Endorphins in human cerebrospinal fluid

Life Sciences, Vol. 31, pp. 1737-1740 Printed in the U.S.A.

Pergamon Press

ENDORPHINS IN H U M A N CEREBROSPINAL FLUID F. Nyberg and L. Terenius

Department of Pharmacology, University of Uppsala, Uppsala, Sweden (Received in final form June 14, 1982 SUMMARY Opiate activity in CSF samples drawn from patients with suspected intracranial hydrodynamic dysfunction has been fractionated on Sephadex G-10 and separated by column electrophoresisin agarose suspension. From the SephadexG-10 chromatography two receptor active fractions (FI and FII) were recovered. Both FI and FII were further resolvedby the electrophoresis. FI separated into at least four components and FII into two components. The study also includes a comparison of the endorphin concentrations in CSF (samples drawn from healthy volunteers) measured by receptorassay with those detected by radioimmunoassay of /~-endorphin, [Met]enkephalin and dynorphin, respectively.The data obtained indicated negligiblequantities of the radioimmunoassayable endorphins in the total CSF opiate activity. The measurements of endorphins in human lumbar cerebrospinal fluid (CSF) was introduced in this laboratory several years ago. A simple chromtographic separation on a Sephadex G-10 column preceeded quantification in a radioreceptorassay (1). This method has been found to provide resuRs which frequently correlate to clinical symptomatology in patients with chronic pain (2) or psychiatric disorder (3). In the course of this work it also became clear that the material active in the receptorassay was not attributable to any of the three principle classes of endorphins viz, the enkephalins,/3-endorphin or dynorphin (4). In another paper (5) we have shown that endorphin activity measured with the original procedure (1) actually represents several active substances. Sensitive structural assignments suggest that they are related to the enkephalin and dynorphin systems. The enkephalin precursor, prepro-enkephalin has been identified (6--7) and known to contain no less than six [Met]enkephalin sequences and one [Leu]enkephalin sequence. During the processing of this precursor, it is likely that longer fragments with opioid activity are formed; in fact, such fragments have already been isolated from the adrenals (9, 10) or pituitary gland (11). The purpose of the present paper is to compare the characteristics and concentration of the endorphins identified by receptorassay with those of/3-endorphin, [Met]enkephalin and dynorphin, respectively. MATERIAL AND METHODS Reference peptides were purchased from Peninsula Labs., San Carlos, USA, except for the enkephalins and [Leu]enkephalin-Arg6 and [Met]enkephalin-Arg6 Phe 7 which were obtained from Bacbem, Bubendorf, Switzerland. Tritium-labelled compounds were purchased from Amersham, England, and purified by high l~erformance liquid chromatography (HPLC) if necessary. Labelling of dynorphin (1--17) and ~-endorphin with lzsI followed the chloramine T procedure. Labelled peptides were adsorbed onto a small SP-Sephadex ion exchanger column in dilute pyridine-formiate buffer which was thoroughly washed to remove free 12sIand then eluted with stepwise increases in ionic strength. Chromatographic media, Sephadex products and agarose (Agarose C) used as a support in electrophoresis were from Pharmacia, Fine Chemicals, Uppsala, Sweden. All solvents and other chemicals were of reagent grade. CSF samples of 50 ml each were obtained from three patients with suspected intracraniai hydrodynamic dysfunction and who underwent exploratory studies of CSF dynamics. All patients were drug-free. One of them (H. N., see Fig 1) was found to have a defective blood-brain barrier. The sample was obtained by lumbar puncture over a period of about 30 minutes, with the patients in a supine position. Samples of 12.5 ml CSF were also obtained from a series of healthy volunteers by lumbar puncture of the subject in a lateral recumbent position. These procedures had been approved by the Ethical Committee of the Ume~t University. The CSF samples were run without any processing through Sephadex G-10 columns (5 x 100 cm for the 50 ml volumes and 2 x 50 cm for 4 ml aliquots of volunteer CSF). Both columns operated at a flow rate of 60 ml/h and fractions of 20 and 5 ml, respectively, were collected. In all experiments the Fraction I and Fraction II material defined as in (5) for large scale runs and as in (1) for 4 ml runs, respectively, was pooled and lyophilized. 0024-3205/82/161737-04503.00/0 Copyright (e) 1982 Pergamon Press Ltd.

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Endorphins in Human CSF

Vol. 31, No.s 16 & 17, 1982

Electrophoresis in columns with a dilute (0.18070 w/v) agarose suspension was performed as described by Hjert6n (12). The agarose was dissolved in boiling water and after 1--2 hours cooling, the gel was gently stirred to form a homogenous suspension. A concentrated ammoniumformiate buffer was added to the suspension in proportions suitable to make the final buffer 0.10 M and of pH 2.7. A column of 0.9 x 65 cm was prepared and the previously lyophilized sample, dissolved in 0.5 ml agarose suspension was applied. Electrophoresis was run for 6 h at 1 000 V and 10 mA current. After termination of the electrophoresis, fractions of 0.4 ml were sucked out from the top end of the column with a fine plastic tubing attached to a syringe. The fractions were diluted with 1 ml of water and the agarose was removed by centrifugation for 5 min in a Beckman Microfuge B. The fractions were iyophilized and tested in the radioreceptorassay. Reference peptides used to calibrate the column were recovered in 70070 or higher yields when run in the femtomole to nanomole range. The radioreceptorassay was performed as described previously (1, 3) using synaptic plasma membranes from whole rat brain without cerebellum and tritium-labelled dihydromorphine as the competing radioligand. Each run included a calibration curve with [Met]enkephalin and the binding activity of the tested fractions was expressed in [Met]enkephalin equivalents. Reference peptides in logaritmic dilutions were tested in an identical fashion. Radioimmunoassays were carried out with overnight incubations, which were terminated by the addition of dextrane-coated charcoal as described elsewhere (13). The total assay volume was 0.225 ml. Assays for [Met]enkephalin were run in the following buffer (0.1 M Na phosphate, pH 7.4, 0.15 M NaC1, 0.93070 EDTA, 0.1°70 gelatin) while assays for dynorphin and/3-¢ndorphin were run in a slightly modified buffer (0.2 M Na-phosphate, pH 7.4, 0.15 M NaC1, 0.02070 Naazide, 0.1 070gelatin, 0.01 070BSA, 0.1 070Triton-X). Crossreaction of the [Met]enkephalin antiserum was approximately 0.8070 with [Leu]enkephalin, about 1070for [Met]enkephalin-Arg6, -Lys 6 and -Arg6Phe 7 and less then 0.01% for the dynorphin, its fragments and ~-endorphin. The dynorphin antiserum was raised against dynorphin (1--13) but tested with the iodinated (1--17) sequence as radioligand. Crossreaction with the (1--13) fragment was 100%, with the (1--8) fragment, enkephalin penta- and hexa-peptides and ~-endorphin less than 1070.Finally, the ~-endorphin antiserum showed less than 107o crossreactivity against all mentioned peptides. RESULTS The first part of the study involved fractionation of 50 ml samples of CSF from individual patients. Chromatography of such samples on a Sephadex G-10 column ehited with dilute acetic acid reveals a complex pattern. Material competing with dihydromorphine for opioid receptors elutes close to the void volume, in the Fraction I (FI) region and after the salt peak in the Fraction II (FII) region (5). Very little active material elutes as the enkephalins (5). The FI and FII material isolated from CSF of three individual subjects were further fractionated by column electrophoresis (Fig. 1). FI material appeared in four main regions as previously shown for a large pool of CSF from various neurologic and psychiatric patients (5). Following the terminology previously introduced (5), opioid activity separated into the FIA, FIB, FIC and FID regions. In the previous work FIA and FIC represented the major components. In the three patients shown here (Fig. I a), the situation seems more complex: in patient K. B., FIC is by far the dominating component, in patient H. N., FIA and FIC are about equal and in patient S. N. FIA is the dominating species. There is in fact an indication for a reciprocal relationship between Fraction IA and IC. FIB definitely appears to separate into two components whereas our previous study (5) showed one dominating component (of low mobility). The FII material (Fig I b) appeared to be chemically less complex in agreement with previous results (5). FIIA and FIIB activity was identified in all three patients. The relative concentrations of the peaks varied, being higher in FIIB, patients K. B. and S. N., or higher in FIIA, patient H. N. (Fig. 1 b). The second part of the study involved measurement of FI and FII endorphins by radioreceptorassay and [Met]enkephalin, dynorphin (1--13, 1--17) and/3-endorphin by radioimmunoassay, in individual CSF samples from healthy volunteers. Levels of FI and FII activity are comparable to those previously described (1--3). Levels obtained in radloimmunoassays were negligible (Table 1). To allow quantitative comparisons between levels obtained in the receptorassay and the radioimmunoassays, some reference peptides were tested in the receptor assay. The following ICs0 values (nM) were obtained: [Met]enkephalin, 7.5; [Met]enkephalin-Arg*, 7.0; [Met]enkephalin -Arg*Phe 7, 18. DISCUSSION The results presented in this paper raise the question of which endorphins actually are released by the neurons and which pcptides are the functionally important species. It seems particularly puzzling that levels of the enkephalins and dynorphin (1--17) are so low, since they occur in large quantities in brain tissue. It is, of course, possible that enkephaiin-related peptides with Cterminal elongation with a few amino acids are metabolically more resistant than the enkephalins themselves and dynorphin (1--17) and therefore reach the CSF. However, the very exis-

Vol.

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Endorphins

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FIG. 1 Column electrophoresls o f FI (a) and FII (b) material isolated from 50 ml samples o f CSF in O.18% agarose suspension. Opioid activity was monitored by a radioreceptorassay. The figure shows data from three individual natients. The positions o f some reference l~eDtides are also included.

tence o f a large number o f different opioid peptides in the CSF suggests that these peptides may also elicit an effect and that the signal from the endorphin neurons is chemically very complex. This is not unexpected in view o f the recently obtained information on the structure o f preproenkephalin which contains no less than six [Met]enkephalin sequences and one [Leu]enkephalin sequence. It is likely that the precursor is processed by trypsin-like enzymes at sites with paired basic amino acid sequences which would give rise to [Met]enkephalin and the -Lys 6, -Arg s, -ArgePhe 7 and -ArgeGlyTLeuS fragments, while [Leu]enkephalin may be accompanied by the -Lys 6 sequence. In fact, several o f these peptide sequences have been isolated (9--11) and found to have considerable receptor affinity. The dynorphin precursor is not yet identified and it is therefore not possible to establish whether any o f these short fragments could also derive from the dynorphin system or not. However, the shortest possible fragment which could not derive from the enkephalin precursor, [Leu]enkephalin-Arg s, and on the other hand is part o f the dynorphin sequence, occurs in very small quantities in brain (unpublished observations) and could not be demonstrated in the CSF samples. We would therefore favour the notion that peptides in the FII area derive from the enkephalin precursor, while in the FI area at least one component may derive from the dynorphin system. Direct chemical identification will finally settle this question. We have tentatively assigned FIIA the structure [Met]enkephalin-Lys 6 while the analogous -Arg e fragment could not be detected in measurable quantities (5). The identity o f the FIIB component remains unclear.

TABLE I Opioid activity in human CSF o f healthy volunteers. Data were obtained by receptorassay o f F l and FII material derived f r o m 4 ml CSF f r o m each individual subject and by specific radioimmunoassay after chromatographic fractionation o f I ml CSF f r o m each subject SUBJECT

FI*

FII*

G.A. J.R. D.S. E.L. V.O.

1.5 0.8 <0.4 2.4 1.3

7.2 4.9 6.9 5.2 5.3

* calculated as [Met]-enkephalinequivalents.

[Met]enkephalin (pmole/ml) <0.05 <0.05 <0.05 <0.05 <0.05

Dynorphin

/3-endorphin

<0.02 <0.02 <0.02 <0.02 <0.02

<0.02 <0.02 0.34 <0.02 <0.02

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Vol. 31, No.s 16 & 17, 1982

The composition of the FI material is complex. We can identify at least four, maybe five, different major components. One interesting observation is the apparent inverse relationship between FIA and FIC which might indicate that they represent different stages of processing of a single precursor. We have previously tentatively assigned FIC the dynorphin (1--8) sequence (5). FIA may then represent a larger fragment. However, radioimmunoassay data indicate very low levels o f dynorphin (1--13) or (1--17). Previous data on the presence of dynorphin immunoreactive material were based on the use of another antiserum raised against dynorphin (1--13). We have later found that this antiserum recognizes a component of neural tissue extracts and CSF distinct from any of the dynorphin (1--8), (1--13) and (1--17) fragments (I. Christensson et al, unpublished.) We are presently trying to establish its identity. Since the dynorphin precursor is yet unknown, it is not possible to speculate about the nature of this fragment. Radioimmunoassay analysis showed very low levels of all the studied species, [Met]enkephalin, dynorphin (1--13; 1--17) or ~3-endorpin. The dynorphin antiserum was not very selective and the j3-endorphin antiserum would also recognize the N-acetylated derivative which has been found to occur in brain (14). ~-endorphin has been found to produce very long-lasting analgesia if introduced into the cerebral ventricles of cats, its virtual absence can therefore hardly be explained by rapid degradation. It should be remembered, however, that 13-endorphin projections do not extend to the spinal cord and low levels in lumbar CSF samples might be expected just on this basis. On the other hand, dynorphin occurs in the spinal cord (15) and could be expected to reach CSF spaces. It has been found to have very feeble if any analgesic activity (16) indicating that it may be very susceptible to metabolic breakdown. It seems clear from this, by no mean extensive discussion, that there must be a complex relationship between the anatomically defined distribution of the neurons containing opioid peptides, the technical constraints associated with the CSF sampling procedure (site of sampling and sample volume) and the functional state in the neurons we attempt to investigate. The data presented here indicated the necessity to perform extensive chemical characterization of opioid peptides of the CSF as also indicated in work made by others (17). Such work is in progress. Finally, our data indicate large variations between patients with similar disease history and similar sampling protocol. The functional significance of these differences remain to be determined. Professor J. Ekstedt and Dr B. Almay are gratefully acknowledged for supplying the CSF samples. We are also indebted to Ms I. Hansson, Ms I. Eriksson, Ms M. Einarsson and Mr P. Le Grev6s for expert technical assistance. The work was supported by the National Institute on Drug Abuse, Washington (Grant No DA 1503) and the Swedish Medical Research Council (Grant No 25X-3766).

REFERENCES 1. L. TERENIUS, and A. WAHLSTROM, Life Sci 16, 1759--1764 (1975). 2. L. TERENIUS, Front Horm. Res 8, 162--177 (1981). 3. L. H. LINDSTROM, G. BESEV, L. M. GUNNE, R. SJOSTROM, L. TERENIUS, A. WAHLSTROM and B. WISTEDT, In: Endorphins and opiate antagonists in psychiatry (Eds. Shah and Donald), Plenum, New York and London 1981, in press. 4. A. WAHLSTROM and L. TERENIUS, Febs Left. 118, 241--244 (1980). 5. F. NYBERG, A. WAHLSTROM, B. SJOLUND and L. TERENIUS, Brain Res., in press. 6. M. NODA, Y. FURUTANI, H. TAKAHASHI, M. TOYOSATU, T. MIROSE, S. INAYAMA, S. NAKANISHI and S. NUMA, Nature 295, 206---208 (1982). 7. U. GUBLER, P. SEEBURG, B. J. HOFFMAN, L. P. GAGE And S. UDENFRIEND, Nature 295, 206--208 (1982). 8. M. COMB, P. H. SEEBURG, J. ADELMAN, L. EIDEN and E. HERBERT, Nature295, 206--208 (1982). 9. A. S. STERN, R. V. LEWIS, S. KIMURA, J. ROSSIER, S. STEIN and S. UDENFRIEND, Biochem. Biophys. Res. Comm. 205, 698--705 (1981). 10. D.L. KILPATRICK, B. N. JONES, K. KOJIMA and S. UDENFRIEND, Biochem. Biophys. Res. Comm. 103, 698--705 (1981). 11. B.R. SEIZINGER, V. HOLLT and A. HERZ, Biochem. Biophys. Res. Comm. 102, 197--205 (1981). 12. S. HJERTI~N, J. Chromatogr. 12, 510--526 (1963). 13. L. BERGSTROM and L. TERENIUS, Europ. J. Pharmacol 60, 439--352 (1979). 14. S. ZAKARIAN and D. G. SMYTH, Nature 296, 250---252 (1982). 15. L.J. BOTTICELLI, B. M. COX and A. GOLDSTEIN, Proc. Natl. Acad. Sci 78, 7783--7786 (1981). 16. H.J. FRIEDMAN, M. F. JEN, J. K. CHANG, N. M. LEE and H. H. LOH, Europ. J. Pharmacol. 69, 357--360 (1981). 17. T. FURUI, N. KAGEYAMA, T. HAGA, A. ICHIYAMA and M. FUKUSHIMA, Pain 9, 63--72 (1981).