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Dynorphin-converting enzyme in human cerebrospinal fluid. A negative correlation between activity and levels of derived

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Claes Post Henning Værøy Linköping University Akershus universitetssykehus

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The user has requested enhancement of the downloaded file. s155 -CONVERTING ENZYME IN HUMAN CEREBROSPINAL FLUID. A negative correlation between activity and levels of prodynorphin derived opioid peptides. Madeleine ThSmwall, Susanne Schwarzmayr, Bela Almay *, Claes Post **, Henning VaerCy *** and Fred Nyberg. Dept. of Pharmaceutical Biosciences, Division of , University of Uppsala, P.O. Box 591, S-751 24 UPPSALA, Sweden.* Dept. of Neurology, University of Ume~, S-801 87 UMEA, Sweden.** Astra Draeo AB P.O. Box 34, S-221 20 LUND, Sweden.*** Dept. of Psychiatry, Gjcvik Fylkessykehus, N-2800 GJI~VIK, Norway. In this work we have studied the dynorphin convertase (DCE) in cerebrospinal fluid (CSF) collected from patients with different states of chronic pain. The assessment of the enzyme activity was accompanied with measurements of the CSF level of prodynorphin derived opioid peptides. In CSF from patients with fibromyalgia the DCE activity was found to be significantly decreased as compared to that of control samples. In contrast a significant increase was found for the level of immunoreactive . In CSF from patients with idiopathic pain a significant negative correlation between all prodynorphin derived peptides and DCE activity was observed. The results suggest a regulatory function on the level of the dynorphin convertase. Table I Propertiesof CSF Dynorphin-convertingenzyme.(3) Previous studies have demonstrated the pretence of a specific convertase acting Mw ~40000 on prodynorphin derived opioid peptides Inhibited by PMSF and DFP, classifying DCE as a serine protease in human cerebrospinal fluid. The pH-optimum: -7.4 enzyme has a molecular weight of around Isoeletric point: -6.9 40 kdalton and was shown to hydrolyze Cleavage sites: Arg-Arg/Arg-Lys the substrate peptides at their paired basic Specificity: Dynorphin A Kin=t30 paM amino acid residues. In accordance with Specific activity: 2.4 mUImg enzyme protein its sensitivity towards protease inhibitors Kin--160 l.tM as PMSF and DFP it was classified as a Specific activity: 0.4 mU/mg enzyme protein serine protease (table I). The CSF samples studied in this work were obtained by lumbar puncture from 25 different subjects with chronic pain, where 9 patients were diagnosed with idiopathic pain and 16 with fibromyalgia. Controls were 11 urological patients with conditions without pain. The frozen CSF sample (2 ml) was thawed and directly separated on a disposable reversed phase octadeca-silyl silica gel, SepPak, cartridge. Before use, the cartridge was successively washed with 5 ml methanol, 5 ml methanol containing 0.04 % trifluoroacetic acid (TFA) and 10 ml H20/0.04 % TFA. The samples were then applied onto the cartridges. After washing with 4 ml H20/0.04 % TFA and 20 % methanol/0.04 % TFA, elution was performed with 4 ml 100 % methanol containing 0.04 % TFA. The fractions were evaporated and subjected to tryptic treatment prior to analysis by radioimmunoassay (RIA) as described elsewhere (2). The converting activity was monitored by following the formation of Leu--Arg6 from synthetic dynorphin A. The incubations were performed in 50 mM Tris/l-lC1, pH 7.4 in Eppendorf tubes in a final volume of 50 lxl. In order to prevent further degradation of the product the protease inhibitors , captopril and phosphoramidon (inhibitors of , -converting enzyme and neutral endopeptidase EC.3.4.24.11, respectively) were added in a final concentration of 20 ~tM. The incubations were allowed to proceed for 120 rain at 37oc and the reactions were terminated by adding cold methanol. Samples were evaporated and the released product was quantified by RIA.

The results showed that levels of DCE activity are significantly decreased in CSF collected from patients with fibromyalgia as compared to control samples (fig. la). Further, in a previous study (4) it was found that CSF levels of dynorphin A are significantly increased in fibromyalgia patients (fig. lb). It seems that in these patients the enzyme activity is lowered at the expense of increased peptide levels. In patients with idiopathic pain the activity of DCE was found to be elevated and the enzyme activity correlated negatively with the peptide concentration (fig. 2). S156 m

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Control Flbromyalgla Control Flbromyelgla n=l 1 .=16 n=6 n=32 Fig. 1. a) Dynorphin-eonverting enzyme activity in CSF from patients with fibromyalgia, expressed as rate of formation of Leu-enk-Arg6 ( pmol/h*ml CSF) from dynorphin A ('t)<0.05) b) Dynorphin A levels in CSF from patients with fibromyalgia, (fmol/ml CSF). (*p<0.05)

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Fig.2. Regressionanalysis between levels of LEA-PP (fmol/ml) and DCE activity (pmol Leu-enk-Arg6/h*ml CSF). (1)<0.02, n=9) It appears from this study that in patients with fibromyalgia, a decrease in DCE activity is contrasted by an increase in the substrate peptide. These results combined with the negative correlation between prodynorphin derived peptides and DCE in CSF from patients with idiopathic pain may indicate a regulatory role of enzyme in pain processing pathways within the CNS. The present results suggest that the assessment of the CSF activity in the prodynorphin system (~cluding measurements of DCE) may have a clear potential in the differential diagnosis of chronic pain syndromes. A chaUange to future work would be to follow up these studies on a broader scale, also to include patients with other pain diagnosis. (This study was supported by the Swedish Medical Research Council, Grant No. 9459).

. Nyberg, F., Nordstrtm, K. and Terenius, L. Biochem. Biophys. Res. Commun. (1985), 131, 1069-1074.

. Silberring, J. and Nyberg, F. J. Biol. Chem. (1989), 264, 11082. 3. Demuth, H-U. and Nyberg, F. J. Enzyme Inhibition (1991), 4, 299-306. 4. VaerCy, H., Nyberg, F. and Terenius, L. Pain (1991), 46, 139-143.

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