation. Therefore, it is likely to result from hydrolysis of
phosphatidylethanolamine by a specific
phospholipase C. The response of the PMEs appears to be regulated by a cAMP-independent process, suggesting the existence of an alternative transduction pathway controlled by
MSH.