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 ,uM), MSH induced a transient (=60-min) rise in the cellular concentration of 3',5'-cyclic adenosine monophosphate (cAMP), which coincided in time with an equivalent decrease (-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 ('-2-fold increase in total PME signal area); one signal has been assigned to phosphoethanolamine. The levels of the PMEs remained high for 24 hr and declined slowly (410 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 ,uM) and isobutylmethylxanthine (0.2 mM), although effective in stimulating the production ofcAMP, 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.cAMP serves as a second messenger for numerous hormonally regulated processes, and its levels are tightly controlled by the relative rates of the adenylate cyclase and cAMP phosphodiesterase reactions (1-3). Melanocyte-stimulating hormone (MSH; melanotropin) controls melanin synthesis in pigment cells by cAMP-mediated (4) regulation of the levels and activity of tyrosinase, the rate-limiting enzyme in melanin synthesis (5). The cAMP response to stimulation by MSH was studied in M2R mouse melanoma cells (6), using [3H]-adenine labeling (7) and conventional techniques (8). cAMP accumulation was stimulated to a surprisingly high extent by MSH (10-1000 nM) and a low dose (2 ,uM) of forskolin under conditions where cAMP phosphodiesterase activity was inhibited by 3-isobutyl-1-methylxanthine (IBMX). This dose of forskolin has a negligible effect when used alone but induces a strong synergistic effect in combination with hormones (9), including MSH (6). Twenty minutes after stimulation, approximately 45% of the cell's adenine nucleotide pool was converted into cAMP (6). Intracellular cAMP concentrations are generally believed to fluctuate in the micromolar range, presumably without significantly affecting cellular ATP levels (10, 11). However, the inclusion of IBMX and forskolin together with MSH in t...

M2R melanoma tumors in male C57 black mice were used to correlate magnetic resonance (MR) images with the corresponding histologic slices and to determine if analysis of the achievable correlation can provide a basis for predicting gross histologic features with MR imaging alone. The MR imaging sections obtained at 4.7 T were each 680 microns thick, with an in-plane resolution of 195 microns. The distribution of melanin within the histologic slices correlated well with the high-signal-intensity regions on the T1-weighted images (T1WIs), while these regions had low signal intensity on the T2-weighted images (T2WIs), providing evidence that melanin or melanin-associated paramagnetic species are responsible for the observed proton relaxation rate enhancement. Viable melanoma cells typically showed intermediate signal intensity on T2WIs, T1WIs, and proton-density images. Necrosis typically had high signal intensity on T2WIs, T1WIs, and proton-density images. Quantitation of the MR imaging results, followed by statistical analysis, demonstrated statistically significant differences between melanin-rich, viable-melanoma, and necrotic regions on MR images.

Melanocortins appear to be involved as regulators in an ever growing number of physiological processes in cells and tissues of diverse functions. While such trends are apparent also in the case of other peptide hormones, it appears that melanocortin receptors can be regarded as unique among G-protein-linked receptors due to their special need for extracellular Ca2+ which may relate to some, yet undetermined selectivity of their actions. The physiological role that Ca2+ may be playing and the diverse signaling mechanisms regulated, as well as the nature of the cell-specific responses elicited in melanocortin-sensitive cells/tissues, have yet to be elucidated. Likewise, it will be of interest to establish the relationship of melanocortins to processes like growth and differentiation of cells, as well as to higher, more complex processes such as those regulated in the CNS.

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