Gary Davidson

Melatonin receptors on ovine pars tuberalis: characterization and autoradiographicai localization.

The functional significance of the pars tuberalis (PT) of the mammalian adenohypophysis has remained an enigma (1, 2). One view of its function is that it acts as an auxiliary gland to support the endocrine role of the pars distalis (PD) (2), as it has been shown to contain immunocytochemically identifiable thyrotrophs and gonadotrophs (1). Many of the cells of the PT are, however, ultrastructurally unique suggesting an independent function for this tissue. Our recent demonstration that the PT of the rat is a major binding site for the ligand iodomelatonin lends further support to this idea (3). We have utilized the highly specific ligand [125l]melatonin, and have demonstrated that it binds exclusively, with very high affinity, to the PT but not the PD of the adult sheep adenohypophysis. These findings support the conclusion that the PT has a distinct role in relation to melatonin action and seasonal reproduction.

Melatonin Receptor Sites in the Syrian Hamster Brain and Pituitary. Localization and Characterization Using [125|]lodomelatonin*

A high‐affinity, discretely localized melatonin receptor has been characterized and mapped within the brain and pituitary of the Syrian hamster using the high specific activity ligand [125|]iodomelatonin and a combination of in vitro autoradiography and membrane homogenate receptor assays. Specific binding of radioligand was found in regions of the epithalamus and hypothalamus in the brain and the pars tuberalis of the pituitary. Excitatory amino‐acid lesions destroyed [125|]iodomelatonin binding within the brain, demonstrating that binding sites are located on neurons. Analysis of [125|]iodomelatonin binding to membrane homogenates of the pars tuberalis revealed a linear relationship between specific ligand binding and the amount of tissue. The time‐course of specific binding at 37°C reached equilibrium after 30 min and remained stable thereafter. The addition of increasing concentrations of [125|]iodomelatonin alone and in the presence of 1 μM melatonin showed that specific binding reached equilibrium at 80 to 100 pM. Analysis of the saturation isotherm using a one‐site binding model was consistent with a single receptor site with a Kd of 29.3 (±5.9 SEM) pM and Bmax of 2.54 (±0.19 SEM) fmol/mg protein.

Guanine Nucleotides Regulate the Affinity of Melatonin Receptors on the Ovine Pars tuberalis

The effect of guanine nucleotides and related analogues on the binding of 2-[125I]-melatonin to membranes prepared from ovine pars tuberalis was studied. Dose-dependent inhibition of 2-[125I]-melatonin binding was observed, with an order of potency of GTP gamma S much greater than Gpp(NH)p greater than GTP = GDP. GMP, cyclic GMP and ATP had negligible effects. Analysis of saturable binding revealed that GTP gamma S (1 microM) promoted an apparent reduction in receptor density of about 50%, without a concomitant change in receptor affinity. These results are consistent with a melatonin receptor existing in an equilibrium between high- and low-affinity states, with GTP and related analogues able to cause a shift in the equilibrium in favour of the lower-affinity form. The sensitivity of 2-[125I]-melatonin binding to guanine nucleotides implies the presence of a melatonin receptor on the ovine pars tuberalis, the action of which is mediated via a G protein.

Both Pertussis Toxin‐Sensitive and Insensitive G‐Proteins Link Melatonin Receptor to Inhibition of Adenylate Cyclase in the Ovine Pars Tuberalis

Bordetella pertussis toxin (islet activating protein, IAP) has been used to investigate the G‐proteins involved in mediating the action of the melatonin receptor. Melatonin inhibits iorskolin‐stimulated cyclic AMP production in ovine pars tuberalis (PT) cells. In cells treated with IAP for 16 h this response is attenuated in a dose‐dependent manner, but not abolished. IAP catalyses the incorporation of [32 P‐ADP]ribose into a 41 kd protein present in PT membranes, but this labelling can be reduced if PT cells are preincubated with IAP for 16 h. Treatment of crude membrane preparations with IAP (20 /ig/ml) suppresses the binding of 2‐[125 l]iodomelatonin by 20%, whereas 1 mM GTP alone reduces binding by 40%, and in combination with IAP its effect is additive (60% inhibition). Therefore, these results indicate that the melatonin receptor acts via two G‐proteins, one pertussis toxin‐sensitive and the other pertussis toxin‐insensitive.

Melatonin Regulates the Synthesis and Secretion of Several Proteins by Pars Tuberalis Cells of the Ovine Pituitary

The pars tuberalis (PT) of the pituitary may be an important target for melatonin action, but the secretory output of the melatonin‐responsive cells is unknown. Using [35S]methionine, protein synthesis and secretion have been studied in primary cultures of ovine PT cells, and analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. Only 4% of the labelled proteins appeared in the medium with the majority retained in the cells. Stimulation of the cells with 10μM forskolin increased the accumulation of several labelled proteins in the medium without corresponding changes in the cell (72, 62, 44, 39, 29, 24, 23, 18 and 14 kd). Two‐dimensional gel electrophoresis showed the proteins to have mildly acidic isoelectric points. Melatonin (1 μM) counteracted the stimulatory effect of forskolin on all but one (23 kd) of these secreted proteins. Immunoprecipitation showed this to be prolactin. Furthermore, melatonin alone appeared to have an inhibitory effect on the synthesis and release of proteins into the medium. The synthesis and secretion of the melatonin‐responsive proteins was not inhibited by actinomycin D (1 μg/ml), indicating control at the translational level. This contrasts with the regulation of prolactin which is actinomycin D‐sensitive. Pulse‐chase experiments demonstrated that it requires 30 min for the secretory proteins to appear in the medium, consistent with intracellular processing and packaging prior to secretion. The secretory proteins labelled in the ovine PT, and responsive to melatonin, did not appear to be specific to the PT, as a similar profile of labelled secretory proteins was produced in primary cultures of pars distalis cells. However, melatonin had no effect on the synthesis and secretion of proteins by the pars distalis. These results demonstrate that in the ovine PT melatonin regulates the synthesis and export of several secretory proteins. These are possibly packaging proteins of secretory granules, similar to the granin family of proteins. Thus, the results confirm that melatonin‐responsive cells are secretory cells and further imply that the PT‐specific product is not a protein.

Ultrastructure of melatonin-responsive cells in the ovine pars tuberalis

SummaryFunctional receptors for melatonin have been localized and characterized on the pars tuberalis (PT) of a number of mammalian species, but the cell-type responsive to melatonin is unknown. The ultrastructure of the ovine pars tuberalis has been examined and these findings correlated with the functional response of the gland to melatonin. This study revealed that two secretory cell types predominate in the ovine PT, which differ in the abundance of dense-core granules. The most abundant of the cells are either agranular or very sparsely granulated and represent 90% of the total population, with the remaining 10% being composed of cells with abundant dense-core vesicles. Few follicular cells were observed. This ratio of secretory cell-types persisted in primary culture, with the two types non-separable by Percoll gradient centrifugation. Using forskolin, as a non-specific stimulant of adenylate cyclase, melatonin was shown to inhibit the formation of cyclic AMP by 80–90% in cells both before and after Percoll centrifugation. The results demonstrate that the agranular secretory cells of the ovine pars tuberalis are the melatonin responsive cell-type of this gland.

Phospholipases and melatonin signal transduction in the ovine pars tuberalis

The potential role of phospholipases in mediating melatonin-dependent inhibition of adenylyl cyclase was investigated in pars tuberalis (PT) cultures. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated the release of choline metabolites and increased the transphosphatidylation reaction. The calcium ionophore A23187 stimulated the release of arachidonic acid from cultures. These observations demonstrate phospholipase A and D activities within PT. Phosphatidic acid inhibited forskolin-stimulated production of cyclic AMP both in PT cells and in membrane preparations. This indicates that melatonin could inhibit adenylyl cyclase by increasing phosphatidic acid levels through activation of cellular phospholipases. Melatonin did not stimulate the release of arachidonic acid or choline from PT cultures, nor did it increase intracellular levels of hydrophobic second messengers or stimulate transphosphatidylation. Therefore melatonin does not stimulate phospholipase A and D pathways in PT cells. However, these pathways are present in the PT and their activation could potentially modulate the cellular actions of melatonin.

p72, a Marker Protein for Melatonin Action in Ovine Pars tuberalis Cells: Its Regulation by Protein Kinase A and Protein Kinase C and Differential Secretion Relative to Prolactin

The function of the pars tuberalis as a mediator of the action of melatonin remains elusive. As a direct method of assessing the potential role of secretory proteins, ovine pars tuberalis cells have been cultured and radiolabelled with 35S-methionine, and the accumulation of specific radioactive products in the medium, measured after separation by SDS-PAGE and fluorography. The synthesis and secretion of a number of labelled proteins are increased by forskolin (1 microM) and inhibited dose dependently by melatonin (IC50, 300 pM), although consistently a 72-kD protein (p72), is the most intensely labelled of these. Thus, 72 acts as a useful marker of cellular activity for melatonin, whereas prolactin (p23) provides a melatonin non-responsive marker in ovine pars tuberalis cell cultures. The synthesis and secretion of p72 and other melatonin-sensitive proteins is regulated through the cyclic AMP/protein kinase A second-messenger pathway, as analogues of cyclic AMP mimic the action of forskolin, yet 1,9-dideoxyforskolin, a forskolin analogue that is not active on adenylate cyclase, has no effect. However, the phorbol ester, phorbol-12,13-myristate acetate, also regulates the synthesis and secretion of the same profile of proteins as forskolin indicating a potential role for protein kinase C, which occurs through an independent rather than a synergistic pathway. The differential effects of nocadazole (1 microM) and extracellular calcium depletion upon p72 and prolactin secretion indicates that p72 is secreted by a calcium and microtubule independent pathway, in contrast to prolactin. These observations in conjunction with the absence of dense-core storage vesicles in melatonin-responsive cells of the ovine PT are consistent with constitutive secretion of p72 from the latter and regulated secretion of prolactin from melatonin non-responsive cells. Using immunoprecipitation de novo synthesis and secretion of either LH or LH-like proteins from ovine pars tuberalis cells could not be detected under the conditions used. The absence of 125I-(Des-Gly10[D-Ala6]-LHRH-ethylamide) binding over most, but not all, of the ovine pars tuberalis supports the contention that the majority of the cells of the ovine pars tuberalis are not gonadotrophs. These results provide further support for the unique function for the pars tuberalis.

Interaction of Forskolin and Melatonin on Cyclic AMP Generation in Pars Tuberalis Cells of Ovine Pituitary

The pineal indoleamine, melatonin, acts on specific secretory cells of the pars tuberalis of the sheep pituitary. Using pars tuberalis cells in primary culture melatonin inhibited forskolin‐stimulated cyclic AMP production in both a time ‐ and dose‐dependent manner, but the nature of the melatonin response was critically dependent upon the stimulatory concentration of forskolin used. Forskolin alone stimulated dose‐dependent cyclic AMP accumulation, which reached an equilibrium state after 15 min. This was maintained for up to 3 h, indicating a lack of desensitization to forskolin. Melatonin (1 μM) inhibited this response by greater than 80% at all doses. However, 100 μM forskolin reduced both the affinity and the number of the melatonin receptors, relative to untreated controls. Consistent with this, melatonin was 100 times less potent at inhibiting forskolin‐stimulated cyclic AMP production, when titrated against 100 μM forskolin as compared to 1 μM forskolin. The response to 1 μM forskolin could be potentiated by 10 μM phorbol 12,13 myristate acetate, but not by calcium ionophore (A23187). This provides evidence for the interaction of the phosphatidylinositol pathway with the cyclic AMP system in these cells. Nevertheless melatonin can inhibit both the potentiated and non‐potentiated response with equal effect.

Neuropeptide Y (NPY) Innervation of the Ovine Pineal Gland

A dense network of neuropeptide Y (NPY)‐like immunoreactive (NPY‐LI) fibres was revealed in the ovine pineal gland at the light microscope level. The dorsal and peripheral regions of the gland contained the most dense concentration of NPY‐LI fibres with relatively few fibres in the mid‐region and almost none in the pineal stalk. The effect of NPY in conjunction with isoproterenol (ISO) on cyclic AMP (cAMP) accumulation and noradrenaline (NA) on melatonin synthesis was investigated using in vitro techniques. NPY had no effect on the stimulation of cAMP or melatonin synthesis by the adrenergic agonists.