L. Dillen

Posttranslational processing of proenkephalins and chromogranins/secretogranins

Posttranslational processing of peptide-precursors is nowadays believed to play an important role in the functioning of neurons and endocrine cells. Both proenkephalins and chromogranins/secretogranins are considered as precursor molecules in these tissues, resulting in posttranslationally formed degradation products with potential biological activities. Among the proteins and peptides of neuronal and endocrine secretory granules, the enkephalins and enkephalin-containing peptides have been most extensively studied. The characterization of the post-translationally formed degradation products of the proenkephalins have enabled the understanding of their processing pathway. Chromogranins/secretogranins represent a group of acidic glycoproteins, contained within hormone storage granules. The biochemistry, biogenesis and molecular properties of these proteins have already been studied for 25 years. The chromogranins/secretogranins have a widespread distribution throughout the neuroendocrine system, the adrenal medullary chromaffin granules being the major source of these storage components. Recent data provide evidence for a precursor role for all members of the chromogranins/secretogranins family although also several other functions have been proposed. In this review, some of the methods applied to study proteolytic processing are described. In addition, the posttranslational processing of chromogranins/secretogranins and proenkephalins, especially the biochemical aspects, will be discussed and compared. Recent exciting developments on the generation and identification of potential physiologically active fragments will be covered.

Chromogranin A processing in sympathetic neurons and release of chromogranin A fragments from sheep spleen

Chromogranin A (CGA) has been localized to the large dense cored vesicles (LDV) of sympathetic neurons. SDS‐PAGE and immunoblotting of soluble LDV proteins from ox and dog adrenergic neuronal cell bodies, axons and nerve terminals, revealed an increasing number of CGA‐immunoreactive forms, consistent with proteolytic processing during axonal transport. Splenic nerve electrical stimulation (10 Hz, 2 min) revealed that, apart from CGA. these CGA‐processing products are released from the sheep spleen. The secretion of CGA‐derived fragments from sympathetic neurons might suggest a role in the regulation of synaptic transmission.

Mass spectrometer characterization of bovine chromaffin granule peptides related to chromogranin B

Peptides were extracted from the lysate of isolated bovine chromaffin granules. Following reversed-phase HPLC purification, the fractions were analyzed by FAB/MS. The presence of methionine-enkephalin and leucine-enkephalin was indicated by their chromatographic retention time and by the m/z value of their protonated molecules. As to five new peptides related to chromogranin B, prominent protonated molecules were observed at m/z 1746, 1446, 1333, 977 and 901. Trypsinolysis resulted in a common loss of a component with mass 545, pointing to a structural relationship and a common precursor molecule. The peptide showing a (M+H)+ ion at m/z 1746 could be identified as a novel, recently reported, neuropeptide derived from chromogranin B, whereas the other peptides with (M+H)+ ions at m/z 1446, 1333, 977 and 901 could be characterized as smaller fragments of this peptide. Peptidase-guided sequence analysis and MS/MS analysis provided sequence information.

Characterization of serotonin receptors and lack of effect of antidepressant therapy on monoamine functions in various regions of the rabbit brain

The effects of single and long-term administration of the antidepressants imipramine, desimipramine, amitriptyline, zimelidine and maprotiline were studied in the rabbit brain. Special attention was given to the brain serotonin (5-HT) receptors. Our results show that in different areas of the rabbit brain, the binding sites for 5-HT display pharmacological characteristics very similar to those of the 5-HT1 and 5-HT2 receptors described for the rat brain. No significant correlation could be shown between the distribution of either of the receptors and the distribution of serotonergic nerve terminals (as measured by the 5-HT content and the [3H]5-HT accumulation). Addition of antidepressants to rabbit brain slices, in vitro, caused an inhibition of the [3H]5-HT accumulation. The compounds only weakly inhibited the binding of [3H]5-HT and [3H]ketanserin as compared to the inhibition caused by serotonergic agonists and antagonists. The [3H]5-HT accumulation in brain slices was markedly reduced 2 h after a single i.p. injection of imipramine. After a two-week administration of the antidepressants, the specific binding of neither [3H]5-HT nor [3H]ketanserin was significantly altered. The simultaneous determination of monoamine metabolites and of dopamine-beta-hydroxylase in the cerebrospinal fluid of these treated rabbits did not reveal any significant difference from the control animals.

Simultaneous determination of the three major monoamine metabolites in cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection

A simple method is described for the simultaneous determination of the three monoamine metabolites, 4-hydroxy-3-methoxyphenylacetic acid, 4-hydroxy-3-methoxyphenylethyleneglycol and 5-hydroxyindole-3-acetic acid, in cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection. Quantitation is accomplished by the standard addition technique. Chromatographic peak heights are corrected for volume effects by comparison with the signal obtained for an added auxiliary reference substance. Sample preparation is kept to a minimum, involving precipitation of proteins by means of perchloric acid and subsequent neutralization. The reproducibility was estimated to be 10%. For one of the metabolites, 4-hydroxy-3-methoxyphenylethyleneglycol, a correlation between the results obtained by this method and a mass fragmentographic method was made, and a satisfactory correlation (r = 0.904, slope = 0.914, intercept 3.26 ng/ml) found. The sensitivity of the method is in the picogram range. The methodology has been applied to measure biogenic amine metabolites in both rabbit and human cerebrospinal fluid. The levels found are in agreement with previously reported values.

Study of the enzymatic degradation of vasostatin I and II and their precursor chromogranin a by dipeptidyl peptidase IV using high-performance liquid chromatography/electrospray mass spectrometry

The interaction of dipeptidyl peptidase IV with structurally related proteins differing in chain length, namely vasostatin I and II and their precursor protein chromogranin A, was examined using high-performance liquid chromatography in combination with electrospray mass spectrometry. Suitable analytical procedures were developed involving the use of reversed-phase high-performance liquid chromatography for purification of the enzymatic degradation products and a peptide mapping procedure for evaluating the enzymatic degradation of the large precursor protein chromogranin A. While vasostatin I was found to be a substrate for dipeptidyl peptidase IV, no N-terminal cleavage of Leu-Pro could be noted for chromogranin A. With respect to vasostatin II, N-terminal degradation was only observed after degradation in the C-terminal domain to proteins containing < or = 78 amino acids. The specificity of the N-terminal release of Leu-Pro was proved by addition of a DPP IV specific inhibitor.

Determination of dopamine-β-hydroxylase in cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection

Abstract An improved method is described for the measurement of dopamine-β-hydroxylase (DβH) activity in cerebrospinal fluid, which is based on an incubation with dopamine at a saturated substrate concentration and quantitation of the reaction product noradrenaline, by high-performance liquid chromatography with electrochemical detection using 3,4-dihydroxynorephedrine as internal standard. Sample workup consists in an ion pair extraction to isolate the catecholamines from the rather complex incubation medium, a cation ion exchange to eliminate the bulk amount of dopamine, and alumina adsorption to concentrate the sample prior to high-performance liquid chromatography. The methodology was used to evaluate some of the characteristics of DβH in cerebrospinal fluid and the stability of the enzyme. The procedure was also employed to determine the change in the DβH following drug administration. Intravenously administered yohimbine caused an increase in DβH activity in cerebrospinal fluid of rabbits as expected from its known α 2 -antagonist properties.

Mass spectrometric identification of phosphorylated vasostatin II, a chromogranin A-derived protein fragment (1–113)

Vasostatin II, an N-terminal chromogranin A-derived protein (CGA1-113), was purified from bovine chromaffin granule lysate and characterized by electrospray mass spectrometry (ES/MS) as being partially phosphorylated. The phosphorylation site was determined to be at the Ser81 position by mass spectrometric peptide mapping and tandem mass spectrometric analysis. This phosphorylation site is close to the processing site (...QKK78HSS(p)81...) yielding vasostatin I, an N-terminal CGA-derived peptide comprising residues 1-76, suggesting that phosphorylation at Ser81 is involved in the formation of vasostatin I in chromaffin cells.

Effects of the α2-antagonist idazoxan on monoaminergic parameters measured in the cerebrospinal fluid of rabbits

The alpha 2-antagonist idazoxan was administered intravenously to rabbits. The increase in central noradrenergic, dopaminergic and serotonergic activity was followed as a function of time by determining neuronal parameters in the cerebrospinal fluid (CSF) and was compared with changes previously determined after yohimbine. These parameters include the enzyme dopamine-beta-hydroxylase (D beta H), the noradrenergic metabolites 3-methoxy-4-hydroxyphenylmandelic acid (VMA) and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), the dopaminergic metabolite 3-methoxy-4-hydroxyphenylacetic acid (HVA) and the serotonergic metabolite 5-hydroxyindole acetic acid (5-HIAA). Control experiments with physiological saline were also performed. D beta H activity increased to 211% in control experiments, and to 570 and 530%, respectively after yohimbine and idazoxan. Compared to the control experiments yohimbine was able to elevate VMA, MHPG and HVA concentrations, but 5-HIAA levels were reduced. Idazoxan caused increased MHPG concentrations, slight increases in VMA, little effect on HVA and no effect on 5-HIAA levels. We conclude that idazoxan was as potent as yohimbine as an alpha 2-antagonist in our in vivo experiments and that idazoxan shows a much greater selectivity with regard to the noradrenergic system.

Evaluation of sample work-up methods and internal standards for the determination of catecholamines in urine by HPLC with electrochemical detection

Two sample work-up methods: (I) one consisting of adsorption of the catecholamines onto alumina followed by ion pair extraction and (II) another consisting of isolation by cation exchange and subsequent adsorption onto alumina, have been evaluated for the assay of urinary catecholamines by means of HPLC with electrochemical detection. With the aim of achieving high precision, two internal standards, i.e. dihydroxybenzylamine and epinine, have been compared. The results indicate that clean HPLC chromatograms are obtained with both work-up methods and that the highest precision (RSD < 4%) is achieved with method II and with epinine as internal standard, whereas the lowest precision is obtained with method I and with dihydroxybenzylamine.