Novel naphthalenic ligands with high affinity for the melatonin receptor.
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Animals↗
Biomedical subjects
P J Morgan
Publications and source records attributed to P J Morgan.
Characterization and autoradiographic localization of the epidermal growth factor receptor in the jejunum of neonatal and weaned pigs.
Receptors for epidermal growth factor (EGF) were characterized on the intestinal membranes of newborn, sucking and weaned pigs. 125I-labelled EGF (125I-EGF) binding to membrane homogenates was time-dependent, saturable, linearly correlated to membrane protein and reversible. Analysis of saturation curve data revealed a single class of 125I-EGF binding sites in both newborn and weaned pigs. Receptor levels tended to be higher in weaned than in newborn pigs; the converse was true for the receptor affinity. In contrast, virtually no binding sites were found on the intestinal membranes of sucking pigs. Autoradiography in vitro of jejunal sections of newborn and weaned pigs demonstrated 125I-EGF receptors on both microvillar and basolateral surfaces of enterocytes, suggesting that luminal EGF could influence developmental processes in the intestine either directly or indirectly following transcytosis of the ligand.
Animals↗
Melatonin receptor mRNA expression in Xenopus oocytes: inhibition of G-protein-activated response.
Melatonin is the major endocrine product of the pineal gland in the mammalian brain and plays a variety of roles in photoperiodic functions. In order to investigate melatonin receptors, poly(A)+ RNA was extracted from pars tuberalis of the ovine pituitary and injected into oocytes of Xenopus laevis. After 3-5 days of incubation, functional melatonin receptors were expressed. Receptors were revealed by their inhibitory effect upon oscillatory currents resulting from AlF4-induced activation of G-proteins in the oocyte membrane under voltage clamp conditions. The effect of melatonin was dose-dependent, non-desensitizing and was not observed in uninjected oocytes.
Aluminum↗
Ultrastructure of melatonin-responsive cells in the ovine pars tuberalis.
Functional 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.
Animals↗
Stimulation of inositol phosphate turnover and calcium influx in ovine pars tuberalis (PT) cells: effect of melatonin.
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Aluminum↗
Autoradiographical localization of oxytocin binding sites on ovine oviduct and uterus throughout the oestrous cycle.
A highly specific oxytocin receptor ligand, 125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH9(2)] vasotocin (125I-OTA), was used to localize high affinity oxytocin receptors in ovine uterine and oviduct tissues throughout the oestrous cycle. The pattern of binding revealed by in vitro autoradiography correlated well with the results of the homogenate receptor assays using the same ligand and with previous binding assays using the tritiated ligand. At oestrus, specific 125I-OTA binding was evident on the luminal epithelium of the caruncular and intercaruncular regions, on the epithelial cells lining the secretory uterine glands and in the stroma underlying the caruncular epithelium. In the myometrium diffuse labelling was evident in the outer longitudinal smooth muscle layer. At Day 4 of the cycle, binding to the stroma was diffuse and virtually absent from the glandular epithelium. No specific binding was evident in either tissue at Day 12 of the luteal phase, but by Day 14, prior to the decrease in peripheral progesterone concentrations, binding was again apparent on the luminal epithelium only. Specific binding to the oviduct was localized to the smooth muscle layer of the isthmus region of oestrous ewes and was not detected at any other stage of the oestrous cycle. These studies extend our knowledge of the distribution of oxytocin binding sites in uterine and oviduct tissues throughout the oestrous cycle and suggest that oxytocin has an important role in stimulating oviduct and uterine motility at a time crucial to successful egg collection and/or sperm embryo transport.
Animals↗
Beta-adrenergic stimulation increases cAMP and melatonin production in ovine pinealocyte cultures.
An ovine pinealocyte suspension culture method, using glands from young sheep as a source of cells, has been developed to study mechanisms of pineal regulation in this species. Cell suspensions are obtained by enzymatic digestion (preliminary trypsinization followed by collagenase/pronase treatment) of pineal glands and these cells may be usefully retained in culture for up to 48 hr. Initial characterization of cyclic adenosine monophosphate and melatonin production responses to adrenergic stimuli (norepinephrine, phenylephrine, isoproterenol) in the absence and presence of antagonists (propranolol, prazosin) by pinealocytes in suspension culture indicate that a beta-adrenergic receptor-mediated mechanism is primarily involved in transduction of the adrenergic signal to melatonin synthesis. This is in agreement with data from earlier work on the sheep using short-term tissue incubations but contrasts with in vivo evidence suggesting a predominant alpha 1-adrenergic receptor-mediated mechanism.
Adrenergic alpha-Agonists↗
Intracellular signalling in the ovine pars tuberalis: an investigation using aluminium fluoride and melatonin.
The effect of aluminium fluoride (AlF4-) has been studied on inositol phosphate accumulation, calcium mobilization, cyclic AMP production and [2-125I]iodomelatonin binding in ovine pars tuberalis cells. These cells have high-affinity receptors for, and respond to, melatonin through inhibition of forskolin-stimulated adenylate cyclase. In the presence of 10 mM LiCl, AlF4- stimulated the net accumulation of inositol monophosphate and inositol bisphosphate. Consistent with these findings, AlF4- increased intracellular calcium; although this response was attenuated in calcium-depleted medium, indicating that the calcium response comprises both intracellular and extracellular components. Melatonin was ineffective on either basal or AlF4(-)-stimulated turnover of inositol phosphates. In concordance with the inositol phosphate response, melatonin had no effect on either the AlF4(-)-stimulated or the basal calcium levels. AlF4- blocked the increase in cyclic AMP stimulation by 1 microM forskolin, being as effective as melatonin, achieving approximately 90% inhibition. AlF4- also attenuated the binding of [2-125I]iodomelatonin to ovine pars tuberalis membranes by 15%. At the concentration used, these results are consistent with the interpretation that AlF4- activates many G protein-mediated responses, and thus imply that the inhibitory pathway for cyclic AMP predominates over the stimulatory arm, whereas there can only be a stimulatory pathway linked to phosphoinositide metabolism in ovine pars tuberalis cells.
Aluminum↗
Characterization and autoradiographical localization of oxytocin receptors within the ovine endometrium in relation to post-partum corpus luteum function.
The induction of ovulation in early post-partum ewes is associated with a high incidence of premature luteal regression which is independent of the suckling stimulus but dependent on the stage post partum. The aim of the present study was to determine whether oxytocin receptors are present on uterine endometrium early in the luteal phase and hence ascertain whether oxytocin-induced uterine prostaglandin F2 alpha release is a possible mechanism involved in the premature regression of these post-partum corpora lutea. Ovarian and uterine tissues were collected on day 4 of the the cycle in ewes induced to ovulate at either 21 or 35 days post partum (n = 4 per group). A further four cyclic ewes were similarly synchronized to ovulate and acted as controls. Corpora lutea from the 21-day post-partum group were significantly (P less than 0.01) smaller, had a lower progesterone content and a reduced capacity to secrete progesterone in vitro than corpora lutea from 35-day post-partum or control ewes. A highly specific oxytocin receptor ligand 125I-labelled d(CH2)5[Tyr(Me)2, Thr4,Tyr-NH29]-vasotocin was used to localize and characterize high affinity oxytocin receptors in uterine endometrium (dissociation constant 145 pmol/l). Oxytocin receptor concentrations in endometrium from ewes induced to ovulate at 21 days post partum were on average five-fold higher (P less than 0.05) than in 35-day post-partum and control groups.
Animals↗
Interactions of a model block copolymer drug delivery system with two serum proteins and myoglobin.
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Butadienes↗
Central melatonin receptors: implications for a mode of action.
The influence of melatonin on circadian and photoperiodic functions in numerous species is well documented. It is known that the effect of melatonin on circadian rhythmicity is mediated via the suprachiasmatic nucleus (SCN), the biological clock of the brain. It is not known however where the photoperiodic effects of melatonin are mediated. Evidence from brain lesioning and melatonin implant studies point to a site in or near the medial hypothalamus. In contrast to these studies, melatonin receptors have been reported in widespread areas of the brain, the pituitary and in peripheral tissues. The characteristics of the reported melatonin receptors vary widely between studies and consequently no definitive description of a physiologically relevant melatonin receptor has received universal recognition. This review marshals recent evidence for the localization and characterization of the melatonin receptor and discusses these findings in the context of the known effects of the hormone in different species.
Animals↗
Evidence for dual adrenergic receptor regulation of ovine pineal function.
Both alpha 1- and beta-adrenergic receptors are present on ovine pineals. In the rat these two receptors interact so that activation of the alpha 1-receptor potentiates the beta-receptor-mediated changes in cyclic AMP and the correlated changes in pineal N-acetyltransferase (EC 2.3.1.87). Here we investigate possible interactions between alpha 1- and beta-receptors through changes in cyclic AMP and assess the importance of each receptor to the melatonin response in ovine pineal punches/slices in vitro. The adrenergic agonists isoproterenol (ISO), noradrenaline (NA), and phenylephrine (PHE) stimulated dose-dependent changes in cyclic AMP with the order of potency of ISO greater than NA greater than PHE, consistent with their relative binding affinities for the beta-receptor. The beta-receptor antagonist, propranolol, showed dose-dependent inhibition of the ISO effect, whereas the alpha 1-selective antagonist, prazosin, had no effect. The S-shape of the stimulation and inhibition curves for ISO reflects cyclic AMP changes mediated by the beta-receptor only without interaction through the alpha 1-receptor. Each of the adrenergic agonists stimulated indistinguishable dose-dependent increases in melatonin release. The ability of PHE to stimulate changes in melatonin release in the absence of concomitant changes in cyclic AMP indicates an important role for the alpha 1-receptor. Prazosin inhibits this response, substantiating this conclusion. However, as propranolol is also inhibitory, it seems that the alpha 1-receptor response is absolutely dependent upon a small level of beta-receptor stimulation, thus providing evidence for adrenergic receptor interaction at a step other than cyclic AMP.
Adrenergic Agonists↗
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.
Animals↗
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.
Animals↗
Adrenergic and VIP stimulation of cyclic AMP accumulation in ovine pineals.
In vitro autoradiography of [125I]cyanopindolol ([125I]cyp) binding to sections of ovine pineal reveals a uniform distribution of beta-adrenergic receptors throughout the gland. Norepinephrine (NE) stimulated cyclic AMP production in pineal slices in both a time- and dose-dependent manner, producing a maximal two-fold increase. NE, isoproterenol (ISO) and epinephrine (E) stimulate cyclic AMP (cAMP) production with equal potency. NE stimulation of cAMP was totally blocked by propranolol (beta antagonist) but only partially blocked by practolol (a beta 1 preferential antagonist) indicating a mixed population of beta 1 and beta 2 receptor subtypes. Displacement of [125I]cyp binding by either practolol or zinterol (preferential beta 2 agonist) revealed IC50s of 1.3 x 10(-5) M and 9.95 x 10(-8) M respectively, confirming a mixed population of beta 1 and beta 2 receptors. A range of peptides previously localised within the pineal by immunocytochemistry were tested at a concentration of 10(-5) M for their effect on cyclic AMP production in pineal homogenates. Only vasoactive intestinal peptide (VIP) was effective showing a dose-dependent stimulation. ISO and VIP stimulation of cAMP were additive indicating action via independent receptors.
Animals↗
Stimulation of melatonin synthesis in ovine pineals in vitro.
Static and superfused pineal slices (750 micron) have been used to study the control of melatonin synthesis by ovine pineals. Static incubates show a time-dependent accumulation of melatonin in the medium; this is significantly increased by stimulation with norepinephrine (NE) (10(-5) M), reaching 300% above control levels after 4 h. Perifused pineal slices show a rapid rise in melatonin release within 12-18 min in response to NE stimulation. This reaches a 3.5-4.5-fold increase in melatonin released within 30 min. Withdrawal of NE is associated with a rapid return to prestimulated levels within 12-18 min. These time-course characteristics compare favorably to those changes seen in vivo. The formation of [14C]melatonin from [14C]-tryptophan shows a linear increase with time. In the presence of NE (10(-5) M), the rate of synthesis is increased, albeit after an initial time lag of at least 30 min. The latter may reflect an N-acetyltransferase-independent mechanism of synthesis and release. In static incubations, propranolol (10(-5) M) inhibited NE-induced melatonin production by about 60%, but prazosin (10(-5) M) had no effect. As dibutyryl cyclic AMP (10(-3) M) stimulated melatonin production, it is concluded that beta-receptors are of primary importance to the control of melatonin production, as in the rat. The role of alpha 1-receptors is less clear, but the stimulatory action of phorbol 12-myristate 13-acetate on melatonin release implicates a receptor linked to phosphatidylinositol turnover.
Animals↗
Demonstration of melatonin-binding sites on the pars tuberalis of the rat.
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.
Animals↗