Participation of ACTH1-10 and ACTH4-10 on the melatonin modulation of benzodiazepine receptors in rat cerebral cortex.

In this study, we examined the effect of intracerebroventricular (i.c.v) injection of melatonin and/or ACTH1-10 and ACTH4-10 on [3H]flunitrazepam binding sites in the cerebral cortex of hypophysectomized rats. Hypophysectomy increased the Bmax (maximum number of binding sites) of benzodiazepine (BNZ) receptors for at least 7 days after surgery, without changing KD (dissociation constant). The i.c.v. injection of melatonin to hypophysectomized rats significantly increased Bmax, whereas the same doses of melatonin were ineffective in sham-operated animals. In both cases, KD values were unchanged. The i.c.v. injection of ACTH1-10 to hypophysectomized animals significantly increased Bmax, an effect that was enhanced by simultaneous i.c.v. injection of ACTH1-10 + melatonin, reaching higher values of Bmax than the i.c.v. injection of these hormones individually. No significant changes in KD values were found after ACTH1-10 and/or melatonin administration. However, the i.c.v. injection of ACTH4-10 to hypophysectomized rats did not change Bmax, although it significantly increased KD values, indicating a decrease in the BNZ binding affinity. Melatonin injection counteracted this effect of ACTH4-10, returning KD to the control value. Moreover, although the lower dose of i.c.v. melatonin used, 10 ng, was unable to modify Bmax of BNZ binding in the ACTH4-10-injected group, the higher dose, 20 ng, significantly increased Bmax. The results suggest that these ACTH-derived peptides can modulate the effect of melatonin on brain benzodiazepine receptors.

Influence of the behaviorally active peptides ACTH1-10 and ACTH4-10 on the melatonin modulation of 3H-flunitrazepam receptor binding in the rat cerebral cortex.

Possible interactions of ACTH-like peptides with melatonin regulation of central-type benzodiazepine (BNZ) receptors have been studied by means of high-affinity 3H-flunitrazepam binding to rat cerebral cortex membrane preparations. Intracerebroventricular injections of melatonin produce a dose-dependent increase in Bmax in pinealectomized rats, without changes in KD. Analogous effects were obtained after intracerebroventricular injection of melatonin in adrenalectomized and in adrenalectomized plus pinealectomized rats, which indicated the lack of participation of adrenal steroids in this response. Moreover, intracerebroventricular injection of ACTH1-10 induced a similar dose-dependent increased Bmax in sham-operated animals, whereas pinealectomy, but not adrenalectomy, partially counteracted this effect of ACTH1-10 administration. Besides, simultaneous injection of ACTh1-10 plus melatonin reverses the effects of pinealectomy, resulting in an additive effect of both compounds on Bmax. The response obtained when using ACTh4-10 was somewhat different, because no dose response was obtained in any experiment. Although lack of endogenous melatonin partially reduced the increasing effect of ACTH4-10 on Bmax, there were no additive effects at the different doses used. The results strongly suggest that ACTH-like peptides, in addition to melatonin, play a role in regulating central-type rat BNZ receptors.

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

The effect of intracerebroventricular (i.c.v.) injection of melatonin and/or beta-endorphin on the [3H]flunitrazepam binding sites in the cerebral cortex of pinealectomized or superior cervical ganglionectomized rats was studied. Pinealectomy decreased the maximum concentration of benzodiazepine receptors (Bmax) without affecting the dissociation constant (KD), while melatonin, ineffective in control animals, counteracted the effect of pinealectomy. Intracerebroventricular injection of beta-endorphin increases Bmax in both control and pinealectomized animals, the effect being significantly higher in the latter. Simultaneous i.c.v. injection of melatonin + beta-endorphin did not further increase Bmax in any group, whereas i.c.v. injection of naloxone significantly blocked the effects of melatonin and/or beta-endorphin administration. Pineal sympathetic denervation produced a significant increase in Bmax and KD, whereas i.c.v. injection of melatonin further increased the former, restoring KD to control values. Neither i.c.v. administration of beta-endorphin or melatonin + beta-endorphin significantly modified the ganglionectomy-dependent increase in Bmax, although both treatments restored KD to control values. Naloxone administration had no effect on beta-endorphin- and melatonin + beta-endorphin-treated ganglionectomized groups, but counteracted the increased effect of melatonin on Bmax in ganglionectomized animals.

Suppressive effect of simultaneous injection of ACTH1-10 and beta-endorphin on brain [3H]flunitrazepam binding.

Seven-day hypophysectomized rats were intracerebroventricularly (i.c.v.) injected with beta-endorphin, ACTH1-10 or beta-endorphin+ACTH1-10 (10-20 ng of each compound) and the [3H]flunitrazepam ([3H])FNZ) binding to the rat cerebral cortex of hypophysectomized rats was assayed one hour later. The i.c.v. injection of ACTH1-10 (10-20 ng) or beta-endorphin (10-20 ng) significantly increased [3H]FNZ binding to a similar extent. The effect of i.c.v. injection of ACTH1-10 on brain binding was blunted by simultaneous beta-endorphin administration at the same doses. The i.c.v. naloxone injection (10-20 ng) did not modify the effect of ACTH1-10 (10 ng) on [3H]FNZ binding, but counteracted, in a dose-related manner, the blocking effect of beta-endorphin on ACTH1-10-dependent brain [3H]FNZ binding. The results suggest the existence of an opioid-melanopeptide integration to control brain benzodiazepine receptors.

Similarity between the effects of suprachiasmatic nuclei lesions and of pinealectomy on gonadotropin release in ovariectomized, sulpiride-treated and melatonin-replaced rats.

The aim of this study was to compare the effects of pineal indole treatments on LH and FSH release in pinealectomized and suprachiasmatic lesioned and ovariectomized rats rendered hyperprolactinemic by acute sulpiride treatment. Pinealectomy or suprachiasmatic nuclei lesions in female rats both decreased plasma LH and FHS at 10, but not at 20 d after surgery, whereas the daily afternoon administration of melatonin effectively restored levels of both gonadotropins to control values. In ovariectomized rats, pinealectomy or suprachiasmatic nuclei lesions were ineffective in counteracting the high plasma levels of LH and FSH. However, sulpiride treatment in both pinealectomized and suprachiasmatic nuclei lesioned and castrated female rats significantly decreased the levels of LH and FSH, an effect which was counteracted by daily afternoon melatonin administration. Other pineal indoles tested, i.e., 5-hydroxy- and 5-methoxytryptophol, were ineffective in regulating gonadotropin levels. The results suggest that the pineal gland, through its hormone melatonin, can modulate gonadotropin secretion by acting on a dopamine mechanism independent of hypothalamic suprachiasmatic areas.

Intracerebroventricular injection of naloxone blocks melatonin-dependent brain [3H]flunitrazepam binding.

The pineal hormone melatonin modulates the brain benzodiazepine binding sites and its circadian rhythm. In the present study the effects of intracerebroventricular (i.c.v.) administration of naloxone (10-20 ng), alone or in association with melatonin and/or beta-endorphin, on [3H]flunitrazepam ([3H]FNZ) binding to the rat cerebral cortex of hypophysectomized rats was investigated. Melatonin (10-20 ng), beta-endorphin (10-20 ng), and melatonin + beta-endorphin (10-20 ng of each compound) all increased [3H]FNZ binding to a similar extent and in a dose-related manner. The effects of melatonin (10 ng) on [3H]FNZ binding were prevented by simultaneous injection with the specific opioid antagonist naloxone. Naloxone also blocks, although to a lesser extent, the effects of beta-endorphin and of melatonin + beta-endorphin injections. Moreover, naloxone blocks the hypophysectomy-dependent increase in [3H]FNZ binding. These results implicate the modulation of melatonin-dependent changes on brain benzodiazepine receptors by opioid peptides.

Characterization of ouabain high-affinity binding to rat cerebral cortex. Modulation by melatonin.

High-affinity [3H]ouabain binding to membrane preparations of rat cerebral cortex was examined using a rapid filtration procedure. At 37 degrees C, binding reached equilibrium in about 60 min. Scatchard analyses of the data at equilibrium revealed a single population of binding sites with a dissociation constant of KD = 3.1 +/- 0.36 nM and a binding site concentration of Bmax = 246.4 +/- 18.4 fmol/mg protein. Kinetic analyses of the association and dissociation curves indicated a kinetic KD = 4.6 nM, which is in good agreement with the value obtained at equilibrium. When various digitalis compounds were tested for their ability to inhibit [3H]ouabain binding, the following Ki values (nM) were obtained: ouabain (3.9); digoxin (18); acetyl-digitoxin (66); k-strophanthin (95); digitoxin (236). When melatonin was added to the incubation medium, the ability of ouabain to inhibit [3H]ouabain binding increased in a dose-related manner to yield the following Ki values (nM): melatonin 10 nM (2); melatonin 20 nM (1.2); melatonin 40 nM (0.8). These data suggest the existence in the rat cerebral cortex of high-affinity ouabain binding sites which may be a locus for the molecular action of melatonin.

Modulation by pineal gland of ouabain high-affinity binding sites in rat cerebral cortex.

To investigate the participation of the pineal gland and its hormone melatonin on Na(+)-K(+)-ATPase (the sodium pump) in rat brain, we used Scatchard plots to analyze the changes in rat cerebral cortex of [3H]ouabain high-affinity binding in groups of intact, pinealectomized (PX), and sham-PX rats. Only one type of binding site, with a dissociation constant of approximately 3 nM and site number (Bmax) of approximately 250 fmol/mg protein, was apparent with our assay conditions. PX or sham-PX rats (subjected to surgery 15 days earlier) were killed at six different time intervals during the 24-h cycle. Intact and sham-PX animals showed a similar biphasic pattern in diurnal rhythm of ouabain binding, with a minimal concentration of binding sites at 1600 h and a maximal concentration at 0400 h. Pinealectomy induced a significant increase in Bmax at all time intervals studied, with the largest rise appearing at night and coinciding with the nocturnal peak, whereas the daytime minimum was blunted. Time-dependent experiments indicated that the Bmax of ouabain high-affinity binding in PX rats attained maximal values at 7 days after surgery and decreased somewhat 7 days later, while sham-PX animals showed only a small transient increase in Bmax up to 7 days after surgery, with values returning to normal by the 15th day. Melatonin administration at a single subcutaneous dose of 25 micrograms/kg body wt given 3 h before death was enough to counteract the PX-induced increase of ouabain high-affinity binding. Melatonin was able to enhance the binding of [3H]ouabain to its receptor site, increasing binding affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Pinealectomy increases ouabain high-affinity binding sites and dissociation constant in rat cerebral cortex.

The effect of the pineal gland on the ouabain high-affinity binding sites (Kd = 3.1 +/- 0.4 nM, Bmax = 246.4 +/- 18.4 fmol/mg protein) in rat cerebral cortex was studied. Pinealectomy increased Bmax (940.7 +/- 42.8 fmol/mg protein) and Kd (7.6 +/- 1.5 nM) while melatonin injection (100 micrograms/kg b.wt.) counteracted these effects, restoring kinetic parameters (Kd = 1.9 +/- 0.05 nM; Bmax = 262.2 +/- 29.6 fmol/mg prot) to control values. Melatonin activity on ouabain binding in vitro did not depend upon a direct effect on the binding sites themselves. However, in competition experiments, melatonin increased binding affinity of ouabain as shown by the decreased IC50 values.

Influence of the pituitary-adrenal axis on benzodiazepine receptor binding to rat cerebral cortex.

The role of the pituitary-adrenal axis on receptor binding and diurnal rhythmicity of benzodiazepines (BNZ) was assessed in the rat cerebral cortex. Groups of intact, adrenalectomized (ADx) and/or hypophysectomized (HPx) rats were killed at six different time intervals during the 24-hour cycle. BNZ binding was estimated by Scatchard analysis of 3H-flunitrazepam high-affinity binding to rat cerebral cortex. Intact and sham ADx animals show a similar pattern in diurnal thythm of BNZ binding, with a maximal concentration at midnight. Bilateral ADx induced a significant increase in Bmax at all time intervals studied, the largest rise appearing at midnight. HPx alone led to a slightly smaller rise in Bmax than in ADx rats, while HPx performed in ADx rats did not modify the response to ADx alone. Bmax of BNZ binding in ADx rats reached maximal values at 3-7 days after surgery, and decreased somewhat at 15 days post-ADx. Corticosterone administration at a single dose of 5 mg i.p. 24 h before sacrifice returned Bmax to normal values in ADx as well as in ADx plus HPx rats. The corticosterone effect is not exerted on the BNZ binding sites themselves, as revealed by the lack of effect of this glucocorticoid in vitro. These findings indicate that BNZ receptors in rat cerebral cortex can be modified by the adrenal gland, with corticosterone as a primary effector.

Lesions in suprachiasmatic nuclei simulate effects of pinealectomy on prolactin release in ovariectomized and sulpiride-treated female rats.

Previous studies indicate that the pineal gland alters prolactin secretion, and it was suggested that at least part of the effect of the pineal hormone melatonin on prolactin release may be mediated by the hypothalamic structures. In this study, pinealectomy and lesions of the suprachiasmatic nuclei were found to alter serum levels of prolactin in the same direction, an effect that was counteracted by daily afternoon melatonin administration. Melatonin, but not other pineal indoles, also prevented sulpiride-induced prolactin secretion in pinealectomized or suprachiasmatic nuclei-lesioned and ovariectomized rats, which suggested that the pineal gland can modulate prolactin secretion by acting through a dopamine mechanism independent of hypothalamic suprachiasmatic structures.