Stimulation of cAMP and phosphomonoester production by melanotropin in melanoma cells: 31P NMR studies.

Abstract

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...

Cyclic 3′‐5′‐adenosine monophosphate binds to annexin I and regulates calcium‐dependent membrane aggregation and ion channel activity

“…It is possible that the affected membranes may be having an effect on the interaction between cAMP and AnxI. However, a survey of the literature indicates that while the resting intracellular concentration of cAMP may be in the micromolar range, quantitative conversion of cytosolic millimolar concentrations of ATP to cAMP has been observed in stimulated adipocytes [22] and melanocytes [23] using HPLC and NMR, respectively. The quantitative flow of ATP to cAMP has also been the subject of extensive studies by mass spectrometry [24].…”

Stimulation of fructose 1,6‐bisphosphate production in melanoma cells by α‐melanocyte‐stimulating hormone

“…This phenomenon was further delineated by 31 P-NMR spectroscopy of cultured M2R cells, which showed that A-melanotropin stimulation under these conditions indeed elevated cellular cAMP to sub-millimolar concentrations coinci-dental with an equimolar decline in cellular ATP. Furthermore, this treatment of the cells acutely induced the production of three additional peaks in the phosphomonoester region of the 31 P-NMR spectrum, an effect that was not mimicked by forskolin, a potent stimulator of cAMP [8]. Phosphomonoester stimulation in C6 glioma cells by the β agonist, isoproterenol, was later reported by others using 31 P-NMR, suggesting a more widespread occurrence of this phenomenon [9].…”

“…M2R cell cultures on microcarrier beads (Ϸ3Ϫ6ϫ10 7 cells/2 ml of beads) were perfused in the spectrometer under conditions identical to those present in the culture incubator using 70 ml of culture medium in a closed loop, as previously described [8]. Following base-line recording, the cells were stimulated under conditions identical to those described for the 32 P i -labeling experiments above and the resulting 31 P and 13 C spectra were recorded.…”

“…For NMR studies of perfused cultures, the cells were grown on microspheres (Biosilon Nunc) as previously described [8,20] by seeding approximately 3Ϫ4ϫ10 6 cells/ml of beads. After culturing for 3 days a cell density of about 1.5Ϫ3ϫ10…”

Tyrosinase isoenzymes in mammalian melanocytes

“…The binding of the hormone to these receptors would lead to a transient increase in the intracellular cAMP levels [8,91 by stimulation of adenylate cyclase activity [lo, 111. Although cAMP might not be the only second messenger stimulated by MSH [12], it is generally agreed that its increased levels are at least partially responsible for tyrosinase stimulation since the dibutyryl derivative of cAMP [ 13, 141, theophylline [14-171 and isobutylmethylxanthine [14, 161 are able to mimick the hormone action, and even to achieve larger activations of the enzyme. However, the molecular mechanisms coupling the increase in intracellular cAMP to tyrosinase activation are unknown, and several possible explanations have been proposed.…”