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Presence of opioid peptides in a neuroblastoma X glioma hybrid cell line
319
European Journal of Pharmacology, 65 (1980) 319--320 © Elsevier/North-Holland Biomedical Press
Rapid communication PRESENCE OF OPIOID PEPTIDES IN A NEUROBLASTOMA X GLIOMA HYBRID CELL LINE THOMAS GLASER *, DIETRICH VAN CALKER, KARIN HUBNER, CHRISTINE STADTKUS and BERND HAMPRECHT
Physiologisch-Chemisches Institut der Universit~it, Wiirzburg, F.R.G. Received 18 June 1980, accepted 23 June 1980
Neuroblastoma x glioma hybrid cells 108CC15 display many neuronal properties (for review see Hamprecht 1977). Besides excitable membranes and the capacity to synthesize acetylcholine (ACh) they possess a number of receptors for neurohormones. Some hormones like prostaglandin E1 (PGE~), adenosine and secretin stimulate the formation of adenosine 3',5'-cyclic monophosphate (cAMP) in the hybrid cells. Other hormones such as the opioids (among them the enkephalins), noradrenaline, ACh and somatostatin inhibit the elevation by PGE~ of the level of cAMP. Therefore, the hybrid cells can be used to detect the presence of substances, which inhibit the formation of cAMP. Employing the hybrid cells as a test system we have detected several factors in acidic extracts of the same hybrids, which inhibit the stimulation by PGEI of the level of cAMP (T. Glaser et al., in preparation). We now report that opioid peptides were among the factors extracted from neuroblastoma x glioma hybrid cells 108CC15. Cells were grown in Petri dishes (145 mm in diameter) as described (Hamprecht, 1977). After 4 days in culture, cells were washed twice before they were extracted by I M acetic acid. After addition of [3H]Leu-enkephalin (40 000 cpm) as recovery standard, the extract was lyophilized, dissolved in water, * To whom correpondence should be sent: Physiologisch-Chemisches Institut, Koellikerstr. 2, D 8700 Wiirzburg, F.R.G.
centrifuged and poured on a column filled with Bio-Beads SM2. After washing with buffer, opioids were eluted with 50% ethanol. The fraction containing opioids was chromatographed on a Sephadex LH-20 column (T. Glaser et al., in preparation). Fractions containing radioactivity were pooled and tested for opioid activity. Extracts thus purified are called partially purified extracts. Hybrid cells containing 720 mg of protein were extracted and the extract was purified as described above. With the aid of a radioimmunoassay involving antibodies prepared against Leu-enkephalin, 0.1 pmol of Leuenkephalin equivalents was found per mg hybrid cell protein in the extract; the amount was calculated from the data shown in fig. 1, panel A. The antibodies cross-react with Metenkephalin, but not with the endorphins or a number of other peptide hormones (Van Calker, 1977). The extract was also tested for its ability to inhibit the elevation by PGEI of the level of cAMP in the hybrid cells. Fig. 1, panel B, shows that similar to Leu-enkephalin (fig. 1B, curve a) the extract (curve b)inhibits the formation of cAMP stimulated by PGEI. In addition, the effect of both Leu-enkephalin (curve c) and the partially purified extract (curve d), is blocked by the opioid antagonist naloxone (10 pM). Besides the cross reaction of the extract with the anti-Leu-enkephalin antiserum, the effect of the extract on the formation of cAMP further indicates that opioid-like material is present in extracts from
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hybrid cells. The concentration of opioids in the extract solution determined by radioimmunoassay very well agrees with the concentration f o u n d in the cAMP assay. Opioid extracted from the hybrid cells also competes with [3H]naloxone for binding to opioid receptors in membranes of hybrid cells (T. Glaser et al., in preparation). Further purification of extracts by high performance liquid chromatography renders two active fractions, with the same retention times as Met- and Leu-enkephalin. In addition, the Met-enkephalin-like material, but not the Leu-enkephalin-like material is inactivated by t r e a t m e n t with cyanogen bromide. These results suggest t h a t the two active fractions correspond to Met- and Leu-enkephalin. Under the assumption t h a t the hybrid cells are homogenous, the finding of two active fractions suggests t h a t both enkephalins can be present in one cell. A c o m m o n precursor for both Met- and Leu-enkephalin has recently been described (Kimura et al., 1980). Our results suggest that the hybrid cells can be used as a model system for studying the biosynthetic pathways of the enkephalins. To our knowledge, this is the first time t h a t a cell line is reported to synthesize opioids that may be identical with the enkephalins. In contrast to our findings it has been reported (Giagnoni et al., 1977) that hybrid cells 108CC15 do n o t contain measurable opioid-like material.
E
References
20
c 10
20
extract (~JI)
Fig. 1. Opioid activity in a partially purified extract from hybrid cells. A. Competition with [3H]Leuenkephalin of opioid-like material for binding to anti-Leu-enkephalin-antiserum. B. Inhibition of the elevation by PGEI of the level of cAMP by Leu-enkephalin (curve a) and hybrid cell extract (curve b) and suppression of the effects of Leuenkephalin (curve c) and hybrid cell extract (curve d) by 10 p.M naloxone.
Giagnoni, G., S.L. Sabol and M. Nirenberg, 1977, Synthesis of opiate peptides by a clonal pituitary tumor cell line, Proc. Natl. Acad. Sci. U.S.A. 74, 2259. Hamprecht, B., 1977, Structural, electrophysiological, biochemical and pharmacological properties of neuroblastoma x giioma hybrids in cell culture, Int. Rev. Cytol. 49, 99. Kimura, S., R.V. Lewis, A.S. Stern, J. Rossier, S.
Stein and S. Udenfriend, 1980, Probable precursors of [Leu]enkephalin and [Met]enkephalin in adrenal medulla: Peptides of 3-5 kilodaltons, Proc. Nat. Acad. Sci. U.S.A. 77, 1681. Van Calker, D., 1977, Ph.D. thesis, University of Munich.
European Journal of Pharmacology, 65 (1980) 319--320 © Elsevier/North-Holland Biomedical Press
Rapid communication PRESENCE OF OPIOID PEPTIDES IN A NEUROBLASTOMA X GLIOMA HYBRID CELL LINE THOMAS GLASER *, DIETRICH VAN CALKER, KARIN HUBNER, CHRISTINE STADTKUS and BERND HAMPRECHT
Physiologisch-Chemisches Institut der Universit~it, Wiirzburg, F.R.G. Received 18 June 1980, accepted 23 June 1980
Neuroblastoma x glioma hybrid cells 108CC15 display many neuronal properties (for review see Hamprecht 1977). Besides excitable membranes and the capacity to synthesize acetylcholine (ACh) they possess a number of receptors for neurohormones. Some hormones like prostaglandin E1 (PGE~), adenosine and secretin stimulate the formation of adenosine 3',5'-cyclic monophosphate (cAMP) in the hybrid cells. Other hormones such as the opioids (among them the enkephalins), noradrenaline, ACh and somatostatin inhibit the elevation by PGE~ of the level of cAMP. Therefore, the hybrid cells can be used to detect the presence of substances, which inhibit the formation of cAMP. Employing the hybrid cells as a test system we have detected several factors in acidic extracts of the same hybrids, which inhibit the stimulation by PGEI of the level of cAMP (T. Glaser et al., in preparation). We now report that opioid peptides were among the factors extracted from neuroblastoma x glioma hybrid cells 108CC15. Cells were grown in Petri dishes (145 mm in diameter) as described (Hamprecht, 1977). After 4 days in culture, cells were washed twice before they were extracted by I M acetic acid. After addition of [3H]Leu-enkephalin (40 000 cpm) as recovery standard, the extract was lyophilized, dissolved in water, * To whom correpondence should be sent: Physiologisch-Chemisches Institut, Koellikerstr. 2, D 8700 Wiirzburg, F.R.G.
centrifuged and poured on a column filled with Bio-Beads SM2. After washing with buffer, opioids were eluted with 50% ethanol. The fraction containing opioids was chromatographed on a Sephadex LH-20 column (T. Glaser et al., in preparation). Fractions containing radioactivity were pooled and tested for opioid activity. Extracts thus purified are called partially purified extracts. Hybrid cells containing 720 mg of protein were extracted and the extract was purified as described above. With the aid of a radioimmunoassay involving antibodies prepared against Leu-enkephalin, 0.1 pmol of Leuenkephalin equivalents was found per mg hybrid cell protein in the extract; the amount was calculated from the data shown in fig. 1, panel A. The antibodies cross-react with Metenkephalin, but not with the endorphins or a number of other peptide hormones (Van Calker, 1977). The extract was also tested for its ability to inhibit the elevation by PGEI of the level of cAMP in the hybrid cells. Fig. 1, panel B, shows that similar to Leu-enkephalin (fig. 1B, curve a) the extract (curve b)inhibits the formation of cAMP stimulated by PGEI. In addition, the effect of both Leu-enkephalin (curve c) and the partially purified extract (curve d), is blocked by the opioid antagonist naloxone (10 pM). Besides the cross reaction of the extract with the anti-Leu-enkephalin antiserum, the effect of the extract on the formation of cAMP further indicates that opioid-like material is present in extracts from
320 3-
A
2" T,
&
1!
10
210
extract (pl) - log [(leu-enkelohalin)(mol I 1 )} oo
10
-II'
9 ~
8 i
7
i
100- B 80-
.
j.
c~
40- / / 2
20" / / .E
hybrid cells. The concentration of opioids in the extract solution determined by radioimmunoassay very well agrees with the concentration f o u n d in the cAMP assay. Opioid extracted from the hybrid cells also competes with [3H]naloxone for binding to opioid receptors in membranes of hybrid cells (T. Glaser et al., in preparation). Further purification of extracts by high performance liquid chromatography renders two active fractions, with the same retention times as Met- and Leu-enkephalin. In addition, the Met-enkephalin-like material, but not the Leu-enkephalin-like material is inactivated by t r e a t m e n t with cyanogen bromide. These results suggest t h a t the two active fractions correspond to Met- and Leu-enkephalin. Under the assumption t h a t the hybrid cells are homogenous, the finding of two active fractions suggests t h a t both enkephalins can be present in one cell. A c o m m o n precursor for both Met- and Leu-enkephalin has recently been described (Kimura et al., 1980). Our results suggest that the hybrid cells can be used as a model system for studying the biosynthetic pathways of the enkephalins. To our knowledge, this is the first time t h a t a cell line is reported to synthesize opioids that may be identical with the enkephalins. In contrast to our findings it has been reported (Giagnoni et al., 1977) that hybrid cells 108CC15 do n o t contain measurable opioid-like material.
E
References
20
c 10
20
extract (~JI)
Fig. 1. Opioid activity in a partially purified extract from hybrid cells. A. Competition with [3H]Leuenkephalin of opioid-like material for binding to anti-Leu-enkephalin-antiserum. B. Inhibition of the elevation by PGEI of the level of cAMP by Leu-enkephalin (curve a) and hybrid cell extract (curve b) and suppression of the effects of Leuenkephalin (curve c) and hybrid cell extract (curve d) by 10 p.M naloxone.
Giagnoni, G., S.L. Sabol and M. Nirenberg, 1977, Synthesis of opiate peptides by a clonal pituitary tumor cell line, Proc. Natl. Acad. Sci. U.S.A. 74, 2259. Hamprecht, B., 1977, Structural, electrophysiological, biochemical and pharmacological properties of neuroblastoma x giioma hybrids in cell culture, Int. Rev. Cytol. 49, 99. Kimura, S., R.V. Lewis, A.S. Stern, J. Rossier, S.
Stein and S. Udenfriend, 1980, Probable precursors of [Leu]enkephalin and [Met]enkephalin in adrenal medulla: Peptides of 3-5 kilodaltons, Proc. Nat. Acad. Sci. U.S.A. 77, 1681. Van Calker, D., 1977, Ph.D. thesis, University of Munich.