Amino acid incorporation during morphine intoxication

Neurochem. Int. Vol. 10, No. I, pp. 83-88, 1987 Printed in Great Britain. All rights reserved

0197-0186/87 $3.00+0.00 © 1987 Pergamon Journals Ltd

AMINO ACID INCORPORATION DURING MORPHINE INTOXICATION ELECTROPHORETIC SEPARATION OF EXTRACELLULAR PROTEINS FROM A BRAIN STEM ASTROGLIAL AND NEURONAL CONTAINING CO-CULTIVATION SYSTEM ELISABETH HANSSON* and LARS Rt3NNB.~CK*t~ Institute of Neurobiology* and Department of Neurologyt, University of Grteborg, Grteborg, Sweden (Received 25 April 1986; accepted 23 June 1986) Abstraet--Extracellular proteins from brain stem astroglial enriched primary cultures were resolved with gel electrophoresis after labelling the cultures with 3H/14C (experimental/control) valine. A protein fraction with a subunit molecular weight (mol. wt) of approx. 40,000 increased in aH/~4C valine labelling after incubating the cultures for 1 h in 10 -6 or 10 -5 M morphine or after incubation for 4 h in 10 -7 M morphine. After incubating the cultures for up to 4 h in 10 6 or 10 -5 M morphine proteins with subunits of mol. wt: ~ 15,000, 40,000 and 80,000 increased in labelling while subunits with mol. wt: ~65,000 and I00,000 decreased in labelling. After co-cultivating the astroglial-enriched primary brain stem cultures for 1 week with fetal (neuronal containing) brain stem cultures, incubation in l0 -7 or 10 6 M morphine for 1 h increased extracellular 3H/14C labelling of electrophoretically resolved material with mol. wt ~15,000 and 40,000. After incubation in the same concentrations for 2 h an additional 80,000 mol. wt fraction increased in 3H/14C labelling. After incubation in 10 -6 M morphine for 4 h the same result as above was obtained, the changes in the 15,000 and 80,000 mol. wt fractions being blocked by prior addition of 10 6M naloxone hydrochloride. Incubation in I0 5 M morphine did not result in significant changes in the co-cultivation system used. It is suggested that extracellular proteins, mainly astroglial-derived, might serve functions during morphine intoxication. Co-cultivation of astroglial-enriched and neuronal containing cultures might be one model system to study macromolecular communication between these cell types.

Several experimental approaches have suggested that ongoing protein synthesis is essential for morphine tolerance and physical dependence to develop although the acute effects of morphine can be mediated even after inhibition of brain protein synthesis (see e.g. Lee et al., 1979; Hitzemann et al., 1979; Clouet, 1971; Spoerlein and Scrafani, 1967; Loh et al., 1969). It has, however, been difficult to identify specific macromolecules that participate in long-term opiate actions. One reason is the complex structural and functional architecture of brain. Metabolic events in different cell types of various brain regions might simply average out, leaving little detectable evidence of specific effects over overall brain metabolism.

These circumstances can lead to an underestimation of protein changes after morphine treatment when separating heterogeneous protein populations (see e.g. Hahn and Goldstein, 1971; Franklin and Cox, 1972). Defined groups of proteins should be selected and separated with improved methodologies (see also Rrnnbfick et al., 1983b). Extracellular fluid proteins might be important for nervous tissue function. Such proteins are probably involved in the communication between cells and have been shown to change in amount during nerve activation, e.g. during acquisition of new behaviours (see Shashoua, 1976, 1979). In previous studies (Hansson and Rrnnb/ick, 1983, 1985) changes in the synthesis and/or release of proteins from astroglial-enriched cultures to the incubation medium have been demonstrated after morphine treatment. The results indicate that some pro-

:~Address correspondence to: Dr Lars Rrnnb/ick, Institute of Neurobiology, University of Grteborg, P.O.B. 33 031, S-400 33 Grteborg, Sweden. 83

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ELISABETH HANSSON a n d LARS RONNBACK

teins participate in cellular functions during morphine action. The experimental conditions have been extended and a co-culture system consisting of neuronal and astroglial cells has been developed (Hansson, 1986). Neuron-containing and astroglialenriched cultures were grown in close apposition to each other, yet physically separate, allowing easy separation of the cultures for subsequent analyses• Macromolecular communication between different cell types can thus be studied. In this paper changes in extracellular proteins after morphine treatment have been evaluated in cultures from brain stem, a region with opiate receptors in the rat (Goodman et al., 1980). EXPERIMENTAL PROCEDURES

Cultivation systems Brain stem primary cultures from newborn rat were cultivated for 14 days, as described by Hansson et al. (1984). The cells reached confluence after ~ 7 days of culture. Petri dishes, 50mm in diameter (Nunc A/S, Denmark) were used. Co-cultivation system: Primary cultures of brain stem from 15-17 day embryonic rat were cultivated for 10 days on poly-L-lysine (Pettmann et al., 1979) (Sigma Fine Chemicals, St. Louis, U.S.A.) coated lids of Petri dishes (35 mm in diameter; Nunc). The culture medium (Hansson et al., 1984) was supplemented with 30 mM glucose and 5/~g insulin per ml (Sigma). The cultures were treated with 10 5 M cytosine-1-/3-D-arabinofuranoside (Sigma) on day 6 and were co-cultivated on day 10 with 3 day-old primary brain stem cultures from newborn rat (Fig. 1). Medium was changed 3 times a week, and the experiments were done after 7 days of co-cultivation (see also Hansson, 1986). Morphine treatments: Twenty hours prior to the experiments, the fetal calf-serum was removed by repeated changes of the culture medium. Morphine chloride (Apoteksbolaget, Sweden; 10-7-10-5M) was added for 1, 2 or 4 h to the cultures in the incubator. In some experiments 10 6M naloxone hydrochloride (Apoteksbolaget, Sweden) was added prior to 10 6M morphine chloride and incubated for 4h. Five /~Ci [3H]valine (L-[3,4-3H]valine 56.8 Ci/mmol; 1 mCi/ml NEN, Boston, Mass.) and 5/iCi [~4C]valine (D-L valine, 1J4C 37.5 mCi/mmol) were added to the drug-treated and control cultures respectively. After incubation, the culture media were carefully removed. Media from 3 Petri dishes were pooled and passed through Millipore filter papers (mesh diameter

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Fig. 1. Schematic drawing of the co-cultivation system consisting of fetal neuron containing cultures and newborn astroglial-enriched cultures. Fetal cell material was cultivated for 10 days on poly-e-lysinecoated lids of Petri dishes (35 mm in diameter). Primary cultures from newborn rat were grown for 3 days in Petri dishes (50 mm in dia), and the fetal culture was transferred to this latter culture upsidedown and placed on a U-formed glass-rod I mm in diameter.

0.45 #m). The samples were treated as described in Hansson and R6nnbfick (1985). Statistical analysis was made according to Student's t-test. As small groups were tested, the significances were confirmed with the Wilcoxon signed rank test. RESULTS

Morphological characterization of fetal and newborn brain stem cultures before and after co-cultivation The brain stem cultures from fetal rat contained polygonal cells on day 10. The cells had a diameter of approx. 16 ~tm, containing one central nucleus with two or more nucleoli, and had processes extending from the cell bodies. The cells were regarded as astroglia due to immunohistochemical staining with antibodies to the glial fibrillary acidic protein (GFAp) (see e.g. Hansson et al., 1984). Among the polygonal cells were cells of different sizes with soma (10-25/~m in diameter) containing one central nucleus with only one visible nucleolus and a number of large processes extending from the soma (about 40-60% of the total cell number). The cells were regarded as neurons due to immunohistochemical staining with antibodies against the neuron-specific enolase (NSE) (see Hansson, 1986). Other cells in the cultures, such as ependymal cells, endothelial cells, macrophages, oligoblasts and mesenchymal cells were found in minor proportions (see also Hansson et al., 1984).

Extracellular proteins in brain culture during morphine treatment Prior to co-cultivation the primary brain stem cultures from newborn rat (3 day-old) contained scattered round or polygonal cells with central nuclei and with few processes. The cultures were not confluent and different cell types could not be separated. After 4 days of co-cultivation with fetal brain stem cultures containing neuronal (NSE-positive) cells, the cultures (totally 7 day-old) from newborn rat had become confluent, although not dense. Most cells were polygonal (dia. ~ 16 pm), contained one central

85

nucleus and had processes extending from the cell soma. These cells bound antibodies to G F A p . N o NSE-positive cells were seen in the primary cultures from newborn rat. After 7 days of co-cultivation, the GFAp-positive cells had even more processes extending from the cell soma and even branching (Fig. 2b). N o NSE-positive cells were seen. After co-cultivation with neuronal containing cultures, the cells in the astroglial-enriched cultures thus extended long processes which were not seen in the absence of cocultivation (Figs 2a,b).

Fig. 2a. Primary brain stem culture from newborn rat cultivated for 10 days (no co-cultivation) as described in the text. The cells are polygonal and spindle-shaped with few or no visible processes. Phase contrast.

Fig. 2b. Primary brain stem culture from newborn rat, grown for 3 days and co-cultivated for 7 days with fetal brain stem cultures containing neuronal (NSE-positive) cells, as described in the text and in Fig. I. Most cells are polygonal with large processes extending from the soma.

86

EL]SABETH HANSSON and

Protein changes in serum free media o f primary brain stem cultures after incubation with morphine There was a slight increase in dpm 3H/dpm]4C (experimental/control) of serum-free incubation media material separated on electrophoresis in gel regions No. 15 (~40,000 tool. wt) after incubating the cultures in 10 7M morphine for 4h. No significant differences in 3H/~C profiles were obtained after incubating the cultures in 10 7 M morphine for BRAIN STEM CULTURES

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1 or 2 h (Fig. 3). When the morphine dose was increased to 1 0 6 M or 10 5M gel region 15 increased in 3H over ~4C incorporation already after 1 h of incubation. After incubation for 2 h there was an increase in incorporation in gel regions Nos 15 and 27 ( ~ 15,000 mol. wt) and after incorporation for 4 h there were increases in gel regions No. 5 (~80,000 mol. wt) 15 and 27, and decreases in gel regions Nos 3 ( ~ 100,000 mol. wt) and 7 (~65,000 tool. wt). Incubation in 10 6M naloxone hydrochloride prior to 10 6M morphine chloride for 4 h did not result in any significant changes compared to those of 10 6M morphine alone (Fig. 3).

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After incubating the cultures for 1 h in 10 7 or 1 0 6 M morphine the incorporation of 3H over ]4C increased in gel regions Nos 15 and 27 (mol. wt as above). No significant effects were seen at a concentration of 10 5 M morphine. After incubation for 2 h there was an additional increase in 3H/]4C labelling in gel region No. 5. When the cultures had been incubated in 10 6M morphine for 4 h there were increases in labelling of gel slices Nos 5, 15 and 27, and decreases in gel slices Nos 3 and 7. The changes in gel slices Nos 5 and 27 were blocked by prior addition of 10 6M naloxone hydrochloride. Incubation in 10 5M morphine did not result in significant changes in this system (Fig. 4).

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The incorporation of radioactive amino acid into electrophoretically resolved protein bands changed in amount in extracellular material of primary astroglial-enriched cultures from brain stem after incubating the cultures with morphine. The affected protein bands corresponded to mol. wt of the subunits of approx. 15,000, 40,000, 65,000, 80,000 and 100,000. The results are in agreement with a previous study (Hansson and R6nnbfick, 1985). Even when the gels were sectioned into 32 slices, some protein bands or slices might contain up to 10 or probably more protein species. It is thus obvious that changes in one functionally important protein fraction might be hidden by changes in the opposite direction of other proteins within the same slice• It might therefore be valuable to separate the same group of proteins according to various characteristics to reduce the risk for artifactual over- or underestimation of protein changes.

Extracellular proteins in brain culture during morphine treatment BRAIN STEM CO-CULTIVATION 3H reline morphine

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the astroglial cells in both cultures are even biochemically differentiated by the co-cultivation with neuronal cells. The naloxone blockade of some of the protein changes caused by morphine in the cocultivation system might indicate some form of macromolecular communication between the cell types. So far we have not identified opiate receptors on astroglial cells (Hansson and Rrnnb/ick, 1983) which is in agreement with Van Calker and Hamprecht (1980), although contradictory results exist (Rougon et al., 1983). Although much more work remains to establish the role of the identified proteins in morphine action on the cellular level and although at present there are large difficulties in obtaining extracellular fluid material from small brain regions in vivo, the results demonstrate that this line of research might be fruitful for further understanding of macromolecular responses in opiate actions. Acknowledgements--The technical assistance of Maj-Britt

Magnusson and Tomas Machek is highly appreciated• The work was supported by the Swedish Medical Research Council (grant No. 25X-06005).

30

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Fig. 4. Experimental conditions similar as in Fig. 3 for brain stem primary cultures co-cultivated with neuronal containing primary brain stem cultures• Interestingly there was both a concentration dependence and a time sequence dependence for the appearance of the protein changes in this study• The data support and extend previous results from our laboratory (Rrnnb/ick et al., 1983a; Hansson and Rrnnb/ick, 1983; Rrnnb/ick and Hansson, 1985), where dose and time dependent changes in protein synthesis have been shown after morphine treatment both in rico and in culture systems. The extracellular proteins can be derived from the astroglial-enriched or from the neuronal containing culture. However, probably most changes must be of astroglial origin as similar qualitative changes after morphine were obtained from the astroglial cultures as from the co-cultivation culture system. In view of the differences between the two experimental set-ups concerning e.g. the extracellular fluid protein changes after low morphine concentrations, it might be that

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