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R. L. SUTHERLAND and M. W. SIMPSON-MORGAN

SUMMARY

A competitive binding technique is described for the estimation of the thyroxine (T4)-binding properties of serum proteins in dilute blood serum and lymph. When used in conjunction with an assay for total T4 the following parameters can be estimated: the number of functionally different T4 binding proteins, their individual association constants and binding capacities for T4, the amount of T4 which is bound to each binding species, and the concentration of unbound (free) T4.

Both human and sheep serum have three functionally different T4-binding proteins. The association constants for the three human proteins were 9·5 × 109, 1·6 × 108 and 3·1 × 105 1/mol for T4-binding globulin (TBG), T4-binding prealbumin (TBPA) and serum albumin, respectively. The corresponding sheep proteins, TBG, TBP-2 and albumin, had association constants of 8·9 × 109, 1·4 × 108 and 3·5 × 1051/mol. Human TBG had a mean binding capacity of 21·3 μg/100 ml and that of ovine TBG was 12·8 μg/100 ml. The other specific binding proteins (TBPA in man and TBP-2 in sheep) had mean binding capacities of 307 and 359 μg/100 ml respectively.

Two functionally different T4-binding proteins were identified in rat serum.

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R. L. SUTHERLAND and M. W. SIMPSON-MORGAN

Electrophoretic studies employing a variety of media and buffer systems have repeatedly shown that tracer thyroxine (T4) moves with two distinct protein bands in sheep serum. These bands correspond to thyroxine-binding globulin (TBG) and serum albumin (Annison, 1960; Farer, Robbins, Blumberg & Rail, 1962; Refetoff, Robin & Fang, 1970). It has been pointed out by Gordon & Coutsoftides (1969) that such electrophoretic techniques are unlikely to depict closely T4 binding in vivo. For this reason we have developed a competitive-binding technique, using Sephadex G-25, which has enabled the measurement of the T4-binding properties of sheep serum proteins at physiological pH. This technique is similar in principle to that previously described by Pearlman & Crépy (1967).

Sephadex G-25 binds T4 in a highly predictable way. When a constant amount of G-25 is in contact with a constant volume of buffer, at equilibrium, the T4 present

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R. L. SUTHERLAND and M. W. SIMPSON-MORGAN

The concentration of circulating plasma thyroxine (T4) appears to have little effect on the maximal binding capacity of plasma thyroxine-binding globulin (TBG). Several studies in man have shown that thyrotoxicosis results in normal or slightly lowered levels of TBG, while hypothyroidism leads to a normal or slightly increased TBG binding capacity (Oppenheimer, Squef, Surks & Hauer, 1963; Inada & Sterling, 1967; Gordon, Kleinerman, Ehrenfeld & Ehrenfeld, 1971). In contrast the binding capacities of corticosteroid-binding globulin (CBG) and sex steroid-binding globulin (SBG) have been shown to be markedly affected by the level of circulating thyroid hormone. In the rat the binding capacity of CBG was markedly depressed by thyroid-ectomy and enhanced by chronic administration of T4 (Labrie, Raynaud & Fortier, 1965). Similarly, Dray, Mowszowicz, Ledru, Crépy, Delzant & Sebaoun (1969) administered T4 to normal human subjects and observed an increase of SBG concentration. Hyperthyroidism has been shown to

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L. M. Williams and P. J. Morgan

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.

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R. L. SUTHERLAND, M. R. BRANDON and M. W. SIMPSON-MORGAN

SUMMARY

When plasma proteins are diluted with buffer the ionic strength and ionic composition of that buffer affects the interactions between thyroxine (T4) and its plasma protein-binding sites. Increases in phosphate, chloride or barbiturate ion concentration from 50 to 200 mmol/l caused a significant decrease in the affinity of plasma proteins for T4, and a concurrent increase in the concentration of unbound T4. These results cannot be completely accounted for by changes in ionic strength since at the same ionic strength different anions caused quantitatively different effects on unbound T4 concentration. The degree of depression of T4 binding by the three anions studied was in the order barbiturate > chloride > phosphate.

The results of a systematic study on the composition of diluent buffer systems indicated that when a 50 mm-sodium phosphate–100 mm-NaCl buffer (pH 7·4) was used as a plasma diluent, there were unlikely to be gross changes in the T4-binding properties of plasma proteins with dilution.

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P Dicks, C J Morgan, P J Morgan, D Kelly and L M Williams

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

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S. SYKES, L. M. MORGAN, J. ENGLISH and V. MARKS

A rat intestinal perfusion technique has been used to assess the ability of a number of monosaccharides, monosaccharide analogues and disaccharides to stimulate intestinal release of immunoreactive gastric inhibitory polypeptide (GIP).

Perfusates containing glucose, sucrose, galactose, maltose, 3-O-methylglucose or α- or β-methylglucoside at concentrations of 100 mmol/l in Krebs–Ringer phosphate buffer (KRP) produced significant stimulation of GIP release compared with the control perfusions with KRP alone (P < 0·02). Mannose, 6-deoxygalactose, 2-deoxyglucose, myoinositol, fructose or lactose (100 mmol/1 of each) did not stimulate GIP release compared with controls. There was no significant difference in the ability of sucrose, maltose or β-methylglucoside (100 mmol/1 of each) to release GIP compared with 100 mmol glucose/1, but galactose, 3-O-methylglucose and α-methylglucoside (100 mmol/1 of each) produced significantly lower GIP responses than did glucose (P <0·02). Addition of 5 mmol phloridzin/1 to a perfusate containing 50 mmol glucose/1 prevented intestinal absorption of glucose and abolished the GIP response.

The molecular configuration of monosaccharides which have the ability to stimulate GIP release agreed well with the structural requirements for active transport by the sodium-dependent hexose pathway.

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J. M. Wallace, P. J. Morgan, R. Helliwell, R. P. Aitken, M. Cheyne and L. M. Williams

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 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

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R. M. Elliott, L. M. Morgan, J. A. Tredger, S. Deacon, J. Wright and V. Marks

ABSTRACT

The acute effects of different macronutrients on the secretion of glucagon-like peptide-1(7–36)amide (GLP-1(7–36)amide) and glucose-dependent insulinotropic polypeptide (GIP) were compared in healthy human subjects. Circulating levels of the two hormones were measured over a 24-h period during which subjects consumed a mixed diet. In the first study, eight subjects consumed three equicaloric (375 kcal) test meals of carbohydrate, fat and protein. Small increases in plasma GLP-1(7–36) amide were found after all meals. Levels reached a maximum 30 min after the carbohydrate and 150 min after the fat load. Ingestion of both carbohydrate and fat induced substantial rises in GIP secretion, but the protein meal had no effect. In a second study, eight subjects consumed 75 g glucose or the equivalent portion of complex carbohydrate as boiled brown rice or barley. Plasma GIP, insulin and glucose levels increased after all three meals, the largest increase being observed following glucose and the smallest following the barley meal. Plasma GLP-1(7–36)amide levels rose only following the glucose meal. In the 24-h study, plasma GLP-1(7–36)amide and GIP concentrations were increased following every meal and remained elevated throughout the day, only falling to fasting levels at night. The increases in circulating GLP-1(7–36)amide and GIP levels following carbohydrate or a mixed meal are consistent with their role as incretins. The more sustained rises observed in the daytime during the 24-h study are consistent with an anabolic role in lipid metabolism.

Journal of Endocrinology (1993) 138, 159–166

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S McNulty, I L Schurov, P J Morgan and M H Hastings

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