Localization and characterization of melatonin receptors in the rabbit spinal cord

ELSEVIER

Neuroscience Letters 204 (1996) 77-80

NtUROSCIEHGf LETTERS

Localization and characterization of melatonin receptors in the rabbit spinal cord Qi W a n a, M i n g x i a L i a o b, G r e g o r y M. B r o w n b, S h i u F u n P a n g a,b,* aDepartment of Physiology, University of Hong Kong, 5 Sassoon Road, Hong Kong, Hong Kong bClarke Institute of Psychiatry, 250 College Street, Toronto, Ontario, MST I R8, Canada Received 30 October 1995; revised version received 20 December 1995; accepted 21 December 1995

Abstract

Melatonin receptors in the rabbit spinal cord were studied. Using in vitro quantitative autoradiography we have localized and characterized 2-[125I]iodomelatonin([125I]MEL)binding sites in the central gray substance (lamina X) of the rabbit spinal cord. Saturation study revealed a single class of high affinity binding sites in the central gray substance with an equilibrium dissociation constant (Kd) of 38.8 ± 5.25 pM and a maximum number of binding sites of 5.69 ± 0.84 fmol/mg protein in the mid-light period. These [t25I]MEL binding sites were highly specific for melatonin. Coincubation with 10/~M or 50/~M guanosine 5'-O-(3-thiotriphosphate) produced a significant change in Kd. These results suggest that melatonin receptors in the rabbit spinal cord are coupled to a guanine-nucleotidebinding protein (G-protein). Our studies suggest that melatonin exerts a direct action on the rabbit spinal cord.

Keywords: Melatonin; 2-[12-';I]Iodomelatonin;Pineal gland; Central nervous system; Central gray substance; G-protein

Melatonin (N-acetyl-5-methoxytryptamine) is a pineal hormone. It acts as a photoperiodic signal and plays an important role in synchronizing biological rhythms and neuroendocrine functions in vertebrates [4,18], including the regulation of nociception [1,10,20], body temperature [2,21,22], blood pressure and vascular smooth muscle activity [25]. Melatonin is known 1:o exert its physiological effects through high affinity melatonin receptors [7,16,19]. Using 2-[t25I]iodomelatonin ([125I]MEL), a biologically active agonist of melatonin, high affinity [125I]MEL binding sites have been demonstrated in various regions of avian and mammalian brain [7,8,19]. Recent studies demonstrated that high affinity melatonin receptors exist in the dorsal gray horn (laminae I-IV) and central gray substance (lamina X) of the., chicken spinal cord [23,24], an integral part of the central nervous system (CNS). It was proposed that melatonin may play a role in the regulation of pain and thermal transmission, autonomic function and visceral reflex in spinal cord function [9,23,24]. In order to further investigate melatonin actions on the mammalian spinal cord, in this study we have localized and char* Corresponding author. Tel.: +852 28199260; fax: +852 28559730.

acterized [t25I]MEL binding sites in the rabbit spinal cord by in vitro quantitative autoradiography. [125I]MEL with specific activity of 2200 Ci/mmol was purchased from NEN (Dupont, Boston, MA, USA). Melatonin, guanosine 5'-0-(3-thiotriphosphate) (GTP),S) and other chemicals were obtained from Sigma (St. Louis, MO, USA). 2-Iodomelatonin was obtained from Research Biochemical, Natick, MA, USA. 6-Chloromeiatonin was kindly donated by Eli-Lilly, Indianapolis, IN, USA. Male New Zealand White rabbits (Oryctolagus cuniculus; 6 weeks old) were adapted to a 12 h light/12 h dark cycle (light on 0300-1500 h) for 2 weeks. Illumination was provided by ceiling-mounted fluorescent lamps. Light intensity was 200-300 lux. Water and food were available ad libitum. Following adaptation, the animals were decapitated at mid-light (0830-0900 h) and their spinal cords were collected and frozen in liquid nitrogen within 5 min and stored at -70°C. Autoradiographic studies were performed as described [24]. Saturation studies were carried out in consecutive sections which were incubated with [125I]MEL (5l l0pM). For kinetic studies, the consecutive sections were incubated with 30pM [125I]MEL for 5-200 min at 22°C. Dissociation was initiated by the addition of

0304-3940/96/$12.00 © 199.6 Elsevier Science Ireland Ltd. All fights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 12321-X

78

Q. Wan et aL / Neuroscience Letters 204 (1996) 77-80

(a)

8

TB

. (b)

~ 0 s ~

=,,0

.'---"'~ 6 ,,d~4

=

t /I

=

g~' so.

O' 0.00

0.04

0.08

0.12

[tzSI]MEL(nM)

Fig. 1. Distribution of 2-[125I]iodomelatonin binding in coronal sections of the rabbit spinal cord. (A,C,D) Representative autoradiograms of total binding in cervical, thoracic and lumbar segments. The dark areas in the central gray substance (lamina X) are the specific binding sites of 2-[1251]iodomelatonin. (B) Representative autoradiogram of non-specific binding of the cervical segment defined with 1/zM melatonin. 1/zM melatonin at 80 min of incubation. In competition studies, consecutive sections were incubated with 35 pM [t25I]MEL and increasing concentrations (10 -t2 to 10-6 M) of various compounds. To observe the effects of GTPyS on the binding parameters of [125I]MEL, consecutive sections were incubated with [125I]MEL (10-120 pM) in the presence of 10/zM or 50/zM GTPyS. Using the M C I D - M I computerized image processing and analysis program (Brock University, St. Catharines, Ont., Canada), optical density and dpm/mg polymer values of the standards were used to generate a standard curve, and the dpm/mg protein Was obtained based on the experimental relationship between the polymer and protein standards [13]. The mean optical densities measured' from the autoradiograms were then transformed to corresponding values of dpm/mg protein. Results from the saturation studies and kinetic studies were analyzed as reported [24]. T h e method for calculating the inhibition constants (Ki) was described by Cheng and Prusoff [6]. AutoradiographiCal studies demonstrated that the specific binding of [t25I]MEL was only localized in the central gray substance (lamina X) throughout all segments of the rabbit spinal cord. There was no difference in the density and distribution of [125I]MEL binding sites among the cervical, thoracic and lumbar segments (Fig. 1). Scatchard analysis of saturation studies revealed a single class of high affinity binding sites with an equilibrium dissociation constant (Kd) of 38.8 _ 5.25 pM (n = 8), a maximum number of binding sites (Bmas) of 5.69-+ 0.84 fmol/mg protein ( n = 8) and a Hill coefficient of 1.02 +_ 0:02 (n = 8) in the mid'light period. A representative saturation study is demonstrated in Fig. 2. Specific binding of [125I]MEL elevated with increasing concentrati0ns of the radioligand and reached a plateau at 0.08 nM (Fig. 2a), indicating that the binding is saturable. Scat-

1

2

3

4

5

6

Bound (frnol/mg protein)

Fig. 2. (a). A representative saturation study of specific [125I]MEL binding in the rabbit spinal cord. TB, total binding; NSB, non-specific binding; SB, specific binding (TB- NSB). (b). Scatchard transformation of the data in (a), with a correlation coefficient (r) of 0.96, an equilibrium binding constant of 35.6 pM and a total number of binding sites of 5.58 fmol/mg protein. (c) Hill plot with a coefficient of 0.97. chard plot shows a linear regression (Fig. 2b). Fig. 2c is a Hill plot. A representative kinetic study is illustrated in Fig. 3. Specific binding increased during the first 30 min and equilibrated after approximately 7 0 - 9 0 min. with a Kl equal to 0.975 X 109/M per min. Following equilibrium with 55 pM [125I]MEL for 80 min at 22°C, 1/zM melatonin was added. Displacement of most of the specific binding of [125I]MEL occurred within 120 min, with a K-1 of 1.44 x 10-2/min and a Ka ( K _ I l K I ) of 14.8 pM. The value of K a derived in the kinetic studies was 11.0 _+ 3.09 pM (n = 3), which was consistent with that determined from the saturation studies (38.8 _+5.25 pM). Inhibition constants (Ki) of various compounds on [125I]MEL binding sites are listed in Table 1. Significant

21 / ®0

~oeiation

1

-nmo ( m~[assoeialion, _i n(c)) 0 1 os 1

o

so .iqr~O(min;SO

200 ---,i~ l 50

i 1O0

~xg_ 150

200

.time [dissociation] (min)

Fig. 3. (a) Time-course of [1251]MEL association and dissociation binding in the rabbit spinal cord in a representative experiment. Consecutive sections were incubated with 30 pM [125I]MELat 22°C for the times indicated and dissociation was initiated by the addition of 1/xM melatonin after a preineubation of 80 min. (b) Pseudo-ftrst-order plot of the association data. (c)First-order plot of the dissociation data. B, ligand-receptor complex at different time points; Be, iigand-receptor complex at equilibrium. In this study, association rate constant (KI) was 0.975 x 109/M per min, dissociation rate constant (K_I) was 1.44 x 10-2/min, and equilibrium dissociation constant (K_IIKI) was 14.8 pM.

Q. Wan et al. / Neuroscience Letters 204 (1996) 77-80

inhibition of [125I]MEL binding by melatonin and its analogs have the following order of potency: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin. Other compounds had little or no significant inhibitions on [125I]MEI., binding in the rabbit spinal cord. Table 2 shows the effects of 10 or 50/~mol/l GTPTS on the [125I]MEL binding of rabbit spinal cord. GTPyS significantly increased (P < 0.05) the Kd without affecting the Bmax (P < 0 . 0 5 ) . Hill coefficients were close to unity in all the cases tested (Table 2), suggesting a single class of binding sites for [1251]MEL in the rabbit spinal cord in the presence or absence of GTP/S. In the present studies, the [125I]MEL binding site of rabbit spinal cord exhibits kinetic and pharmacological characteristics of a melatonin receptor. The binding was saturable, reversible, specific and of high affinity. Linearity of Scatchard plots and the unity of Hill coefficients indicate that a single class of [125I]MEL binding sites exists in the rabbit spinal cord. The non-hydrolyzable GTP analog, GTI~S, decreased the binding affinity of [125I]MEL binding sites in the rabbit spinal cord. It has been suggested that the change of binding affinity may be due to the destabilization of hormone-receptor-G-protein interaction by GTP analogs [11]. Our results suggest that rabbit spinal [125I]MEL binding sites are linked to a G-protein. GTPyS has been shown to increase Kd and decrease Bmax in the rat cerebral arteries of circle of Willis, chicken kidney and pigeon spleen [3,15,17], and to decrease Bmax without change in Kd in the ovine pars tuberalis and rat suprachiasmatic nucleus [8,12]. The effect of GTPyS on the rabbit spinal cord is consistent with that in the hamster brain, rabbit cortex and chicken lung in which Kd increases without change in Bmax w e r e reported [5,15]. The various responses of [125I]MEL binding to GTI~S in different tissues may partly explain the different physiological effects of melatonin on different systems of various species. The present results obtained from the rabbit spinal cords are the first demonstration of melatonin receptors in the mammalian spinal cord. This study further supports our earlier finding that melatonin has a direct effect on the Table 1 Inhibition constants (K) of melatonin, its analogs and other neurotransmitters on 2-[ 125l]iodomelatonin binding in the rabbit spinal cord

2-1odomelatonin Melatonin 6-Chloromelatonin 6-Hydroxymelatonin N-Acetylserotonin 5-Methoxytryptamine 5-Methoxytryptophol 3-Acetylindole

n

Ki (nM)

3 3 3 3 3 3 3 3

0.09 ± 0.02 0.17 ± 0.03 1.43 ± 0.05 12.3 ± 0.06 429 ± 50.4 515 __-87.6 1945 ± 352 > 10 000

n, the number of animals; data shown are the mean ± SEM.

79

Table 2 Effectsof GTP),Son the bindingaffinity(Kd),densities (Bmax)and Hill coefficientsof 2-[1251]iodomelatoninbinding sites in rabbit spinal cord n

Kd (pM)

Bmax(fmol/ mg protein)

Hill coefficient

Control GT~S

3 3

35.2 ± 4.75 53.6 ± 6.27*

5.75 ± 0.91 5.64 + 0.85

0.98 ± 0.03 0.98 ± 0.03

(lO/,tM) GTPTS (50#M)

3

65.8 ± 6.84 *,#

5.59 ± 0.89

0.99 ± 0.02

n, the number of animals; data shown are the mean ± SEM. *P < 0.05, GTPyS versus control, Student's t-test. #P < 0.05, 50#M GTP~S versus 10/zM GTPyS, Student's t-test.

spinal cord [23,24]. Throughout all the segments of rabbit spinal cord, putative melatonin receptors are only localized in the central gray substance (lamina X). Therefore, it is quite possible that melatonin plays a role in regulating the mechanisms of nociception, thermal transmission and visceral reflex of rabbit spinal cord, because (1) lamina X receives afferent input similar to that of laminae I and II: nociceptive and visceral responses have been reported for cells of lamina X in mammals [26]; (2) melatonin is involved in the regulation of circadian rhythms of nociception and has an analgesic property [1,10,20]; and (3) melat0nin affects thermoregulation and has a hypothermic property. In addition, it is also involved in regulating the circadian variations of body temperature [2,9, 14,21,22]. In our earlier studies, putative melat0nin receptors in the chicken spinal cord are shown to distribute mainly in the dorsal gray horns (laminae I-IV) and the central gray substance (lamina X) [24]. In contrast to the chicken spinal cord, melatonin receptors in the rabbit spinal cord are localized only in the lamina X. The discrepancy may reflect different mechanisms of melatonin action on the avian and mammalian spinal cords. However, melatonin actions on the chicken and rabbit spinal cord may be similar as lamina X and laminae I-II have similar functions [26]. The identification and characterization of melatonin receptors in the rabbit spinal cord indicates that melatonin plays a role in the rabbit cord function. The distribution of melatonin receptors in the central gray substance (lamina X) implies a modulatory effect of melatonin on the mechanisms of sensory transmission and/or visceral reflex of the spinal cord. This study was supported in part by CRCG, RGC, Neuroendocrine Research Fund and Clarke Foundation grant to S.F. Pang. G.M. Brown is an OMHF Research Associate. The authors would also like to thank K.F.L. Tsang for assistance. [1] Bar-Or, A. and Brown, G.M., Pineal involvement in the diurnal rhythm of nociception in the rat, Life Sci., 44 (1989) 1067-1075.

80

Q. Wan et aL / Neuroscience Letters 204 (1996) 77-80

[2] Cagnacci, A., Eiliott, J.A. and Yen, S.S.C., Melatonin: a major regulator of the circadian rhythm of core temperature in human, J. Clin. Endocdnol. Metab., 75 (1992) 447-452. [3] Capsoni, S., Viswanathan, M., Oliveira, A.M. and Saavedra, J.M., Characterization of melatonin receptors and signal transduction system in rat arteries forming the circle of Willis, Endocrinology, 135 (1994) 373-378. [4] Cardinali, D.P., A mammalian pineal hormone, Endocr. Rev., 2 (1988)327-346. [5] Carlson, L.L., Weaver, D.R. and Reppert, S.M., Melatonin signal transduction in hamster brain: inhibition of adenylate cyclase by a pertussis toxin-sensitive G protein, Endocrinology, 125 (1989) 2670-2676. [6] Cheng, Y.C. and Prusoff, W.H., Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (IC5o) of an enzymatic reaction, Biochem. Pharmacol., 22 (1973) 3099-3108. [7] Dubocovich, M.L., Shankar, G. and Mickel M., 2-[1251]Iodomelatonin labels sites with identical pharmacological characteristics in hamster brain and chicken retina, Ear. J. Pharmacol., 162 (1989) 289-299. [8] Gauer, F., Masson-Pevet, M. and Pevet, P., Effect of constant light, pinealectomy and guanosine triphosphate gamma-S on the density of melatonin receptors in the rat suprachiasmatic nucleus: a possible implication on melatonin action, J. Neuroendocrinol., 4 (1992) 455-459. [9] Kachi, T., Pineal action on the autonomic system, Pineal Res. Rev., 5 (1987) 217-264. [10] John, T.M., Brown, M.C., Wideman, L. and Brown, G.M., Melatonin replacement nullifies the effect of light-induced functional pinealectomy on nociceptive rhythm in the rat, Physiol. Behav., 55 (1993) 735-739. [!1] Lefkowitz, R.J., Caron, M.G., Michel, T. and Stadel, T.M., Mechanism of hormone receptor-coupling: the fl-adrenergic receptor and adenylate cyclase, Fed. Prec., 41 (1982) 2664--2670. [12] Morgan, P.J., Lawson, W., Davidson, G. and Howell, H.E., Guanine nucleotides regulate the affinity of melatonin receptors on ovine pars tuberalis, Neuroendocrinology, 50 (1989) 359-362. [13] Nazarali, A.J., Gutkind, J.S., and Saavedra, J.M., Calibration of 1251-polymer standards with 125I-brain paste standards for use in quantitative receptor autoradiography, J. Neurosci. Methods, 30 (1989) 247-253.

[14] Oshima, 1., Yamada, H., Goto, M., Sato, K. and Ebihara, S., Pineal and retinal melatonin is involved in the control of circadian locomotor activity and body temperature rhythms in the pigeon, J. Comp. Physiol., 166 (1989) 217-226. [15] Pang, S.F., Ayre, E.A., Poon, A.M.S., Pang, C.S., Yuan, H., Wang, Z.P., Song, Y. and Brown, G.M., Effects of guanosine 5'O-(3-thiotriphosphate) on 2-[125I]iodomelatonin binding in the chicken lung, brain and kidney: hypothesis of different subtypes of high affinity melatonin receptors, Biol. Signals, 2 (1993) 2736. [16] Pang, S.F., Dubocovich, M.L. and Brown, G.M., Melatonin receptors in peripheral tissues: a new area of melatonin research, Biol. Signals, 2 (1993) 177-180. [17] Poon, A.M.S., Wang, X.L. and Pang, S.F., Characteristics of 2[125I]iodomelatonin binding sites in the pigeon spleen and modulation of binding by guanine nucleotides, J. Pineal Res., 14 (1993) 169-177. [18] Reiter, R.J., Pineal melatonin: cell biology of its synthesis and of its physiological interactions, Endocr. Rev., 12 ( 1991) 151- 180. [19] Reppert, S.M., Weaver, D.R., Rivkees, S.A. and Stopa, E.G., Putative melatonin receptors in human biological clock, Science, 242 (1988) 78-81. [20] Rosenfeld, J.P. and Rice, P.E., Diurnal rhythms in nociceptive thresholds of rats, Physiol. Behav., 23 (1979) 419-420. [21] Saarela, S. and Reiter, R.J., Function of melatonin in thermoregulatory processes, Life Sci., 54 (1993) 295-31 I. [22] Strassmann, R.J., Quails, C.R., Lisansky, E.J. and Peake, G.T., Elevated rectal temperature produced by all-night bright light is reversed by melatonin infusion in men, J. Appl. Physiol., 7 (1991) 2178-2182. [23] Wan, Q. and Pang, S.F., [1251]Iodomelatonin binding sites in the chicken spinal cord: binding characteristics and diurnal variation, Neurosci. Lett., 163 (1993) 101-104. [24] Wan, Q. and Pang, S.F., Segmental, coronal and subcellular distribution of 2-[1251]iodomelatonin binding sites in the chicken spinal cord, Neurosci. Lett., 180 (1994) 253-256. [25] Weekley, L.B., Melatonin-induced relaxation of aorta: interaction with adrenergic agonists, J. Pineal Res., ! 1 (1991) 28-34. [26] Willis, W.D. and Coggeshall, R.E, Sensory Mechanisms of the Spinal Cord, Plenum Press, New York. 1991, pp. 153-215.