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Characterization of the effects of acute ethanol administration on the release of β-endorphin peptides by the rat hypothalamus
European Journal of Pharmacology, 180 (1990) 21-29
21
Elsevier EJP 51287
Characterization of the effects of acute ethanol administration on the release of fl-endorphin peptides by the rat hypothalamus Christina Gianoulakis Douglct~ Hospital Research Centre and Department of Psychiatry, McGill University, Verdun, Quebec, Canada H4H 1 R3
Received 6 February 1990, accepted 13 Februa~ 1990
In the present studies the direct effect of ethanol on the release of fl-endorphin by the rat hypothalamus was investigated. When various concentrations of ethanol (10-120 raM) were added into the incubation medium, it was noticed that though low concentrations of ethanol (10, 20 and 30 raM) induced a pronounced increase in the release of fl-endorphin-like peptides from the hypothalamus, high concentrations of ethanol (40, 60 and 120 raM) induced a less pronounced increase. Exposure of hypothalamus to depolarizing concentrations of potassium chloride (following washing of the ethanol), provoked a significant release of /3-endorphin-like peptides, regardless of the ethanol concentration the tissues were exposed prior to the stimulation with the potassium chloride. Chromatographic analysis of the incubation media with Sephadex-G-75 revealed that the hypothalamus released mainly fl-endorphin-sized peptides. Analysis of the fl-endorphin-sized peptides with reverse-phase high performance liquid chromatography indicated the presence of fl-endorphin-(1-31) as well as non-acetyl and acetyl fl-endorphin-(1-27). Thus ethanol exerts a biphasic effect on the release of fl-endorphin-like peptides by the rat hypothalamus, with low concentrations inducing a dose-dependent increase, reaching maximum at 20 mM ethanol, and with higher concentrations of ethanol inducing a less pronounced increase in the release of fl-endorphin-like peptides, leading to an inverted U-shaped dose response relationship of ethanol and release of fl-endorphin-like peptides from the rat hypothalamus.
Hypothalamus; fl-Endorphin release; Ethanol (in vitro)
I. In~'oduction A number of studies have indicated that in human acute alcohol intake may induce both an anxiolytic and an anxiogenic effect (Lukas and Mendelson, 1988; Pohorecky, 1981). In humans, usually moderate consumption of alcohol can relieve stress and anxiety, while excessive drinking can produce depression and anxiety (Pohorecky,
Correspondence to: C. Gianoulakis, Douglas Hospital Research Centre, 6875 LaSalle Blvd., Verdun, Quebec, Canada H4H IR3.
1981). In rodents, both acute and chronic ethanol treatment increase circulating levels of adrenal corticotropin (ACTH) and adrenal corticosteroid hormones (Guaza et al., 1983; Rivier and Vale, 1988; Tabakoff et al., 1978). This increase in A C T H and corticosterone has been shown to be due to the increased release of adrenal corticotropin-releasing factor (CRF) from hypothalamic neurons by ethanol (Redei et al., 1988). In addition to the CRF-mediating effect, ethanol has a direct effect on the release of A C T H by the anterior pituitary (Redei et al., 1986; Keith et al., 1986). C R F has been shown to increase the release
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
22 of fl-endorphin, dynorphin and [MetS]enkephalin from hypothalamic slices (Nikolarakis et al., 1986; Nikolarakis et al., ]989). Since fl-endorphin, a potent endogenous opioid peptide and ACTH share the same high molecular weight precursor molecule, the pro-opiomelanocortin (POMC) (Crine et al., 1978), and are secreted concomitantly by the pituitary gland under a number of physiological conditions (Guillemin et al., 1978), ethanol may influence the release of fl-endorphin from the pituitary gland and the hypothalamus. A number of reports have suggested that acute and chronic ethanol treatment in vivo and in vitro alters the activity of the endogenous opioid system, opioid peptides and receptors (Gianoulakis, 1983; Gianoulakis and Barcomb, 1987; Gianoulakis et al., 1988; Seizinger et al., 1984; Charness et al., 1983). In fact, it has been proposed that many of the effects of ethanol may be mediated by the endogenous opioid system (Altshuler et al., 1980; Triana et al., 1980). Chronic and acute ethanol treatment increased the release of fl-endorphin by the anterior and neurointermediate lobes of the rat pituitary gland (Gianoulakis et al., 1981; 1983; 1988; Gianoulakis and Barcomb, 1987; Seizinger et al., 1984). Although ethanol may alter the release of fl-endorphin peptides in the brain, very little is known about its effects on the secretion of fl-endorphin peptides from neurons, due to the difficulty in studying the secretion of brain peptides in vivo. An alternative in investigating the release of brain peptides is the use of in vitro systems. An increased release of fl-endorphin-like peptides from brain neuronal systems could provide direct evidence that ethanol may induce some of its mood influencing effects by its effects on the release of fl-endorphin peptides, fl-Endorphin peptides may interact with brain opiate receptors and mediate some of the effects of ethanol, such as the mild analgesia and euphoria, while at the same time, may modify the activity of the brain dopaminergic system. In the present studies, we address the following questions: (1) what is the effect of acute in vitro exposure of hypothalamus to various concentrations of ethanol, on the release of fl-endorphin-like peptides; (2) what are the forms of the fl-endorphin peptides released from the hypothalamus.
2. Materials and methods
2.1. Tissue preparation and release experiments Male Sprague-Dawley rats (250-300 g body weight) were killed by decapitation. Immediately following killing the hypothalamus was dissected and placed in well aerated incubation medium. Hypothalamic blocks were dissected as described by Glowinski and Iversen (1966). The tissues were placed in a holder having a polypropylene meshscreen base (500 /~M meshsize, Small Parts Inc., Miami, Florida), and the holder was inserted into a teflon vial with 1 ml of well aerated Krebs Ringer bicarbonate (KRB) medium containing 0.01% bovine serum albumin, 30/~g/ml bacitracin and 10 - 6 M bestatin. The vials were incubated in a Dubnoff metabolic shaking bath at 3 7 ° C under an atmosphere of 95% 0 2 and 5% CO s . Following 15 min of incubation the holder with the tissue was quickly removed from the first vial, washed and placed in the next vial containing fresh aerated medium and incubated for 15 min The first 30 rain of the preincubation period was followed by eight 15 min incubations. The tissues were exposed to various concentrations of ethanol (10-120 mM) in the fourth 15 min incubation period and to 50 mM potassium in the seventh 15 rain incubation period. To maintain the isomolarity of the medium, the increased concentration of KCI was associated with decreased concentration of NaC1.
2.2. Estimation of fl-endorphin-like immunoreactivity Appropriate alliquots from each incubation medium were used for estimation of the content of immunoreactive fl-endorphin. The specificity of the fl-endorphin antiserum and the procedure used for the radioimmunoassay have been described previously (Gianoulakis et al., 1981). In brief, a 1 : 30000 final dilution of the antiserum was used. The antiserum is specific for the C-terminus of fl-endorphin. It gave almost 100% cross-reactivity with bovine fl-lipotropin (fl-LPH) and a-Nacetylated fl-endorphin and 70% cross-reactivity with fl-endorphin-(1-27). This antiserum did not
23 cross-react with ACTH, a-melanotropin (a-MSH) or the bovine fl-LPH fragments 61-65, 62-67 and 80-84. Thus, the antiserum will recognize POMC, fl-LPH, fl-endorphin-(1-31) and fl-endorphin-(127) in both a-N-acetylated or non-acetylated forms, but it will not recognize et-endorphin-(flendorphin-(1-16) and 3,-endorphin (fl-endorphin(1-17)). The intra- and interassay coefficients of variation were 8.9 and 10.1%, respectively.
2.3. Characterization of the released fl-endorphinlike peptides To characterize the forms of fl-endorphin peptides released into the incubation medium under the various conditions tested, incubation media from four experiments were acidified at the end of each 15 min incubation period to a final concentration of 0.2 N HCI, and were pooled to represent the release of fl-endorphins under: (a) basal conditions (second and third 15 min incubation period); (b) ethanol exposure (fourth 15 min incubation period) and (c) potassium-stimulated release (seventh 15 rain incubation period). The pooled incubation media were evaporated to dryness in a Speed Vac evaporating centrifuge (Savant Co.) connected to a freeze dryer (Labconco). The dried media were reconstituted in 0.5 ml of 50% acetic acid and were fractionated by gel filtration on a Sephadex G-75 column (50 x 0.5 cm) in 50% acetic acid. Fractions of 0.5 ml were collected and the content of immtmoreactive fl-endorphin was estimated in each fraction. The elution positions of fl-LPH and fl-endorphin were determined using standard ovine fl-LPH and fl-endorphin-(1-31). The fractions at the position of fl-endorphin were pooled, dried in a Speed Vac evaporating centrifuge and redissolved in 0.1% trifluoroacetic acid, 24% acetonitrile solution, and further analyzed with reverse-phase HPLC. For reverse phase HPLC analysis a /~ Bondapak C-18 column (30 cm × 3.0 mm, Waters MiUipore) was used. The peptides were eluted with a linear gradient of 24-36% acetonitrile, 0.1% trifluoroacetic acid (Dennis et al., 1983). Fractions of 1.0 ml were collected. From each fraction 0.5 ml was dried trader vacuum in a Speed Vac evaporating centrifuge and subsequently assayed for the content of
immunoreactive B-endorphin using an antiserum specific for the C-terminus of B-endorphin as described previously. The remaining 0.5 ml were also dried under vacuum and immunoassayed using an antiserum specific for the a-N-acetyl terminus of B-endorphin. This antiserum was purchased from Peninsula Laboratories, Belmont, CA (RAS 8640) and shows a 100% cross-reactivity with N-acetyl B-endorphin-(1-27), 0.07% cross-reactivity with camel and human fl-endorphin-(l-31), 0.02% cross-reactivity with human B-LPH and 0.001% cross-reactivity with [MetS]enkephalin. In our laboratory the sensitivity of this antiserum was about 20 pg/tube. The intra and interassay coefficients of variation were 4.6 and 9.8%, respectively. The elution positions of the synthetic fl-endorphin standard peptides were determined in separate experiments by applying 2-4 /~g of the standard peptides, dissolved in 0.1% TFA, 24% acetonitrile, on the HPLC column and eluting with 24-36% linear acetonitrile gradient (Dennis et al., 1983).
2.4. Statistical analysis Results are expressed as the mean ± S.E. of immunoreactive fl-endorphin content in the medium of each 15 min incubation period. Significant difference between the content of fl-EPLIR in the incubation medium prior to exposure to ethanol (spontaneous release) and during the ethanol exposure were determined by the twotailed paired t-test. A value of P ~<0.05 was considered significant.
3. Results Figure 1 shows the spontaneous and potassium (50 mM potassium chloride)-stimulated release of /3-endorphin-like peptides from the rat hypothalamus. As is clearly shown the spontaneous release of fl-endorphin-like peptides remained relatively constant for the duration of the experiment and the tissue was able to respond to the exposure of depolarizing concentrations of potassium chloride, added in the seventh incubation period, by a significant increase in the release of immunoreactive fl-endorphin.
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Fig. 1. The effect of potassium (K " ) (50 mM) on the release of immunoreactive/~-endorphin (ir-fl-endorphin) from rat hypothalamus. Each bar represents the mean + S.E. of five experiments. *Significant difference from spontaneous release. P ~< 0.05.
Figure 2 demonstrates the effect of various concentrations of ethanol on the release of fl-endorphin peptides by the hypothalamus. As is clearly shown ethanol increased the release of fl-endorphin-like peptides. However, the response of the hypothalamic fl-endorphin system to increasing concentrations of ethanol was maximum at 20 mM ethanol and then with increasingly higher concentrations of ethanol it started to decrease gradually towards baseline level reaching a minimum at 60 mM ethanol. Using even higher concentrations of ethanol (120 mM), a small increase in the release of hypothalamic flendorphin-like peptides was observed. Following removal of the ethanol from the incubation medium, the release of/3-endorphin-like peptides returned to pre-stimulation levels. Addition of 50 mM K + in the seventh 15 rain incubation period induced an increased release of fl-endorphin-like peptides which was estimated to be about 2.5 times than the spontaneous release, regardless of the prior exposure to ethanol.
To characterize the forms of fl-endorphin-like peptides released into the incubation media under the various conditions tested, incubation media from four experiments were acidified at the end of each 15 min incubation periods and were pooled to represent the release of fl-endorphin-like peptides under (a) basal conditions, (b) ethanol exposure and (c) K + exposure. The pooled media were analyzed by Sephadex G-75 chromatography. The results of the Sephadex G-75 chromatographic analysis of the media are shown on fig. 3. The major forms of fl-endorphin-like peptides released by the hypothalamus had an apparent molecular weight of B-endorphin. Furthermore, smaller quantities of /3-endorphin-like peptides with higher molecular weight than fl-LPH were also observed. The HPLC profiles of the fl-endorphin sized peptides in the incubation media under basal conditions, as well as in the presence of ethanol or 50 mM potassium, were qualitatively similar, however, the absolute concentrations of the peptides were higher in the media of hypothalami exposed to low concentrations of ethanol (10, 20 and 30 mM) or 50 mM potassium. Figure 4 shows the HPLC analysis of the fl-endorphin-sized peptides released by the hypothalamus in the presence of 10 mM ethanol. It clearly shows the presence of fl-endorphin-(1-3]) as well as of several other peptides such as non-acetylated and acetylated fl-endorphin-(1-27). To determine whether the fl-endorphin-(1-27) forms represent breakdown products of the fl-endorphin-(1-3]) released from the hypothalamus, exogenous standard fl-endorphin-(]-3]) peptide was incubated in the presence and absence of hypothalamic tissue under identical conditions as in the release experiments. These incubation media were subjected to Sephadex G-75 and HPLC analysis. Figure 5 shows the HPLC profile of exogenous fl-endorphin-(]-3l) incubated for 15 rain in medium with 10 mM ethanol in the absence (fig. 5A) and presence (fig. 5B) of hypothalamic tissue. It Ls clearly seen that in the absence of hypothalamic tissue there was no generation of shorter fl-endorphin forms, while in the presence of hypothalamic tissue exogenous j~-endorphin-(1-31) was processed to a number of fl-endorphin-related peptides. A known quantity of ~25I-fl-endorphin-
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Fig. 2. The effect of various concentrations of ethanol (ETOH) and 50 mM potassium (K + ) on the release of immunoreactive fl-endorphin (ir-/~-endorphin) by the hypothalamus. Each bar represents the mean+S.E., n = number of distinct experiments. *Significant difference from spontaneous release. P ~<0.05.
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Fig. 3. Gel filtration chromatographic separation of/g-endorphin-like peptides in incubation media of hypothalamus, at basal, ethanol exposure and potassium (K + )-stimulated conditions. Media were chromatographed on Sephadex G-75 and eluted with 50% acetic acid. Fractions, 0.5 ml were collected and analyzed for content of fl-endorpbin-like immunoreactivity. Arrows at the top of the figures show the positions were standard ovine fl-LPH and/?-endorphin eluted.
26
B- Endorphin ;
4. Discussion
,
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The present results clearly indicate that the effect of ethanol on the release of fl-endorphin peptides by the hypothalarnus depends on the c o n c e n t r a t i o n o f e t h a n o l used. L o w c o n c e n t r a t i o n s o f e t h a n o l i n c r e a s e t h e r e l e a s e o f /3-end o r p h i n p e p t i d e s , a n d a m a x i m u m i n c r e a s e is o b tained with 20 mM ethanol. However, the increase
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T h e m e c h a n i s m b y w h i c h e t h a n o l e x p o s u r e in vitro induces increased release of /3-endorphin p e p t i d e s b y t h e h y p o t h a l a m u s is n o t clear. H o w -
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3=5 4L5 55 65 7'5 T I M E IN M I N U T E S Fig. 4. Ehition profiles on reverse-phase HPLC of the fl-endorphin-sized peptides obtained from Sephadex G-75 chromatography of incubation media of hypothalamus in the response to 10 mM ethanol. The peptides were eluted with a linear gradient of 24-36% acetonitrile in 0.1% trifluoroacetic acid and a flow rate of 1 ml per min 1.0 ml fractions were collected. 0.5 ml of each fraction were evaporated to dryness and immunoassayed with an antiserum directed to the C-terminal of ~-endorphin (e I). The remaining 0.5 ml were evaporated to dryness and Lmmunoassayed with an antiserum specific for the acetylated forms of fl-endorphin ( t - . . . . -t). The acetonotrile gradient is indicated by the dotted line (. . . . . . ). Arrows at the top of the figure show the positions of standard peptides which were run separately.
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(1-31) p u r i f i e d w i t h a C - 1 8 S e p - P a k c a r t r i d g e ( W a t e r s S c i e n t i f i c ) a n d H P L C , w a s u s e d t o estimate the recovery of/~-endorphin after Sephadex G - 7 5 a n d H P L C . A n 88% r e c o v e r y w a s o b t a i n e d a f t e r S e p h a d e x G - 7 5 a n d a 77% r e c o v e r y a f t e r HPLC.
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TIME IN M I N U T E S Fig. 5. The HPLC profile of exogenous fl-endorphin-(l-31) incubated for 15 min in incubation medium containing 10 mM ethanol in the absence (A) and presence (B) of hypothalamic tissue under identical conditions as the release experiments. The peptides were eluted with a linear gradient of 24-36% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml per min and were detected by UV absorption at 214 nm. The acetonitrile gradient is indicated by the dotted line (. . . . . . ). Arrows at the top of the figure show the positions of standard peptides which were run separately.
27 ever, a number of hypotheses may be proposed, some of which are supported with experimental evidence. Thus, in the hypothalamic tissue ethanol stimulates the release of CRF which may stimulate the release of fl-endorphin. In support of this hypothesis reports show that ethanol increases the release of CRF by the hypothalamic neurons (Redei et al., 1988), and that CRF increases the release of fl-endorphin, dynorphin and [MetS]en kephalin by hypothalamic slices (Nikolarakis et al., 1986; Nikolarakis et al., 1989). Furthermore, it was shown that the effect of various concentrations of ethanol on the release of CRF from hypothalamic neurons is similar to the ethanol effects on the release of fl-endorphin peptides, observed in the present studies, with low concentrations of ethanol stimulating the release of CRF and high concentrations of ethanol inducing a smaller increase or no increase in CRF release (Redei et al., 1988) by the hypothalamus. Another mechanism by which ethanol may increase the release of fl-endorphin by the hypothalamic neurons could be by inhibiting the release of dopamine. Though in human fetal hypothalamus dopamine was shown to stimulate the release of fl-endorphin (Rasmussen et al., 1987), in the rat hypothalami dopamine has an inhibitory effect on fl-endorphin release (Verrnes et al., 1985). Ethanol has been shown to modify the activity of the brain dopaminergic system and the specific effect depends on the dose of ethanol administered and how long after ethanol administration the investigations were carried out (Badawy et al., 1983; K.iianma and Tabakoff, 1983). Ethanol at low doses decreases the release of dopamine, while ethanol at high doses increases the release of dopamine (Kiianma and Tabakoff, 1983). Thus low concentrations of ethanol will have a stronger stimulatory effect on the release of hypothalamic fl-endorphin peptides than high concentrations of ethanol. Further studies using dopamine receptor agonists and antagonists in combination with ethanol may clarify the possible mechanisms of action of ethanol on the hypothalamic fl-endorphin system. It is of interest that the ascending region of the dose-response curve of the effect of ethanol on the release of fl-endorphin by the hypothalamus
coincides with concentrations of ethanol observed in the ascending phase of the blood ethanol curve in human subjects (0-100 mg/dl), usually observed within 15-30 rain following ingestion of 0.50 or 0.75 g ethanol per kg body weight (Schuckit, 1988). During this time interval, about 30 min after ethanol consumption, human subjects usually exhibit a feeling of euphoria, relaxation and general well-being (Lukas and Mendelson, 1988). However, when the blood alcohol content exceeds these concentrations, a number of individuals may exhibit depression and anxiety (McGuire et al., 1966). Thus, it appears that concentrations of ethanol which induce mild euphoria and relaxation also induce a pronounced release of hypothalamic CRF as well as fl-endorphin peptides from the hypothalamus. Numerous studies on the tissue content of the fl-endorphin-related peptides in the hypothalamus, anterior and intermediate lobes of the pituitary gland have shown that the post-translational processing of POMC to fl-LPH and fl-endorphin differs in these tissues (Crine et al., 1978; Akil et al., 1981; Zakarian and Smyth, 1982). In the hypothalamus, the final maturation product of POMC is fl-endorphin-(1-31) (Zakarian and Smyth, 1982). Thus, in the present studies it was observed that the major proportion of the fl-endorphin-like peptides released by the hypothalamus had a molecular weight similar to fl-endorphin. Furthermore, following HPLC analysis it was noticed that in addition to fl-endorphin-(1-31) shorter forms of fl-endorphin were present in the incubation media, as non-acetyl and acetyl fl-endorphin-(1-27). These fl-endorphin fragments could represent either products released from the hypothalamus, or breakdown products of fl-endorphin-(1-31) after its release from the tissue. Although fl-endorphin(1-31) is the major fl-endorphin peptide present in the hypothalamus, the presence of acetyl and non-acetyl fl-endorphin-(1-27) and /3-endorphin(1-26) in extracts of hypothalamic tissue has been reported (Emeson and Eipper, 1986) providing support for the possible release of the fl-endorphin-(1-27) forms detected in the incubation media from the hypothalamus. Furthermore, in the present studies incubation of hypothalamus with exogenous fl-endorphin-(1-31) led to break-
28 down of the exogenous fl-endorphin-(l-31), indicating that processing of fl-endorphin-(1-31) could also take place following its release from the hypothalamic tissue a n d that the two peptidase inhibitors, bestatin and bacitracin, which were present in the i n c u b a t i o n m e d i u m could not block completely the b r e a k d o w n of the fl-endorphin-(1-31). The physiological significance of either the release or the generation of different molecular weight forms of fl-endorphin by the h y p o t h a l a m u s is not known. A n u m b e r of studies have recently shown that shorter f l - e n d o r p h i n fragments may act as antagonists to fl-endorphin-(1-31) (Suh et al., 1987). Thus these fl-endorphin forms detected in the i n c u b a t i o n media m a y have a specific r o l e in the hypothalamus. In conclusion, the results from the present studies indicate that the effect of ethanol on the release of fl-endorphin-like peptides from the hypothalamus depends on the c o n c e n t r a t i o n of ethanol used. H y p o t h a l a m u s exhibits an increased release of fl-endorphin-like peptides following exposure to ethanol. This increased release is more pron o u n c e d at low c o n c e n t r a t i o n s of ethanol and may be involved in m e d i a t i n g some of the effects of ethanol, as mild euphoria a n d analgesia. High concentrations of ethanol induce a smaller increase in the release of h y p o t h a l a m i c 13endorphin-like peptides, a n d this m a y be partially responsible for the depression a n d anxiety an individual may feel following excessive ethanol consumption.
Acknowledgements The author wishes to thank Mrs. D. Vetro and Mrs. J. Currie for preparation of the manuscript. This work was supported by the Medical Research Council of Canada, Grant MA-6923.
References Akil, H., Y. Ueda and H.L. Liu, 1981, A sensitive coupled HP&C/RIA technique for separation of endorphins: Multiple forms of fl-endorphin in rat pituitary intermediate vs anterior lobe, Neuropeptides 1,429.
Altshuler, H.L., P.E. Philips and D.E. Feinhandler, 1980, Alteration of ethanol self-administration by naltrexone, Life Sci. 26, 679. Badawy, A.B., D.L. Williams and M. Evans, 1983, Role of tyrosine in the acute effects of ethanol on rat brain catecholamine synthesis, Pharmacol. Biochem. Behav. 18 (Suppl. 1), 389. Charness, M.E., A.S. Gordon and I. Diamond, 1983, Ethanol modulation of opiate receptors in cultured neural cells, Science 222, 1246. Crine, P., C. Gianoulakis, N.G. Seidah, F. Go~ard, P.D. Pezalla, M. Lis and M. Chr6tien, 1978, Biosynthesis of fl-endorphin from fl-lipotropin and a larger molecular weight precursor in rat pars intermedia, Proc. Natl. Acad. Sci. U.S.A. 75, 4719. Dennis, M., C. Lazure, N.G. Seidah and M. Chretien, 1983, Characterization of fl-endorphin immunoreactive peptides in rat pituitary and brain by coupled gel and reverse-phase high performance liquid chromatography, J. Chromatogr. 266, 163. Emeson, R.B. and B.A. Eipper, 1986, Characterization of proACTH/endorphin-derived peptides in rat hypothalamus, J. Neurosci. 6, 837. Gianoulakis, C., 1983, Long-term ethanol 'alters the binding of 3H-opiates to brain membranes, Life Sci. 33, 725. Gianoulakis, C. and A. Barcomb, 1987, Effect of acute ethanol in vivo and in vitro on the fl-endorphin system in the rat, Life Sci. 40, 19. Gianoulakis, C., J.S.D. Chan, H. Kalant and M. Chr~tien, 1983, Chronic ethanol treatment alters the biosynthesis of fl-endorphin by the rat neurointermediate lobe, Can. J. Physiol. Pharmacol. 61,967. Gianoulakis, C., W.D. Hutchison and H. Kalant, I988, Biosynthesis and post-translational processing of B-endorphin following ethanol withdrawal, Endocrinology 122, 817. Gianoulakis, C., N. Woo, J.N. Drouin, N.G. Seidah, H. Kalant and M. Chr6tien, 1981, Biosynthesisof/Lendorphin by the neurointermediate lobes from rats dependent on morphine or alcohol, Life SCi. 29, 1973. Glowinski, J. a~d L. Iversen, 1966, Regional studies of catecholamines in the rat brain - I. The disposition of [~Hlnorepinephrine, [3H]dopamine and [3H]DOPA in various regions, J. Neurochem. 13, 655. Guaza, C., A. Torrellas and S. Borrell, 1983, Adrenocortical response to acute and chronic ethanol administration in rats, Psychopharmacology 79, 173. Guillemin, R., T. Vargo, L Rossier, S. Minick, C. Rivier and W. Vale, 1978, B-Endorphin and adrenocorficotropia are secreted concomitantly by the pituitary gland, Biopolymers 17, 1927. Keith, L.D., J.C. Crabbe, L.M. Robertson and J.W. Kendall, 1986, Ethanol stimulated endorphin and corticotropin in vitro, Brain Res. 367, 22. Kiianma, K. and B. Tabakoff, 1983, Neurochemical correlates of tolerance and strain differences in the neurochemieal effects of ethanot, Pb.armacol. Biochem. Behav. 18 (Suppl. 1), 383.
29 Lukas, S.E. and J.H. Mendelson, 1988, Electroenephalographic activity and plasma ACTH during ethanol-induced euphoria, Biol. Psychiat. 23, 141. McGuire, M.T., J.H. Mendelson and S. Stein, 1966, Comparative psychosocial studies of alcoholic and non-alcoholic subjects undergoing experimentally induced ethanol intoxication, Psychosom. Med. 28, 13. Nikolarakis, K.E., O.F.X. Almeida and A. Herz, 1986, Stimulation of hypothalamic fl-endorphin and dynorphin release by corticotropin-relea.sing factor (in vitro), Brain Res. 399, 152. Nikolarakis, K.E., O.F.X. Almeida and A. Herz, 1989, Multiple factors influencing the in vitro release of [MetS]-enkephalin from rat hypothalamic slices, J. Neurochem. 52, 428. Pohorecky, L.A., 1981, The interaction of alcohol and stress (review), Neurosci. Behav. Rev. 5, 209. Rasmussen, D.D., J.H. Liu, P.L. Wolf and S.S.C. Yen, 1987, Neurosecretion of human hypothalamic immunoreactive fl-endorphin: In vitro regulation by dopamine, Neuroendocrinology 45, 197. Redei, E., J.B. Branch, S. Gholami, E.Y.R. Lin and A. Newman-Taylor, 1988, Effect of ethanol on CRF release in vitro, Endocrinology 123, 2736. Redei, E., J.B. Branch and A. Newman-Taylor, 1986, Direct effect of ethanol on adrenocorticotropin (ACTH) release in vitro, J. Pharmacol. Exp. Ther. 237, 59.
Rivier, C. and W. Vale, 1988, Interaction between ethanol and stress on ACTH and fl-endorphin secretion, Alcohol. Clin. Exp. Res. 12, 206. Schuckit, M.A., 1988, Reactions to alcohol in sons of alcoholics and controls~ Alcohol. Clin. Exp. Res. 12, 465. Seizinger, B.R., K. Bovermann, V. Hollt and A. Herz, 1984, Enhanced activity of the fl-endorphinergic system in the anterior and neurointermediate lobe of the rat pituitary after chronic treatment with ethanol liquid diet, J. Pharmacol. Exp. Ther. 230, 455. Suh, H.H., L.F. Tseng and C.H. Li, 1987, Beta-endorphin-(1-27) antagonizes beta-endorphin induced hypothermia in mice, Peptides 8, 123. Tabakoff, B., R.C. Jaffe and R.F. Ritzmann, 1978, Corticosterone concentration in mice during ethanol drink and withdrawal, J. Pharm. Pharmacol. 30, 371. Triana, E., J.F. Richard and P.E. Strokes, 1980, The relationship between endorphins and alcohol induced sub-cortical activity, Am. J. Psychiat. 127, 491. Vermes, I., F.J.H. Tilders and J.C. Stoof, 1985, Dopamine inhibits the release of immunoreactive fl-endorphin from rat hypothalamus in vitro, Brain Res. 326, 41. Zakazian, S. and D. Smyth, 1982, Beta-endorphin is processed differentially in specific regions of rat pituitary and brain, Nature 296, 250.
21
Elsevier EJP 51287
Characterization of the effects of acute ethanol administration on the release of fl-endorphin peptides by the rat hypothalamus Christina Gianoulakis Douglct~ Hospital Research Centre and Department of Psychiatry, McGill University, Verdun, Quebec, Canada H4H 1 R3
Received 6 February 1990, accepted 13 Februa~ 1990
In the present studies the direct effect of ethanol on the release of fl-endorphin by the rat hypothalamus was investigated. When various concentrations of ethanol (10-120 raM) were added into the incubation medium, it was noticed that though low concentrations of ethanol (10, 20 and 30 raM) induced a pronounced increase in the release of fl-endorphin-like peptides from the hypothalamus, high concentrations of ethanol (40, 60 and 120 raM) induced a less pronounced increase. Exposure of hypothalamus to depolarizing concentrations of potassium chloride (following washing of the ethanol), provoked a significant release of /3-endorphin-like peptides, regardless of the ethanol concentration the tissues were exposed prior to the stimulation with the potassium chloride. Chromatographic analysis of the incubation media with Sephadex-G-75 revealed that the hypothalamus released mainly fl-endorphin-sized peptides. Analysis of the fl-endorphin-sized peptides with reverse-phase high performance liquid chromatography indicated the presence of fl-endorphin-(1-31) as well as non-acetyl and acetyl fl-endorphin-(1-27). Thus ethanol exerts a biphasic effect on the release of fl-endorphin-like peptides by the rat hypothalamus, with low concentrations inducing a dose-dependent increase, reaching maximum at 20 mM ethanol, and with higher concentrations of ethanol inducing a less pronounced increase in the release of fl-endorphin-like peptides, leading to an inverted U-shaped dose response relationship of ethanol and release of fl-endorphin-like peptides from the rat hypothalamus.
Hypothalamus; fl-Endorphin release; Ethanol (in vitro)
I. In~'oduction A number of studies have indicated that in human acute alcohol intake may induce both an anxiolytic and an anxiogenic effect (Lukas and Mendelson, 1988; Pohorecky, 1981). In humans, usually moderate consumption of alcohol can relieve stress and anxiety, while excessive drinking can produce depression and anxiety (Pohorecky,
Correspondence to: C. Gianoulakis, Douglas Hospital Research Centre, 6875 LaSalle Blvd., Verdun, Quebec, Canada H4H IR3.
1981). In rodents, both acute and chronic ethanol treatment increase circulating levels of adrenal corticotropin (ACTH) and adrenal corticosteroid hormones (Guaza et al., 1983; Rivier and Vale, 1988; Tabakoff et al., 1978). This increase in A C T H and corticosterone has been shown to be due to the increased release of adrenal corticotropin-releasing factor (CRF) from hypothalamic neurons by ethanol (Redei et al., 1988). In addition to the CRF-mediating effect, ethanol has a direct effect on the release of A C T H by the anterior pituitary (Redei et al., 1986; Keith et al., 1986). C R F has been shown to increase the release
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
22 of fl-endorphin, dynorphin and [MetS]enkephalin from hypothalamic slices (Nikolarakis et al., 1986; Nikolarakis et al., ]989). Since fl-endorphin, a potent endogenous opioid peptide and ACTH share the same high molecular weight precursor molecule, the pro-opiomelanocortin (POMC) (Crine et al., 1978), and are secreted concomitantly by the pituitary gland under a number of physiological conditions (Guillemin et al., 1978), ethanol may influence the release of fl-endorphin from the pituitary gland and the hypothalamus. A number of reports have suggested that acute and chronic ethanol treatment in vivo and in vitro alters the activity of the endogenous opioid system, opioid peptides and receptors (Gianoulakis, 1983; Gianoulakis and Barcomb, 1987; Gianoulakis et al., 1988; Seizinger et al., 1984; Charness et al., 1983). In fact, it has been proposed that many of the effects of ethanol may be mediated by the endogenous opioid system (Altshuler et al., 1980; Triana et al., 1980). Chronic and acute ethanol treatment increased the release of fl-endorphin by the anterior and neurointermediate lobes of the rat pituitary gland (Gianoulakis et al., 1981; 1983; 1988; Gianoulakis and Barcomb, 1987; Seizinger et al., 1984). Although ethanol may alter the release of fl-endorphin peptides in the brain, very little is known about its effects on the secretion of fl-endorphin peptides from neurons, due to the difficulty in studying the secretion of brain peptides in vivo. An alternative in investigating the release of brain peptides is the use of in vitro systems. An increased release of fl-endorphin-like peptides from brain neuronal systems could provide direct evidence that ethanol may induce some of its mood influencing effects by its effects on the release of fl-endorphin peptides, fl-Endorphin peptides may interact with brain opiate receptors and mediate some of the effects of ethanol, such as the mild analgesia and euphoria, while at the same time, may modify the activity of the brain dopaminergic system. In the present studies, we address the following questions: (1) what is the effect of acute in vitro exposure of hypothalamus to various concentrations of ethanol, on the release of fl-endorphin-like peptides; (2) what are the forms of the fl-endorphin peptides released from the hypothalamus.
2. Materials and methods
2.1. Tissue preparation and release experiments Male Sprague-Dawley rats (250-300 g body weight) were killed by decapitation. Immediately following killing the hypothalamus was dissected and placed in well aerated incubation medium. Hypothalamic blocks were dissected as described by Glowinski and Iversen (1966). The tissues were placed in a holder having a polypropylene meshscreen base (500 /~M meshsize, Small Parts Inc., Miami, Florida), and the holder was inserted into a teflon vial with 1 ml of well aerated Krebs Ringer bicarbonate (KRB) medium containing 0.01% bovine serum albumin, 30/~g/ml bacitracin and 10 - 6 M bestatin. The vials were incubated in a Dubnoff metabolic shaking bath at 3 7 ° C under an atmosphere of 95% 0 2 and 5% CO s . Following 15 min of incubation the holder with the tissue was quickly removed from the first vial, washed and placed in the next vial containing fresh aerated medium and incubated for 15 min The first 30 rain of the preincubation period was followed by eight 15 min incubations. The tissues were exposed to various concentrations of ethanol (10-120 mM) in the fourth 15 min incubation period and to 50 mM potassium in the seventh 15 rain incubation period. To maintain the isomolarity of the medium, the increased concentration of KCI was associated with decreased concentration of NaC1.
2.2. Estimation of fl-endorphin-like immunoreactivity Appropriate alliquots from each incubation medium were used for estimation of the content of immunoreactive fl-endorphin. The specificity of the fl-endorphin antiserum and the procedure used for the radioimmunoassay have been described previously (Gianoulakis et al., 1981). In brief, a 1 : 30000 final dilution of the antiserum was used. The antiserum is specific for the C-terminus of fl-endorphin. It gave almost 100% cross-reactivity with bovine fl-lipotropin (fl-LPH) and a-Nacetylated fl-endorphin and 70% cross-reactivity with fl-endorphin-(1-27). This antiserum did not
23 cross-react with ACTH, a-melanotropin (a-MSH) or the bovine fl-LPH fragments 61-65, 62-67 and 80-84. Thus, the antiserum will recognize POMC, fl-LPH, fl-endorphin-(1-31) and fl-endorphin-(127) in both a-N-acetylated or non-acetylated forms, but it will not recognize et-endorphin-(flendorphin-(1-16) and 3,-endorphin (fl-endorphin(1-17)). The intra- and interassay coefficients of variation were 8.9 and 10.1%, respectively.
2.3. Characterization of the released fl-endorphinlike peptides To characterize the forms of fl-endorphin peptides released into the incubation medium under the various conditions tested, incubation media from four experiments were acidified at the end of each 15 min incubation period to a final concentration of 0.2 N HCI, and were pooled to represent the release of fl-endorphins under: (a) basal conditions (second and third 15 min incubation period); (b) ethanol exposure (fourth 15 min incubation period) and (c) potassium-stimulated release (seventh 15 rain incubation period). The pooled incubation media were evaporated to dryness in a Speed Vac evaporating centrifuge (Savant Co.) connected to a freeze dryer (Labconco). The dried media were reconstituted in 0.5 ml of 50% acetic acid and were fractionated by gel filtration on a Sephadex G-75 column (50 x 0.5 cm) in 50% acetic acid. Fractions of 0.5 ml were collected and the content of immtmoreactive fl-endorphin was estimated in each fraction. The elution positions of fl-LPH and fl-endorphin were determined using standard ovine fl-LPH and fl-endorphin-(1-31). The fractions at the position of fl-endorphin were pooled, dried in a Speed Vac evaporating centrifuge and redissolved in 0.1% trifluoroacetic acid, 24% acetonitrile solution, and further analyzed with reverse-phase HPLC. For reverse phase HPLC analysis a /~ Bondapak C-18 column (30 cm × 3.0 mm, Waters MiUipore) was used. The peptides were eluted with a linear gradient of 24-36% acetonitrile, 0.1% trifluoroacetic acid (Dennis et al., 1983). Fractions of 1.0 ml were collected. From each fraction 0.5 ml was dried trader vacuum in a Speed Vac evaporating centrifuge and subsequently assayed for the content of
immunoreactive B-endorphin using an antiserum specific for the C-terminus of B-endorphin as described previously. The remaining 0.5 ml were also dried under vacuum and immunoassayed using an antiserum specific for the a-N-acetyl terminus of B-endorphin. This antiserum was purchased from Peninsula Laboratories, Belmont, CA (RAS 8640) and shows a 100% cross-reactivity with N-acetyl B-endorphin-(1-27), 0.07% cross-reactivity with camel and human fl-endorphin-(l-31), 0.02% cross-reactivity with human B-LPH and 0.001% cross-reactivity with [MetS]enkephalin. In our laboratory the sensitivity of this antiserum was about 20 pg/tube. The intra and interassay coefficients of variation were 4.6 and 9.8%, respectively. The elution positions of the synthetic fl-endorphin standard peptides were determined in separate experiments by applying 2-4 /~g of the standard peptides, dissolved in 0.1% TFA, 24% acetonitrile, on the HPLC column and eluting with 24-36% linear acetonitrile gradient (Dennis et al., 1983).
2.4. Statistical analysis Results are expressed as the mean ± S.E. of immunoreactive fl-endorphin content in the medium of each 15 min incubation period. Significant difference between the content of fl-EPLIR in the incubation medium prior to exposure to ethanol (spontaneous release) and during the ethanol exposure were determined by the twotailed paired t-test. A value of P ~<0.05 was considered significant.
3. Results Figure 1 shows the spontaneous and potassium (50 mM potassium chloride)-stimulated release of /3-endorphin-like peptides from the rat hypothalamus. As is clearly shown the spontaneous release of fl-endorphin-like peptides remained relatively constant for the duration of the experiment and the tissue was able to respond to the exposure of depolarizing concentrations of potassium chloride, added in the seventh incubation period, by a significant increase in the release of immunoreactive fl-endorphin.
24 HYPOTHALAMUS < ,< "J -r I0 n )...
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Fig. 1. The effect of potassium (K " ) (50 mM) on the release of immunoreactive/~-endorphin (ir-fl-endorphin) from rat hypothalamus. Each bar represents the mean + S.E. of five experiments. *Significant difference from spontaneous release. P ~< 0.05.
Figure 2 demonstrates the effect of various concentrations of ethanol on the release of fl-endorphin peptides by the hypothalamus. As is clearly shown ethanol increased the release of fl-endorphin-like peptides. However, the response of the hypothalamic fl-endorphin system to increasing concentrations of ethanol was maximum at 20 mM ethanol and then with increasingly higher concentrations of ethanol it started to decrease gradually towards baseline level reaching a minimum at 60 mM ethanol. Using even higher concentrations of ethanol (120 mM), a small increase in the release of hypothalamic flendorphin-like peptides was observed. Following removal of the ethanol from the incubation medium, the release of/3-endorphin-like peptides returned to pre-stimulation levels. Addition of 50 mM K + in the seventh 15 rain incubation period induced an increased release of fl-endorphin-like peptides which was estimated to be about 2.5 times than the spontaneous release, regardless of the prior exposure to ethanol.
To characterize the forms of fl-endorphin-like peptides released into the incubation media under the various conditions tested, incubation media from four experiments were acidified at the end of each 15 min incubation periods and were pooled to represent the release of fl-endorphin-like peptides under (a) basal conditions, (b) ethanol exposure and (c) K + exposure. The pooled media were analyzed by Sephadex G-75 chromatography. The results of the Sephadex G-75 chromatographic analysis of the media are shown on fig. 3. The major forms of fl-endorphin-like peptides released by the hypothalamus had an apparent molecular weight of B-endorphin. Furthermore, smaller quantities of /3-endorphin-like peptides with higher molecular weight than fl-LPH were also observed. The HPLC profiles of the fl-endorphin sized peptides in the incubation media under basal conditions, as well as in the presence of ethanol or 50 mM potassium, were qualitatively similar, however, the absolute concentrations of the peptides were higher in the media of hypothalami exposed to low concentrations of ethanol (10, 20 and 30 mM) or 50 mM potassium. Figure 4 shows the HPLC analysis of the fl-endorphin-sized peptides released by the hypothalamus in the presence of 10 mM ethanol. It clearly shows the presence of fl-endorphin-(1-3]) as well as of several other peptides such as non-acetylated and acetylated fl-endorphin-(1-27). To determine whether the fl-endorphin-(1-27) forms represent breakdown products of the fl-endorphin-(1-3]) released from the hypothalamus, exogenous standard fl-endorphin-(]-3]) peptide was incubated in the presence and absence of hypothalamic tissue under identical conditions as in the release experiments. These incubation media were subjected to Sephadex G-75 and HPLC analysis. Figure 5 shows the HPLC profile of exogenous fl-endorphin-(]-3l) incubated for 15 rain in medium with 10 mM ethanol in the absence (fig. 5A) and presence (fig. 5B) of hypothalamic tissue. It Ls clearly seen that in the absence of hypothalamic tissue there was no generation of shorter fl-endorphin forms, while in the presence of hypothalamic tissue exogenous j~-endorphin-(1-31) was processed to a number of fl-endorphin-related peptides. A known quantity of ~25I-fl-endorphin-
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Fig. 3. Gel filtration chromatographic separation of/g-endorphin-like peptides in incubation media of hypothalamus, at basal, ethanol exposure and potassium (K + )-stimulated conditions. Media were chromatographed on Sephadex G-75 and eluted with 50% acetic acid. Fractions, 0.5 ml were collected and analyzed for content of fl-endorpbin-like immunoreactivity. Arrows at the top of the figures show the positions were standard ovine fl-LPH and/?-endorphin eluted.
26
B- Endorphin ;
4. Discussion
,
200
The present results clearly indicate that the effect of ethanol on the release of fl-endorphin peptides by the hypothalarnus depends on the c o n c e n t r a t i o n o f e t h a n o l used. L o w c o n c e n t r a t i o n s o f e t h a n o l i n c r e a s e t h e r e l e a s e o f /3-end o r p h i n p e p t i d e s , a n d a m a x i m u m i n c r e a s e is o b tained with 20 mM ethanol. However, the increase
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3=5 4L5 55 65 7'5 T I M E IN M I N U T E S Fig. 4. Ehition profiles on reverse-phase HPLC of the fl-endorphin-sized peptides obtained from Sephadex G-75 chromatography of incubation media of hypothalamus in the response to 10 mM ethanol. The peptides were eluted with a linear gradient of 24-36% acetonitrile in 0.1% trifluoroacetic acid and a flow rate of 1 ml per min 1.0 ml fractions were collected. 0.5 ml of each fraction were evaporated to dryness and immunoassayed with an antiserum directed to the C-terminal of ~-endorphin (e I). The remaining 0.5 ml were evaporated to dryness and Lmmunoassayed with an antiserum specific for the acetylated forms of fl-endorphin ( t - . . . . -t). The acetonotrile gradient is indicated by the dotted line (. . . . . . ). Arrows at the top of the figure show the positions of standard peptides which were run separately.
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(1-31) p u r i f i e d w i t h a C - 1 8 S e p - P a k c a r t r i d g e ( W a t e r s S c i e n t i f i c ) a n d H P L C , w a s u s e d t o estimate the recovery of/~-endorphin after Sephadex G - 7 5 a n d H P L C . A n 88% r e c o v e r y w a s o b t a i n e d a f t e r S e p h a d e x G - 7 5 a n d a 77% r e c o v e r y a f t e r HPLC.
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TIME IN M I N U T E S Fig. 5. The HPLC profile of exogenous fl-endorphin-(l-31) incubated for 15 min in incubation medium containing 10 mM ethanol in the absence (A) and presence (B) of hypothalamic tissue under identical conditions as the release experiments. The peptides were eluted with a linear gradient of 24-36% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml per min and were detected by UV absorption at 214 nm. The acetonitrile gradient is indicated by the dotted line (. . . . . . ). Arrows at the top of the figure show the positions of standard peptides which were run separately.
27 ever, a number of hypotheses may be proposed, some of which are supported with experimental evidence. Thus, in the hypothalamic tissue ethanol stimulates the release of CRF which may stimulate the release of fl-endorphin. In support of this hypothesis reports show that ethanol increases the release of CRF by the hypothalamic neurons (Redei et al., 1988), and that CRF increases the release of fl-endorphin, dynorphin and [MetS]en kephalin by hypothalamic slices (Nikolarakis et al., 1986; Nikolarakis et al., 1989). Furthermore, it was shown that the effect of various concentrations of ethanol on the release of CRF from hypothalamic neurons is similar to the ethanol effects on the release of fl-endorphin peptides, observed in the present studies, with low concentrations of ethanol stimulating the release of CRF and high concentrations of ethanol inducing a smaller increase or no increase in CRF release (Redei et al., 1988) by the hypothalamus. Another mechanism by which ethanol may increase the release of fl-endorphin by the hypothalamic neurons could be by inhibiting the release of dopamine. Though in human fetal hypothalamus dopamine was shown to stimulate the release of fl-endorphin (Rasmussen et al., 1987), in the rat hypothalami dopamine has an inhibitory effect on fl-endorphin release (Verrnes et al., 1985). Ethanol has been shown to modify the activity of the brain dopaminergic system and the specific effect depends on the dose of ethanol administered and how long after ethanol administration the investigations were carried out (Badawy et al., 1983; K.iianma and Tabakoff, 1983). Ethanol at low doses decreases the release of dopamine, while ethanol at high doses increases the release of dopamine (Kiianma and Tabakoff, 1983). Thus low concentrations of ethanol will have a stronger stimulatory effect on the release of hypothalamic fl-endorphin peptides than high concentrations of ethanol. Further studies using dopamine receptor agonists and antagonists in combination with ethanol may clarify the possible mechanisms of action of ethanol on the hypothalamic fl-endorphin system. It is of interest that the ascending region of the dose-response curve of the effect of ethanol on the release of fl-endorphin by the hypothalamus
coincides with concentrations of ethanol observed in the ascending phase of the blood ethanol curve in human subjects (0-100 mg/dl), usually observed within 15-30 rain following ingestion of 0.50 or 0.75 g ethanol per kg body weight (Schuckit, 1988). During this time interval, about 30 min after ethanol consumption, human subjects usually exhibit a feeling of euphoria, relaxation and general well-being (Lukas and Mendelson, 1988). However, when the blood alcohol content exceeds these concentrations, a number of individuals may exhibit depression and anxiety (McGuire et al., 1966). Thus, it appears that concentrations of ethanol which induce mild euphoria and relaxation also induce a pronounced release of hypothalamic CRF as well as fl-endorphin peptides from the hypothalamus. Numerous studies on the tissue content of the fl-endorphin-related peptides in the hypothalamus, anterior and intermediate lobes of the pituitary gland have shown that the post-translational processing of POMC to fl-LPH and fl-endorphin differs in these tissues (Crine et al., 1978; Akil et al., 1981; Zakarian and Smyth, 1982). In the hypothalamus, the final maturation product of POMC is fl-endorphin-(1-31) (Zakarian and Smyth, 1982). Thus, in the present studies it was observed that the major proportion of the fl-endorphin-like peptides released by the hypothalamus had a molecular weight similar to fl-endorphin. Furthermore, following HPLC analysis it was noticed that in addition to fl-endorphin-(1-31) shorter forms of fl-endorphin were present in the incubation media, as non-acetyl and acetyl fl-endorphin-(1-27). These fl-endorphin fragments could represent either products released from the hypothalamus, or breakdown products of fl-endorphin-(1-31) after its release from the tissue. Although fl-endorphin(1-31) is the major fl-endorphin peptide present in the hypothalamus, the presence of acetyl and non-acetyl fl-endorphin-(1-27) and /3-endorphin(1-26) in extracts of hypothalamic tissue has been reported (Emeson and Eipper, 1986) providing support for the possible release of the fl-endorphin-(1-27) forms detected in the incubation media from the hypothalamus. Furthermore, in the present studies incubation of hypothalamus with exogenous fl-endorphin-(1-31) led to break-
28 down of the exogenous fl-endorphin-(l-31), indicating that processing of fl-endorphin-(1-31) could also take place following its release from the hypothalamic tissue a n d that the two peptidase inhibitors, bestatin and bacitracin, which were present in the i n c u b a t i o n m e d i u m could not block completely the b r e a k d o w n of the fl-endorphin-(1-31). The physiological significance of either the release or the generation of different molecular weight forms of fl-endorphin by the h y p o t h a l a m u s is not known. A n u m b e r of studies have recently shown that shorter f l - e n d o r p h i n fragments may act as antagonists to fl-endorphin-(1-31) (Suh et al., 1987). Thus these fl-endorphin forms detected in the i n c u b a t i o n media m a y have a specific r o l e in the hypothalamus. In conclusion, the results from the present studies indicate that the effect of ethanol on the release of fl-endorphin-like peptides from the hypothalamus depends on the c o n c e n t r a t i o n of ethanol used. H y p o t h a l a m u s exhibits an increased release of fl-endorphin-like peptides following exposure to ethanol. This increased release is more pron o u n c e d at low c o n c e n t r a t i o n s of ethanol and may be involved in m e d i a t i n g some of the effects of ethanol, as mild euphoria a n d analgesia. High concentrations of ethanol induce a smaller increase in the release of h y p o t h a l a m i c 13endorphin-like peptides, a n d this m a y be partially responsible for the depression a n d anxiety an individual may feel following excessive ethanol consumption.
Acknowledgements The author wishes to thank Mrs. D. Vetro and Mrs. J. Currie for preparation of the manuscript. This work was supported by the Medical Research Council of Canada, Grant MA-6923.
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