Elizabeth Erdelyi

Simultaneous determination of indole- and catecholamines in tissues using a weak cation-exchange resin☆☆☆

During the past decade, a number of procedures have been used for routine assay of catecholamines and the indoleamines, in various tissues. Many of these procedures do not allow simultaneous assay of the amines, or, if they do, as in the case of some solvent extraction procedures, do not allow for separation of amines from their precursor amino acids (l-6). The successful application of the weak cation-exchange resins by some authors (7-13) for one or two amines made it possible for us to adapt and improve those methods and, at the same time, to study various parameters of the procedure which have not been previously reported in order to increase the versatility of the assay method. The procedure can be used for simultaneous determination of norepinephrine (NE), epinephrine (E) , dopamine (DA), serotonin, (5-HT) and 5-methoxytryptamine (5-MT). Basically, the methods involve the deproteinieation of the tissue with either perchloric acid or ethanol, adsorption of the amines on the weak cation-exchange resin Bio-Rex 70, elution, and spectrofluorometric determination of the amines. The advantages of the method described in this paper over other published ones are: (a) the purification steps of the amines are greatly reduced, requiring only 2.5 hr to run up to 50 samples through the columns, (b) the recovery of the amines is higher, (c) one can determine quantitatively 20 ng of any of the four amines in one tissue sample, and (d) the method is highly reproducible.

Microdialysis of extracellular endogenous opioid peptides from rat brain in vivo.

The combination of microdialysis and a highly sensitive radioimmunoassay was developed in order to monitor the in vivo extracellular levels of endogenous opioid peptides from discrete regions of the rat brain. The radioimmunoassay cross-reacts 100% with peptides with alpha N-acetyl Tyr.Gly.Gly.Phe-Met or -Leu at the N terminus and thus recognizes all known endogenous opioid peptide fragments following acetylation of the sample. The assay was conducted on solid phase with antibody bound via protein A to 96-well plates and provided a limit of detection of approximately 0.2 fmol. A variety of dialysis membranes were evaluated with respect to their efficiency in recovering opioid peptides in vitro. Custom-made probes (4 mm active length) manufactured from polyacrylonitrile membranes and commercially available polycarbonate membrane probes proved most suitable with relative recoveries for [Met]- and [Leu]enkephalin in the range 6-10% at a flow rate of 2.7 microliters/min. Probes implanted in the globus pallidus/ventral pallidum of halothane/N2O anaesthetized rats recovered approximately 1.5 fmol of immunoreactive opioid material per 30-min sample in the absence of peptidase inhibitors. The majority of this immunoreactivity co-eluted with [Met]- and [Leu]enkephalin on reverse-phase high-performance liquid chromatography. A 2-min pulse of 100 mM K(+)-containing artificial cerebrospinal fluid in the perfusion medium during a 30-min sampling period increased the recovered immunoreactive material to 43.9 fmol +/- 12.4 S.E.M. A second stimulation 3 h later also resulted in elevated levels with an S2:S1 ratio of 0.64 +/- 0.03. The second stimulation was completely blocked by perfusion of a 10 mM EGTA-containing medium, basal release on average remaining unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual determination of extracellular cholecystokinin and neurotensin fragments in rat forebrain: Microdialysis combined with a sequential multiple antigen radioimmunoassay

Microdialysis was combined with a highly sensitive sequential multiple antigen radioimmunoassay to simultaneously measure extracellular cholecystokinin and neurotensin fragments from discrete regions of the rat brain in vivo. The assay was conducted in 96-well plates and provided a limit of detection for both peptides of 0.1 fmol. Dialysis membranes composed of polyacrylonitrile, Cuprophan and polycarbonate were evaluated in vitro using both radiolabelled peptides and radioimmunoassay. Polycarbonate probes were implanted in the posterior medial nucleus accumbens-septum, medial caudate nucleus or medial prefrontal cortex of halothane-N2O-anaesthetized rats. Cholecystokinin immunoreactivity levels were generally above the assay detection limits (0.1-0.7 fmol) in 30-min samples from all three regions under basal conditions. Recovered basal amounts of neurotensin immunoreactivity were detectable in the nucleus accumbens-septum in approximately 50% of experiments (0.1-0.2 fmol) but were not measured in the caudate nucleus or prefrontal cortex. In the nucleus accumbens-septum, a 10-min pulse of 200 mM K(+)-containing artificial cerebrospinal fluid in the perfusion medium during a 30-min sampling period increased the recovered cholecystokinin and neurotensin immunoreactivity to 9.7 fmol +/- 1.9 S.E.M. and 5.8 +/- 1.6 S.E.M., respectively. A second stimulation following a 2.5-h interval produced similar elevations with S2:S1 ratios of 0.62 +/- 0.07 and 0.68 +/- 0.07 for cholecystokinin and neurotensin, respectively. In a separate series of experiments the second stimulation of both peptides was prevented by perfusion of a 10 mM EGTA-containing medium. Similar results were obtained in the caudate nucleus for cholecystokinin, but K(+)-induced elevations in neurotensin immunoreactivity were much smaller (0.5 fmol) in this brain region and calcium dependency was not established. Sequential K+ stimulations at 50, 100 and 200 mM produced progressively greater increases in recovered cholecystokinin and neurotensin immunoreactivity from the nucleus accumbens-septum and of cholecystokinin immunoreactivity from the prefrontal cortex. No neurotensin immunoreactivity was detected in the prefrontal cortex following K+ stimulation. Large post mortem increases in the recovered amounts of cholecystokinin and neurotensin immunoreactivity were observed. This effect was significantly attenuated by EGTA although there was a large calcium-independent component of the cholecystokinin immunoreactivity. On reverse-phase high-performance liquid chromatography the major cholecystokinin-immunoreactive peak co-eluted with sulphated cholecystokinin octapeptide. Neurotensin-immunoreactive material co-eluted with neurotensin (1-13), neurotensin (1-12), neurotensin (1-11), neurotensin (1-10) and neurotensin (1-8). These results further demonstrate the potential of microdialysis for studying neuropeptide release and metabolism in vivo when combined with sufficiently sensitive assay procedures.

Regulation of both preproenkephalin mRNA and its derived opioids by haloperidol — a method for measurement of peptides and mRNA in the same tissue extract

The goal of this study was to delineate the effects of dopamine antagonists on the regulation of preproenkephalin mRNA and opioid peptides in the rat brain. We have developed a method whereby both mRNA and peptides can be efficiently measured in the same tissue extract, thus reducing the effects of intraspecies variation, differences in dissection and the number of animals required for statistical significance. A sub-chronic dose of haloperidol (3 mg/kg given i.p. in 100 microliters DMSO daily for 5 days) produced a 1.8-fold increase (P less than 0.001) in striatal preproenkephalin mRNA levels when compared to animals injected with vehicle dimethyl sulfoxide (DMSO) employing the same schedule. Total opioid peptides as measured by a radioimmunoassay directed to the N-terminus of enkephalins and endorphins were elevated 1.6 fold (P less than 0.001) in the rat striatum. However in other brain regions examined no increases were observed either in preproenkephalin mRNA or the tissue levels of opioid peptides. Analysis of the opioid-like immunoreactive peptides by reverse-phase HPLC analysis showed no dramatic changes in the ratios of the various opioid peptides between haloperidol and vehicle injected animals. Naive animals showed no statistical differences in opioid peptide levels compared to the haloperidol treated animals. There was a statistically significant decrease (30%) in the opioid peptide content of the animals injected with vehicle daily for 5 days when compared with the animals merely sacrificed, or those given acute injections (either with haloperidol or vehicle) the day of sacrifice.(ABSTRACT TRUNCATED AT 250 WORDS)

Neural differentiation in the OTT-6050 mouse teratoma: Enzymatic and immunofluorescence characterization of a tumor fraction showing melanogenesis in neuroepithelial cells

A pigmented tumor fraction, designated IB-9, obtained following cellular dissociation and elutriation procedures applied to the solid transplants of the OTT-6050 mouse teratoma cell line, was characterized enzymatically and by immunofluorescence for the presence of tyrosinase and tyrosine hydroxylase (TH). Enzymatic assays of the pigmented tumors were compared with those obtained on non-pigmented teratoma-derived tumors, on pigmented tumors obtained from the mouse melanoma B16 line as a control for tyrosinase activity, and on whole brains of adult 129/J mice as a control for TH activity. All the teratoma-derived tumors, including the IB-9 fraction, showed a predominance of TH over tyrosinase activity. The levels of TH activity appeared independent of the presence or the extent of melanin pigment. All pigmented teratoma-derived tumors showed low levels of tyrosinase activity. On the basis of the enzymatic assays, the IB-9 tumors were divided into two groups: group I, which showed low enzyme activity, almost certainly entirely tyrosinase; and group II, in which the enzyme activity appeared largely due to TH, with presumably a very low background of tyrosinase activity. Immunofluorescence demonstrated the localization of TH activity to non-pigmented cells of the IB-9 fraction, whereas the pigmented cells showed absence of TH activity. These findings, taken in conjunction with the presence by electron microscopy of premelanosomes and melanosomes, indicate that pigment formation associated with melanosomal differentiation in the neural cells of IB-9 with the histologic patterns of primitive CNS neuroepithelium results from tyrosinase activity only and is therefore unrelated to the metabolic pathways involved in catecholamine synthesis and degradation. It is suggested that, at this stage of differentiation and in this system, the expression of catecholamine synthesis via tyrosine hydroxylase in neuroepithelial cells, and of melanin pigment via tyrosinase, are probably mutually exclusive.

Measurement of Total Opioid Peptides in Rat Brain and Pituitary by Radioimmunoassay Directed at the α‐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 ˜15 fmol of β‐endorphin per incubation tube, i.e., ˜ 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 (β‐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.

Peptide E and Its Products, BAM 18 and Leu-Enkephalin, in Bovine Adrenal Medulla and Cultured Chromaffin Cells: Release in Response to Stimulation

Peptide E is a 25 amino acid opioid peptide which, if cleaved at the sole double basic (Lys‐Arg) typical processing site, would generate two opioid fragments, the amino‐terminal fragment BAM 18 and the carboxy‐terminal fragment Leu‐enkephalin. We have analysed extracts of bovine adrenal medulla in order to quantify these three opioid peptides (peptide E, BAM 18, and Leu‐enkephalin). Here we present evidence that BAM 18 and Leu‐enkephalin were present in similar amounts, whereas peptide E was present at a higher concentration. This is consistent with previous observations showing a preferential accumulation of larger peptides in the bovine adrenal, and also with the Lys‐Arg bond being the principal site of cleavage of peptide E. However, when bovine adrenal chromaffin cells were maintained in culture for several days, Leu‐enkephalin was found to be present in much greater amounts than was BAM 18‐like immunoreactivity. The molar amounts of peptide E still exceeded the estimated levels of BAM 18 and Leu‐enkephalin. We provide evidence that under conditions of basal release BAM 18 and peptide E were released, whereas Leu‐enkephalin was released in much smaller amounts, if at all. On stimulation with nicotine results were consistent with an increased release of all three peptides with a preferential stimulation of Leu‐enkephalin release. Under all conditions, the molar amounts of peptide E released apparently exceeded that of the other peptides. The results are discussed in terms of the regulation of partial proteolysis and the fate of peptide E.

Candidate Opioid Peptides for Interaction with the Immune System

Endogenous opioids are a recently discovered group of peptides which have been implicated as modulators of a number of biological systems. Both opiate receptors and their peptide ligands are widely distributed throughout the CNS and are additionally located in some peripheral sites. The classical effects of opiates as analgesics have perhaps overshadowed many other important biological activities of this group of neuroactive peptides. This book provides strong indications that endogenous opioid peptides play a crucial role in the regulation of the immune system.

ELECTROPHORETIC STUDY OF 5-HYDROXYTRYPTOPHAN DECARBOXYLASE FROM BRAIN AND LIVER IN SEVERAL SPECIES

—An electrophoretic investigation in acrylamide gels of 5‐hydroxytryptophan decarboxylase, obtained mostly from mouse, rat, and beef brain and also from beef and human liver, showed electrophoretic differences between species. With the exception of the rat, only one molecular species was found (the same in beef brain and liver). In the rat, polymers form spontaneously and are, at least in part, disaggregated by urea and by triton. Mouse‐rat or beef‐rat molecular hybrids form in the admixtures. No electrophoretic differences were found in five mice strains that were investigated. Techniques of electrophoretic analysis and of assay of 5‐hydroxytryptophan decarboxylase are described, which can be easily applied to other enzymes, provided a substrate is available in radioactive form.

Synthetic N-dimethyl β-endorphin, a stabilized opioid peptide

Abstract A procedure for the dimethylation of the amino groups of human β-endorphin by reductive methylation is described. A single product with the theoretical maximum degree of methylation was produced and the consequences of dimethylation on proteolytic attack are reported. The derivative was shown to be resistant to tryptic digestion and to attack by leucine aminopeptidase. The nonmethylated β-endorphin was rapidly degraded by incubation with pituitary homogenate; under these conditions methylated β-endorphin was degraded at a slower rate. In the presence of bacitracin, the methylated peptide was essentially resistant to degradation by the pituitary homogenate. The methylated peptide may be expected to have a longer in vivo half-life.