Evidence for a calcium/calmodulin involvement in density-dependent melanogenesis in murine B16 melanoma cells.

Previous work from our laboratory has shown that both cyclic AMP and calcium/calmodulin appear to be involved in the regulation of melanogenesis in murine B16 melanoma cells. In these cells as in murine Cloudman S91 cells, melanogenic responsiveness to melanocyte-stimulating hormone (MSH) varies with cell density in culture. Our objective in this study was to learn more about the intracellular systems involved in the control of melanogenesis, particularly the role played by calcium. The melanogenic response to alpha MSH was compared to the response to drugs affecting intracellular free calcium and calmodulin over a range of cell densities in B16F1 cells. alpha MSH-stimulated melanin production was extremely density-dependent but alpha MSH-stimulated cyclic AMP production was independent of cell density. The melanogenic response to agents that increased intracellular calcium (A23187) or inhibited intracellular calmodulin varied with cell density. A drug (TMB8) that lowered intracellular free calcium, however, increased melanogenesis independently of cell density. At high cell density it was found that an elevation in calcium decreased melanogenesis, whereas agents that reduced calcium or inhibited calmodulin activity increased melanogenesis. At low cell density, however, the inhibitory response to A23187 was lost and in some experiments even stimulated melanogenesis. These data suggest that the calcium/calmodulin signalling system has an inhibitory influence on melanogenesis, and its expression, which depends upon cell density, may also modulate the response to stimulatory agents such as alpha MSH.     

Calcium Plays a Complex Role in the Regulation of Melanogenesis in Murine B16 Melanoma Cells

To learn more of the role of calcium in the regulation of melanogenesis, we have used direct manipulation of medium calcium and pharmacological modulation of intracellular calcium to examine the consequences on unstimulated and cyclic AMP elevated tyrosinase activity and melanin synthesis and distribution in B16 melanoma cells. In unstimulated cells, calcium is clearly inhibitory to tyrosinase activity. However, in cells stimulated with cAMP-elevating agents the requirement for extracellular calcium was changed such that cells required a minimum of 0.4–0.6 mmol medium calcium for maximum tyrosinase response to these agents. Paradoxically, pharmacologically increasing intracellular calcium in cAMP-stimulated cells with ionophore inhibited tyrosinase activity, and the calcium-lowering agent TMB8 and the calcium channel blocker verapamil both stimulated tyrosinase activity. When melanin synthesis was measured in cAMP-stimulated cells, TMB8 was found to significantly increase the sensitivity and the maximum melanogenic response to α-MSH, suggesting the presence of at least one level of endogenous calcium inhibitory control operative in these cells. In addition, TMB8 changed the distribution of melanin between the cell and the medium such that, in the presence of α-MSH and TMB8, significantly more melanin was secreted into the medium. These data suggest that calcium is required for several steps in melanogenesis, having an apparently inhibitory effect on pre-tyrosinase activity in unstimulated cells, but also showing evidence of a positive role in cyclic AMP-stimulated tyrosinase activity, as well as a further possible inhibitory role in melanin movement or secretion.     

Investigation of the Regulation of Pigmentation in α-Melanocyte-Stimulating Hormone Responsive and Unresponsive Cultured B16 Melanoma Cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to αMSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1°) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to αMSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro αMSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1° cells. αMSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1° cells to produce melanin in response to αMSH is not due to a lack of αMSH receptors or cAMP response to αMSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1° and F1 cells.     

Effects of ruthenium red, A23187 and D-600 on steroidogenesis in Y-1 cells.

The effects of the calcium antagonists ruthenium red and D-600 and the cation ionophore A23187 on steroidogenesis were investigated. Steroidogenesis triggered by corticotrophin and cyclic AMP was inhibited by each of the agents. Incubation of Y-1 cells with an excess of ethyleneglycol-bis-(beta-amino-ethylether)-N,N'-tetraacetic acid (EGTA) abolished the steroidogenic response to corticotrophin while the response to cyclic AMP was unaffected. The ability of ruthenium red and D-600 (1 . 10(-5) M), and A23187 (6 . 10(-6 M) to inhibit a response which does not require the presence of extracellular calcium (cyclic AMP induced steroidogenesis) suggests that they are altering intracellular calcium. Neither of the calcium antagonists nor the cation ionophore inhibited the steroidogenic response to exogenous pregnenolone, thereby suggesting that the cells were still viable. Only when A23187 was used in the presence of a 15-fold increase in extracellular calcium (4.8 mM) was the response to pregnenolone diminished. The data are interpreted as a further indication that, in intact cells, intracellular calcium plays a role in the steroidogenic pathway.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Investigation of the regulation of pigmentation in alpha-melanocyte-stimulating hormone responsive and unresponsive cultured B16 melanoma cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to alpha MSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1 degree) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to alpha MSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro alpha MSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1 degree cells. alpha MSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1 degree cells to produce melanin in response to alpha MSH is not due to a lack of alpha MSH receptors or cAMP response to alpha MSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1 degree and F1 cells.     

Stimulation of melanotic expression in murine melanoma cells exposed to polyamine antimetabolites

An exposure of cultured Cloudman S91 melanoma cells to inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG), distinctly promoted the expression of differentiated biochemical functions of the tumor cells. Slight to moderate growth inhibition produced by the compounds was associated with a stimulation of melanogenesis, as reflected by a striking enhancement of tyrosinase (EC 1.10.3.1) activity and an increase in cellular melanin content. Both antimetabolites acted synergistically with α-melanotropin (MSH), as regards the stimulation of melanogenesis. Exposure of the melanoma cells to MSH resulted in most experiments in a marked decrease of the intracellular polyamine pools, usually involving all three polyamines (putrescine, spermidine and spermine). The DFMO-induced stimulation of melanogenesis was totally suppressed by the administration of putrescine, whereas the MSH-stimulated tyrosinase activity was not influenced by the diamine. Although many recent reports indicate that terminal differentiation is accompanied by a distinct stimulation of polyamine biosynthesis, our results suggest that in certain cells polyamine deprivation may lead to an enhanced expression of differentiated phenotype.     

Stimulation of hamster adipocyte cyclic 3':5'-nucleotide phosphodiesterase activity by ionophore A23187 and calcium.

Incubation of hamster isolated fat cells with the ionophore A23187 and calcium for 20 minutes caused 30-40% increases in the cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) activity of adipocyte homogenates when either 0.6 micron cyclic AMP or 0.6 micron cyclic GMP was the enzyme substrate. The stimulation of adipocyte cyclic AMP phosphodiesterase activity by A23187 and calcium was not antagonized by the adrenergic receptor blocking agents phentolamine and propranolol. The changes in enzyme activity produced by the ionophore and calcium were not associated with elevated intracellular cyclic AMP levels. Furthermore, A23187 and calcium acted to enhance adipocyte phosphodiesterase activity before, but not after, homogenization of the fat cells. These data suggest that the phosphodiesterase activity of hamster isolated fat cells may, at least in part, be regulated by fluctuations in intracellular calcium concentrations.     

Inhibition of tyrosinase activity and protein synthesis in melanoma cells by calcium ionophore A23187

Calcium ionophore A23187 lowers basal levels of tyrosinase and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. Ionophore at a concentration of 10(-6) g/ml causes a 50% reduction in basal levels of tyrosinase and inhibits the MSH stimulated level of enzyme. Ionophore A23187 also inhibits the PGE1 mediated stimulation of tyrosinase, as well as the rise in enzyme activity observed in cells exposed to either theophylline (1 mM) or dbcAMP (10(-4)M). Ionophore does not affect basal levels of cyclic AMP nor the elevated levels produced by either MSH or PGE1, suggesting then, that the antagonistic activity of A23187 is localized to a point in the pathway of tyrosinase activation distal to the formation of cAMP. Ionophore causes a rapid and marked (greater than 50%) inhibition of cellular protein synthesis and it is possible that this calcium mobilizing compound may exert its inhibitory effects on tyrosinase activity by causing a general reduction in cellular translation. Since the inhibition of protein synthesis occurs in cells exposed to ionophore in either the presence or absence of calcium in the medium, it seems, likely that the ionophore may exert its effects by causing the release of calcium from intracellular sites.     

Inhibitors of intracellular cyclic AMP accumulation affect differentiation of sporogenous mutants of Dictyostelium discoideum

Abstract Sporogenous mutants of Dictyostelium discoideum strain V12M2 were used to determine whether the intracellular levels of cyclic AMP or other second messengers regulate differentiation. Increasing external concentrations of cyclic AMP promoted spore formation. Caffeine and progesterone, which lower intracellular cyclic AMP levels by different mechanisms, blocked spore formation and favored stalk cell formation. In contrast, differentiation of both spore and stalk cells occurred normally in the presence of agents that disrupt calcium/calmodulin or protein kinase C-based second messenger systems. The data are in accord with the view that (1) intracellular cyclic AMP is essential for terminal differentiation of both cell types, and (2) higher levels are required for formation of spores than for stalk cells.     

Inhibition of ACTH secretion in mouse pituitary tumor cells by activation of muscarinic cholinergic receptors

Hormonally stimulated secretion of ACTH from AtT-20 mouse pituitary tumor cells is a cyclic AMP-mediated process. The presence of inhibitory cholinergic muscarinic receptors on these cells was recently reported, and in this study, the relationship between the activation of these receptors and the consequent inhibition of cyclic AMP formation and ACTH secretion was investigated. The muscarinic agent, oxotremorine, antagonized both cyclic AMP synthesis and ACTH secretion in response to corticotropin-releasing factor (CRF), vasoactive intestinal peptide, a 27-amino acid peptide with an N-terminal histidine and a C-terminal isoleucine amide, and forskolin. Other muscarinic agents, carbachol and bethanechol, had similar inhibitory effects. The cholinomimetics reduced basal (unstimulated) ACTH secretion without decreasing basal cyclic AMP levels, and also antagonized hormone release in response to cyclic AMP-independent agonists such as K+, A-23187, and phorbol ester. Scopolamine reversed the inhibitory effects of the muscarinic agents on basal and stimulated ACTH secretion and cyclic AMP formation. Increasing the extracellular calcium concentration reversed the muscarinic antagonism of basal and CRF-stimulated hormone release without affecting the cyclic AMP response. Pertussis toxin pretreatment attenuated the inhibitory effects of the muscarinic agents on forskolin-stimulated cyclic AMP synthesis and ACTH secretion as well as the inhibitory effect of carbachol on basal ACTH release. The data suggest that cyclic AMP is an essential mediator in the ACTH secretory pathway, but that an alternate cyclic AMP-independent ACTH pathway also exists in the clonal cells, and that both pathways may be modulated by a common postcholinergic receptor mechanism.     

Calmodulin modulates prolactin secretion in vitro: studies with calmodulin containing liposomes

The control of prolactin secretion by Ca calmodulin and cyclic AMP was studied. Ca++ ionophore A23187 stimulated both cyclic AMP accumulation and prolactin release by primary culture of anterior pituitary cells in vitro. The increase of cyclic AMP formation by A23187 preceded that of prolactin release. To test the calmodulin involvement in these processes we used either selective calmodulin antagonist, the naphthalene sulphonamide derivative W7, or calmodulin containing liposomes. W7 dose dependently inhibited both basal or A23187 stimulated cyclic AMP accumulation and prolactin secretion. Insertion of Ca calmodulin within the cells stimulated prolactin secretion without modifying cyclic AMP accumulation. W7 inhibited the Ca calmodulin containing liposomes stimulation of prolactin release. These results suggest that calmodulin participates to the process of prolactin release.     

Inhibition of Tyrosinase Activity and Protein Synthesis in Melanoma Cells by Calcium lonophore A23187

Calcium ionophore A23187 lowers basal levels of tyrosinase and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. lonophore at a concentration of 10^–6 g/ml causes a 50% reduction in basal levels of tyrosinase and inhibits the MSH stimulated level of enzyme. lonophore A23187 also inhibits the PGEi mediated stimulation of tyrosinase, as well as the rise in enzyme activity observed in cells exposed to either theophylline (1 mM) or dbcAMP (10^–4M). lonophore does not affect basal levels of cyclic AMP nor the elevated levels produced by either MSH or PGEi, suggesting then, that the antagonistic activity of A23187 is localized to a point in the pathway of tyrosinase activation distal to the formation of cAMP. lonophore causes a rapid and marked (> 50%) inhibition of cellular protein synthesis and it is possible that this calcium mobilizing compound may exert its inhibitory effects on tyrosinase activity by causing a general reduction in cellular translation. Since the inhibition of protein synthesis occurs in cells exposed to ionophore in either the presence or absence of calcium in the medium, it seems, likely that the ionophore may exert its effects by causing the release of calcium from intracellular sites.     

Melanocyte-stimulating hormone affects melanogenic differentiation of quail neural crest cells in vitro

Quail neural crest cells were treated in vitro with alpha-melanocyte-stimulating hormone (alpha-MSH) or dibutyryl cyclic AMP (dbcAMP) plus theophylline. These treatments increased the proportion of melanocytes to total cells in crest cell outgrowth cultures. Pigmentation of neural crest cell clusters proceeded more rapidly when cultures were treated with alpha-MSH or dbcAMP plus theophylline than when untreated. In clonal cell cultures, the proportion of pigmented colonies to total colonies was increased by MSH treatment. From these results, MSH seems not only to accelerate melanogenic differentiation but also to affect the state of commitment of neural crest cells to melanogenic differentiation in vitro, and this action of MSH appears to be mediated by cAMP.     

Expression of melanocyte stimulating hormone receptors correlates with mammalian pigmentation, and can be modulated by interferons

The relationship between melanogenesis and the expression of melanocyte stimulating hormone (MSH) receptors on the surface of melanocytes was examined using sublines generated from the melanotic JB/MS melanoma. JB/MS cells were propagated in long term culture to allow for phenotypic drift in their characteristics of differentiation, and then were cloned; the cloned cells ranged from well differentiated and pigmented to undifferentiated and amelanotic. Spontaneous and MSH-induced melanogenesis in these different lines was measured and correlated with the number of MSH receptors expressed. After 6 months of in vitro culture, the ability of the cells to respond to MSH was significantly reduced, as were the number of MSH receptors expressed; the cells had reduced pigmentation and were relatively undifferentiated histologically. Subsequently, clonally-derived pigmented cells were found to have numbers of surface MSH receptors (approximately 60,000 per cell) and levels of melanogenic activity similar to the original JB/MS cell line. However, an amelanotic clone had an even more dramatically reduced level of pigmentation which correlated with a further decrease in the expression of MSH receptors (less than 1,000 per cell) and the production of a potent melanogenic inhibitor. We also examined the responses of these various sublines to alpha, beta, and gamma-interferons and found significant heterogeneity in their abilities to respond to these cytokines. This study clearly shows that there is a direct correlation between melanogenesis and the expression of MSH receptors on the surface of melanocytes, and that melanogenic inhibitors may be critically involved in the regulation of mammalian pigmentation.     

The effect of alphaMSH on beta-adrenergic receptor mechanisms in the rat pineal.

The effects of αMSH on beta adrenergic receptor response of rat pineal cells were studied in vitro. Responses measured were membrane hyperpolarization, measured with a micro-electrode, and cyclic AMP formation, measured by radio immunoassay. Normal resting membrane potential of pineal cells is approximately -40mV. Addition of NE produces a dose-dependent hyperpolarization of these cells. The addition of αMSH in vitro produces a very slight, but significant, depolarization, and markedly attenuates subsequent NE responses. αMSH has no effect on the cyclic AMP content of pineal glands, but again attenuates the action of NE in producing increased cellular cyclic AMP. These results suggest that αMSH may modulate pineal responsiveness.     

Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin

The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP). Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively). Two inhibitors of the calcium-dependent regulatory protein, calmodulin [trifluoperazine, 40 microM and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl]imidazolium chloride, ( R24571 ) 20 microM] significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml. Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml). A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml). These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.     

Neurotensin regulation of TSH secretion in the rat

The ionophore A23187 (6.7 microM) increased the rates of formation of prostaglandins and cyclic AMP in suspensions of thioglycollate-elicited rat peritoneal macrophages. Both effects were inhibited by the calmodulin blocker trifluoperazine (50 microM) and the calcium channel blocker verapamil (500 microM). Inhibitors of phospholipase A2 and cyclo-oxygenase also blocked both actions of A23187. The stimulated prostaglandin formation was markedly reduced when the cells were preincubated with 8-bromo-cyclic AMP (1mM), dibutyryl cyclic AMP (1mM) or cholera toxin (500ng/ml). Addition of exogenous arachidonic acid (30 microM) alleviated this inhibition. We propose that the effect of A23187 on macrophages includes a 'self-limiting' mechanism whereby newly-synthesized prostaglandins can inhibit, via cyclic AMP, a step(s) prior to the transformation of arachidonic acid and thus modulate their own production.     

Effect of Forskolin and Prostaglandin E1 on Stimulus Secretion Coupling in Cultured Bovine Adrenal Chromaffin Cells

Treatment of adrenal chromaffin cells with forskolin (0.1-10 microM) stimulated cyclic AMP levels, reduced the maximal stimulation of release of noradrenaline by nicotine, and increased release in response to elevated external potassium and the calcium ionophore A23187. The presence of the phosphodiesterase inhibitor Ro 20-17-24 with forskolin potentiated both the stimulation of cyclic AMP and the inhibition of nicotine-induced noradrenaline release. Dibutyryl cyclic AMP, and the elevation of cyclic AMP with prostaglandin E1, also attenuated nicotine-stimulated release. However, when the stimulation of intracellular cyclic AMP production by prostaglandin E1 was potentiated by low levels of forskolin, there was not a concomitant potentiation of effect on noradrenaline release. Dideoxyforskolin, an analogue of forskolin which does not stimulate adenylate cyclase, inhibited both potassium- and nicotine-stimulated release, probably by a mechanism unrelated to the action of forskolin in these experiments. Using Fura-2 to estimate free intracellular calcium levels, both forskolin and dideoxyforskolin (at 10 microM) reduced the calcium transient in response to nicotine. These results support a model in which elevation of cyclic AMP inhibits the activation of nicotinic receptors, but augments stimulus secretion coupling downstream of calcium entry. The data, however, do not indicate a simple relationship between total intracellular cyclic AMP levels and the attenuation of nicotinic stimulation of release.     

Calcium dependence of beta-adrenoceptor mediated cyclic AMP accumulation in human lymphocytes

In intact human lymphocytes, cyclic AMP accumulation in response to isoproterenol was inhibited by 5 mM EDTA, by deletion of calcium ions from the medium and by 1 mM lanthanum chloride, but not by 1 microM verapamil or by 10 microM nifedipine. A23187 caused a modest increase in cyclic AMP content. Exposure of lymphocytes to 5 microM 1-isoproterenol desensitized the cells to subsequent beta-adrenergic stimulation, reducing cyclic AMP accumulation. With higher concentrations of 1-isoproterenol (50 microM), receptor density was reduced as well. None of the above agents attenuated losses in agonist-stimulated cyclic AMP accumulation induced by treatment with 5 microM isoproterenol for 90 min. These data suggest that calcium ions, both those present in the extracellular medium and those bound to the plasma membrane, are required for isoproterenol-stimulation of adenylate cyclase. In addition, it appears that neither the presence of extracellular calcium ions nor full activation of adenylate cyclase are required for desensitization.