ABSTRACT

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.

Abstract

To define the hormonal influences that are directly involved in the hair follicle cycles of animals with differing patterns of fibre growth and moulting, we have investigated the possible presence of IGF-I and melatonin receptors on the dermis and hair follicles of cashmere and Angora goats, sampled in February, March and June, using quantitative in vitro autoradiography. The presence of IGF-I receptors in the dermis of both breeds of goat was determined using cryostat sections incubated with 50 pm ¹²⁵I-labelled IGF-I in the presence or absence of 50 nm IGF-I. Sections of the growing tip of deer antlers containing the cartilaginous zone, a tissue known to contain high concentrations of specific IGF-I receptors, were used as a positive control. As the production of antler velvet uniquely involves the generation of hair follicles de novo, the presence of IGF-I receptors in the velvet-producing region was also investigated. In both breeds of goat, specific ¹²⁵I-IGF-I binding was localised over the inner and outer root sheath, the matrix, the germinal matrix, the dermal papilla and the sebaceous glands and satisfied the basic kinetic criteria considered to be representative of a specific IGF-I receptor. Analysis of saturation isotherms using a one-site binding model revealed dissociation constants (K _d) in the range 0·1–0·9 nm and theoretical maximal numbers of binding sites (B _max) between 21·4 and 45·6 fmol/mg tissue. K _d and B _max values derived from cashmere and Angora goats sampled at different times of the year did not differ significantly between breeds or sampling times. Specific ¹²⁵I-IGF-I binding was also localised to the developing follicles on the deer antler dermis. The presence of melatonin receptors within the goat dermis was also investigated. Sections were incubated with 100 pm 2-[¹²⁵I]iodomelatonin with or without 0·1 μm melatonin, along with sections of sheep pars tuberalis which are known to contain high levels of high-affinity melatonin receptors. No displaceable 2-[¹²⁵I]iodomelatonin binding was found on any sections of the cashmere or Angora skin analysed. It is therefore concluded that melatonin receptors are not present on the hair follicles or associated structures. IGF-I receptors are present on the hair follicle and sebaceous gland and may be involved in the growth of both seasonally and non-seasonally produced fibre and in the development of antler velvet.

Journal of Endocrinology (1996) 151, 55–63

ABSTRACT

In-vivo microperfusion was used to localize the reabsorptive defect responsible for the hypercalciuria of diabetes mellitus and to investigate possible causative factors. Unidirectional proximal calcium absorption was not significantly different in rats made diabetic with streptozotocin compared with controls, providing evidence against the involvement of this nephron segment in the phenomenon. Calcium absorption by the loop of Henle, was however, significantly (P<0·01) lower in diabetic animals (32·1 ±1·2 vs 40·4±0·6 pmol/min). Based on our knowledge of calcium movements within the loop, it is likely that the reabsorptive defect resides within the thick ascending limb. The calcium lesion was found to be independent of acute changes in intraluminal glucose concentration and could not be corrected by acute insulin treatment. The study also provides new information on the relationship between intratubular glucose and fluid movements in the rat nephron. In diabetic rats a proximal perfusate containing 30 mmol glucose/l resulted in fluid absorption comparable with that seen in control rats perfused with 5 mmol glucose/l. However, intraluminal glucose had a stimulatory effect on fluid absorption in the loop of Henle of diabetic rats (10·7 ±0·5 vs 7·9±0·4 nl/min; P<0·01).

Journal of Endocrinology (1991) 131, 373–380

Abstract

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

Abstract

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

ABSTRACT

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 F_2α 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 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 < 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-NH₂ ⁹]-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 < 0·05) than in 35-day post-partum and control groups.

Journal of Endocrinology (1991) 128, 253–260