DEVELOPMENT OF a RADIOIMMUNOASSAY FOR Α-Melanocyte-Stimulating HORMONE IN THE RAT

Thody, A. J.; Penny, R.J.; Clark, Del; Taylor, Christine

doi:10.1677/joe.0.0670385

Cited by 43 publications

(12 citation statements)

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“…Antibodies to synthetic «-MSH (Ciba-Geigy Ltd.) which had been conjugated to rabbit serum albumin by the ethyl carbodiimide method were raised in rabbits. Cross-reactivity to «-MSH and several MSH analogues was tested as reported previously [Thody et al, 1975b]. The antiserum chosen for the assay was used at an overall dilution of 1:52,800 and showed no appreciable cross-reactivity to the MSH analogues tested (table I).…”

Section: Methodsmentioning

confidence: 99%

“…Synthetic a-MSH (Ciba) was labelled with I25I (IMS 30 Amersham) using the chloramine-T method of G reenwood et at. [1963] modified as described previously [Thody et at., 1975b]. Concentration and immunoreactivity of the labelled material was judged by assaying serial dilutions of the label against a standard displace ment curve.…”

Section: Methodsmentioning

confidence: 99%

“…Once parallel displacement of the labelled a-MSH by serial dilutions of homogenate had been demonstrated, subsequent assays were performed on 200 /d of a single 1:12,000 dilution in 6 replicates. After incubation for 2 days at 4°C, bound and free hormone were separated using the dextran coated charcoal method as previously described [Thody et al, 1975b]. The standard curve was plotted as logit B/B0 against logn standard concen tration, and under the specified conditions of the assay a straight line plot resulted ( fig.…”

Section: Methodsmentioning

confidence: 99%

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An Improved Radioimmunoassay for α-Melanocyte-Stimulating Hormone (α-MSH) in the Rat: Serum and Pituitary α-MSH Levels after Drugs Which Modify Catecholaminergic Neurotransmission

Penny¹,

Thody²

1978

Neuroendocrinology

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A modification of our previous radioimmunoassay (RIA) for α-melanocyte-stimulating hormone (α-MSH) is described that permits the measurement of circulating levels in the rat without the need for an extraction procedure. Using this method, serum and neurointermediate lobe () immunoreactive α-MSH levels were measured in rats after administration of haloperidol, 2-bromo α-ergocryptine (CB 154), and α-methyl-p-tyrosine (α-MPT). Haloperidol caused a rapid increase, α-MPT a slow increase, and CB 154 a rapid decrease in serum α-MSH. At the time intervals studied none of the drugs had any significant effect on α-MSH content. It was concluded from the results of the above drug treatments that modulation of dopaminergic neurotransmission both pre- and postsynaptically produces changes in serum immunoreactive α-MSH levels. This supports the suggestion that circulating α-MSH in the rat is under an inhibitory control by a catecholaminergic system.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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An Improved Radioimmunoassay for α-Melanocyte-Stimulating Hormone (α-MSH) in the Rat: Serum and Pituitary α-MSH Levels after Drugs Which Modify Catecholaminergic Neurotransmission

Penny¹,

Thody²

1978

Neuroendocrinology

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“…However, the way in which this peptide enters the CSF is not yet clear. a-MSH is a major melanotrophic peptide of the pars intermedia of the rat [4,26] and is normally released into the systemic circulation [19,26,271. Prolactin and possibly other pituitary hormones are thought to enter the CSF via the circulation by filtration at the choroid plexus [3] but it would seem unlikely that much a-MSH follows this route because no correlation was found between immunoreactive a-MSH levels in plasma and CSF.…”

Section: Discussionmentioning

confidence: 99%

Plasma and cerebrospinal fluid α-MSH levels in the rat after hypophysectomy and stimulation of pituitary α-MSH secretion

Thody

Rotte

Greidanus

1979

Brain Research Bulletin

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This lack of correlation between a-MSH levels in the CSF and plasma suggests that the systemic circulation does not deliver u-MSH to the CSF. The apparently normal levels of u-MSH in the hypothalamus after hypophysectomy suggests that this tissue is able to synthesize u-MSH and it is possible that the hypothalamus is a source of the a-MSH in the CSF.Cerebrospinal fluid a-MSH ~ER~~R~~~IN~ FLXJED fCSF3 is likely to serve as an important route for many hormones that modulate bmin function. A number of deferent peptide hormones have been found in the CSF [l, 3, 5-7, 9, 12, 241 and while those of hy~~thal~ic origin such as vasopressin, oxytocin and the releasing hormones enter the CSF directly, the way in which pituitary hormones such as ACTS reach the CSF is not yet clear.weight. ~y~~hy~ctorny was performed by the pam~h~~ge~ approach and con&met3 at autopsy by examination of the seUa turcia.Melanocyte-stimulating hormone (MSH) and related peptides are known to affect brain function both in man and ex~e~me~tal animals 12, 11, 28, 291. While it is generally accepted that these melanotropic peptides are produced by the pituitary gland bioassayable MSH and immunoreactive MSH peptides have been found in different regions of the brain and shown to persist after removal of the pituitary [S, 14, 1.5, 17, 18,2%23]. This suggests that the brain, as weif as the pituitary, is capable of producing MSH peptides. fn this study we report the presence of ~mmunnrea~tive cu-MSH in the CSF of the rat and present data which suggest that at teast some of this peptide is of brain rather t.han pituitary origin.Rats were anaesthetized with urethane (1.1 g/Kg IP) and polyethylene cannulae inserted into the cistema magna for the withdrawal of CSF [7]. Approximately SO-100 1,t1 CSF were wi~d~aw~~ centrifuged and stored at -20°C until assayed.(1) Polyethylene cannulae were inserted into the jugular vein and advanced to the heart. Approximately 500 ~1 blood were withdrawn and replaced by an equal votume of 0.5% salineiheparin solution.(2) In experiment 2 {see below) the rats were decapitated and trunk blood collected into tubes containing lithium sequestrene,.All blood samples were centrifuged and the plasma stored at -20°C. METHODMale Wistar rats were obtained from TNO, Zeist, The Rats were decapitated and the brain immediately reNetherlands or bred in the Depa~ment of Dermatology, moved. The hypothalamus was dissected and this and the Newcastle, and were used when approximately 180 g in remainder of the brain minus the olfactory lobes were rinsed

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“…The initial studies were those of McGuire et al [ll], followed by those of Abe et al [12], Thody et a/. [13], Goos and Jenks [14], and Usategui et al [21]. With the exception of the last report, sensitivity and (or) specificity were insufficient for an exact determination of a-melanotropin in peripheral plasma.…”

Section: Discussionmentioning

confidence: 99%

Specific Antibodies against alpha-Melanotropin for Radioimmunoassay. Parallelism between the Immunochemical Cross-Reactions of Melanotropin Structural Analogues and Their Biological Activity

et al. 1977

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The purpose of this experimental investigation was to provide antibodies against a-melanotropin with a high serum titer and with great specificity. To this end, rabbits were immunised with a complexantigen consisting of human serum albumin containing a rather low number of a-melanotropin molecules attached uniquely through the y-carboxyl-group of (glutamic acid)-5 (probably mostly to s-amino groups of albumin lysine residues). In such a complex-antigen the groups chemically characteristic of a-melanotropin (C-terminal valine amide and N-terminal IV-acetylserine) are expected to be maximally exposed to the surroundings.The antisera had high antibody titers (1 : 125000 up to 1 :900000). Their specificitywas directed towards the C-terminal part of a-melanotropin. The cross-reaction with adrenocorticotropin was small (about 0.1 x) and negligible with other peptide hormones, including P-melanotropin. The sensitivity of the assay was 40 pg/ml and the detection limit for a-melanotropin about 3 pg. Radioiodination of a-melanotropin with the lactoperoxidase method yielded a monoiodinated tracer with a specific activity of about 1500 Ci/mmol.The biological activity of structural analogues and fragments of r-melanotropin (assay in vifro with skins of Rana pipiens) paralleled their activity in the radioimmunoassay. This may indicate similar structural requirements of the melanophore receptor and antibody populations. r-Melanotropin is a tridecapeptide with the following primary structure : Ac . Ser-Tyr-Ser-Met-Glu-HisPhe-Arg-Trp-Gly-Lys-Pro-Val . NH2, ascertained by degradation and chemical synthesis [l]. It is an important pigmentary hormone of lower vertebrates and is secreted in the Pars intermedia of the pituitary [2]. r-Melanotropin also displays biological activity in mammals, influencing sebum secretion, central nervous functions (memory, behaviour), and foetal growth (review [3]).Investigations into the physiology and pharmacology of a-melanotropin require efficient, sensitive, and specific methods for the detection and determination of the hormone at very low concentrations. Bic,?ssays with the isolated skin of Rana pipiens [4,5] or Anolis carolinensis [6,7] are quite sensitive (about 10 pg),.~ This report has partially been presented during the postersession at the 1st International Symposium on Melanotropin, Amsterdam. July [14][15][16] 1976.Ahhrc~iutions. Amino-acids and peptides: according to the tentative rules and recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature, Eur. J . Biochern. 27, 201 -207 (1972).but cannot distinguish between a-melanotropin, /Imelanotropin, adrenocorticotropin, and other melanophore-dispersing peptides. Furthermore, unknown compounds in certain sera [S] and catecholamines interfere with the bioassay. On the other hand, radioimmunochemical methods exhibit good structural specificity and high sensitivity. They permit direct and specific determinations of peptide hormones in serum, provided that cross-reactions can be excludedThe amino acid sequence of a-melanotropi...

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DEVELOPMENT OF a RADIOIMMUNOASSAY FOR Α-Melanocyte-Stimulating HORMONE IN THE RAT

Cited by 43 publications

References 0 publications

An Improved Radioimmunoassay for <i>α</i>-Melanocyte-Stimulating Hormone (<i>α</i>-MSH) in the Rat: Serum and Pituitary <i>α</i>-MSH Levels after Drugs Which Modify Catecholaminergic Neurotransmission

An Improved Radioimmunoassay for <i>α</i>-Melanocyte-Stimulating Hormone (<i>α</i>-MSH) in the Rat: Serum and Pituitary <i>α</i>-MSH Levels after Drugs Which Modify Catecholaminergic Neurotransmission

Plasma and cerebrospinal fluid α-MSH levels in the rat after hypophysectomy and stimulation of pituitary α-MSH secretion

Specific Antibodies against alpha-Melanotropin for Radioimmunoassay. Parallelism between the Immunochemical Cross-Reactions of Melanotropin Structural Analogues and Their Biological Activity

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