J.P.H. Burbach

COMBINATION OF HIGH PRESSURE LIQUID CHROMATOGRAPHY AND RADIOIMMUNOASSAY IS A POWERFUL TOOL FOR THE SPECIFIC AND QUANTITATIVE DETERMINATION OF ENDORPHINS AND RELATED PEPTIDES

A method for the separation and subsequent quantification of endorphins and related peptides was developed. Separation of the peptides was achieved by high pressure liquid chromatography on a reversed phase column. By virtue of the high resolving capacity of this system peptides differing in only one amino acid residue could be separated easily. For quantification of the isolated peptides specific radioimmunoassay systems were used. The combination of the two techniques was applied to determine specifically a number of endorphin-like peptides in rat pituitary gland. For the first time the presence of des-tyrosine-endorphins in addition to known endorphin fragments is demonstrated.

α-Endorphin, γ-endorphin and their Des tyrosine fragments in rat pituitary and brain tissue

Abstract Enkephalins, endorphins and related peptides were determined in pituitary and brain tissue of rats which were killed by decapitation or microwave irradiation. The tissues were heated in 1M acetic acid prior to homogenization and the levels of the various peptides were measured by means of a combination of HPLC and radioimmunoassays. Enkephalin levels in pituitary and brain of irradiation-killed rats were much higher as compared to those in tissue of rats sacrificed by decapitation. Similar data were obtained with respect to pituitary levels of γ-endorphin, des-Tyr-γ-endorphin and des- Tyr-α-endorphin. However, brain levels of α- and γ-endorphin and their respective des-Tyr-fragments were not different with the two methods of sacrifice used. The concentrations of β-endorphin in the pituitary gland were similar in rats killed by microwave irradiation and decapitation, but irradiation showed higher β-endorphin levels in the brain than decapitation. These results suggest that β-endorphin fragments like α- and γ-endorphin and des-Tyr-α- and des-Tyr-γ-endorphin are endogenous peptides in the rat pituitary gland and the brain.

Autoradiographic localization of binding sites for the arginine-vasopressin (VP) metabolite, VP4–9, in rat brain

A discrete neuro-anatomical pattern of binding sites was observed for a principal metabolite of arginine-vasopressin (VP4-9) after incubation of tissue sections with [35S]VP4-9 and autoradiography. [35S]VP4-9-labeled binding sites were highly concentrated in the pineal gland, the nucleus tractus solitarii (nts), the arcuate nucleus region (an) and the organum vasculosum lamina terminalis (ovlt). The distribution of these sites is distinctly different from the putative VP and oxytocin receptor systems in rat brain. It is possible that the VP4-9 binding sites are also involved in memory processes and/or that the VP metabolite exerts additional effects on the brain.

Oxytocin biotransformation in the rat limbic brain: Characterization of peptidase activities and significance in the formation of oxytocin fragments

The enzymatic conversion of oxytocin by brain peptidases has been studied. Oxytocin was incubated with synaptosomal plasma membranes (SPM) isolated from the rat brain. Qualitative studies using a microdansylation technique revealed two types of oxytocin converting peptidases, e.g. aminopeptidase and C-terminal cleaving peptidase activities. Both enzyme activities were quantitated using [14C]oxytocin labeled at either the tyrosine-2 or the glycinamide-9 residue. Radiolabeled products were separated by high-voltage paper electrophoresis or high-pressure liquid chromatography. The aminopeptidase activity was optimally active at pH 6.9 with a Michaelis constant (Km) of 6.1 x 10(-5) M. The pH optimum of the C-terminal cleaving peptidase activity was pH 6.0 with Km = 1.3 x 10(-5) M. Subcellularly, highest amino-peptidase activities were associated with SPM, synaptosomal and microsomal preparations, while the C-terminal cleaving peptidase prevailed in the cytosol and mitochondrial fractions. The regional distribution of both peptidases showed differences between several brain areas and indicated the medial basal hypothalamus as a locus of high oxytocin biotransformation. In the course of this investigation an oxytocin fragment of unknown structure was detected in the digests and its accumulation was studied together with the determination of peptidase activities. It is suggested that the SPM-associated peptidases may have a role in the modulation of oxytocin action in the brain.

Sites of behavioral and neurochemical action of ACTH-like peptides and neurohypophyseal hormones

In order to localize the site of action of neuropeptides in relation to their effects on behavior and memory various approaches have been used. As a result of studies using rats bearing lesions in different areas of the limbic system as well as of studies in which neuropeptides were locally applied into various areas of the brain it appeared that the limbic system (amygdala, hippocampus, septum and some thalamic areas) plays an essential role in the effect of vasopressin and ACTH and their derivatives on behavior and memory. Neurochemical studies generally indicate that changes occur in catecholamine utilization in these various limbic regions upon administration of these neuropeptides. It can be concluded that the effects of vasopressin in the terminal regions of the coeruleo-telencephalic noradrenalin system correlate with its effects on consolidation of memory. It is likely that the effects of vasopressin on other transmitter systems (e.g. dopamine in the amygdala and serotonin in the hippocampus) correspond with the effect of this neuropeptide on retrieval processes. In addition, regional differences in biotransformation of the neurohypophyseal hormones suggest that different patterns of behaviorally active fragments of these peptides may be present locally in the brain.

Biotransformation of endorphins by a synaptosomal plasma membrane preparation of rat brain and by human serum

β-Endorphin (β-LPH 61–91), γ-endorphin (61–77), des-tyrosine-γ-endorphin (62–77), α-endorphin (61–76), and β-LPH 61–69 either labeled with [125I] at the N-terminal 61-tyrosine residue or unlabeled were incubated with a crude synaptosomal plasma membrane fraction of rat brain or in human serum. At different time intervals the release of [125I]-tyrosine or the change in immunoreactivity of the endorphins was determined. The cSPM preparation displayed both high aminopeptidase and endopeptidase activities. In contrast, human serum mainly contained aminopeptidase activity. The data suggest that functional endorphin metabolism may occur at the synaptosomal plasma membrane. These membranes may potentially be involved in the formation of behaviorally active endorphin fragments.

Stimulation of pro-opiomelanocortin gene expression by glucocorticoids in the denervated rat intermediate pituitary gland

The effect of frontal deafferentation of the medial basal hypothalamus on pro-opiomelanocortin (POMC) gene expression was studied in the intermediate lobe (IL) and anterior lobe (AL) of the pituitary gland in the absence and presence of corticosterone (CORT). The lesion in the basal hypothalamus removed neural inputs to the IL and induced the glucocorticoid receptor (GR) in this tissue. The GR was visualized in the denervated IL by immunocytochemistry. Induction of the GR had a slow onset and was detectable at 3 weeks after lesion, but not at one week after the lesion. In order to study the effect of IL denervation on pro-opiomelanocortin (POMC) gene expression, the level of messenger RNA specifically encoding POMC was measured 1 and 3 weeks after lesion, in the IL and AL. In adrenalectomized (ADX) animals, the changes in POMC mRNA levels were not significant 1 week after lesion in the IL. Three weeks after denervation there was a 3-fold decrease in POMC mRNA in the IL in ADX rats which was blocked by chronic CORT replacement via subcutaneously implanted pellets. In the AL, CORT reduced the level of POMC gene expression in both the lesioned and control animals. It is concluded that (1) removal of neural input induces GR in the denervated IL cells; (2) with the appearance of the GR, POMC gene expression in the IL becomes sensitive to circulating glucocorticoids; (3) under these conditions, CORT may stimulate POMC gene expression in the IL as opposed to its inhibitory effect in the AL.

Conversion of Des-tyrosine-γ-endorphin by brain synaptic membrane associated peptidases: Identification of generated peptide fragments

Abstract Des-tyrosine-γ-endorphin, a β-endorphin fragment with neuroleptic-like properties, was digested with a cSPM fraction of rat brain. A profile of metabolites and a time course of conversion were obtained by HPLC analysis of the digests. Quantitative amino acid analysis and a second HPLC fractionation step which was designed to separate and to identify very similar des-tyrosine-γ-endorphin fragments, combined with dansyl end group determination allowed the characterization of β-LPH 65–77, β-LPH 66–77 and β-LPH 62–73 as main conversion products. In the digests the C-terminal leucyl peptides β-LPH 67–77 and β-LPH 68–77 as well as the N-terminal glycyl peptides β-LPH 62–74 and β-LPH 62–76 were minor components. The data indicate the involvement of several types of peptidase activities in the conversion process. It is suggested that these peptidases have a role in mediating in vivo des-tyrosine-γ-endorphin effects. In addition, this study points to the capacity of the brain to gene-rate small peptides with neuroleptic-like properties.

β-Endorphin biotransformation in brain: Formation of γ-endorphin by a synaptosomal plasma membrane associated endopeptidase distinct from cathepsin D

cSPM preparations of rat brain contain a peptidase activity which generates γ-endorphin from β-endorphin. Some properties of this enzyme were studied and compared with those of cathepsin D. Maximal accumulation of γ-endorphin upon digestion of β-endorphin with a cSPM preparation was found at neutral pH values. The activity of cathepsin D, forming γ-endorphin and β-LPH 78–91 was limited to acidic pH values. The SPM associated peptidase was not inhibited by the specific cathepsin D inhibitor pepstatin. The peptidase activity remained associated with SPM preparations, which were purified extensively by sucrose density centrifugation. It is concluded that the SPM associated peptidase which generates γ-endorphin from β-endorphin is distinct from cathepsin D. Such an enzyme may have a physiological function in the formation of β-endorphin fragments in the brain.

In situ hybridization of oxytocin messenger RNA: Macroscopic distribution and quantitation in rat hypothalamic cell groups

Oxytocin mRNA was detected in the rat hypothalamus by in situ hybridization to a single stranded 35S-labelled DNA probe and the distribution of oxytocin mRNA-containing cell groups was studied at the macroscopic level. Specificity of hybridization was confirmed by comparison to vasopressin mRNA hybridization in parallel tissue sections. Cell groups containing oxytocin mRNA were confined to a set of hypothalamic cell groups, i.c. the supraoptic, paraventricular, anterior commissural nuclei, nucleus circularis and scattered hypothalamic islets. These cell groups displayed similar densities of autoradiographic signals indicating that the oxytocin gene is expressed at approximately the same average level at these various sites.