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Release of β-endorphin from rat hypothalamus in vitro
European Journal of Pharmacology, 55 (1979) 425--428 © Elsevier/North-Holland Biomedical Press
425
Short communication RELEASE OF ~-ENDORPHIN FROM RAT HYPOTHALAMUS IN VITRO HILLMAN OSBORNE *, RYSZARD PRZEW~OCKI **, VOLKER HOLLT and ALBERT HERZ
Department of Neuropharmacology, Max-Planck-Institut fiir Psychiatrie, Kraepelinstrasse 2, D-8000 Miinchen 40, F.R.G. Received 20 March 1979, accepted 20 March 1979
H. OSBORNE, R. PRZEW~OCKI, V. HOLT and A. HERZ, Release of ~-endorphin from rat hypothalamus in
vitro, European J. Pharmacol. 55 (1979) 425--428. The rate of release of ~-endorphin-like immunoreactivity (~-EI) from rat hypothalamic slices was increased 3to 4-fold over the spontaneous release during exposure to a depolarizing concentration of potassium ions. This augmented outflow was abolished in the absence of calcium. The amount of fl-EI released from slices exposed to a second pulse of potassium was reduced by approximately 50% compared to that in the first. 70% of the potassium-induced released immunoreactive material migrated on a calibrated Sephadex G-50 column like synthetic human ~-endorphin. These findings are discussed in terms of a neuronal release of ~-endorphin and may be taken as supportive evidence for a neurotransmitter and/or neurohormonal role of ~-endorphin in the hypothalamus. ~-Endorphin release
Radioimmunoassay
Hypothalamus
1. Introduction Since the initial isolation and characterization of/3-endorphin from the pituitary gland (Bradbury et al., 1976; Li and Chung, 1976), significant amounts of this peptide have been found in various regions of the brain (Rossier et al., 1977). The observation that brain ~-endorphin levels remained unaltered in hypophysectomized rats (Rossier et al., 1977), suggests that brain fl-end0rphin is synthesized within the brain. Recent immunocytochemical studies have revealed the localization of ~-endorphin reactivity in neuronal perikarya in the basal tuberal hypothalamus and in nerve fibres distributed throughout the diencephalon and anterior pons of rat brain (Bloom et al., 1978). The localization of ~-endorphin in discrete neuronal circuits in * Send reprint requests to: H. Osborne at the above address. ** Permanent address: Institute of Pharmacology, Polish Academy of Sciences, Cracow, Poland.
Neurotransmitter candidate
the brain suggests that fi-endorphin may be involved in neuromodulation or transmitter activities. Although the release of ~-endorphin has recently been shown to occur from rat pituitary in vitro (Przewlocki et al., 1978) the release of this peptide from the CNS has not been demonstrated.
2. Materials and methods Hypothalamic tissue, weighing between 40--45 mg, was dissected from male rats (Sprague-Dawley, 220--250g) immediately following decapitation. The tissue was crosscut at intervals of 260 pm by means of a McIlwain chopper. The slices from 12 hypothalami were loaded into a superfusion apparatus and were superfused with Krebs-bicarbonate medium at 37°C at a rate of 1 ml/ min as described previously (Osborne et al., 1978). Unless stated otherwise, the medium was composed as follows (raM): NaC1 118; KC1 4.75; KH2PO4 1.19; CaC12 2.54; MgSO4
426
1.2; NaHCOa 25; glucose 11; 0.1% (w/v). Bovine serum albumin was included in order to minimize adsorption of ~-EI to the apparatus and collection vessels. The medium was gassed with an oxygen/carbon dioxide (95/5 v/v mixture; the pH was 7.4. Sample collections ( 4 m i n ) were lyophilized and were analysed for fl-EI using a radioimmunoassay procedure as described previously (HSllt et al., 1978a). The sensitivity of detection of the RIA was 7 femtomoles/0.5 ml t u b e volume for ~-endorphin. The antiserum also recognized human /3-1ipotropin which showed a crossreactivity of 100% on a molar basis. Determination of ~-EI in hypothalamic slices was done according to the procedure of HSllt et al. (1978b). The potassium-stimulated outflow from slices o f 5 0 h y p o t h a l a m i was lyophilized, redissolved in 1 ml of RIA buffer and subjected to gel-filtration on a Sephadex G-50 superfine column (95 × 0.9 cm). The column was eluted with RIA buffer at a flow rate of 5 ml/h at 6°C. 0.87 ml fractions were collected and duplicate 0.35 ml aliquots assayed for immunoreactive ~-endorphin. The column was calibrated with Dextran blue (void volume), synthetic human fl-endorphin (Paesel, Frankfurt, FRG) and highly purified human ~-lipotropin (a generous gift from Dr. C.H. Li, San Franciso, USA).
H. OSBORNE ET AL.
a
25"
20.
15,
d 12 16 20 2/, 28 32 36 /,0 4Z, 48 52 Time (rain)
Fig. 1. Release of ~-endorphin immunoreactivity (fmoles rain -1, ordinate) from rat hypothalamus slices (a) in Krebs-bicarbonate medium and (b) in calcium-free Krebs-bicarbonate medium. Elevation of potassium ion concentration of the medium to 47 mM is indicated by the black bar. Each point represents the mean +-S.D. of 6--8 determinations.
3. Results
The outflow of ~-EI increased 3- to 4-fold over that of the spontaneous efflux values on application of a depolarizing concentration of potassium ions for 8 min as shown in fig. la. The efflux of ~-EI returned to values approaching the basal ones 12 min after reapplication of normal Krebs solution. The outflow of ~-EI resulting from the potassium stimulation pulse was equivalent to approximately 2% of the total tissue ~-EI content (32.3 + 8.6 pmoles/g). In several experiments increases o f 8 - t o 10-fold over basal levels were observed. In the absence of calcium
from the superfusion medium, the effect of an increase in potassium ions was totally abolished as shown in fig. l b . The omission of calcium ions did not markedly influence basal efflux levels. In experiments in which a second potassium pulse was applied 20 min after the first, the release of ~-EI amounted to approximately 50% of that obtained in the preceding stimulation (n = 4). Fig. 2 shows the elution profile of the immunoreactive materials released on potassium stimulation after chromatography on Sephadex G-50. Material comigrating with
~-ENDORPHIN RELEASE IN VITRO Vo
A "6 2 -=
/th-LPH
427
r~h-'E
VT
50"
o
E
f ~ 25" E E_
30
40
sb
s0
?V
froction number
Fig. 2. Sephadex G-50 elution profile o f ~-endorphin immunoreactive materials released during potassium stimulation. The potassium-stimulated outflow of ~-EI from slices of 50 hypothalami was separated on a calibrated Sephadex G-50 column as described in Materials and methods. The position of the void volume (V0), human ~-lipotropin (~3h-LPH), and synthetic human /3-endorphin (~h-E) is shown at the the t o p of the figure.
human ~-endorphin represented the predominant (70%) immunoreactive component. In addition to fl-lipotropin, eluted between fractions 32-36, a Significant amount of material was observed which eluted immediately after the void volume (fraction 26-31); this may represent the putative precursor molecule for /~-endorphin/fl-lipotropin proposed by Mains et al. (1977).
4. Discussion The experiments reported here demonstrate both an evoked release of ~-EI from the hypothalamus in response to an increase in potassium ions and the fact that this release is dependent on calcium. The dependence of the evoked release on calcium ions is similar to the experimental conditions required for the release of putative neurotransmitters from the central nervous system and is consonant with the hypothesis that ~-endorphin may fulfil a neurotransmitter
and/or neurohormonal role in the hypothalamus. The intraneuronal distribution of fl-endorphin in the central nervous system in discrete well delineated fibre pathways (Bloom et al., 1978) adds further support to this hypothesis. The finding that only about 2 % of the tissue content of /3-EI was released in response to depolarization by high potassium is indicative of a limited tissue store of readily releasable/I-El and as such is in keeping with the observations on the release of other suspected neurotransmitters. Further evidence of a limited tissue store of releasable/3-EIwas obtained in experiments in which a second potassium-stimulating pulse, applied 20 min after the first,resulted in a reduced/~-EI outflow of approximately 50%. It is of interest to note that in the intermediate lobe of rat pituitary, which has the highest tissue concentration of /3-endorphin, high potassium ions were ineffective in releasing/3-EI (Przewl~ocki et al., 1978). Gel-filtration of the immunoreactive components revealed that/3-endorphin comprised 70% of the immunoreactivity released. Evidence was also found for ~-lipotropin (10% of total immunoreactivity) and a larger molecular component (about 10% of total immunoreactivity) which might correspond to the putative precursor molecule of/~-lipotropin/fl-endorphin (fig. 2) suggested by Mains et al. (1977). Although we cannot preclude that the released ~-endorphin resulted from enzymatic cleavage of either of these larger molecular components during the experiment, chromatographic separation of hypothalamic tissue extracts, in which enzymatic degradation was prevented by boiling intact tissue with 0.1 N HC1, revealed that 80% of the immunoreactive materials behaved like synthetic human ~-endorphin (data not shown).
Acknowledgement These investigations were supported by Deutsche Forschungsgemeinschaft, Bonn.
428
References Bloom, F., E. Battenberg, J. Rossier, N. Ling and R. Guillemin, 1978, Neurons containing ~-endorphin in rat brain exist separately from those containing enkephalin: Immunocytological studies, Proc. Nat. Acad. Sci. U.S.A. 3, 1591. Bradbury, A.F., D.G. Smith and C.R. Snell, 1976, Lipotropin: Precursor to two biologically active peptides, Biochem. Biophys. Res. Commun. 69, 950. HSllt, V., R. Przewtocki and A. Herz, 1978a, Radioimmunoassay of ~-endorphin. Basal and stimulated levels in extracted rat plasma, Naunyn-Schmiedeb. Arch. Pharmacol. 3 0 3 , 1 7 1 . HSllt, V., R. Przewt'ocki and A. Herz, 1978b, ~-Endorphin-like immunoreactivity in plasma, pituitaries and hypothalamus of rats following treatment with opiates, Life Sci. 23, 1057.
H. OSBORNE ET AL. Li, C.H. and D. Chung, 1976, Isolation and structure of an untriakontapeptide with opiate activity from camel pituitary glands, Proc. Nat. Acad. Sci. U.S.A. 73, 1145. Mains, R.E., B.A. Eipper and N. Ling, 1977, Common precursor to corticotropins and endorphins, Proc. Nat. Acad. Sci. U.S.A. 74, 3014. Osborne, H., V. HSllt and A. Herz, 1978, Potassiuminduced release of enkephalins from rat striatal slices, European J. Pharmacol. 48, 219. Przew~ocki, R., V. HSllt and A. Herz, 1978, Release of ~-endorphin from rat pituitary in vitro, European J. pharmacol. 51,179. Rossier, J., T.M. Vargo, M. Scott, N. Ling, F.E. Bloom and R. Guillemin, 1977, Regional dissociation o f ~-endorphin and enkephalin contents in rat brain and pituitary, Proc. Nat. Acad. Sci. U.S.A. 74, 5162.
425
Short communication RELEASE OF ~-ENDORPHIN FROM RAT HYPOTHALAMUS IN VITRO HILLMAN OSBORNE *, RYSZARD PRZEW~OCKI **, VOLKER HOLLT and ALBERT HERZ
Department of Neuropharmacology, Max-Planck-Institut fiir Psychiatrie, Kraepelinstrasse 2, D-8000 Miinchen 40, F.R.G. Received 20 March 1979, accepted 20 March 1979
H. OSBORNE, R. PRZEW~OCKI, V. HOLT and A. HERZ, Release of ~-endorphin from rat hypothalamus in
vitro, European J. Pharmacol. 55 (1979) 425--428. The rate of release of ~-endorphin-like immunoreactivity (~-EI) from rat hypothalamic slices was increased 3to 4-fold over the spontaneous release during exposure to a depolarizing concentration of potassium ions. This augmented outflow was abolished in the absence of calcium. The amount of fl-EI released from slices exposed to a second pulse of potassium was reduced by approximately 50% compared to that in the first. 70% of the potassium-induced released immunoreactive material migrated on a calibrated Sephadex G-50 column like synthetic human ~-endorphin. These findings are discussed in terms of a neuronal release of ~-endorphin and may be taken as supportive evidence for a neurotransmitter and/or neurohormonal role of ~-endorphin in the hypothalamus. ~-Endorphin release
Radioimmunoassay
Hypothalamus
1. Introduction Since the initial isolation and characterization of/3-endorphin from the pituitary gland (Bradbury et al., 1976; Li and Chung, 1976), significant amounts of this peptide have been found in various regions of the brain (Rossier et al., 1977). The observation that brain ~-endorphin levels remained unaltered in hypophysectomized rats (Rossier et al., 1977), suggests that brain fl-end0rphin is synthesized within the brain. Recent immunocytochemical studies have revealed the localization of ~-endorphin reactivity in neuronal perikarya in the basal tuberal hypothalamus and in nerve fibres distributed throughout the diencephalon and anterior pons of rat brain (Bloom et al., 1978). The localization of ~-endorphin in discrete neuronal circuits in * Send reprint requests to: H. Osborne at the above address. ** Permanent address: Institute of Pharmacology, Polish Academy of Sciences, Cracow, Poland.
Neurotransmitter candidate
the brain suggests that fi-endorphin may be involved in neuromodulation or transmitter activities. Although the release of ~-endorphin has recently been shown to occur from rat pituitary in vitro (Przewlocki et al., 1978) the release of this peptide from the CNS has not been demonstrated.
2. Materials and methods Hypothalamic tissue, weighing between 40--45 mg, was dissected from male rats (Sprague-Dawley, 220--250g) immediately following decapitation. The tissue was crosscut at intervals of 260 pm by means of a McIlwain chopper. The slices from 12 hypothalami were loaded into a superfusion apparatus and were superfused with Krebs-bicarbonate medium at 37°C at a rate of 1 ml/ min as described previously (Osborne et al., 1978). Unless stated otherwise, the medium was composed as follows (raM): NaC1 118; KC1 4.75; KH2PO4 1.19; CaC12 2.54; MgSO4
426
1.2; NaHCOa 25; glucose 11; 0.1% (w/v). Bovine serum albumin was included in order to minimize adsorption of ~-EI to the apparatus and collection vessels. The medium was gassed with an oxygen/carbon dioxide (95/5 v/v mixture; the pH was 7.4. Sample collections ( 4 m i n ) were lyophilized and were analysed for fl-EI using a radioimmunoassay procedure as described previously (HSllt et al., 1978a). The sensitivity of detection of the RIA was 7 femtomoles/0.5 ml t u b e volume for ~-endorphin. The antiserum also recognized human /3-1ipotropin which showed a crossreactivity of 100% on a molar basis. Determination of ~-EI in hypothalamic slices was done according to the procedure of HSllt et al. (1978b). The potassium-stimulated outflow from slices o f 5 0 h y p o t h a l a m i was lyophilized, redissolved in 1 ml of RIA buffer and subjected to gel-filtration on a Sephadex G-50 superfine column (95 × 0.9 cm). The column was eluted with RIA buffer at a flow rate of 5 ml/h at 6°C. 0.87 ml fractions were collected and duplicate 0.35 ml aliquots assayed for immunoreactive ~-endorphin. The column was calibrated with Dextran blue (void volume), synthetic human fl-endorphin (Paesel, Frankfurt, FRG) and highly purified human ~-lipotropin (a generous gift from Dr. C.H. Li, San Franciso, USA).
H. OSBORNE ET AL.
a
25"
20.
15,
d 12 16 20 2/, 28 32 36 /,0 4Z, 48 52 Time (rain)
Fig. 1. Release of ~-endorphin immunoreactivity (fmoles rain -1, ordinate) from rat hypothalamus slices (a) in Krebs-bicarbonate medium and (b) in calcium-free Krebs-bicarbonate medium. Elevation of potassium ion concentration of the medium to 47 mM is indicated by the black bar. Each point represents the mean +-S.D. of 6--8 determinations.
3. Results
The outflow of ~-EI increased 3- to 4-fold over that of the spontaneous efflux values on application of a depolarizing concentration of potassium ions for 8 min as shown in fig. la. The efflux of ~-EI returned to values approaching the basal ones 12 min after reapplication of normal Krebs solution. The outflow of ~-EI resulting from the potassium stimulation pulse was equivalent to approximately 2% of the total tissue ~-EI content (32.3 + 8.6 pmoles/g). In several experiments increases o f 8 - t o 10-fold over basal levels were observed. In the absence of calcium
from the superfusion medium, the effect of an increase in potassium ions was totally abolished as shown in fig. l b . The omission of calcium ions did not markedly influence basal efflux levels. In experiments in which a second potassium pulse was applied 20 min after the first, the release of ~-EI amounted to approximately 50% of that obtained in the preceding stimulation (n = 4). Fig. 2 shows the elution profile of the immunoreactive materials released on potassium stimulation after chromatography on Sephadex G-50. Material comigrating with
~-ENDORPHIN RELEASE IN VITRO Vo
A "6 2 -=
/th-LPH
427
r~h-'E
VT
50"
o
E
f ~ 25" E E_
30
40
sb
s0
?V
froction number
Fig. 2. Sephadex G-50 elution profile o f ~-endorphin immunoreactive materials released during potassium stimulation. The potassium-stimulated outflow of ~-EI from slices of 50 hypothalami was separated on a calibrated Sephadex G-50 column as described in Materials and methods. The position of the void volume (V0), human ~-lipotropin (~3h-LPH), and synthetic human /3-endorphin (~h-E) is shown at the the t o p of the figure.
human ~-endorphin represented the predominant (70%) immunoreactive component. In addition to fl-lipotropin, eluted between fractions 32-36, a Significant amount of material was observed which eluted immediately after the void volume (fraction 26-31); this may represent the putative precursor molecule for /~-endorphin/fl-lipotropin proposed by Mains et al. (1977).
4. Discussion The experiments reported here demonstrate both an evoked release of ~-EI from the hypothalamus in response to an increase in potassium ions and the fact that this release is dependent on calcium. The dependence of the evoked release on calcium ions is similar to the experimental conditions required for the release of putative neurotransmitters from the central nervous system and is consonant with the hypothesis that ~-endorphin may fulfil a neurotransmitter
and/or neurohormonal role in the hypothalamus. The intraneuronal distribution of fl-endorphin in the central nervous system in discrete well delineated fibre pathways (Bloom et al., 1978) adds further support to this hypothesis. The finding that only about 2 % of the tissue content of /3-EI was released in response to depolarization by high potassium is indicative of a limited tissue store of readily releasable/I-El and as such is in keeping with the observations on the release of other suspected neurotransmitters. Further evidence of a limited tissue store of releasable/3-EIwas obtained in experiments in which a second potassium-stimulating pulse, applied 20 min after the first,resulted in a reduced/~-EI outflow of approximately 50%. It is of interest to note that in the intermediate lobe of rat pituitary, which has the highest tissue concentration of /3-endorphin, high potassium ions were ineffective in releasing/3-EI (Przewl~ocki et al., 1978). Gel-filtration of the immunoreactive components revealed that/3-endorphin comprised 70% of the immunoreactivity released. Evidence was also found for ~-lipotropin (10% of total immunoreactivity) and a larger molecular component (about 10% of total immunoreactivity) which might correspond to the putative precursor molecule of/~-lipotropin/fl-endorphin (fig. 2) suggested by Mains et al. (1977). Although we cannot preclude that the released ~-endorphin resulted from enzymatic cleavage of either of these larger molecular components during the experiment, chromatographic separation of hypothalamic tissue extracts, in which enzymatic degradation was prevented by boiling intact tissue with 0.1 N HC1, revealed that 80% of the immunoreactive materials behaved like synthetic human ~-endorphin (data not shown).
Acknowledgement These investigations were supported by Deutsche Forschungsgemeinschaft, Bonn.
428
References Bloom, F., E. Battenberg, J. Rossier, N. Ling and R. Guillemin, 1978, Neurons containing ~-endorphin in rat brain exist separately from those containing enkephalin: Immunocytological studies, Proc. Nat. Acad. Sci. U.S.A. 3, 1591. Bradbury, A.F., D.G. Smith and C.R. Snell, 1976, Lipotropin: Precursor to two biologically active peptides, Biochem. Biophys. Res. Commun. 69, 950. HSllt, V., R. Przewtocki and A. Herz, 1978a, Radioimmunoassay of ~-endorphin. Basal and stimulated levels in extracted rat plasma, Naunyn-Schmiedeb. Arch. Pharmacol. 3 0 3 , 1 7 1 . HSllt, V., R. Przewt'ocki and A. Herz, 1978b, ~-Endorphin-like immunoreactivity in plasma, pituitaries and hypothalamus of rats following treatment with opiates, Life Sci. 23, 1057.
H. OSBORNE ET AL. Li, C.H. and D. Chung, 1976, Isolation and structure of an untriakontapeptide with opiate activity from camel pituitary glands, Proc. Nat. Acad. Sci. U.S.A. 73, 1145. Mains, R.E., B.A. Eipper and N. Ling, 1977, Common precursor to corticotropins and endorphins, Proc. Nat. Acad. Sci. U.S.A. 74, 3014. Osborne, H., V. HSllt and A. Herz, 1978, Potassiuminduced release of enkephalins from rat striatal slices, European J. Pharmacol. 48, 219. Przew~ocki, R., V. HSllt and A. Herz, 1978, Release of ~-endorphin from rat pituitary in vitro, European J. pharmacol. 51,179. Rossier, J., T.M. Vargo, M. Scott, N. Ling, F.E. Bloom and R. Guillemin, 1977, Regional dissociation o f ~-endorphin and enkephalin contents in rat brain and pituitary, Proc. Nat. Acad. Sci. U.S.A. 74, 5162.