pars tuberalis of the pituitary, which adheres closely to the ventral edge of the hypothalamic median eminence ( Ivanov et al. 2002 , Graham et al. 2003 ). Specific central receptors for NMU (NMU-R2) are highly localised to the hypothalamic
Ruben Nogueiras, Sulay Tovar, Sharon E Mitchell, Perry Barrett, D Vernon Rayner, Carlos Dieguez and Lynda M Williams
Shona H Wood
prime site of action is the pituitary pars tuberalis. LP activation of TSHβ leads to an increase in deiodinase 2 ( DIO2 ) activity in adjacent ependymal cells (tanycytes), which express the TSH receptor. DOI2 converts inactive T 4 to active T 3
Sayaka Aizawa, Takafumi Sakai and Ichiro Sakata
Introduction The pars tuberalis (PT), which comprises the rostral part of the anterior lobe of the pituitary gland that surrounds the median eminence as a thin cell layer, has characteristics different from those of the pars distalis (PD). The
L. M. Williams and P. J. Morgan
Melatonin-binding sites have previously been identified in the suprachiasmatic nucleus (SCN) and median eminence (ME) of the rat. We have further investigated the localization of melatonin-binding sites in the rat hypothalamus and pituitary using the ligand [125I] iodomelatonin and in-vitro autoradiography. The presence of specific melatonin-binding sites in the SCN is confirmed; however the second area of melatonin binding is identified as the pars tuberalis of the pituitary and not the ME as previously described. No other areas which bound melatonin were found in either the pituitary or the hypothalamus.
E. CAMERON and C. L. FOSTER
The cytological appearance of the pars tuberalis both at the light- and the electron-microscopical levels is described. Two main types of cell, one, which is designated the tuberalis cell, having a rounded contour, pale cytoplasm with a few small secretory granules of about 100 nm in diameter, many polyribosomes and a relatively large ovoid nucleus; and the other, the interstitial cell, with long processes often encircling tuberalis cells, an irregular nucleus and cytoplasm with abundant microfilaments about 10 nm in diameter were seen. Beaded nerve fibres and neurosecretory material were demonstrated in the pars tuberalis with chrome alum—haematoxylin, a finding confirmed at the electron-microscopical level where nerve fibres which appeared to be making contact with tuberalis cells and containing numerous microvesicles of about 50 nm in diameter were observed.
M Guerra and EM Rodriguez
The cell types of the pars tuberalis (PT) are the follicular cells, the pars distalis cells and the so-called PT-specific cells. The latter are distinct endocrine cells displaying melatonin receptors. Although the nature of the secretory product(s) of the PT-specific cells has not yet been clarified, the function of these cells has started to be unfolded. For practical reasons, previous authors have designated the, as yet, unidentified PT hormone(s) as tuberalin(s). PT-specific cells synthesise the common alpha subunit of the pars distalis glycoprotein hormones, and it has been suggested that tuberalin would correspond to the beta chain of a specific glycoprotein secreted by these cells. The aims of the present investigation were to identify the compounds secreted by the specific cells of bovine PT, and to establish their cellular and subcellular distribution. For this purpose, proteins secreted into the culture medium of PT explants were separated by electrophoresis and used to raise antibodies. Two of these proteins, with an apparent molecular mass of 21 and 72, generated antibodies (Ab-21 and Ab-72) that differentially immunoreacted with PT-specific cells. These two antibodies were used for immunoblotting of conditioned medium and of PT explants, and for light and electron microscopy immunocytochemistry. In immunoblots, Ab-21 reacted with compounds of 21, 22, 47 and 52 kDa, whereas Ab-72 revealed a compound of 72 kDa only. Ab-72 immunoreactive material corresponded to a protein, here designated as tuberalin I, secreted by a small population of PT-specific cells (type 2 cells), and stored in 140 nm secretory granules. Immunoreactive tuberalin I was missing from bovine pars distalis and from rat PT. The predominant population of PT-specific cells (type 3 cells) secreted and stored, within 280 nm secretory granules, an Ab-21 immunoreactive protein, here designated as tuberalin II. All cells of rat PT immunoreacted with Ab-21. In the cells of bovine and rat PT, immunoreactive tuberalin II was mostly confined to a paranuclear spot; this spot also bound wheat germ agglutinin and reacted with an antibody against the alpha chain of glycoprotein pars distalis hormones. It is suggested that tuberalin II would correspond to the beta chain of a specific glycoprotein secreted by type 3 PT-specific cells. In bovine PT, the cells displaying immunoreactive tuberalins I and II did not react with any of the antibodies against pars distalis hormones.
S McNulty, I L Schurov, P J Morgan and M H Hastings
Treatment of ovine pars tuberalis (oPT) cultures with forskolin activates adenylyl cyclase, leading to increased levels of cyclic AMP, activation of protein kinase A, phosphorylation of the calcium/cyclic AMP response-element binding protein and the increased synthesis and secretion of several proteins. Simultaneous treatment with melatonin inhibits or reverses these effects of forskolin. In the neonatal rat pituitary, the inhibitory effects of melatonin are mediated by changes in membrane potential.
This study therefore investigated whether the inhibitory action of melatonin in oPT cultures is also dependent on the modulation of plasma membrane potential. Treatment of cultures with the ionophore valinomycin selectively permeabilised the cell plasma membrane to potassium, thereby causing membrane hyperpolarisation. In cultures of oPT, valinomycin inhibited in a concentration-dependent manner (maximal effect 2 μm) the stimulatory action of forskolin (1 μm) on intracellular levels of cyclic AMP, indicating that the activity of adenylyl cyclase in this tissue is sensitive to hyperpolarisation of the plasma membrane. However, increasing the extracellular concentration of potassium from 5 mm to 100 mm, which would depolarise the plasma membrane, had no effect on the inhibitory action of melatonin (1 μm) in forskolin-stimulated cultures. This indicated that melatonin could be effective in cells with sustained depolarisation. To test directly whether integrity of the plasma membrane is essential for melatonin to inhibit adenylyl cyclase, cultures were treated with the cholesterol-chelating agent saponin (50 μg/ml). Saponin increased cellular permeability to trypan blue and enhanced the release of the cytoplasmic enzyme lactate dehydrogenase to the extracellular medium, demonstrating that cell plasma membranes had been permeabilised, thereby abolishing membrane polarity. In cultures pretreated with saponin there was a tendency for levels of cyclic AMP to be reduced. However, permeabilisation did not block the forskolin-stimulated increases in cyclic AMP levels nor did it alter the ability of melatonin to inhibit the production of cyclic AMP in forskolin-stimulated cultures.
This study demonstrated that, while it is possible to inhibit the stimulatory actions of forskolin in the oPT by increasing the permeability of cells to potassium and thereby hyperpolarising them, melatonin is able to inhibit cyclic AMP in permeabilised cells and so can act independently of changes in membrane potential.
Journal of Endocrinology (1995) 145, 471–478
D G Hazlerigg, M H Hastings and P J Morgan
The pars tuberalis (PT) of the anterior pituitary is characterized by the presence of a high concentration of melatonin receptors, and acute exposure of cells from this tissue to melatonin inhibits the accumulation of cyclic AMP (cAMP) stimulated by forskolin. Conversely, exposure of ovine PT (oPT) cells to melatonin for periods of up to 16 h causes a progressive increase in subsequent basal and forskolin-stimulated production of cAMP. These observations are consistent with the possibility that the PT is involved in the mediation of melatonin-dependent phenomena in mammals. If the chronic effects of exposure to melatonin are indeed functionally significant, then one would anticipate that those responses of oPT cells known to be dependent upon levels of cAMP would also show an enhanced response to stimulation following prolonged exposure to the hormone. In the present study, the activation of cAMP-dependent protein kinase and the synthesis of secretory protein by oPT cells were found to be sensitized by prolonged exposure to physiological concentrations of melatonin. In the case of the synthesis of secretory protein this effect of melatonin was confined to those proteins whose synthesis has been shown to be sensitive to melatonin in acute experiments. These observations support the hypothesis that melatonin-induced sensitization modulates the putative biosynthetic and secretory function of the PT.
The present study also examined the mechanism of sensitization of oPT cells by melatonin. The development of sensitization was not affected by simultaneous exposure of oPT cells to forskolin (1 μm) during pretreatment with melatonin. This observation suggests that melatonin-induced sensitization occurs independently of the established acute effects of the hormone on cAMP levels in oPT cells. Since no effects of melatonin upon any other signalling cascade have been observed in these cells, the most plausible explanation for this finding is that sensitization is a direct consequence of prolonged activation of melatonin receptors. Such a mechanism might be linked to the partial down-regulation of melatonin receptors known to occur in oPT cells in response to prolonged exposure to the hormone. In order to test this hypothesis further, the process of recovery from the sensitizing effects of melatonin was examined. The recovery of oPT cells from the sensitizing effects of exposure to melatonin (100 pm, 16 h) took place gradually and, even after an interval of 16 h, cells that had previously been exposed to melatonin for 16 h remained sensitized to approximately 20% of the extent seen immediately following pretreatment with melatonin for 16 h. In contrast to the previously reported insensitivity of the development of sensitization to the protein synthesis inhibitor, cycloheximide, the recovery of oPT cells from melatonin-induced sensitization was completely blocked by cycloheximide (10 μg/ml). Taken together, these observations are consistent with the hypothesis that melatonin-induced sensitization of oPT cells is the result of a reduction in levels of certain as yet unidentified protein(s), involved in the tonic inhibition of adenylate cyclase activity, occurring in parallel with the down-regulation of melatonin receptors, and that, conversely, the resynthesis of these factor(s) is a prerequisite for the return of oPT cells to the desensitized condition.
Journal of Endocrinology (1994) 142, 127–138
G A Lincoln
Previous studies involving the placement of microimplants of melatonin in the brain in sheep exposed to long days have provided evidence that melatonin acts within or close to the mediobasal hypothalamus (MBH) to mediate the effects of daylength on cycles in reproduction, moulting and other seasonal characteristics. To extend these observations, groups of Soay rams have now been treated with micro-implants of melatonin placed in the pars tuberalis (PT) and pars distalis (PD) of the pituitary gland, and in the lateral septum of the forebrain (septum). The PT and septum are potential target sites for the action of melatonin based on the localized binding of iodomelatonin assessed by in situ autoradiography. The animals were initially exposed to alternating 16-week periods of long days (16 h light:8 h darkness; 16L:8D) and short days (8L:16D) to entrain the seasonal cycles. The treatments were started at 10 weeks into a period of long days when the animals had a physiology normally observed in summer (low blood plasma concentrations of FSH and high concentrations of prolactin),and they remained under long days throughout the experiments. In experiment 1, animals received micro-implants of melatonin placed in the PT (n=6) or PD (n=4), or received empty implants in similar sites (n=4) or no surgery (n=4; total control, n=8). In experiment 2, groups of animals received microimplants of melatonin placed in the lateral septum (septum, n=7) or received corresponding control treatments (total control, n=8). The micro-implants consisted of 22 gauge stainless-steel needles with melatonin fused inside the tip. They were inserted bilaterally in the selected sites and left in place for 14 weeks. The biological effects of the treatments were assessed by measuring the changes in the blood plasma concentrations of FSH and prolactin, growth of the testes and moulting of the pelage over a period of 28 weeks (14 weeks treatment and 14 weeks post-treatment).
The administration of melatonin in the PT, but not in the PD or septum, affected the photoperiodically induced cycle in the secretion of FSH and prolactin. In the PT group there was no significant change in the plasma concentrations of FSH during the treatment with melatonin, but there was a significant (P<0·001), ANOVA) decrease in the levels of FSH after the treatment associated with premature regression of the testes. The plasma concentrations of prolactin were significantly (P<0·001, ANOVA) decreased during the treatment with melatonin in the PT group and increased after the treatment with associated changes in the growth and moulting of the pelage in the most responsive animals. The effects of melatonin in the PT were qualitatively similar but less consistent than those previously observed following placement of micro-implants in the MBH (data included for comparison). The results support the conclusion that melatonin acts, at least in part, in the PT to mediate the inductive effects of photoperiod on the timing of seasonal cycles of reproduction and moulting in rams.
Journal of Endocrinology (1994) 142, 267–276
Shona Wood and Andrew Loudon
). Indeed, in some species such as ferrets and seasonal wallabies, melatonin receptors cannot be detected in the brain ( Paterson et al . 1992 , Weaver et al . 1996 , Hinds & Loudon 1997 ). Unexpectedly, the pars tuberalis (PT) of the pituitary gland is