Biomedical subjects

M R Boarder

Publications and source records attributed to M R Boarder.

At least 91 records · Page 5Linked to original sources

PubMed1984

Characterisation of Met-enkephalin(Arg6,Phe7) immunoreactivity in human adrenal and human phaeochromocytoma.

The molecular forms of opioid peptides in human adrenal have not been well characterised. These peptides are predominantly derived from the proenkephalin A precursor, which has the sequence of Met-enkephalin(Arg6,Phe7) as its carboxyl terminus. We have looked in the present study at the subcellular distribution and the molecular form of immunoreactivity to this sequence in post-mortem human adrenal medulla and in phaeochromocytoma. In the human adrenal homogenates, the immunoreactivity distributes on a sucrose gradient in a manner consistent with localisation in chromaffin granules. On chromatography, the immunoreactivity from adrenal medulla is predominantly in the heptapeptide form; the intermediate (3000-4000) molecular weight material is only a minor component of immunoreactivity, in contrast to bovine tissue extracts where this is the major form of immunoreactivity. In the phaeochromocytoma extracts, the heptapeptide sequence again predominates over a minor amount of intermediate sized material. The results are discussed in terms of post-mortem changes, precursor processing and the function of the adrenal medulla.

Adrenal Gland Neoplasms↗

PubMed1983

Measurement of total opioid peptides in rat brain and pituitary by radioimmunoassay directed at the alpha-N-acetyl derivative.

A sensitive assay, which cross-reacts with and is specific for diverse opioid peptides, is described. This is based on the prior acetylation of samples and subsequent radioimmunoassay with an antiserum highly specific for the acetylated NH2 terminus of opioid peptides. The result is a procedure that can be used to investigate multiple forms of opioid peptides in extracts of biological material. The sensitivity of the assay is approximately 15 fmol of beta-endorphin per incubation tube, i.e., approximately 100-fold greater sensitivity than the radioreceptor assay used in our laboratory. The peptide concentration required for 50% displacement of trace ranged from 0.65 nM (beta-endorphin) to 1.6 nM (Met-enkephalin). The assay apparently shows an absolute requirement for a free (or acetylated) NH2 terminus corresponding to either a Leu- or Met-enkephalin sequence. Use of the assay with and without prior acetylation of sample provides a method for estimation of the ratio of acetylated:nonacetylated opioid peptides in crude or fractionated extracts. The procedure is used to investigate the forms of opioid peptide found in rat brain and pituitary.

Acetylation↗

PubMed1982

Acquisition and extinction of continuously and partially reinforced running in rats with lesions of the dorsal noradrenergic bundle.

Local injection of 6-hydroxydopamine was used to selectively destroy the dorsal ascending noradrenergic bundle (DB) in rats. Two lesion procedures were used, differing in the extent of depletion of forebrain noradrenaline they produced (greater than 90% or 77%). In Experiments 1-3 the rats were run in a straight alley for food reward on continuous (CR) or partial (PR) reinforcement schedules. The smaller lesion reduced and the larger lesion eliminated the partial reinforcement acquisition effect (i.e. the faster start and run speeds produced by PR during training) and the partial reinforcement extinction effect (PREE, i.e. the greater resistance to extinction produced by PR training); these changes were due to altered performance only in the PR condition. Abolition of the PREE by the larger DB lesion occurred with 50 acquisition trials, but with 100 trials the lesion had no effect. In Experiment 4 rats were run in a double runway with food reward on CR in the second goal box, and on CR, PR or without reinforcement in the first. The larger lesion again eliminated the PREE in the first runway, but did not block the frustration effect in the second runway (i.e. the faster speeds observed in the PR condition after non-reward than after reward in the first goal box). These results are consistent with the hypothesis that DB lesions alter behavioural responses to signals of non-reward, but not to non-reward itself. They cannot be predicted from two other hypotheses: that the DB mediates responses to reward or that it subserves selective attention. Since septal and hippocampal, but not amygdalar, lesions have been reported to produced similar behavioural changes, it is proposed that the critical DB projection for the effects observed in these experiments is to the septo-hippocampal system.

Animals↗

PubMed1982

Met-enkephalin [Arg6, Phe7] immunoreactivity in bovine caudate and bovine adrenal medulla.

A radioimmunoassay specific for the COOH-terminus of Met-enkephalin [Arg6, Phe7] and a separate assay specific for the COOH-terminus of Met-enkephalin are described. Immunoreactivity by these two assays was compared in bovine caudate and bovine chromaffin granule preparation after Sephadex G75 chromatography in 50% acetic acid. When the assays were applied to the chromatography fractions of the bovine caudate extract, the majority of the immunoreactivity was found in the fractions corresponding to the heptapeptide and the pentapeptide respectively. When the chromaffin granule chromatography fractions were assayed, both of the radioimmunoassays showed that most reactivity was in several peaks in the larger molecular weight fractions. The major peak for the Met-enkephalin [Arg6, Phe7] assay had an apparent molecular weight of 2800, while with the Met-enkephalin assay the dominant peak of immunoreactivity had an apparent molecular weight of 10,000. The presence of authentic Met-enkephalin [Arg6, Phe7] in both caudate and chromaffin granule extracts was confirmed by reverse-phase chromatography of the previously sized fractions. It appears then that the processing of precursors of opioid peptides is directed, in the caudate, to the synthesis and storage of the enkephalins and of Met-enkephalin [Arg6, Phe7]; in the adrenal medulla the major products of precursor processing are a variety of polypeptides of larger sizes.

Adrenal Medulla↗

PubMed1982

Measurement of methionine-enkephalin [Arg6,Phe7] in rat brain by specific radioimmunoassay directed at methionine sulphoxide enkephalin[Arg6,Phe7].

A specific and sensitive radioimmunoassay procedure for Met-enkephalin[Arg6,Phe7] which allows its measurement in regions of the rat brain is described. The antiserum as raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized 125I-labelled Met(O)-enkephalin[Arg6,Phe7]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg6,Phe7]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg6,Phe7]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.

Animals↗

PubMed1982

Opioid peptides in human plasma: evidence for multiple forms.

Studies were designed to assess whether the enkephalin-containing peptides and proteins present in the chromaffin granules of the adrenal medulla and in other secretory tissues, such as the neurohypophysis, could be found circulating in human blood. We analyzed human plasma acid acetone extracts chromatographed on Sephadex G-75 in acetic acid and found evidence for the existence of opioid peptides of several different molecular weights and a large number of peptides and small proteins which generate opioid activity after tryptic digestion. These compounds are different from and present in much greater quantities than previously described opioid peptides in human plasma, and are separate from dynorphin-immunoreactive compounds, which we also report in the blood. Expressed in leucine-enkephalin equivalents on a radioreceptor assay, we found 63.2 +/- 6.5 (n = 4; mean +/- SEM) pmol/ml plasma. One active peak from the Sephadex G-75 chromatography of human plasma (apparent mol wt, 3000) was examined by reverse phase high pressure liquid chromatography, tryptic digestion, and Sephadex G-50 chromatography. The results were consistent with the notion that this opioid active peptide contains an enkephalin sequence at its N-terminal, followed by a basic residue. Mild stress (2 min of deep knee bends) produced a 2-fold elevation in overall circulating opioid activity. The possibility is considered that the large enkephalin-containing peptides may have an endocrine function, independent of a role as enkephalin precursors.

Adult↗

PubMed1982