María I. Vacas

The sympathetic superior cervical ganglia as peripheral neuroendocrine centers

The superior cervical ganglia (SCG) provide sympathetic innervation to the pineal gland, cephalic blood vessels, the choroid plexus, the eye, carotid body and the salivary and thyroid glands. Removal of the ganglia brings about several neuroendocrine changes in mammals, including the disruption of water balance in pituitary stalk-sectioned rats, and the alteration of normal photoperiodic control of reproduction in hamsters, ferrets, voles, rams and goats. These effects are commonly attributed to pineal denervation. However pinealectomy does not always mimic ganglionectomy in its neuroendocrine sequelae. This paper discusses several examples illustrating the lack of homology of ganglia and pineal removal, including the prolactin release brought about by gonadal steroids in spayed rats, the changes in drinking behaviour caused by ganglionectomy and the control of goitrogenic response to methylmercaptoimidazole in rats. All these examples indicate that SCG removal, at least as far as for neuroendocrinologists and pineal experimenters are concerned, should not be considered simply as “pineal denervation”. A functionally relevant link between SCG and the hypothalamus may occur in rats inasmuch as ganglionectomy depresses norepinephrine uptake and increases the number and responses afα-adrenoceptors in medial basal hypothalamus. Lastly the SCG are active points of concurrency for hormone signals, as revealed by the metabolic changes induced by steroid and anterior pituitary hormones in these structures even in the absence of intact preganglionic connections, as well as by the existence of putative receptors for some of the hormones, namely, estradiol, testosterone and corticosteroids. The SCG appear to constitute a peripheral neuroendocrine center.

Melatonin increases cGMP and decreases cAMP levels in rat medial basal hypothalamus in vitro

Measurement of cyclic nucleotide accumulation in rat medial basal hypothalamus (MBH) incubated in vitro with methoxyindoles indicated that melatonin (10(-8) M or greater) increased significantly cGMP and depressed cAMP levels. Only the effect on MBH cAMP was shared by 5-methoxytryptophol which exhibited a greater activity than melatonin. 6-Fluoromelatonin (10(-7) M) increased cGMP, whereas the effect of 6-hydroxymelatonin was not significant. Both 6-fluoro- and 6-hydroxymelatonin (10(-5) M) depressed MBH cAMP accumulation while only 6-fluoromelatonin affected it at 10(-7) M concentrations. These data suggest that melatonin and 5-methoxytryptophol affect differently cyclic nucleotide content of MBH at physiological concentrations.

Acute superior cervical ganglionectomy depresses the postcastration of gonadotropins in male rats

In male rats, superior cervical ganglionectomy (SCGx) delayed the rise of serum FSH and LH induced by orchidectomy by 24 h. SCGx resulted in a decrease of median eminence norepinephrine (NE) content 16 h after surgery and in an increase of medio-basal hypothalamic cAMP synthesis and receptor occupancy. These data indicate that NE release from degenerating terminals originating in the superior cervical ganglion neurons modifies the regulatory mechanisms controlling the rise of gonadotropins after orchidectomy in rats.

Neuroendocrine integrative mechanisms in mammalian pineal gland: Effects of steroid and adenohypophysial hormones on melatonin synthesis in vitro

The time course for the decrease in norepinephrine concentration of rat pineal explants in culture indicated a significant fall starting at the 4th hour and completed after 16-24 h of incubation. Significant decreases of serotonin and 5-hydroxyindoleacetic acid (HIAA) levels in tissue, an increase of HIAA/serotonin ratio, and an increase of melatonin production rate in vitro were also observed as a function of the incubation time. Estradiol (10(-7)-10(-5) M) increased rat pineal melatonin content, testosterone (10(-5) M) decreased it and progesterone was devoid of activity when incubated with explants for up to 6 h. The in vitro stimulatory effect of estradiol on rat pineal methoxyindole synthesis was blocked by propranolol but not by phentolamine; propranolol also blocked the increase of nuclear estradiol-receptor complex produced by estrogen exposure of pineal explants. TSH (1-100 ng/ml), growth hormone (10-100 ng/ml) and LH (10 ng/ml) augmented rat pineal melatonin content while 100 ng/ml of FSH decreased it significantly. Prolactin exerted a biphasic effect on rat pineal explants, the lowest concentration augmenting melatonin content while the high concentration depressed it. Deep, intermediate and superficial segments of guinea-pig pineal glands showed an increase in melatonin concentration after a 6-h incubation in the presence of 10(-7)-10(-5) M estradiol.

In vitro effect of neuropeptide Y on melatonin and norepinephrine release in rat pineal gland.

Summary1.To study neuropeptide Y (NPY) effect on melatonin production, rat pineal explants were incubated for 6 hr with 10–1000 nM NPY in the presence or absence of 10µM norepinephrine (NE). Melatonin content in the pineal gland and media was measured by radioimmunoassay (RIA).2.NPY (10–1000 nM) increased melatonin production and, at 10 or 100 nM concentrations (but not 1000 nM), enhanced NE stimulation of melatonin production.3.NPY (1000 nM) impaired3H-labeled transmitter release induced by a K+ depolarizing stimulus in rat pineals incubated with3H-NE.4.These results suggest that NPY affects both pre- and postsynaptic pineal mechanisms.

Cellular and molecular mechanisms controlling melatonin release by mammalian pineal glands

Summary1.The pineal gland is regulated primarily by photoperiodic information attaining the organ through a multisynaptic pathway initiated in the retina and the retinohypothalamic tract.2.Norepinephrine (NE) released from superior cervical ganglion (SCG) neurons that provide sympathetic innervation to the pineal acts through alpha1- and beta1-adrenoceptors to stimulate melatonin synthesis and release.3.The increase in cyclic AMP mediated by beta1-adrenergic activation is potentiated by the increase in Ca2+ flux, inositol phospholipid turnover, and prostaglandin and leukotriene synthesis produced by alpha1-adrenergic activation.4.Central pinealopetal connections may also participate in pineal control mechanisms; transmitters and modulators in these pathways include several neuropeptides, amino acids such as gamma-aminobutyric acid (GABA) and glutamate, and biogenic amines such as serotonin, acetylcholine, and dopamine.5.Secondary regulatory signals for pineal secretory activity are several hormones that act on receptors sites on pineal cells or at any stage of the neuronal pinealopetal pathway.

Binding sites for [3H]‐melatonin in human platelets

Abstract: A number of in vitro effects of melatonin on human platelets were revealed in previous studies. In order to examine whether high affinity binding sites for [3H]‐melatonin were present in membrane preparations of human platelets, a rapid filtration procedure through Whatman GFB paper was employed. Maximal melatonin binding was attained within 3 hr at 0°C. Scatchard analysis indicated a single population of binding sites with a dissociation constant (Kd) = 4.1 ± 0.5 nM and maximal number of binding sites (Bmax) = 24.2 ± 1.9 fmol/mg protein (mean ± SEM of five experiments). When various indole analogs were tested for their ability to inhibit [3H]‐melatonin binding, the following Ki (nM) were obtained: 6‐chloromelatonin (11.4), 2‐iodomelatonin (22.0), melatonin (24.7), 5‐methoxytryptophol (49.9), N‐acetylserotonin (68.9), 6‐hydroxymelatonin (78.2), 5‐methoxytryptamine (184). Serotonin was a potent inhibitor of [3H]‐melatonin binding with a Ki = 20.6 nM. Except for 2‐methylserotonin and α‐methylserotonin, a number of serotonin agonists and antagonists tested did not affect melatonin binding to platelet membranes. Binding experiments carried out at either 0800 or 2000 did not reveal time‐dependent differences in Kd or Bmax. The results suggest that high affinity melatonin acceptors are present in human platelets.

Binding Sites for Melatonin in Bovine Pineal Gland

High-affinity binding of melatonin to crude membrane preparations of bovine pineal gland was examined by a rapid filtration procedure through Whatman GFB paper. Maximal melatonin binding was attained in 60 min at 37 degrees C, in 2 h at 25 degrees C and in 5 h at 0 degree C; at 25 and 37 degrees C it was 36 and 42% of that at 0 degree C. Specific binding was thermolabile and decreased following incubation with trypsin; it was also pH dependent, the maximum being observed at physiological pH. Melatonin binding was inhibited by the addition of monovalent or divalent ions to the incubation buffer. Subcellular fractionation studies indicated that 39 and 50% of binding was located in the pellets at 900 and 27,000 g whereas 11% was detected in the microsomal pellet. Scatchard analysis revealed a single population of binding sites with Kd = (7.0 +/- 1.5) 10(-7) M (mean +/- SEM, n = 4); binding site concentration ranged from 185 to 356 fmol/mg of protein. When various melatonin analogues were tested for their ability to inhibit (3H)-melatonin binding, the following Ki values (10(-7) M), were obtained: N-acetyl-serotonin 120, serotonin 130, 2-methyl indole 154, 5-hydroxytryptophol 218, 5-methoxytryptamine 266, 5-methoxytryptophol 660, tryptamine 1,740, 5-hydroxyindole acetic acid 3,455, 5-methoxyindole acetic acid 12,690, 5-hydroxytryptophan 13,600, and 6-hydroxymelatonin 55,550. Those results suggest that melatonin receptors may be present in the pineal gland.

Characterization of a cytosol progesterone receptor in bovine pineal gland

The possible existence of pineal progesterone (P) receptors was assessed in 105,000 g supernatants of bovine pineal glands incubated with 3H-P in the presence of 10 microM cortisol. By charcoal adsorption analysis specific binding (defined as the difference between 3H-P binding in the absence and presence of 1,000-fold excess of unlabeled P) attained its maximum after 60 min at 37 degrees C. Binding was abolished by heating the cytosol or by preincubating it with trypsin. A single population of binding sites with a Kd = 1.64 nM and binding site concentration = 34 fmol/mg of cytosol protein was detectable by Scatchard analysis. In competition experiments the B50 progesterone values (nM) were: P (2), 20 alpha-dihydroprogesterone (12), 20 beta-dihydroprogesterone (25), estradiol (158), testosterone (675) and corticosterone (greater than 1,000). After sucrose gradient centrifugation of pineal cytosol incubated with 3H-P a discrete peak of radioactivity in the 7-8 S region of the gradient was observed, and disappeared following incubation with excess unlabeled P. These results indicate that P receptors are present in the bovine pineal gland.

Origins of the sympathetic projections to rat thyroid and parathyroid glands

The present study aimed to characterize the localization and pathways of sympathetic neurons innervating the thyroid and parathyroid glands. In rats subjected to unilateral superior cervical ganglionectomy or unilateral decentralization of the superior cervical ganglion 7 days earlier, ipsilateral depression of thyroid norepinephrine (NE) and epinephrine content to 6-16% of the contralateral intact lobe was observed. In both groups of animals neuronal [3H]NE uptake by the ipsilateral thyroid lobe was suppressed. In unilaterally decentralized rats pineal catecholamine levels remained within normal values whereas in unilaterally ganglionectomized rats a 74% decrease of pineal NE and E content was found. Unilateral section of the external carotid nerve abolished, as did unilateral superior cervical ganglionectomy, neuronal [3H]NE uptake in the ipsilateral thyroid lobe. In contrast external carotid nerve section did not modify the neuronal uptake of [3H]NE in the pineal gland. Either unilateral superior cervical ganglionectomy or external carotid nerve section resulted in similar involution of ipsilateral thyroid lobes of hypophysectomized rats. These results indicate that postganglionic sympathetic perikarya innervating the thyroid-parathyroid territory are located in the middle and/or inferior cervical ganglia and send their axons through the SCG and the external carotid nerve to these glands.