Diurnal changes in melatonin binding sites of hamster and rat brains. Correlation with neuroendocrine responsiveness to melatonin

Neuroecience Letters, 15 t'1979"b259-263 © Elsevier!North-Holland Scientific JPubli~eJmLtd.

259

DIURNAL CHANGES IN MELATONIN BINDING SITES OF HAMSTER AND RAT BRAINS. CORREI~TION WITH NEUROENIK)CRINE ~NSIVENE88 TO MELATONIN MARgA I. VACAS* and DiP. CARDINALI** Cenfro de Estudios Formacoldgicos y de Pti~ipios Naturules (CEFAPRIN), Obligado 2490, 1423 Buenoe Aires (A~ntfna) (Received July 27th, 1979) (Accepted August 8th, 1979)

SUMMARY

A diurnalvariation in neuroendocdne sensitivity to melatonin is known to occur in hamsters ~md rats.The present experiments were carried out to examine the pou~Jity that affinityand/or number of melatonin binding sites in brain could chang~ a ~ d i n g l y at the two times when exogenous

melatonin is known to be ineffective or effective to produce neuroendocrine clumges, i.e., at 07 : 00 or 20 : 00 h (lights on from 07 : 00 to 21 : 00 h daily). The rmmber of melatonin binding sites in b ~ t e r and rat brains was at 20 : 00 h 3 4 - J 6 % h ~ than at 07 : 00 h, without changing their towards [ ~ ] m e l a t o n i n ( ~ : gd = 53 nM; rat: gd = 73--77 riM). These ~ ~ in melatonin receptor d e n n y may play a role in daily changes in ~nsitivlty ~ the exogenous methoxyindole.

The a d m l n ~ of the l~.neal~ rne~fin (5-methoxy-Nacety~) to h ~ ~ mrs k followed by v.azkedneuroendoczine change~~ upon the ~ ~ time of injection.Ini~ o ~ a t i o n s on a dhmmlly~iopendent effectof melatonln in the nmmmsl~n ~ were made by ~ uzl H u p ~ [4] who reported ~ the pinee serotonm levels continue t o vary ~ ff ~ are given d ~ y injections of mel~ tontn a t t h e ommt of d~knem; ff h o w l e r n~ktonin is adminktemd in the 8th h of the light period ( w h ~ little ff ~ ~ iJ secreted) the se~ctonin ~ in the ~ ~ ~ blocked.Recent studiesin the golden demonm~_te that ~ inketlo~ of 10 or 25 ~g of melatonm shortly before the end of the light ~ ~ ~ r e g n z ~ n and

~

N~qonJl de I n v e ~ l l ~

C~t#flc~ ~ T ~

260

arrest of the estrous cycle within 7 weeks in animals kept in 14-h photoperiod whereas the same dose injected in the morning hours is ineffective [6,9]. Since we have recently described the occurrence of melatonin receptors (defined by their binding characteristics) in membrane preparations from various regions of the bovine brain [2,3] we thought it was worthwhile to examine the possibility that affinity and/or number of melatonin binding sites in hamster and rat brains could change accordingly at the two times when exogenous melatonin was reported as effective or ineffective to affect the neuroendocrine system (i.e., at the end of the light or dark phase of daily photoperiod, respectively). Sexually mature Wistar male rats or male golden hamsters were kept under light from 07 : 00 to 21 : 00 h daily and were given access to food and water ad libitum The animals were killed by decapitation and individuals brains were dissected out and frozen at -70°C. Crude brain membrane pellets were prepared as described in detail elsewhere [3]. The final precipitates were resuspended in 5 vol. of 50 mM Tris-HCI buffer (pH 7.4), containing 6 mM CaCh and 0.1% ascorbic acid. Aliquots (0.25 ml) of tissue suspension were incubated in triplicate for 5 h at 0° C in darkness with 5-80 nM pH]melatonin (2-aminoethyl-2.pH]melatonin, 31.5 Ci/mmol, New England Nuclear Corp., Boston, MA) with or without a 1000·fold excess of unlabeled melatonin. Protein concentration was determined by the procedure of Lowry et al. [5] using bovine albumin as a standard. In each experiment a parallel series of reaction mixtures containing no tissue frac· tions was run as controls. Binding assays utilized a rapid filtration procedure through Whatman GFB glass fiber filters under vacuum. After washing with ice-cold buffer the radioactivity left on the filter was measured by liquid scintillation spectrometry. A toluene-phosphor solution containing 30% Triton X-I00 (v/v) was used as a scintillation fluid. Quenching was corrected by automatic external standardization. In all the experiments, the differ· ence between the total [3H] melatonin bound and the [3H] melatonin bound in the presence of excess melatonin (non-specific binding) was taken as the amount of specific melatonin binding. Specific binding ranged between 40% and 60% of total binding. The specific binding data were transformed [8] and the line slopes and intercepts were determined by regression anal· ysis. Each experiment was repeated at least twice. Detailed description of thermolability, sensitivity to enzymatic digestion, specificity and distribUtion of melatonin binding sites in brain is published elsewhere [3]. At the incubation used (at O°C for 6 h) binding data are an estimate of total, i.e., occupied and unoccupied, binding sites, since the maximum reached in time course experiments is essentially similar at 0,18 or 37°C [3]. In agreement with previous data on bovine brain [2,3] it was found that the specific [lH]melatonin binding is saturable in hamster and rat brains at both selected times, i.e., at the end of the dark phase (07 : 00 h) or of the light phase (20 : 00 h) of daily photoperiod (Figs. 1 and 2). Plot-

261 HAMSTER

BRAIN

Kd(2OOOh}= 5,5.10 -8 M I.~

{~000

Kd(o~OOh)=S-3.,0JM

(OTOOh) ',~ "

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"

3H-ME~LATONIN(ziOL9 M)

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3H-MELATONIN BOUND(x 10"12M}

lr~g. 1. [ ) H ] l ~ k t o B ~ binding as a function of incxea~r,g concentration of [=H]melatonin in the i ~ u b ~ t i o n ~ of membrax~ prepmations from the b~dr~ of ~ r ~ killed at t h e end o f the dark phase (07 : 00 h) or at the 13t~ h o f the light phase of daily photopeflod C~) : 00 h). q1~e inset presents the data in S c a l d plots for the ~nat~on of d~tion c o n s t a r ~ (Kd) and mcdmal number of b~,nding sites (~). Each point r ~ the mean of triplicate samples; the intrasny coefficient of v~d~tion w u l e ~ than 15%. 07 : O0 h: K d = 53 nM and ~ m n = 3~.0 fmol/mg protein; 20 : 00 h: Kd = 53 ~nd l ~

~ 50.0 fmollmg protein.

RAT

BRAIN

Kd{Z~h )- 77 10°'eM K~OTOOh~- 7.~, 10"6M

!,o #

Fig. 2. [ J H ~ e b t o . ~ b.~nd~_ ~ ~ ~ , ~ e ~ ~ o ~ from ~ i r m of m ~ k t ] | ~ in the ~ p e ~ t ~ i n F ~ ~, ~ ~ n ~ in ~ , I. C7 : OO h: Kd ~ 7~ nM B ~ ~ 6 2 ~ fmQI/mg p~o~in 20 : 00 h: K d ~ 77 nM and S m ~ ~ 94 f ~ l / ~ g p~ot~i~.

T, k a I , ~ I

flI~CLW[C~ OF [SH]~B~TONIN TO ~ HAMB'II]3~ AND RAT BRAINS T-me- , . t . ~ k , m ~ e ~ at tlseeadof the dm:k((~'; : 00 h)m- ~ RJIJd. ( n -

FRACTIONSOF rZO : O0 h)phase.Men -*

6 ia meh group) i

m

[ ~ H ~

brain Rat brain

b o u d (fa~lmg m'ot-~)

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20 " 0 0 h

12A ~ 1.8

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24.9 :~ 9-.6a

a p < 0.05 i s eompm'ed to animak killed at 07 : 00 h (Student',, test)

ring these results according to the Hill equation m m l t ~ in stndght lines and Hill coefficients of about 1 in all cues, ~ the absence of c o operativity. After, incu with 29 nM [:'H]m e l a t o n i n binding at 2 0 : 0 0 h was 34--56% higher tJum at 07 : (tO h (Table I). Scratched analysk of the ~ curves indicated an appme.~nt s i n ~ population of binding sites with a Kd~alue ofabou~ 50 nM ( ~ ) a n d 70 nM (rat). The Ke~aluee were ~ y the same at the two mulied times, eflthovgh the maximal number of binding sites i n ~ by 48--r52% at the end of the light period (F~gs. I and 2). The foregoing resultu ~ the occuffence of diuma~y.related changes in the number of melatonin receptor s t ~ without changes in the affini~ of the receptor for the radiolabeled ~ Taken t o g e t ~ r with data on the neuroendocri~e sensitivity towarde melatontn in rat, [4] and hamsters [ 1,6,9 ] the preeent repJlte indicate that mc re melatonJn binding sites are available in brain at the time of eemitivity, i.e., at the end of t ~ . light period, than at the time when the endocrine system was refractory to melatonin injections, i.e., at the end of the dark phase of daily photo-

peYotL An important factor regulating the number of bi~ding sites may be the endogenous sec~tion of melatonin which is highest at night and negligible during light hours [10.11]. Exposures to high melat~tin levek d~dring the dark phase of the daily photoperiod may cause desen~izafion of the neuroendocrine system to exogenous melatonin by 'down regulation' of receptor binding sites, whereas during daytime the number of receptor m t ~ may increase as melatonin levels are low and restoration of sensitivity occum at the end of the light phase. Any ~ c e in this d i ~ rhythm leeds to edte,mtion in the ~ to melatonin. This is supported b y the r e f m c t o ~ of the neutoeedoedne system in pinealect o ~ ~ when a ~ ~ o n of melatonin was given at t h e end of theiight~ [7 ]; 3 ~ in~-tior~ of melatonin at 3-h intervals beginning 4 h

263

after the onset o f light induce a n e u r o e n d o c r i n e response (e.g., t~sticular regression) in pinealectomized b u t n o t in i n t a c t animals [9]. These differences in sensitivity of pinealectomized and intact hamsters might reflect a n interaction b e t w e e n endogenous e n d exogenous m e l a t o n i n or a role o f t h e pineal gland in phasing t h e ~esponsiveness of target tissues

to melatonin. It is tempting to speculate that repetitive melatonin injections t o pinealectomized hamsters cause an increase in t h e n u m b e r of receptor sites u p t o a responsive stage. G r e a t e r a m o u n t s o f melatonin, e.g., t h o s e resulting f r o m t h e sum of exogenous and e n d o g e n o u s melatonin after 3

daily injections to intact hamsters, or following the implant of a subcutaneous Silasticcapsule containh~g melatonin in control animals, cause refractoriness to the hormone by 'down regulation' of melatonin binding sites. ACKNOWLEDGEMENTS

These studies w~re supported in part by grants from the Consejo Nacional de Investigaciones Cientificas y Tdcnicas, Argentina (No. 6638), Programa Latinoamericano de InvestigaciSn en ReproducciAn H u m a n a ( P L A I ~ R H 109.206~.76) and the Duro Test Co. North Bergen, NJ. REFERENCE~

1 Bit~lan, E.L., Hamster refractoriness: the role of insensitivity of ~ineal target tissues, Solemn, 202 (1978) 648--650. 2 Carding, D.P., Vae~, M.Io and Estevez-Boyer, E., High affinity binding of melatonin in bovine ~ r d ~ | b m l hypothalamtm, IRCS Medical Sci., 6 {]978) ~57. 3 ?m~dinafi,D.P., V a t s , M.I. and Est~vez-Boyer, E., Sp~cific b~nd~ng,;f mv~tonin in bovine brain, Endocrinology, 1979, in press. 4 F~ke, V~I. and Huppert, L.C., Melatonin action on p~neai w r i ~ wl;h photoperiod, ~ , 202 (1978) 6 4 8 4 5 0 . 5 ~ , O,, ~ o u g . h , N., F~rr, A. and l~ndall, R., Protein m e ~ r ~ e n t with the Folin phenol relgent, J, biol. Chem., 193 (1951) 265--275. 6 Reiter, R~., Bl~k, D~., Johmon, L.Y., Rmieen, P.K., Vaugb~n, M.K. and Waxing, P~., Melatonin inhib~ion of reproduction in the male hanmter: its ,teper~Jcncy on time of day of adminib~mtion and on an infarct and ~ p a t h e t i c a l l y innervated pine~i gland, ~ ~ , 22 (1976) 107~116. 7 P~lt~, R.J., Rudeen, P.K. ~ l m m n , J.W., Vaughan, M.K., Johns~-~, L.Y. and Litt|~, J.C., 8 ~ m ~ o ~ nmJatonin imphnti inhibit reproductive atrop~ y in male hamltez8 induced ~y daily mektonh~ injection, ~ . Re~. Comm., ~ (1977) 35--44. 8 8e~teherd, G., The a t ~ of p ~ o ~ fo~ ~ n~lecu_l~ and i ~ , Ann, N.Y. Aead~&d., 51 (1949) 660--670. 9 Tamarkln, L., Holii~ter, C.W., L~febv~, N.G. and C-~,ldnmn, B.D., Melaton~n inductto~ of gomtdal q ~ in pinetlectomized Sy~.~ har~ter~, Science, 198 (1977) 95~-955.

10 Tm~tarki,,L., ~ ,

S~. Rod Kl~h% D.C.,Regul~tir~of pinealmeb~t~nin in ~he

11 Wtlki~m~ M , A~n~tt, Jo, ~ k ~ , # ~nd de ~ , D., D ~ \ r , a t ~ n o~ a d~kind~ ~ ~f ~ N~ety! ~ ~ a~ivlty arid~ u l t ~ n ~ r~io~ ~ y of ~ ~ m~ , ~ and pituitary ~ of the w~e rat,J. ~ . , 72 (~ 977) 243~24~,