Melatonin counteracts pinealectomy-dependent decreases in rat brain [3H]flunitrazepam binding through an opioid mechanism

Neuroscience Letters. 164 (1993) 149 153 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.01)

149

NSL 10056

Melatonin counteracts pinealectomy-dependent decreases in rat brain [3H]flunitrazepam binding through an opioid mechanism Maria Dolores Gomar, Begofia Fern'andez, Jos6 Luis Castillo, Carmen Maria del Aguila, Dario Acufia-Castroviejo* Deparlamento de Fisiologia. E du Medicina, Univer.w(hut de GramuhA E-18012 Granada, Spain (Received 19 April 1903: Revised version received 24 Scptember 1993: Accepted 24 September 1993) K O words': Melatonin; fl-Endorphim Opioid receptor: Benzodiazepine binding: Naloxone; Pineal gland: Superior cervical ganglion The effect of intracerebroventricular (i.e.v.) injection of melatonin and/or fl-endorphin on the ['H]flunitrazepam binding sites in the cerebral cortex of pinealectomized or superior cervical ganglionectomized rats ~ as studied, Pinealectomy decreased the maximum concentration of benzodiazepine receptors (B.,~,) without affecting the dissociation constant (Kt0, while melatonin, ineffective in control animals, counteracted the effect of pinealectomy. lntracerebroventricular injection of fl-endorphin increases B..... in bolh control and pinealectomized animals, the effect being significantly higher in the latter. Simultaneous i.c,v, injection of melatonin */%endorphin did not further increase B..... in any group, whereas i.c.v, injection of naloxone signilicantly blocked the effects of melatonin and/or fl-endorphin administration. Pineal sympathetic denervation produced a significant increase in B,,,, and K~> whereas i.c.v, injection of melatonin further increased the former, restoring K D to control values. Neither i.c.v, administration offl-endorphin or melatonin + fl-endorphin significantly moditied the ganglionectomy-dependent increase in B....... although both treatments restored K D to control values. Naloxone administration had no effect on fl-endorphin- and melat(min + fl-endorphin-treated ganglioneclomized groups, but counteracted the increased efl\'ct of melatonin on Bin,,, in gangli,mectomized animals.

Opioid actions include influences on hypothalamic neurotransmitters, neurohypophyseal secretions, and direct roles as neurotransmitters [9]. Conversely, opioid functions are themselves influenced by a variety of endocrine and neural systems [9]. The pineal gland and its hormonal products, including melatonin (N-acetyl-5methoxytryptamine, aMT), is one of the systems which has significant effects on opioid functions [20]. Pinealectomy abolishes the day night rhythm of morphine analgesia [15]. and disrupts the daily rhythm of the [MetS]enkephalin [18] and fl-endorphin [14] content in the anterior hypothalamus-preoptic areas. Treatment of mice with aM'I" results in a dose-dependent analgesic effect that can be reversed by naloxone [17]. Naloxone also attenuates the nocturnal elevation of aMT. and blocks plasma aMT increased after morphine administration [12]. The pineal in turn, influences a wide ranue of hormonal and neuroendocrine functions [8], which have led to the proposal that the pineal is involved in the "line tuning' and integration of neural and endocrine activities through its hormone aMT ]23]. One of these activities may be the regulation of GABAA benz,~diazcpi,w

(GABA-BNZ) receptor complex in the central nervous system (CNS). Pinealectomy, a surgical procedure that causes proconvulsant activity in several animal species, disrupts the circadian rhythm of GABA and BNZ receptors in rat brain, whereas aMT administration has been shown to restore GABA and BNZ receptor levels [3, 4]. These effects of aMT on brain BNZ receptors may account for its anticonvulsant properties [5, 6] since the BNZ receptor antagonist flumazenil counteracts aMT anticonvulsant activity [15]. Moreover, opioid receptors have also been implicated in seizure activity [26], and aMT-induced behavioral changes such as analgesia, anxiolysis, locomotor activity depression and increased latency for convulsions [15, 27] were blunted by administration of naloxone or flumazenil [15]. Collectively, these results are compatible with the view that aMT, the opioid system and benzodiazepines are closely related drugs sharing a common activity in the brain. Since several effects of aMT appear to be produced through an opioid mechanism, the major objective of the present work was to assess whether the opioid peptide fl-endorphin resembles the effects of aMT on brain BNZ binding. and the involvement of the pineal gland in mediating

*Corresponding author. Fax: (31) (5R) ? Jr354[).

Ill!?~" cl,ai!ges.

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Male Wistar rats (200-230 g) were housed under a 12:12 h light-dark cycle, with lights on at 07.00 h, and with free access to food and tap water. Surgery was performed under equithesin anesthesia (0.5-0.7 ml/rat). Pinealectomy (Px) was performed as previously described [4], and sham-pinealectomy (sham-Px), which was under the same surgical procedure as Px except that the pineal gland was left intact, was used as control. Superior cervical ganglionectomy (Gx) was performed through an incision in the neck, the ganglia being exposed and removed carefully without damage to the adjacent tissue. Sham-ganglionectomy (sham-Gx), made similarly to Gx except that the ganglia were not removed, served as control. At the end of surgery, each rat was stereotaxically implanted with a single stainless steel guide tube (22 gauge) aimed at the lateral cerebral ventricle for subsequent i.c.v, injection of drugs [16]. Seven days after surgery the rats were i.c.v, injected at 09.00 h with saline, aMT, fl-endorphin, aMT +fl-endorphin and/or naloxone (10 ng of each compound, dissolved in 5/tl saline; Sigma Chemical, St. Louis, MO, USA). One hour later, the rats were i.c.v, injected with 2 pl of black ink and sacrificed by cervical dislocation. The brains were removed and the success of the cannula placement could be verified by the presence of ink in the ventricular system. Binding experiments were carried out on crude P2 membrane fractions prepared from cerebral cortexes of individual brains, as previously described [1]. The maximum number of binding sites (B~,x) and dissociation constant (Ko) were calculated by Scatchard analysis using a Ligand-PC program kindly provided by P.J. Munson (Laboratory of Theoretical and Physical Biol-

ogy, NIH, Bethesda, MD). The protein concentration was measured by the Folin phenol reagent method. All results are expressed as the mean _+S.E.M. Statistical analysis included two-way ANOVA followed by Bont~rroni's test. The effects of i.c.v, administration of aMT and fl-endorphin on sham-Px and Px rats are shown in Table I. Pinealectomy significantly decreases BNZ binding sites compared to sham-operated animals, whereas i.c.v. aMT, ineffective in sham-Px animals, counteracts the effect of pinealectomy. The i.c.v, administration of fl-endorphin increases BNZ binding in both sham-Px and Px rats, the effect being significantly higher in the latter. Simultaneous i.c.v, administration of aMT +fl-endorphin did not further increase Bin,~ in any group compared with the injection of each hormone independently. In all groups, the aMT-and/or fl-endorphin-dependent increase in B~x was counteracted by the i.c.v, administration of 10 ng naloxone. In Px animals naloxone blocked all the effects of aMT on Bm,x, whereas the same dose of naloxone only partially blunted the effects of flendorphin or aMT + fl-endorphin. No changes in affinity were found in any experiment. To assess the participation of sympathetic pineal innervation on changes in brain [3H]FNZ binding, a similar set of experiments were made in sham-Gx and Gx rats (Table 1I). Ganglionectomy significantly increased [3H]FNZ binding sites and the dissociation constant. The i.c.v, injection of 10 ng aMT further increased B~ax in Gx rats, but was ineffective in sham-Gx animals. The injection offl-endorphin and of aMT + fl-endorphin caused a significant increase in Bma~in sham-Gx rats, but was ineffective in Gx rats. Moreover, the i.c.v, injection of 10 ng

TABLE 1 E F F E C T O F I N T R A C E R E B R O V E N T R I C U L A R A D M I N I S T R A T I O N O F VEHICLE OR N A L O X O N E ON [3H]FLUNITRAZEPAM BINDI N G TO C E R E B R A L CORTEX IN S H A M - O P E R A T E D A N D P I N E A L E C T O M I Z E D RATS. Values represent the mean + S.E.M. of the maximum concentration ( B ~ 0 and affinity (KD) of benzodiazepine receptors. Statistical analysis was carried out using a two-way ANOVA followed by a Bonferroni's test. Treated groups

Sham-Px + saline Sham-Px + melatonin Sham-Px + fl-endorphin Sham-Px + melatonin + fl-endorphin Px + saline Px + melatonin Px + fl-endorphin Px + melatonin + fl-endorphin

Vehicle (5/.tl saline)

n

B~ax

6 5 5 5 6 8 8 8

610 630 960 1024 450 1343 1388 1470

+ 49 _+ 41 t _+ 57 ** _+ 89"* _+ 56* _+ 87 *'*'~' +_ 76 *'*'@ _+ 93 *'*(~

Naloxone (10 ng/5/ll saline)

KD

Bm.~

X~,

1.3 1.1 2.1 1.8 1.1 2.4 2.3 2.6

590 584 627 613 435 384 670 683

1.0 ~ 0.3 1.6 _+ 0.3 1.2 _+ 0.2 1.5 + 0.4 1.2 _+ 0.2 1.3 + 0.2 1.8 + 0.4 1.6 _+ 0.3

+ 0.3 + 0.2 + 0.5 _+ 0.4 _+ 0.2 + 0.7 + 0.6 + 0.5

*P < 0.001 vs. Sham-Px + saline; *P < 0.001 vs. Px + saline; (*P < 0.001 vs. their respective Sham-Px; *P < 0.01 vs. Sham-Px + saline; ~P < 0.001 vs. Px + saline; #P < 0.001 vs. vehicle.

+ 47 + 44 ~ _+ 52 ~'~ _+ 57 ~'# _+ 58 ~ + 43 *''~ + 66 :'~'# + 61:*"

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TABLE I1 EFFECTS OF I N T R A C E R E B R O V E N T R I C U L A R A D M I N I S T R A T I O N OF N A L O X O N E OR VEHICLE ON [~H]FLUNITRAZEPAM B I N D I N G TO C E R E B R A L CORTEX IN SHAM-OPERATED A N D G A N G L I O N E C T O M I Z E D RATS. Values represent the mean + S.E.M. of the maximum concentration (Bm~0 and affinity (KD) of benzodiazepine receptors. Statistical analysis was carried out as in Table I. Treated groups

Vehicle (5/21 saline)

Naloxone (10 ng/5/~1 saline)

n

B ......

K[)

B ....

K[)

Sham-Gx + saline Sham-Gx + melatonin

6 4

Sham-Gx + fl-endorphin Sham-Gx + melatonin + fl-endorphin Gx + saline Gx + melatonin

4 4 6 8

Gx + fl-endorphin Gcx + melatonin + fl-endorphin

8 8

601+_ 64 614+ 47' 940 ___ 68*'* 985 +_ 77** 1169 + _ 78** 1520 +- 117" 1186 +- 102"* 1188+- 86**

1.3+_0.4 + 1.1 +_0.3' 1.6 + 0.5 + 1.4 +_ 0.5 ~ 4.5+-0,6 2.5 _+ 0.4* 2.6 +- 0.5* 2.8+-0.4*

608_+ 66 595+_ 54 645 + 62; 623 _+ 60; 1130+- 85* 990 +_ 74*: 1070 +- 110" 1010_+ 79*

1.2_+0.2 + 1.0+0.3' 1.3 + 0.3 ~ 1.5 -+ 0.24 4.8_+0.5 , 1.6 +_ 0.4 * 2.t) +- 0.C 2.2+_0.3 ~

*P < 0,001 vs. Sham-Gx + saline; ' P < 0.001 vs. Gx + melatonin; :P < 0,001 vs. vehicle.

naloxone to sham-Gx rats counteracted the effects offlendorphin, but did not modify the effects of Gx per se. Naloxone was also ineffective in fl-endorphin and in aMT + fl-endorphin Gx-treated groups. A increased KD found in Gx rats was blunted after aMT or fl-endorphin treatment. No changes in KD were found in the other groups. The results show that i.c.v, injection of aMT counteracts the Px-decrease in Bma x of [3H]FNZ binding to rat cerebral cortex, without changes in KD. The i.c.v, injection offl-endorphin also increases BNZ binding in shamPx rats, although the effect was significantly higher in the Px group. These results suggest that the Px-dependent lack of aMT increases the responsivenes of the BNZ receptor to aMT andfl-endorphin. The non additive effects of simultaneous aMT+fl-endorphin injection indicate that they act on the same receptor to by a competitive mechanism. Naloxone blocks the effects of aMT, fl-endorphin and a combination of both compounds, demonstrating an opioid effect of aMT. The fact that naloxone was more effective in counteracting the effects of aMT than fl-endorphin may be due to a lower affinity of aMT to the opioid receptor, and the dose of naloxone used was enough to displace aMT but not fl-endorphin [161. The effects of Gx on brain [3H]FNZ binding further demonstrate that Gx does not always simulate pinealectomy. A time-dependent difference between Px and Gx on BNZ binding may exist, and a previous study indicates no changes in 'peripheral-type' BNZ binding sites after 3 weeks Gx in the rat cerebral cortex [28]. The responsiveness of 'peripheral' and 'central' BNZ receptor subtypes to Gx may be not similar, and a significant increase in [3H]GABA~ binding affinity after 15 days (ix

has been reported [4], suggesting a different regulation of the GABAA-BNZ receptor complex and the 'peripheral' BNZ receptor. The lack of effect of naloxone on Gxincreased [3H]FNZ binding suggests that other systems participate in the modulation of BNZ receptors. Pretreatment with BNZ prior to stress, which increases brain norepinephrine (NE) levels, impedes the opioid stress-induced analgesia in rats, whereas administration of the BNZ receptor antagonist flumazenil, blocked these changes [11]. Acute stress resulted in an increase whereas 7 days chronic stress resulted in a significant decrease in the BNZ binding in the cerebral cortex [11, 25]. These data, and the fact that Gx reduces the NE activity [11], point to a relationship between BNZ binding and NE content in the brain, and suggest that the degeneration of sympathetic fibers which project from the SCG to the cerebral cortex [13] may be responsible, at least in part, for the changes in this brain area of B..... after Gx in rats. This degenerative-induced increase in BNZ binding may explain the lack of further effect of fl-endorphin on BNZ binding in Gx rats at the dose used. The aMT effect in Gx rats could be due to changes in receptor sensitivity due to the abnormal brain aMT levels after Gx. Superior cervical ganglionectomy may be responsible for the concomitant decrease in hypothalamic CRH and arginine vasopressin [24], both hormones which tonically enhance hypothalamic and brain fl-endorphin content [22]. Consequently, fl-endorphin decreases after Gx and it cannot be responsible for the increase in BNZ binding found after Gx; this is in agreement with the lack of effect of naloxone on this group. Pinealectomy increases ACTH and corticosterone [2], whereas the effect of glucocorticoid administration to counteract the adrenalec-

152

tomy (ADx)-dependent increases in brain BNZ binding is contradictory [1, 10]. Since the maximum effect of ADx on rat cerebral cortex BNZ binding was found 7 days after ADx [1], decreasing 15 days later, the absence of ADx effects on cerebral cortex BNZ binding previously reported [10] in 3 week ADx rats seems to confirm a time-dependent effect of ADx. Therefore, the Gx-dependent changes in CRH and in turn ACTH and/or steroid levels may be responsible, at least partially, for changes in brain BNZ binding. In this respect, the "peripheral-type' BNZ receptor, currently called "mytochondrial benzodiazepine receptor' (M BR), regulates the entrance of cholesterol to the mytochondria, which is the first step in the synthesis of steroids [21]. It is possible that changes in ACTH (which also regulate the entrance of cholesterol to mytochondria) induced by Gx (and perhaps Px) may interact with MBR. thus modifying neurosteroid synthesis with in turn may alter the GABAABNZ receptor complex [21 ]. To summarize, two interconnected systems are described to modulate brain BNZ receptors, namely, the pineal-endogenous opioid and the superior cervical ganglia systems. The former requires a link between aMT and opioids, a coupling previously reported [10, 16, 20] and further supported by our data. This pineal-opioid connection may constitute a main brain homeostatic mechanism involved in the regulation of biological functions, including the organism's response to stressful events, with the brain BNZ (and GABA) as effectors. Sympathetic innervation provided by SCG may in turn control brain BNZ binding. A common final pathway of both systems may involve changes in MBR activity and neurosteroid synthesis. This work was partially supported by the Junta de Andalucia. We thank Ms. C. Coope for revising the English style of the manuscript. 1 Acufia Castroviejo, D., Fermindez, B., Gomar, M.D., Del Aguila, C.M. and Castillo, J.L., Influence of the pituitary-adrenal axis on benzodiazepine receptor binding to rat cerebral cortex, Neuroendocrinology, 51 (1990) 97-103. 2 Acufia Castroviejo, D., Garcia del Rio, C., Garcia-Torres, L., Luna, J. and Osorio, C., Role of pineal gland in kidney-adrenal homeostasis, Horm. Metab. Res., 16 (1984) 589-592. 3 Acufia Castroviejo, D., Lowenstein, RR., Rosenstein, R. and Cardinali, D.E, Diurnal variations on benzodiazepine binding in rat cerebral cortex: disruption by pinealectomy, J. Pineal Res., 3 (1986) 101-106. 4 Acufia Castroviejo, D., Rosenstein, R., Romero, H.E. and Cardinail, D.P., Changes in gamma-aminobutyric acid high affinity binding to rat cerebral cortex membranes after pinealectomy and melatonin administration to rats, Neuroendocrinology, 43 (1986) 24-31. 5 Albertsom T.E., Peterson, S.L., Stark, L.G., Lakin, M.L. and Winters, W.D,, The anticonvulsant properties of melatonin on kindled seizures in rats, Neuropharmacology, 20 (1981 ) 61-66.

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