Melatonin effect on the cyclic GMP system in the golden hamster retina

BRAIN RESEARCH ELSEVIER

Brain Research 711 (1996) l 12-117

Research report

Melatonin effect on the cyclic GMP system in the golden hamster retina Maria P. Faillace, Maria I. Keller Sarmiento, Ruth E. Rosenstein * Departamento de Fisiologla, Facultad de Medicina, UniL,ersidad de Buenos Aires, CC 243, 1425, Buenos Aires, Argentina Received 6 July 1995; accepted 24 October 1995

Abstract

Melatonin effect on retinal cyclic GMP accumulation, guanylate cyclase activity, cyclic GMP content and cyclic GMP phosphodiesterase activity was examined in the Syrian hamster retina. Melatonin increased significantly cyclic GMP accumulation at picomolar concentrations and in a time-dependent manner. The kinetic analysis of guanylate cyclase activity revealed a significant increase of both apparent Vma,, and Kr, , induced by 10 nM melatonin. The effect of melatonin was higher in the absence, than in the presence of the phoshodiesterase inhibitor (IBMX), suggesting an effect on cyclic GMP catabolism. Phosphodiesterase activity was significantly decreased by melatonin. The results show a dual effect of melatonin on cyclic GMP levels, i.e. by increasing the synthesis and inhibiting the degradation, both resulting in an increase of cyclic GMP levels. Taking into account the key role of cyclic GMP in visual mechanisms, the results would suggest the participation of melatonin in retinal physiology. Keywords: Melatonin; Retina; Cyclic GMP; Golden hamster; Phosphodiesterase; Guanylate cyclasc

1. Introduction

Melatonin (5-methoxy-N-acetyltryptamine) initially characterized in the pineal gland, was later on identified in the retina of several species including humans, as were the melatonin-synthesizing enzymes serotonin N-acetyltransferase and hydroxyindole-O-methyltransferase [4,31]. The ability of the mammalian retina to synthesize melatonin was demonstrated in vitro [1,5] and persisted after pinealectomy [15,24], indicating that the retina sustains its own melatonin levels. While pineal melatonin is secreted to the circulation, retinal melatonin is thought to act locally within the eye [22]. Retinal melatonin has been implicated in photoreceptor disc shedding and phagocytosis [2], melanosome aggregation in pigment epithelium, and cone photoreceptor retinomotor movements [23]. Picomolar concentrations of melatonin selectively inhibit the calcium-dependent release of dopamine from rabbit and chicken retina in vitro through the activation of functionally and pharmacologically characterizable melatonin receptors [9,10]. The most striking feature of melatonin regulation in the

* Corresponding author. Fax: (54) (1) 963-6287. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 1 4 0 5 - 5

pineal gland [16] and the retina [12,13,20] of several species, is the dramatic day-night rhythmicity in its synthesis, leading to increased melatonin levels at night. Pineal melatonin is synthesized and secreted with a precise nocturnal rhythm driven by the suprachiasmatic nucleus master clock [16]. Recently, we demonstrated, in the golden hamster retina, that melatonin levels followed a diurnal rhythm which peaked in the dark-phase [11]. Because in hamster retina, melatonin was mainly influenced by a photic stimulus, it seems feasible that retinal melatonin could play the role of a dark ('not night') signal, relevant information for a light-dark transducer as the retina is. In several tissues such as the brain, melatonin is involved in the regulation of cyclic GMP, inasmuch as the injection of melatonin into the cisterna magna elicited a significant rise in cyclic GMP levels in the cerebrospinal fluid of the rabbit [26]. This effect was also observed in vitro in the rat testis [14] and medial basal hypothalamus [29] as well as in human monocytes [27]. Taking into account the key role of cyclic GMP in the retina, a possible interaction of melatonin with retinal cyclic GMP metabolism would suggest melatonin participation in retinal physiology. Therefore, we considered it worthwhile to examine the effect of melatonin on cyclic GMP and its related enzymes: guanylate cyclase and phosphodiesterase, in the hamster retina.

M.P. Faillaceet al. /Brain Research 711 (1996) 112-117 2. Materials and methods

2.1. Reagents and drugs Bovine serum albumin, 3-isobutyl-l-methylxanthine (IBMX), melatonin and cyclic GMP were obtained from Sigma Chemical Co. (St. Louis, MO, USA) [3H]cyclic GMP, [ce-32p]GTP were purchased from New England Nuclear Corp. (Boston, MA, USA), Alumina and Dowex were purchased from Bio-Rad Laboratories (Richmond, CA, USA) 2.2. AnimaL~ and tissues Male golden hamsters (average weight 120 + 20 g) were purchased from a local dealer, derived from a stock supplied by Charles River Breeding Laboratories (Wilmington, MA, USA). The animals were kept under a photoperiod of 14 h of light-10 h of darkness (lights on at 06.00 h), with free access to food and water. The animals were killed by decapitation, the eyes were enucleated, and the retinas were excised and incubated as described below for each protocol. Killing of animals, extraction of retinas and in vitro incubations (in the case of dark-exposed hamsters at 24.00, 04.00, and 06.00 h) were carried out under dim red light. Retinas were incubated at 37°C for 30 min in 500 /xl of buffer containing 140 mM NaC1, 5 mM KC1, 2.5 mM CaC12, 1 mM MgC12, 10 mM HEPES, 10 mM glucose, with or without 0.5 mM IBMX (adjusted to pH 7.4 with Tris base) in the presence or absence of melatonin ( 1 10,000 pM). Incubations were stopped by removing the medium and by homogenizing the tissue in 1 ml of 1 M perchloric acid. Samples were stored at 4°C until purification. 2.3. Cyclic GMP radioimmunoassay (RIA) Perchloric acid homogenates were centrifuged at 5000 × g for 5 min at 4°C. The supernatants were purified by sequential chromatography on alumina (AG 7, 100-200 mesh) and Dowex 50W-X8 (H + form). Samples were applied on alumina columns (2.5 cm long), previously washed with 2 ml of 200 mM ammonium formate and 16 ml of water, which were sequentially washed with 4 ml of water, 5 ml of 0.6 M HC1 in 95% ethanol, 5 ml of 50% ethanol, and 2 ml of water. Finally, 4 ml of 200 mM ammonium formate was added to elute nucleotides which were collected directly on Dowex columns (4.5 cm long). Cyclic GMP was then eluted with 3 ml of water. Columns were calibrated for recovery using external standards of [3H]cyclic GMP run with each assay. The cyclic GMP content was measured by RIA. Aliquots of cyclic GMPcontaining fractions and standards were acetylated with acetic anhydride/triethylamine. The acetylated samples and the standard curve were mixed with [125I]cyclic GMP

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(15,000-20,000 cpm, specific activity 1900-2000 Ci/mmol) and a rabbit antiserum supplied by Chemicon International, Inc., diluted 1:150, and incubated overnight at 4°C. The antibody complex was precipitated with ethanol at 4°C using 2% bovine serum albumin as a carrier, centrifuged at 2000 × g for 30 min, and separated by aspirating supernatants. The radioactivity was measured in a gamma counter. The standard curve was linear in the range 10-5000 fmol of cyclic GMP. 2.4. Guanylate cyclase activity assay Retinas were incubated for 15 min at 37°C in 500 /zl of the buffer previously described, in the presence or absence of melatonin. At the end on incubation, the medium was removed and the retinas were homogenized in a buffer solution (40 mM Tris-HC1 pH 7.6, containing 1 mM of phenylmethylsulfonyl fluoride (PMSF)). Guanylate cyclase activity was determined as described by Domino et al. [8] with some modifications. Reaction mixtures contained 50 /xl of a buffer stock solution (40 mM Tris-HC1 pH 7.6, 6 mM MnC12, 2 mM IBMX, 2 mM cyclic GMP, 1 m g / m l BSA, 0.6 m g / m l of creatine kinase and 13.24 m g / m l of creatine phosphate, [ a 32P]GTP (700,000 cpm per assay tube, specific activity 1500-3000 Ci/mmol), 25 /zl of 0.5 mM GTP and 25 /xl of the enzyme source. To determine the enzymatic kinetic parameters (Km and Vmax), seven different concentrations of unlabeled GTP (0.025-1 mM) were assayed. Tubes were incubated at 37°C during 15 min. The reaction was stopped by adding 100 /zl of 30 mM EDTA at 4°C and [32p]cyclic GMP was separated from unreacted substrate and other [3ep]-containing compounds, by column (2.5 cm long) chromatography on neutral alumina (AG 7, 100-200 mesh, Bio-Rad Laboratories). Nonenzymatic formation of cyclic GMP was tested by adding buffer instead of the enzyme source or a heat-inactivated enzyme solution. Radioactivity of the samples was determined in a liquid scintillation counter. Recoveries from the columns (usually 70-80%) were determined by adding a tracer amount of [3H]cyclic GMP to each sample, and the values obtained were corrected for recovery. Melatonin did not affect the recovery of cyclic GMP from the columns. 2.5. Phosphodiesterase (PDE) actit~ity assay Retinas were incubated for 15 min at 37°C as previously described, in presence or absence of melatonin. PDE activity was determined by the method of Thompson et al. [28], with minor modifications. Individual retinas were homogenized in 300 /xl of cold distilled water, and centrifuged at 12,000 × g for 15 min at 4°C (PDE source). The reaction contained 40 mM Tris-HC1 (pH 8.0 at 37°C), 8 mM 2-mercaptoethanol, 5 mM MgCI 2, 100 /xM cyclic GMP, 1 mM GTP, [3H]cyclic GMP (80,000 cpm/tube, specific activity 15 Ci/mmol) and 50 ~1 of tissue aliquots

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M.P. Faillace et al. / Brain Research 711 (1996) 112-117

Table 1 Effect of melatonin on cyclic GMP accumulation in golden hamster retina

Control Melatonin 1 pM 100 pM 10,000 pM

12.00 h

20.00 h

24.00 h

04.00 h

06.00 h

34.4 + 2.6 (08)

30.1 __+1.2 (14)

33.5 + 1.7 (15)

28.4 + 1.0 (12)

19.6 + 6.5 (13)

37.7 __+2.6 (17) 37.9 __+1.5 (16) 27.2 _+ 1.3 (13)

28.7 + 3.0 (10) 41.0 + 1.8 * * (11) 38.6 + 1.9 * * (17)

50.2 + 5.1 * * (12) 53.1 _+ 2.6 * * (11) 80.5 + 3.4 * * (11)

36.6 _+ 1.9 * (14) 46.9 + 2.2 * * (12) 44.0 + 1.9 * * (12)

23.7 _+3.8 (11) 13.6 _+ 2.3 (9) 17.1 _+ 2.0 (11)

The hamsters were killed at the times indicated. The retinas were excised and incubated as described in Section 2 with or without melatonin, in the presence of IBMX. Melatonin increased cyclic GMP accumulation significantly at 20.00, 24.00, and 04.00 h with a threshold concentration of 100 pM (20.00 h) and 1 pM (24.00 and 04.00 h). The control values of cGMP levels corresponding to 06.00 h were significantly different from all the other intervals except for 04.00 h ( P < 0.01, by Tukey's test). Data are means + S.E.M. values, expressed as pmol/mg protein. * P < 0.05, * * P < 0.01 by Dunnett's test.

in a f i n a l v o l u m e o f 1 0 0 / x l . T u b e s w e r e i n c u b a t e d at 37°C for 10 min. T h e r e a c t i o n w a s s t o p p e d b y b o i l i n g the t u b e s for 1 m i n . A f t e r chilling, the s a m p l e s w e r e i n c u b a t e d for 10 m i n at 30°C w i t h 5 0 / z l o f 5 ' - n u c l e o t i d a s e (1 m g / m l in distilled water), to a l l o w c o n v e r s i o n o f 5 ' - G M P into guanosine. The unreacted cyclic nucleotides were removed b y a d d i n g 1 m l o f r e s i n ( D o w e x 1 X 8, 2 0 0 - 4 0 0 m e s h , S i g m a C h e m i c a l Co.) plus t h r e e v o l u m e s o f m e t h a n o l . A f t e r c e n t r i f u g a t i o n at 9 0 0 × g for 15 m i n at 4°C, the s u p e r n a t a n t s w e r e c o l l e c t e d a n d the r a d i o a c t i v i t y determ i n e d in a liquid s c i n t i l l a t i o n c o u n t e r . A h e a t - i n a c t i v a t e d e n z y m e s o l u t i o n w a s u s e d as a b l a n k for the assay. In o u r c o n d i t i o n s , P D E a c t i v i t y w a s i n h i b i t e d b y 7 5 % b y 0.5 m M I B M X . T h e i n h i b i t o r y effects o f m e l a t o n i n a n d I B M X o n P D E a c t i v i t y w e r e n o t additive. P r o t e i n w a s d e t e r m i n e d b y the m e t h o d o f L o w r y et al. [18], u s i n g b o v i n e s e r u m a l b u m i n as s t a n d a r d . Statistical a n a l y s i s o f results w a s m a d e b y a S t u d e n t ' s t-test or b y a o n e - w a y or t w o - w a y a n a l y s i s o f v a r i a n c e ( A N O V A ) f o l l o w e d b y a D u n n e t t ' s t-test, as stated. S l o p e s a n d y - i n t e r c e p t s in e n z y m e activities w e r e c a l c u l a t e d b y the m e t h o d o f least squares.

3. Results T a b l e 1 s u m m a r i z e s the results o b t a i n e d w h e n the in v i t r o effect o f m e l a t o n i n o n retinal c y c l i c G M P a c c u m u l a -

Table 2 Effect of melatonin on guanylate cyclase activity in golden hamster retina 20.00 h

24.00 h

04.00 h

Control 63.5 ± 0.7 (17) 49.8 _+0.7 (17) 29.7 _+0.4 (14) 10 nM melatonin 67.4_+1.2 * (19) 53.2+ 1.4 * (17) 28.5 ±0.7 (15) The hamsters were killed at the times indicated. The retinas were excised and incubated as described in Section 2 with or without melatonin (10 nM). Melatonin increased enzyme activity significantly at 20.00 and 24.00 h. The control values at different time points differ significantly ( P < 0.01, by Tukey's test). Shown are the means + S.E.M., expressed as pmol/mg protein/min. Results were analyzed statistically by Student's t-test. * P < 0.05.

[

-5

0

0

lOnMmel

5

10

15

20

30

25

35

1/S ([ GTP ], mM) Fig. 1. Hamster retinal guanylate cyclase activity; graphical determination of the apparent Vmax and g m values in the presence or absence of 10 nM melatonin. Slopes and y-intercepts were calculated by the method of least squares. The points represent mean values from triplicates samples employing 5 hamsters/group; differences among triplicates were less than 10%. Apparent K m (mM) and Vmax (pmol/mg prot/min) values in this experiment were: 0.308, 340 (control); 0.414, 498 (melatonin). tion w a s assessed. In h a m s t e r retinas e x c i s e d at 20.00, 2 4 . 0 0 or 0 4 . 0 0 h, m e l a t o n i n s i g n i f i c a n t l y i n c r e a s e d cyclic G M P a c c u m u l a t i o n , in the p r e s e n c e o f I B M X . M e l a t o n i n w a s i n e f f e c t i v e at 12.00 or 0 6 . 0 0 h, at a n y c o n c e n t r a t i o n tested. M e l a t o n i n effect o n g u a n y l a t e c y c l a s e activity at different t i m e s t h r o u g h o u t the 2 4 - h c y c l e is s h o w n in T a b l e 2. M e l a t o n i n s i g n i f i c a n t l y i n c r e a s e d e n z y m e activity at 2 0 . 0 0 a n d 2 4 . 0 0 h, b e i n g i n e f f e c t i v e at 12.00, 0 4 . 0 0 a n d 0 6 . 0 0 h. Fig. 1 d e p i c t s the k i n e t i c s o f g u a n y l a t e c y c l a s e activity in g o l d e n h a m s t e r r e t i n a s e x c i s e d at 2 4 . 0 0 h, in the presTable 3 Effect of melatonin on retinal guanylate cyclase activity

Control 10 nM melatonin

K m (mM)

Vmax (pmol/mg protein/min)

0.286 + 0.019 (4) 0.441 _+0.025 * (4)

386 _ 20 (4) 507_+ 36 * (4)

Graphical determination of apparent K m and Vmax w a s made as in Fig. l. Shown are the means±S.E.M. Results were analyzed statistically by Student's t-test. * P < 0.05.

M.P. Faillace et aL / Brain Research 711 (1996) 112-117

5°°/ /

~,,,BM×

400

T

withoutIBMX

~'i

~73oo

gs '~ _~ 2oo

I

I pMreel 100pMreel 10000pMmel Fig. 2. Melatonin effect on cyclic GMP content in the absence or presence of the phosphodiesterase inhibitor. The hamsters were killed at 24.00 h, the retinas were excised and incubated as described. Melatonin increased significantly cyclic GMP content at each concentration tested, with or without IBMX. Values represents mean+S.E.M. ( n = 6 to 8 animals /group). The basal levels in the absence or presence of IBMX were expressed as 100% and were: 10.5 + 1.3 and 34.8_+ 1.5 respectively.

I. . . . . .

~ 3.5 .~, ~ 30 o.o

~

m

~

control

J

10 nM mel

Fig. 3. Melatonin effect on phosphodiesterase activity. Melatonin decreased significantly retinal phosphodiesterase activity of hamsters killed at 24.00 h. Values are mean+S.E.M. ( n = 20 to 24 animals/group) * P < 0.05, Student's t-test.

ence (or absence) of 10 nM melatonin. Melatonin significantly increased apparent Vmax as well as K m of guanylate cyclase (Table 3). In order to further define the mechanism of melatonin effect on retinal cyclic GMP content, cyclic GMP levels were measured in the presence or absence of IBMX (Fig. 2). Melatonin, at all concentrations tested, was more effective at increasing cyclic GMP in the absence than in the presence of the phosphodiesterase inhibitor. In view of this result, the effect of melatonin on phosphodiesterase activity was examined (Fig. 3). Melatonin decreased significantly retinal phosphodiesterase activity at 24.00 h.

4. D i s c u s s i o n

A number of key aspects of retinal physiology are currently considered amenable of melatonin regulation: photoreceptor disc shedding and phagocytosis [2], retinomotor movements [23], membrane potential of retinal

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pigment epithelium cells [19] and modulation of dopamine release [9]. The foregoing results demonstrate a significant, time-dependent effect of melatonin on cyclic GMP accumulation in the presence of IBMX, and guanylate cyclase activity. Furthermore, cyclic GMP content and cyclic GMP-phophodiesterase activity were significantly modified by melatonin. Cyclic GMP plays a key role in retinal physiology being the intracellular messenger that links rhodopsin isomerization with changes in membrane permeability upon illumination. In rod photoreceptor ceils, the light response is triggered by an enzymatic cascade that causes cyclic GMP levels to fall: excited rhodopsin ~ rod G-protein (transducin) ~ cyclic GMP phosphodiesterase. This results in the inactivation of plasma membrane channels positively gated by cyclic GMP [21]. Melatonin significantly increased the in vitro accumulation of cyclic GMP, in the presence of IBMX, at a very low concentration (1 pM) and in a time-dependent manner. This effect is compatible with the occurrence of high-picomolar-affinity melatonin binding sites, as have been described in the rabbit retina [3]. Melatonin effect did not seem to be directly related to the illumination conditions to which the animals were exposed, as melatonin was effective both at light (20.00 h) and dark incubations (24.00 and 04.00 h) and was ineffective also under both situations (12.00 h and 06.00 h, respectively). The time-dependent effect of melatonin could be related to a diurnal change in the sensitivity of the tissue, as previously described in the brain [25], currently associated to a diurnal variation of melatonin receptor sites. However the circadian variation of melatonin receptors at a hamster retinal level, has not been described yet. On the other hand, as the control values significantly differed through time, it is possible that the cyclic GMP content in the hamster retina is already experiencing a rhythm in its exposure to melatonin that could account for the different response to the methoxiyndole at different time points. Melatonin effect on cyclic GMP was measured in the presence of the phosphodiesterase inhibitor IBMX, pointing at retinal guanylate cyclase as a target for melatonin action. Two facts support that this is indeed the case. First, small but significant changes in the kinetic parameters of guanylate cyclase were observed in the presence of melatonin, the hormone increasing both apparent Vmax and K m of the retinal enzyme. Second, melatonin was ineffective on guanylate cyclase activity at those times (12.00 and 06.00 h) at which no increase in cyclic GMP was observed. The effect of melatonin on guanylate cyclase activity was evident only at the highest concentration of melatonin tested and even in this case the effect was very small albeit significant. The conditions for the assessment of cyclic GMP accumulation and guanylate cyclase are quite different. For example, guanylate cyclase assay includes all necessary factors for full activity, and some of them could be rate-limiting for cyclic GMP synthesis in the

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M.P. Faillace et al. / Brain Research 711 (1996) 112-117

accumulation assay. This way, melatonin might act on one or more of these limiting factors. This could account for an apparently lower sensitivity to melatonin when guanylate cyclase is determined, and for the apparent lack of effect of melatonin on guanylate cyclase at 04.00 h, a time point at which cyclic GMP accumulates in response to melatonin in the presence of the phophodiesterase inhibitor. Moreover, a clear relationship between cyclic GMP levels, measured in the presence of IBMX, and guanylate cyclase activity is not evident under control conditions (i.e. in the absence of melatonin), as guanylate cyclase activity differed between 20.00 and 24.00 h, whereas cyclic GMP levels did not. The daily variations in cyclic GMP levels and guanylate cyclase activity is being currently investigated in greater detail. In order to disclose whether there was a coupled action of melatonin on guanylate cyclase and cyclic GMP phophodiesterase, melatonin effect on cyclic GMP was assessed in the absence of IBMX. Under that condition, melatonin was more potent than in the presence of the phosphodiesterase inhibitor, suggesting an effect of melatonin on phosphodiesterase activity. That the assumption eventually was correct, was showed by the significant inhibition of phosphodiesterase activity by nanomolar concentrations of melatonin. The present results show a dual action of melatonin on retinal cyclic GMP content, i.e. by increasing its synthesis and inhibiting its breakdown, both leading to an increase of cyclic GMP levels. The intracellular events triggered by melatonin that could explain these effects remain to be established. It has been shown that in brain synaptosomes, melatonin depresses calcium uptake induced by high potassium [30]. There are, at least, two ways by which calcium decreases retinal cyclic GMP concentration, that is, by inhibiting guanylate cyclase [17] and stimulating phosphodiesterase [15], both likely involved in light adaptation mechanism. Therefore, it is tempting to speculate that the effect of melatonin on the enzymes involved in cyclic GMP metabolism could be mediated by inhibition of calcium entry. The in vitro effect of melatonin on calcium uptake in hamster retina is under current investigation. Melatonin effect on cyclic GMP content and related enzymatic activities were assessed in the whole retina. Therefore we could not ascertain the locus of the observed phenomena. Since photoreceptors cells are the most abundant cellular type in the hamster retina, it seems likely that the melatonin effect took place in those cells. It has been proposed that cyclic GMP compartmentation may occur in rod outer segments [6,7] and the possibility that the measured changes induced by melatonin in total cyclic GMP concentration could reflect larger or lesser changes in the region near cyclic GMP-regulated ion channels a n d / o r in other retinal cellular type remains to be tested. It has been postulated that in the retina, melatonin mimics darkness (i.e. by causing aggregation of melanin

pigment granules in the retinal pigment epithelium, activation of disc shedding in rod photoreceptors, and inhibition of dopamine release). The foregoing results suggest that in the hamster retina, melatonin through the increase in cyclic GMP, could also participate in the phototransduction mechanism, as a signal that 'means' darkness at a retinal level.

Acknowledgements The authors wish to thank Dr. Daniel P. Cardinali and Dr. Marcelo A. de las Heras for the helpful discussion of this manuscript, Dr. Eduardo H. Charreau and Dr. Omar P. Pignataro for the iodination of [I25I]cyclic GMP. This research was supported by a grant from Fundaci6n Antorchas, Buenos Aires, Argentina.

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