Cyclic AMP and adrenergic receptors in melanophore responses to methylxanthines

EUROPEAN JOURNALOF PHARMACOLOGY12 (1970) 365-370. NORTH-HOLLANDPUBLISHINGCOMPANY

C Y C L I C AMP A N D A D R E N E R G I C R E C E P T O R S IN M E L A N O P H O R E R E S P O N S E S T O M E T H Y L X A N T H I N E S Joel M.GOLDMAN and Mac E.HADLEY Department of Biological Sciences, University of Arizona, Tucson, Arizona 85721, USA

l~ceived 7 April 1970

Accepted 29 June 1970

J.M.GOLDMAN and M.E.HADLEY, Cyclic AMP and adrenergic receptors in melanophore responses to methylxanthines, European J. Pharmacol. 12 (1970) 365-370. Skins of the lizard Anolis carolinensis darken in response to melanophore stimulating hormone (MSH), methylxanthines (e.g., theophyUine), and catecholamines, the latter through #-adrenergic receptor stimulation. ffAdrenergic blocking agents inhibit MSH, #-adrenergic blocking agents inhibit catecholamines, but neither inhibit methylxanthines. Catecholamines lighten MSH-darkened skins through t~-receptor stimulation but, in contrast, increase the darkening of methylxanthine-darkened skins through #-receptor stimulation. Apparently,a-stimulation dominates in the presence of MSH whereas, in the presence of methylxanthines, #-receptor stimulation dominates. ~-Adrenergic stimulation apparently decreases cyclic AMP levels within melanophores, possibly by stimulation of cyclic AMP phosphodiesterase activity which would lead to cyclic AMP degradation. In the presence of methylxanthines which inhibit phosphodiesterase, t~-stimulation would then have less effect and, therefore, #-stimulation would dominate. Catecholamines MSH (melanophore stimulating hormone) Phosphodiesterase

1. INTRODUCTION Considerable evidence now supports a role for cyclic 3',5'-adenosine monophosphate (cyclic AMP) as the intracellular mediator of the actions of a number of hormones. Levels of this nucleotide depend upon the activity of two enzymes: (1)adenyl cyclase which catalyzes the formation of cyclic AMP and (2)cyclic AMP phosphodiesterase which catalyzes the degradation of cyclic AMP. Methylxanthines, such as caffeine and theophylline, mimic the effects of a number of hormones. Whereas hormones are believed to directly stimulate adenyl cyclase, methylxanthines apparently inhibit phosphodiesterase activity (Butcher and Sutherland, 1962). Both of these mechanisms lead to an intracellular increase in cyclic AMP levels which then mediate the ensuing

Pigmentation Hormone action, mechanism of Adrenergic blocking agents

responses. Methylxanthines mimic the darkening action of melanophore stimulating hormone (MSH) (Lerner and Takahashi, 1956; Novales, 1959; Hadley and Goldman, 1969a) b y Jdlsp~s]hg melanosomes within melanophores. Evidence suggests that both MSH and methylxanthines affect vertebrate melanophores through an increase in cyclic AMP (Bitensky and Burstein, 1965; Novales and Davis, 1967; Abe et al., 1969a; Hadley and Goldman, 1969a; Goldman and Hadley, 1969b). Although MSH and methylxanthines may increase cyclic AMP levels, we have found that catecholamines antagonize the action of MSH but, in contrast, are synergistic with methylxanthine effects on melanophores. In the present communication we demonstrate that the preferential stimulation of either ct- or /~-adrenergic receptors by catecholamines accounts for

366

J.M. GoMman, M.E.Hadley, Melanophore responses to methylxanthines

the opposite effects of these catecholamines on MSHor methylxanthine-darkened skins. We also provide further information on the roles of adrenergic receptors and cyclic AMP in the mechanism of hormone action on vertebrate melanophores.

2. MATERIALS AND METHODS The skin color of the lizard Anolis carolinensis varies from a bright green to a dark brown. Color changes in Anolis result from the intraceUular movement of melanosomes within melanophores. Although other chromatophores are present in the dermis, apparently their contribution to color change is passive (Taylor and Hadley, 1970). Dispersion of the melanosomes from a perinuclear position out into the melanophore processes results in a darkening of the skin to a brown color. Melanosome aggregation from the melanophore processes to a perinuclear position causes lightening of the skin to a green color. Such changes in skin color in response to hormonal or pharmacological stimulation are measured as changes in light reflectance from the outer (epidermal) surface of the skins using a Photovolt Photoelectric Reflection Meter (Photovolt Corporation, New York, N.Y.) as originally described for the frog skin bioassay for MSH (Shizume et al., 1954). An increase in reflectance represents skin lightening whereas a decrease in reflectance indicates skin darkening. Male and female lizards were obtained from the Snake Farm (Laplace, Louisiana). After decapitation, back skins were removed and rinsed several times in Ringer solution (pH 7.4). Skins were then individually mounted on metal rings and held in place by outer overlapping plastic rings (Shizume et al., 1954). These skins were allowed to equilibrate in Ringer solution for one to two hours until they were light in color. An initial reflectance value was obtained for each skin and these skins were then arranged in groups so that the initial average reflectance value for each group was approximately equal. Succeeding average group values were compared with the initial value and recorded as percent changes in reflectance. Hormonal and pharmacological agents were obtained, prepared and utilized as previously described (Goldman and Hadley, 1969a; Goldman and Hadley, 1969b). Statistical comparisons of mean values used Student's t test.

3. RESULTS Melanophore stimulating hormone (MSH) and methylxanthines (caffeine, theophylline and theobromine) darken skins of the lizard, Anolis carolinensis (fig. 1). This darkening is reversible since skins relighten if rinsed in Ringer solution, in the absence of MSH or methylxanthines (fig. 1). Darkening of Anolis skin results from melanosome dispersion whereas skin lightening results from melanosome aggregation. When catecholamines are added to darkened skins, the ensuing response depends on whether the skins have been darkened with MSH or with methylxanthines. Epinephrine lightens MSH-darkened skins but increases the darkening of methylxanthine-darkened skins (fig. 2A). This lightening of MSH-darkened skins by catecholamines is inhibited by a-adrenergic blocking agents (fig. 2B) whereas fl-adrenergic blocking agents enhance this lightening (fig. 2C). In contrast,

60

50

40

30

EO

rO

TNEOPHYLLINE amISKIN DANKENIN@ r ' I S K I N LIGHTrNIRG

CAFFEINE

/1 ill

TH[OBROMINE

I1

MSH

Fh

RINGER CONTROL

Fig. 1. Darkening effect of methylxanthines and MSH

on

Anolis carolinensis skins. Skins were darkened with methyl-

xanthines (5 XIO-a M) or MSH (9XI0-9 g/ml). After the skins were maximally darkened, they were rinsed several times with Ringer solution to allow them to relighten. The solid bars on the left represent the darkening caused by MSH or methylxanthines, whereas the open bars o n t h e right represent relightening of these same skins. The last bar on the right represents a group of skins that remained in Ringer solution as a control group. Vertical lines represent standard errors of the mean for the eight (methylxanthines) or ten (MSH and Ringer) skins per group.

J.M. Goldman, M.E.Hadley, Melanophore responses to methylxanthines

A

367

'11

SO

40

IO

E

E

N[

HE

MM

Up ElIe

liter 4111111" 41flit llfllr MIN

~

I ,i

E Ifllr

lifter

0 I

NE NE Ith~lr ifl~lr

~

E ilflw

E Ilflll,

20

I0

PRO

l l O Dill DC!

Ira(IN DARKENING [ ~ $ K | N LIGHTENING

Fig. 2. Effect of catecholamines on skins darkened with MSH or theophyUine. Skins were darkened with theophylline (THEO, 10-3 M) or MSH (6 X 10-9 g/ml in A; 3 X 10--9 g/ml in B; 2 X 10-9 g/ml in C). To experiments B-E, either an Ot(ergotamine, ERG, 10-s M; Dibenamine, DIB, 10-s M) or a #(dichloroisoproterenol, DCI, 2 X 10-s M; propranolol, PRO, 10-s M) adrenergic blocking agent was also added to one of the two groups. After the skins had darkened, either epinephrine (E) or norepinephrine (NE) was added (10-s M) to the skins. Standard errors of the mean for the seven (experiments B and C) or eight (experiments A, D and E) skins per group are represented by vertical lines. Differences between the two groups in each experiment are significant (p < 0.001 for A, B, C and E;p<0.01 for D).

the synergistic increase in darkening of methylxanthine-darkened skins by catecholamines is enhanced by (~-adrenergic blocking agents (fig. 2D) b u t inhibited b y /3-adrenergic blocking agents (fig. 2E). In fact, in the presence of/3-adrenergic blocking agents, catecholamines lighten methylxanthine-darkened skins (fig. 2E). In addition to darkening induced by MSH and methylxanthines, a third method o f darkening Anolis skins is through catecholamine stimulation of/~-adrenergic receptors (Goldman and Hadley, 1969a). This darkening by catecholamines is inhibited by/~-adrenergic blocking agents but enhanced by r,-adrenergic blocking agents (fig. 3A). MSH-induced darkening is inhibited b y ot-adrenergic blocking agents but not b y /3-adrenergic blocking agents (fig. 3B) (Goldman and Hadley, 1970). In contrast, theophylline-induced darkening is neither inhibited nor enhanced by or- or fl-ad~energic blocking agents (fig. 3C).

,;o ,;o I

MIH ~a

DCI

~SH . NSH . .

TH~ODIN

.

TH[O 1H[O

SKIN OARK[NIH6

Fig. 3. Effect of adrenergic blocking agents on darkening caused by isoproterenol, theophylline or MSH. An 0t- (Dibenamine, DIB, 2 X 10"-s M in A; 10-s M in B and C) or/~(dichloroisoproterenol, DCI, 2 X 10- 5 M; propranolol, PRO, 10"s M) adrenergic blocking agent was added to the skins in one of the three groups in each experiment. Then, isoproterenol (ISO, 10-5 M) was added to the skins in experiment A, MSH (3 X 10-9 g/ml) to experiment B and theophylline (THEO, 10-a M) to experiment C. Vertical lines represent standard errors of the mean for the seven (experiment A) or eight (experiments B and C) skins per group. Differences between groups in experiment A are significant (p <0.01 for ISO and DIB + ISO; p < 0.001 for ISO and DCI + ISO). In experiment B the difference between the MSH group and the DIB + MSH group is significant (p < 0.001) but the difference between the MSH group and the DCI + MSH group is not significant (p > 0.5). Differences between the THEO group and other groups in experiment C are not significant (p > 0.05).

4. DISCUSSION Catecholamines have a dual action on melanophores of Anolis carolinensis. Stimulation of ~-adrenergic receptors causes melanosome dispersion leading to skin darkening whereas ct-adrenergic stimulation causes melanosome aggregation which results in skin lightening (Goldman and Hadley+ 1969a). When skins are darkened with MSH, catecholamines lighten them b y stimulating a-adrenergic receptors (Goldman and Hadley, 1969a). ct-Adrenergic blocking agents inhibit this lightening whereas ~-blocking agents enhance it b y blocking the/l-adrenergie receptors which mediate skin darkening. In contrast, we have demonstrated here that catecholamines increase the darkening o f

368

J.M.Goldman, M.E.Hadley, Melanophore responses to methylxanthines

skins previously darkened with methylxanthines. This synergistic darkening is mediated through stimulation of /3-adrenergic receptors since H-blocking agents block this response and, in fact, convert it to an a-adrenergic response, i.e., skin lightening, a-Blocking agents enhance this darkening by inhibiting the opposing activity of a-adrenergic receptors which mediate lightening. Other workers have found that/3-adrenergic stimulation is synergistic with the effects of methylxanthines in other tissues (Turtle et al., 1967; Turtle and Kipnis, 1967; Weiss et al., 1966) whereas a-adrenergic stimulation is inhibitory to the effects of methylxanthines (Turtle et al., 1967; Turtle and Kipnis, 1967). These and other data (Robison et al., 1967; Abe et al., 1969b) suggest that /3-adrenergic stimulation increases cyclic AMP levels within tissues whereas a-adrenergic stimulation decreases cyclic AMP levels. Data have accumulated indicating that cyclic AMP is the intracellular mediator for melanotropic agents. Cyclic AMP darkens skins of amphibians (Bitensky and Burstein, 1965; Novales and Davis, 1967) and dibutyryl cyclic AMP (a potent analogue of cyclic AMP) darkens amphibian (Goldman and Hadley, 1969b; Bagnara and Hadley, 1969) and reptilian (Hadley and Goldman, 1969a, 1969b) skins. In addition, MSH increases cyclic AMP levels in frog skin (Abe et al., 1969a), whereas such lightening agents as melatonin and norepinephrine inhibit the increase in cyclic AMP due to MSH (Abe et al., 1969a, 1969b). Also, methylxanthines, which inhibit the phosphodiesterase that degrades cyclic AMP, darken amphibian (Lerner and Takahashi, 1956; Novales, 1959; Goldman and Hadley, 1969b) and reptilian (Hadley and Goldman, 1969a) skins. Since catecholamines stimulate both a- and ~-adrenergic receptors, it would appear that in the presence of MSH the effect of a-adrenergic receptor stimulation dominates and this results in skin lightening. In other species of vertebrates we have found (Goldman and Hadley, 1969b; Hadley and Goldman, 1970) that in the absence of readily demonstrable a-adrenergic receptors, catecholamines increase the darkening of MSH-darkened skins through ~-adrenergic stimulation. It is interesting, therefore, that in the presence of methylxanthines, the effect of ~-adrenergic receptor stimulation dominates and this results in skin darkening. Although catecholamines

apparently stimulate both a- and/3-adrenergic receptors, in the presence of methylxanthines the effect of a-adrenergic stimulation is not seen. If a-adrenergic stimulation leads to a decrease in cyclic AMP levels by stimulating cyclic AMP phosphodiesterase, then, in the presence of methylxanthines which inhibit phosphodiesterase, it is reasonable to expect that a-adrenergic stimulation would have a diminished effect and that /3-receptor stimulation would dominate. However, in the presence of/3-adrenergic blockade it is possible that there would be sufficient a-activity left to cause some lightening. This suggestion that a-adrenergic receptor activity involves stimulation of phosphodiesterase is in contrast to the suggestion that a-receptor stimulation inhibits synthesis of cyclic AMP by acting on adenyl cyclase (Robison et al., 1967; Turtle and Kipnis, 1967). In addition, Abe et al. (1969b) discuss why norepinephrine does not lighten theophyllinedarkened frog skins at concentrations that lighten MSH-darkened skins. They suggest that it is possible that either norepinephrine interferes with the action of MSH, but does not affect the basal activity of adenyl cyclase, or that the skin darkening effect of theophylline is related only in part to phosphodiesterase inhibition and that part of the effect results from other actions of theophylline. It would appear that it is just as reasonable to suggest that a-receptor stimulation could stimulate phosphodiesterase rather than inhibiting adenyl cyclase. It is interesting that diazoxide which inhibits phosphodiesterase (Senft, 1968) inhibits insulin release (Seltzer and Allen, 1965; Wong et al., 1967). This inhibition of insulin secretion is abolished by phentolamine, the a-adrenergic blocking agent (Senft et al., 1968). Although it is not clear how the action of diazoxide is mediated by a-receptors, it seems significant that the action of an inhibitor of phosphodiesterase is related to the a-adrenergic receptor. MSH-induced darkening is inhibited by a-adrenergic blocking agents (Goldman and Hadley, 1970) whereas catecholamine-induced darkening is inhibited by /3-blocking agents (Goldman and Hadley, 1969a). In contrast, we have demonstrated here that methylxanthines are not inhibited by adrenergic blocking agents at concentrations which inhibit MSH or catecholamines. These data indicate that although catecholamines darken skins through stimulation of

J.M. Goldman, M.E.Hadley, Melanophore responses to methylxanthines /3-adrenergic receptors and MSH appears to darken them through stimulation o f a component o f the a-adrenergic receptor (Goldman and Hadley, 1970), methylxanthines apparently do not act through stimulation o f adrenergic receptors. The finding that the darkening action of MSH apparently involves interaction with a component of the a-adrenergic receptor is interesting, especially in light o f the catecholamine-induced lightening being also mediated through a-receptors. As we have suggested elsewhere (Goldman and Hadley, 1970), if the action o f MSH were to lead to inhibition o f phosphodiesterase activity there should then follow an increase in the intracellular level o f cyclic AMP which would then cause darkening. It is possible that there is a competitive antagonism between MSH and a-adrenergic stimulation affecting the level of phosphodiesterase activity, a-Adrenergic stimulation would lead to an increase in phosphodiesterase activity thereby decreasing cyclic AMP levels whereas MSH would decrease phosphodiesterase activity thereby causing an increase in cyclic AMP levels. Novales and Novales (1965) obtained data suggesting that epinephrine is a competitive antagonist of MSH on frog skins. By equating phosphodiesterase activity with the a-adrenergic receptor, it is possible to explain how the a-receptor could mediate b o t h MSHinduced darkening (melanosome dispersion) and catecholamine-induced lightening (melanosome aggregation) o f Anolis carolinensis skins.

ACKNOWLEDGEMENTS This study was supported in part by g~ant GB-8347 from the National Science Foundation and a National Science Foundation Institutional Grant to the University of Arizona.

REFERENCES Abe, K., R.W.Butcher, W.E.Nicholson, C.E.Baird, R.A.Liddie t I and G.W.Liddie, 1969a, Adenosine 3,5-monophosphate (cyclic AMP) as the mediator of the actions of melanocyte stimulating hormone (MSH) and norepinephrine on the frog skin, Endocrinology 94, 362. Abe, K., G.A.Robison, G.W.Liddie, R.W.Butcher, W.E. Nicholson and C.E.Bakd, 1969b, Role of cyclic AMP in mediating the effects of MSH, norepinephrine and melatonin on frog skin color, Endocrinology 85,674.

369

Bagnara, J.T. and M.E.Hadiey, 1969, The control of bright colored pigment cells of fishes and amphibians, Am. Zool. 9,465. Bitensky, M.W. and S.R.Burstein, 1965, Effects of cyclic adenosine monophosphate and melanocyte-stimulating hormone on frog skin in vitro, Nature 208, 1282. Butcher, R.W. and E.W.Sutherland, 1962, Adenosine 3',5 'phosphate in biological materials. I. Purification and properties of cyclic 3',5t-nucleotide phosphodizsterase and use of this enzyme to characterize adenosine 3',5'phosphate in human urine, J. Biol. Chem. 237, 1244. Goldman, J.M. and M.E.Hadley, 1969a, In vitro demonstration of adrenexgic receptors controlling melanophore responses of the lizard, Anolis carolinensis, J. Pharmaeol. Exptl. Therap. 166, 1. Goldman, J.M. and M.E.Hadiey, ~1~69b, The /~-adrenergic receptor and cyclic 3',5'-adenosine monophosphate: possible roles in the regulation of melanophore responses of the spadefoot toad, Scaphiopus couchi, Gen. Comp. Endocrinol. 13,151. Goldman, J.M. and M.E.Hadiey, 1970, Evidence for separate receptors for melanophore stimulating hormone and catecholamine regulation of cycle AMP in the control of melanophore responses, Brit. J. Pharmaco!. 39, 160: Hadley, M.E. and J.M.Goldman, 1969a, Physiological color changes in reptiles, Am. Zool. 9,489. Hadley, M.E. and J.M.Goldman, 1969b, Effects of cyclic 3',5'-AMP and other adenine nucleotides on the melanophores of the lizard (Anolis carolinensis), Brit. J. Pharmacol. 37,650. Hadley, M.E. and J.M.Goldman, 1970, Adrenergic receptors and geographic variation in Rana pipiens chromatophore responses, Am. J. Physiol. 219, 72. Lerner, A.B. and Y.Takahashi, 1956, Hormonal control of melanin pigmentation, Rec. Progr. Horm. Res. 12, 303. Novales, R.R., 1959, The effects of osmotic pressure and sodium concentration on the response of melanophores to intermedin, Physiol. Zool. 32, 15. Novales, R.R. and WJ.Davis, 1967, Melanin-dispersing effect of adenosine 3',5'-monophosphate on amphibian melanophores, Endocrinology 81,283. Novales, R.R. and B.J.Novales, 1965, Analysis of antagonisms between pineal melatonin and other agents which act on the amphibian melanophore, ProgL Brain Res. 10, 507. Robison, ~G.A., R.W.Butcher and E.W.Suthedand, 1967, Adenyl cyclase as an adrenergic receptor, Ann. N.Y. Acad. Sci. 139,703. Seltzer, H.S. and E.W.Allen, 1965, Inhibition of insulin secretion in "diazoxide-diabetes', Diabetes 14,439. Senft, G., 1968, Biochemical aspects of the hyperglycemic action of diazoxide, Ann. N.Y. Acad. Sci. 150, 242. Senft, G., R.Sitt, W.Losert, G.Schultz and M.Hoffmann, 1968, Hemmung der insulininkretion dutch a-receptoren stimulierende substanzen, Naunyn-Schmiedebergs Arch. Pharmacol. Exptl. Pathol. 260, 309. Shizume, K., A.B.Lerner and T.B.Fitzpatrick, 1954, In vitro

370

J.M. Goldman, M.E.Hadley, Melanophore responses to methylxanthines

bioassay for the melanocyte stimulating hormone, Endocrinology 54, 553. Taylor, J.D. and M.E.Hadley, 1970, Chromatophores and color change in the lizard, Anolis carolinensis, Z. Zellforsch. Mikroskop. Anat. 104,282. Turtle, J.R. and D.M.Kipnis, 1967, An adrenergic receptor mechanism for the control of cyclic 3',5'-adenosine monophosphate synthesis in tissues, Biochem. Biophys. Res. Commun. 28,797.

Turtle, J.R., G.K.Littleton and D.M.Kipnis, 1967, Stimulation of insulin secretion by theophylline, Nature 213, 727. Weiss, B., J.I.Davies and B.B.Brodie, 1966, Evidence for a role of adenosine 3',5'-monOphosphate in adipose tissue lipolysis, Biochem. Pharmacol. 15, 1553. Wong, K.K., S.Symchowicz, M.S.Staub and l.l.A.Tabachnick, 1967, The in vitro effect of catecholamines, diazoxide, and theophylline on insulin release, Life Sci. 6, 2285.