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Regulation of proopiomelanocortin messenger RNA concentrations by opioid peptides in primary cell cultures of rat hypothalamus
Molecular Brain Research, 10 (1991) 115-121 © 1991 Elsevier Science Publishers B.V. 0169-328X/91/$03.50 ADONIS 0169328X91702863
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Regulation of proopiomelanocortin messenger RNA concentrations by opioid peptides in primary cell cultures of rat hypothalamus Sylvie l'H6reault and Nicholas Barden Molecular Psychogenetics Laboratory, CHUL Research Centre and Laval University, Ste Foy, Que. (Canada)
(Accepted 11 December 1990) Key words: fl-Endorphin; mRNA; Opioid peptide receptor; Corticotropin-releasing factor
The effects of opioid peptides on a 1.1-kb long proopiomelanocortin messenger RNA (POMC mRNA) have been investigated in rat hypothalamic cells maintained in culture. Most opioid peptides exerted an inhibitory control on POMC mRNA steady-state concentrations. fl-Endorphin1_31 caused a 65% maximal inhibitory effect (IC50 = 6.1 x 10-9 M) while slightly less inhibition was caused by Met- and Leu-enkephalin, dynorphin AI_8 and DADLE ([D-AIa2,D-Leus] enkephalin). The effects of fl-endorphinl_3~ and of Met-enkephalin were completely reversed by the t$ opioid antagonist ICI 174,864 while the r-receptor specific antagonist binaltorphimine or the o-receptor specific antagonist DTG (1,3-di(2-tolyl) guanidine) respectively blocked the inhibitory actions of dynorphin A~_8 and of DADLE. The #-receptor specific agonist DAGO ([D-Aia2,N-Me-Phe4,Glys-OL]enkephalin)did not affect POMC mRNA levels. The failure of the dopaminergic D 2 antagonist haloperidol to modify the inhibitory effects of opioid peptides argues for a direct inhibitory opioid peptide modulation of hypothalamic POMC mRNA levels mediated by the 6-, r- and o- (but not #-) receptors in vivo.
INTRODUCTION fl-Endorphin, the most potent known naturally occuring analgesic agent, was first identified and isolated from porcine pituitary 5'6. After the discovery of the opioid properties of fl-endorphin 7'28, this 31-residue peptide was found to originate from a multifunctional prohormone, proopiomelanocortin (POMC), a 31 kDa peptide which is also the precursor of adrenocorticotrophic hormone (ACTH), a- and fl-melanocyte-stimulating hormones (a-MSH and fl-MSH) and fl-lipotropin. POMC m R N A and POMC-derived peptides have been found in various tissues including brain (hypothalamus), pancreas, placenta and testis 2,11,34. However, the regulation of POMC gene transcription, as well as the nature and release of the peptides formed from the precursor have several tissue-specific characteristics 2'16"4°. POMC gene regulation in the pituitary gland has been extensively investigated. The release of a-MSH and fl-endorphin from rat neurointermediate lobe as well as the control of POMC m R N A levels in this tissue have been shown to be under direct tonic inhibition by dopamine 27,32. In contrast with neurointermediate lobe melanotropes, anterior pituitary gland corticotropes are not directly innervated by hypo-
thalamic neurons. Circulating glucocorticoids and hypothalamic factors, which reach the anterior pituitary via the portal capillary system, are thus the major factors regulating the anterior pituitary POMC system. A C T H release is stimulated by corticotropin-releasing factor and inhibited by glucocorticoid feedback action 15'43"4s. Although corticotropin-releasing factor plays a pivotal role in the control of the hypothalamic-pituitary-adrenal axis 1'18'38, endogenous opioids are also involved in this process either directly or via effects on monoaminergic neurotransmitters 2. Buckingham 8 has shown that hypothalamic corticotropin-releasing factor secretion is stimulated by low fl-endorphin concentrations while it is reduced by high concentrations of fl-endorphin. It has also been shown that corticotropin-releasing factorimmunoreactive perikarya and fl-endorphin-immunoreactive fibers are co-localized in the paraventricular nucleus of the hypothalamus 36 and that this hypothalamic fl-endorphin originates from the POMC-containing neurons in the mediobasal hypothalamus 2°. The predominant pathway for processing of hypothalamic POMC leads to the formation of fl-endorphinl_31 and a-MSH. Large amounts of these POMC-derived peptides are found in the hypothalamus 33 and, to a lesser extent, in limbic and
Correspondence: N. Barden, Molecular Psychogenetics Laboratory, CHUL Research Centre and Laval University, 2705 Blvd. Laurier, Ste Foy, Qu6., Canada GIV4G2.
116 brainstem regions 36,39. These regions are innervated by P O M C - c o n t a i n i n g fibres originating from the arcuate nucleus of the hypothalamus and may be involved in control of behavior and nociception as well as hormonal and autonomic functions23"36. In view of the fact that endogenous opioid peptides are present in the hypothalamus t3 and are involved in n e u r o h u m o r a l regulation 1,18,3°, it is important to study the regulation of hypothalamic P O M C m R N A concentrations. Some studies have reported that P O M C m R N A and POMC-related peptides are under an inhibitory dopaminergic 24"25'42"44 and glucocorticoid4 control and under a stimulatory control by serotonin 24'25. Z h a n g et al. found evidence for synaptic association between Met-enkephalin-containing axon terminals and POMCcontaining n e u r o n s in the medio-basal hypothalamus 49 and autoregulation of the P O M C system by the precursor-derived products has been assessed since the discovery of interconnections between P O M C neurons in the medio-basal hypothalamus 1°. We have investigated the possibility that naturally occurring endogenous opioid peptides including flendorphin1_31, Met- and Leu-enkephalins and dynorphin A1 8, a m a j o r dynorphin product in brain regions 45, could regulate the P O M C m R N A levels in primary cultures of rat hypothalamic neurons. In addition, specific opioid receptor agonists and antagonists have been used to characterize the type of opioid receptors involved in the control of the hypothalamic P O M C m R N A levels. Although corticotropin-releasing factor-containing n e u r o n s are often intimately associated with P O M C peptides throughout the brain 49, there is but scant
24 well plates previously treated with gelatin, poly-L-lysine(mol. wt. 90,000) and PBS+ 10% fetal calf serum and maintained in the same chemically defined medium, but without serum, at 37 °C in a humidified CO2:air, 5%:95% atmosphere. The medium was changed 5 days later and then each 2 days thereafter. After 15 days of culture, cells were incubated with test substances dissolved in the incubation medium containing 10-5 M ascorbic acid and bacitracin 30/~g/ml. Control incubations contained medium, ascorbic acid and bacitracin only. Drugs Drugs and suppliers were as follows: fl-endorphin (rat 1-31, Peninsula Lab.); dynorphin A (1-8 porcine, Peninsula Lab.); Met-enkephalin (Sigma); Leu-enkephalin (Peninsula Lab.); DAGO ([D-Ala2,N-Me-Phe4,Glys-0L]-enkephalin acetate, Sigma); DADLE ([D-Ala2,D-Leus]-enkephalin, Peninsula Lab.); ICI 174, 864 (N,N-diallyl-Tyr-Aib(a-iso-butyric acid)-Aib-Phe-Leu, Cambridge Res. Biochem.); Binaltorphimine (Binaltorphimine HCi, RBI); DTG (1,3-di(2-tolyl) guanidine, RBI); haioperidol (Sigma); bacitracin (Sigma). Haloperidol was dissolved in ethanol/HCl 0.01 N and diluted with the medium (DMEM-F12 containing supplements, ascorbic acid and bacitracin). In these experiments, control incubations also contained the same final concentration of ethanol (0.01%). RNA isolation For Northern blot analysis, total RNA was extracted using guanidinium isothiocyanate. RNA was separated on 1% agaroseformaldehyde denaturing gels and blotted onto a nylon filter. For dot blot analysis, cells were scraped from the dishes after 48 h of incubation with the test substance and cytoplasmic RNA was extracted according to the procedure of White and CarterBancroft47. Each cytoplasmic RNA preparation was serially diluted in 10 × standard saline citrate (SSC) and dot blotted onto duplicate nylon filters. RNA was fixed to the membrane by a 2 min UV exposure.
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knowledge about interactions between these two peptidergic systems. For this reason, we have also studied the effect of corticotropin-releasing factor on the P O M C m R N A level content of rat hypothalamic cells. O u r results show an inhibitory peptidergic control of the P O M C m R N A levels by both opioid peptides, mediated by ~c-, 6- and o-receptors (but not by /~receptors) and also by corticotropin-releasing factor.
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MATERIALS AND METHODS Hypothalamic cell culture Female Sprague-Dawley rats were mated during a two day period and embryos, taken between days 16 and 18 of gestation, placed in sterile phosphate-buffered saline (PBS) containing glucose, 0.6%, pH 7.4, before excision of the hypothalamus under a dissecting microscope. Cell cultures were prepared using a modified technique of Faivre-Bauman et a1.17. Hypothalami were collected in antibioticfree Dulbecco's modified Eagle's medium (DMEM:F12, 50:50, pH 7.0), supplemented with insulin (5 ~g/ml), selenium (15 nM), human transferrin (50/tg/ml), glutamine (50 mg/ml), putrescine (50/~M), progesterone (10 nM) and 10% fetal calf serum. The tissue was minced and then mechanically dissociated by passages through an elongated Pasteur pipette. Cells were seeded (1 million cells/well) in
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Fig. 1. POMC mRNA in hypothalamic cells and tissue. Total RNA was extracted from rat hypothalamic tissue (lane 1, 30 pg RNA) or cells maintained in culture during 16 days (lane 2, 20 ag RNA), separated on 1% agarose-formaldehyde denaturing gel, and blotted onto a nylon filter. The northern blot was hybridized with a POMC cRNA probe. Under our hybridization conditions, a single strong band corresponding to the 1.1 kb POMC mRNA, is identified in each lane.
117 cRNA probes Filters were hybridized with a [32p]UTP-labelled cRNA probes prepared by run-off transcription, using T7 RNA polymerase, of a 750 bp cDNA corresponding to most of exon 3 of the rat POMC gene (kindly provided by Dr. J. Drouin) inserted in the vector pGEM-1. Control of the total amount of RNA on blots was achieved by hybridization of duplicate filters with a fl-actin cRNA probe produced from a 1500 bp fl-actin PstI fragment inserted in pGEM-1.
Hybridization Filters were prehybridized for 4 h at 42 °C in the following buffer: 50% (v/v) formamide, 5 x SSC, 8 x Denhardt's, 0.1% SDS, 50 mM phosphate, 200 pg/ml yeast tRNA and 0.025% (W/V) denatured salmon sperm DNA. Hybridization took place in the same buffer containing 2 x 106 cpm/ml of 32p-labeUed cRNA, at 65 °C for 16-20 h. Filters were washed twice (20 min each) in 1 x SSC, 0.1% SDS
at room temperature and twice (1 h each) in 0.1 x SSC, 0.1% SDS at 70 °C. Filters were wrapped in Saran Wrap and exposed to Kodak X-OMAT A R films with (POMC) or without (fl-actin) intensifying screens.
Data analysis Films were analyzed with a Ras Image analysis system (Amersham) and the slope of the linear regression of the optical density of four or more serial dilutions was calculated. Only dilutions which were in the linear range of analysis were used for slope calculation. The ratio of the slope for POMC mRNA relative to the slope of fl-actin mRNA in the same sample is expressed as a percentage of the slope ratio found in the control incubation. Statistical analysis was performed with the Duncan-Kramer test23 after analysis of variance.
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Fig. 2. Opioidergic inhibition of POMC mRNA levels in hypothalamic cells is shown by RNA dot blot hybridization analysis. Hypothalamic cells were maintained in culture for 15 days before incubation for 48 h with an opioid agonist (fl-endorphinl_3,, DADLE, dynorphin A~_s, Leu-enkephalin, Met-enkephalin or DAGO, 10 7 M) added to the medium. Total RNA, extracted by the method of White and Carter-Bancroft, was deposited on duplicate nylon filters by a dot blot procedure and hybridized with either a POMC cRNA probe or a fl-actin cRNA probe. Films were analyzed with a Ras Image analysis system and the slope of the linear regression calculated from four or more serial dilutions which fell within the linear range of analysis. Results are shown as the ratio of the concentrations of POMC mRNA relative to that of fl-actin mRNA (which remains constant in non-dividing neurons) and this fraction was expressed as a percentage of the value found in control incubation (which contains ascorbic acid and bacitracin only). The results shown are the mean + S.E.M. of at least three separate experiments, each of which consisted of between 6 and 12 replicate incubations. The significance of differences between means was evaluated by the Duncan-Kramer test after analysis of variance. **P < 0.01.
On Northern blot analysis, POMC mRNA isolated from cultured hypothalamic cells was identical to that isolated from hypothalamic tissue (Fig. 1) and corresponds to the approximately 1.1 kb POMC mRNA found in pituitary gland n. Several opioid peptides markedly inhibited the accumulation of POMC mRNA in hypothalamic cells. These include fl-endorphin1_31, Met- and Leu-enkephalins, dynorphin AI_ 8 and the o-receptorspecific agonist DADLE but not the/~-receptor-specific agonist DAGO (Fig. 2). fl-Endorphinl_31 appeared to be the most potent of these inhibitory opioid peptides and caused a 65% maximal inhibition of POMC mRNA concentrations with an IC50 of 6.1 × 10-9 M (Fig. 3). Further investigation of receptor-specific actions showed
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118 that the inhibitory effects of enkephalins and of flendorphinl_3] were reversed by the 6-receptor antagonist ICI 174,864 while those of dynorphin and of D A D L E were, respectively, reversed by addition of the K-receptor antagonist, binaltorphimine or of the o-receptor antagonist D T G (Fig. 4). Incubation of hypothalamic cells with receptor-specific antagonists alone did not result in any change in P O M C m R N A concentrations (Fig. 4). We have previously shown 24 that dopamine is a potent inhibitor of P O M C m R N A concentrations in hypothaiamic cells. H o w e v e r , while the effect of dopamine is completely reversed by addition of the dopaminergic antagonist haloperidol, this drug had no effect on the inhibitory action of fl-endorphin]_3~ , dynorphin AI_s or Met-enkephalin (Fig. 5). Incubation of hypothalamic cells with 10 -7 M corticotropin-releasing factor caused a 30.2 + 2.8% (P < 0.05) reduction in P O M C m R N A concentrations while dexamethasone, 10 -7 M, inhibited P O M C m R N A steady-state concentrations by 29.3 +
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Fig. 5. Opioid peptide-induced down regulation of POMC mRNA levels in hypothalamic cells is not mediated by a D 2 dopaminergic mechanism. Haloperidol (10 -5 M) did not reverse the inhibition of POMC mRNA levels caused by 10 -7 M concentrations of flendorphinl_3], dynorphin At_ 8 or Met-enkephalin. After RNA dot blot analysis, the results are expressed as the mean + S.E.M. of two experiments each of which consisted of duplicate incubations. The significance of differences between means was evaluated by the Duncan-Kramer test after analysis of variance. **P < 0.01; *P < 0.05.
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Fig. 4. Pharmacological analysis of opioidergic effects on POMC mRNA levels in hypothalamic cells. Specific opioid-receptor antagonists (ICI 174,864, Binaltorphimine or DTG, 10-5 M) were used in the presence or absence of an opioid agonist (fl-endorphin I 3], dynorphin Al_8, Met-enkephalin or DADLE, 10 7 M) for 48 h. Total RNA was dot blot analyzed as previously described. The results shown are the mean + S.E.M. of at least two separate experiments, each of which consisted of three replicate incubations. The significance of differences between means was evaluated by the Duncan-Kramer test after analysis of variance. **P < 0.01.
As shown by Northern blot analysis of total R N A (Fig. 1), the 1.1 kb P O M C m R N A present in both hypothalamic tissue and cultured neurons is similar to that found in pituitary gland 11. The probe used here is specific for rat P O M C m R N A and the lack of any cross hybridization with 28S or 18S ribosomal R N A , permitted analysis of P O M C m R N A concentrations in cell cultures by dot blot hybridization techniques. Primary cultures of hypothalamic neurons have been well characterized 17 and shown to contain numerous peptide and aminergic neurotransmitters including a - M S H and dopamine 41. On the basis of morphological and electrical properties, these cultured neurons resemble cells in vivo. The apparent decreased level of P O M C gene expression in cultured cells as compared to intact tissue (Fig. 1) may be due either to lower basal levels of expression in vitro compared to in vivo, or decreased viability of neurons derived specifically from the arcuate nucleus compared to other hypothalamic regions. We have shown that opioid peptides exert an inhibitory control over P O M C m R N A levels. In a dose
119 dependent manner, fl-endorphinl_31 appeared to be by far the most potent opioid peptide, followed by enkephalins and dynorphin AI_ s. D A D L E , a o-opiate receptor agonist 3 had a moderate effect while D A G O , a ~-opiate receptor agonist 21 had no significant effect. Although a peptidase inhibitor was present in all incubations, we cannot exclude the possibility that differences in apparent potency stem from different rates of degradation. However, in this respect, it is interesting to note that the more stable analogs containing D-amino acids are the peptides which show the least or no activity. The inhibitory effects of D A D L E , dynorphin and Met-enkephalin were respectively reversed by opioid receptor antagonists specific for o-, r- and 6-receptors TM 37,46. Although fl-endorphinl_31 has been found to be potent at both 6 and/~ opioid receptors, the fact that fl-endorphinl_a:induced inhibition could be totally reversed by a 6-opioid receptor-specific antagonist suggests that POMC mRNA levels in hypothalamic cells are predominantly controlled via 6, r- and o-opioid receptors. This is further supported by the fact that the /~-opioid receptor-specific agonist D A G O did not decrease POMC mRNA concentrations. Thus ~ opioid receptors do not appear to be involved in the opioid peptide control of POMC mRNA concentrations. Although endogenous opioid peptides may be synthesized by hypothalamic cells, they do not appear to be released into the culture medium, since the addition of opioid receptor antagonists alone did not cause any significant change in POMC mRNA levels. The differences in the potency of each opioid peptide to induce an inhibitory effect may be explained by the nature and concentration of the opioid receptor involved, the affinity of the agonist for the receptor as well as, as mentioned above, by the stability of the opioid peptide under assay conditions. Because of this multiplicity of variables, it is difficult to make any direct comparisons of the relative potencies of the opioid peptides used in this study. It is, nevertheless, interesting to compare the effect of Met-enkephalin, which is mediated by the 6-opioid receptor, and the effect of fl-endorphin1_31 which can act at both 6- and/~-receptors but only appears to act through the 6-receptor to modify POMC mRNA levels. The greater apparent potency of fl-endorphinl_31 to decrease POMC mRNA concentrations, in comparison to Met-enkephalin, cannot therefore be ascribed to synergistic actions on both 6- and ~-receptors. This conclusion is further supported by the failure of D A G O , a ~-receptor-specific agonist, to further increase the inhibitory effects of Met-enkephalin on POMC mRNA concentrations (results not shown). Previous studies have shown that acute morphine (a /~-opioid receptor agonist 35) administration does not affect levels of immuno-
reactive fl-endorphin in the hypothalamus 26, although, in a different in vivo study it was found that, during morphine tolerance, the hypothalamic POMC system was down regulated 29. We found no effect of a p-receptorspecific agonist (DAGO) on POMC mRNA levels, which may be compared to the lack of effect of acute morphine on fl-endorphin concentrations. While hypotheses of a down regulation of the hypothalamic POMC system by opioid peptides have been formulated 1°'49, it seems unlikely, from our results, that the chronic morphine effect could be produced by direct action on p-opioid receptors. There remains the possibility that acute and chronic effects of opiate receptor agonists may differ and that chronic stimulation may lead to variations in the expression of opiate receptors themselves. In in vivo studies opioid peptide agonists could act either directly or indirectly and, in this context, it is known that opioid peptides can suppress L H R H , TRH, oxytoxin and corticotropin-releasing factor under certain conditions, and can stimulate prolactin and arginine-vasopressin ls'3°. It appears that a number of the hormonal effects produced by opioid peptides in vivo are mediated through dopaminergic, serotonergic and/or noradrenergic mechanisms 18'19'3°. A previous study 24 has shown that dopamine exerts inhibitory control over POMC mRNA concentrations, while serotonergic action was slightly stimulatory. However, the inhibitory effects of opioid peptides seen in this study could not be reversed by a dopamine receptor blocker, indicating that, in hypothalamic cells in culture, inhibitory opioid peptide actions on POMC mRNA levels are not mediated by the dopaminergic system. The fact that 6-, r- and a-opioid receptors 14 have been detected in the arcuate nucleus of the hypothalamus argues strongly for a direct action of opioid peptides on POMC mRNA levels. Because of the known involvement of hypothalamic POMC in the control of the hypothalamic-pituitaryadrenal axis 8"9'5°, an action which could be exerted via effects on corticotropin-releasing factor, and of the colocalization of corticotropin-releasing factor neurons with fl-endorphin-containing neuron terminals 36, it is important to know if certain components of the hypothalamic-pituitary-adrenal axis exert a regulatory feedback action on hypothalamic POMC mRNA levels. Nikolarakis et al. 33 found that the release of fl-endorphin was maximal 1 h after direct application of corticotropinreleasing factor into the arcuate nucleus of the rat and returned to basal levels after 2 h. We found that corticotropin-releasing factor could decrease POMC mRNA levels after 48 h of incubation with hypothalamic neurons in culture. Dexamethasone, a glucocorticoid analogue, also caused a down regulation of POMC
120 m R N A levels, in a g r e e m e n t with results from in vivo studies of h y p o t h a l a m i c P O M C m R N A c o n c e n t r a t i o n s 4. In s u m m a r y , o u r results d e m o n s t r a t e a negative r e g u l a t i o n of h y p o t h a l a m i c P O M C m R N A c o n c e n t r a tions by o p i o i d peptides m e d i a t e d t h r o u g h 6-, ~c- a n d or-
opioid r e c e p t o r subtypes. This indicates that h y p o t h a lamic P O M C - c o n t a i n i n g n e u r o n s m a y have the ability to a u t o r e g u l a t e their levels of P O M C m R N A b y f o r m a t i o n of its t r a n s l a t i o n p r o d u c t , f l - e n d o r p h i n .
REFERENCES 1 Almeida, O.EX., Nikolarakis, K.E. and Herz, A., Evidence for the involvement of endogenous opioids in the inhibition of luteinizing hormone by corticotropin-releasing factor, Endocrinology, 122 (1988) 1034-1041. 2 Autelitano, D.J., Clements, J.A., Nikolaidis, I., Canny, B.J. and Funder, J.W., Concomitant dopaminergic and glucocorticoid control of pituitary proopiomelanocortin messenger ribonucleic acid and fl-endorpin levels, Endocrinology, 121 (1987) 1689-1696. 3 Barnard, E.A. and Demoliou-Mason, C., Molecular properties of opioid receptors, Br. Med. Bull., 39 (1983) 37-45. 4 Beaulieu, S., Gagn6, B. and Barden, N., Glucocorticoid regulation of proopiomelanocortin messenger ribonucleic acid content of rat hypothalamus, Mol. Endocrinol., 2 (1988) 727-731. 5 Bradbury, A.F., Smyth, D.G. and Snell, C.R., In R. Walter and J. Meienhffer (Eds.), Peptides: Chemistry, Structure and Biology, Ann Arbor Science, MI, 1975, pp. 609-615. 6 Bradbury, A.E, Smyth, D.G. and Snell, C.R., Polypeptide hormones, molecular and cellular aspects, Ciba Found. Symp., 41 (1976) 61-75. 7 Bradbury, A.E, Smyth, D.G., Snell, C.R., Birdsall, N.J.M. and Hulme, E.C., C-fragment of lipotropin has a high affinity for brain opiate receptors, Nature, 260 (1976) 793-795. 8 Buckingham, J.C., Stimulation and inhibition of corticotropinreleasing factor secretion by beta-endorphin, Neuroendocrinology, 42 (1986) 148-152. 9 Caiogero, A.E., Galluchi, W.T., Gold, P.W. and Chrousos, G.P., Multiple feedback regulation loops upon rat hypothalamic corticotropin-releasing hormone secretion: potential clinical implications, J. Clin. Invest., 82 (1988) 676-774. 10 Chen, Y.Y. and Pelletier, G., Demonstration of contacts between proopiomelanocortin neurons in the rat hypothalamus, Neurosci. Lett., 43 (1983) 271-276. 11 Civelli, O., Birnberg, N. and Herbert, E., Detection and quantitation of proopiomelanocortin mRNA in pituitary and brain tissues form different species, J. Biol. Chem., 257 (1982) 6783-6787. 12 Cotton, R., Giles, M.G., Miller, L., Shaw, J.S. and Timms, D., ICI 174864: a highly selective antagonist for the opioid breceptor, Eur. J. Pharmacol., 97 (1984) 331-332. 13 Cuello, A.C., Central distribution of opioid peptides, Br. Med. Bull., 39 (1983) 11-16. 14 Duka, I., Schubert, P., Wuster, M., Siber, R. and Herz, A., A selective distribution pattern of different opiate receptors in certain areas of rat brain as revealed by in vitro autoradiography, Neurosci. Lett., 21 (1981) 119-124. 15 Eberwine, J.H. and Roberts, J.L., Glucocorticoid regulation of proopiomelanocortin gene transcription in rat pituitary, J. Biol, Chem., 259 (1984) 2166-2170. 16 Emeson, R.B. and Eipper, B.A., Characterization of proACTH/endorphin-derived peptides in rat hypothalamus, J. Neurosci., 6 (1986) 837-849. 17 Faivre-Bauman, A., Puymirat, J., Loudes, C. and Tixier-Vidal, A., Differentiated mouse fetal hypothalamic cells in serum-free medium. In Methods for Serum Free Culture of Neuronal and Lymphoid Cells, Alan R. Liss, New York, 1984, pp. 37-56. 18 Grossman, A. and Rees, L.H., The neuroendocrinology of opioid peptides, Br. Med. Bull., 39 (1983) 83-88. 19 Grossman, A. and Besser, G.M., Opiates control ACTH
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29 30 31 32
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through a noradrenergic mechanism, Clin. Endocrinol., 17 (1982) 287-290. Kiss, J.Z., Cassell, M.D. and Palkovits, M., Analysis of the ACTH/fl-end/a-MSH-immunoreactive afferent input to the hypothalamic paraventricular nucleus of rat, Brain Res., 324 (1984) 91-99. Kosterlitz, H.W. and Paterson, S.J., Tyr-D-AIa-GIy-MePheNHCOCHzOH is a selective ligand for the mu opiate binding sites, Br. J. Pharmacol., 73 (1981) 299 P. Kramer, C.Y., Extension of multiple range tests to group means with unequal number of replications, Biometrics, 12 (1956) 307-310. Krieger, D.T., Neuropeptides in neurologic and psychiatric disease. In Martin and Barchas (Eds.), An Overview of Neuropeptides, Raven, New York, 1986, pp. 1-32. L'H6reault, S. and Barden, N., Monoaminergic regulation of proopiomelanocortin messenger RNA concentrations in primary cell cultures of rat hypothalamus, Mol. Brain Res., 10 (1991) in press. Locatelli, V., Petraglia, F., Penalva, A. and Penerai, A.E., Effect of dopaminergic drugs on hypothalamic and pituitary immunoreactive fl-endorphin concentration in the rat, Life Sci., 33 (1983) 1711-1717. Lim, A.T.W. and Funder, J.W., Stress-induced changes in plasma, pituitary and hypothalamic immunoreactive fl-endorphin: effects of diurnal variation, adrenalectomy, corticosteroids, and opiate agonists and antagonists, Neuroendocrinology, 36 (1983) 225-234. Loeffler, J.P., Demeneix, B.A., Kley, N.A. and H611t, V., Dopamine inhibition of pro-opiomelanocortin gene expression in the intermediate lobe of the pituitary, Neuroendocrinology, 47 (1988) 95-101. Loh, Y.P., Eskay, R.L. and Brownstein, M., Alpha-MSH-like peptides in rat brain: identification and changes in level during development, Biochem. Biophys. Res. Commun., 94 (1980) 916-923. Mocchetti, I. and Costa, E., Down-regulation of hypothalamic proopiomelanocortin system during morphine tolerance, Clin. Neuropharmacol., 9 Suppl. 4 (1986) 125-127. Morley, J.S., The endocrinology of the opiates and opioid peptides, Metabolism, 30 (1981) 195-209. Morley, J.S., Chemistry of opioid peptides, Br. Med. Bull., 39 (1983) 5-10. Munemura, M., Eskay, R.L., Kebabian, J.W. and Long, R., Release of a-melanocyte-stimulating hormone from dispersed cells of the intermediate lobe of the rat pituitary gland: involvement of catecholamines and adenosine 3",5"-monophosphate, Endocrinology, 106 (1980) 1795-1803. Nikolarakis, K.E., Almeida, O.F.X. and Herz, A., Stimulation of hypothalamic fl-endorphin and dynorphin release by corticotropin-releasing factor in vitro, Brain Res., 399 (1986) 152-155. Ogawa, N., Panerai, A., Lee, S., Forsbach, G., Havlicek, V. and Friesen, H.G., fl-Endorphin concentration in the brain of intact and hypophysectomized rats, Life Sci., 25 (1979) 317-326. Paterson, S.J., Robson, L.E. and Kosterlitz, H.W., Classification of opioid receptors, Br. Med. Bull., 39 (1983) 31-36. Pilcher, W.H. and Joseph, S.A., Co-localization of CRF-ir perikarya and ACTH-ir fibers in rat brain, Brain Res., 299 (1984) 91-102. Portoghese, ES., Lipkowski, A.W. and Takemori, A.E., Binaltorphimine and nor-binaltorphimine, potent and selective kappa opioid receptor antagonists, Life Sci., 40 (1987) 1287-1292.
121 38 Rivier, C., Rivier, J. and Vale, W., Inhibition of adrenocorticotrophic hormone secretion in the rat by immunoneutralization of corticotropin-releasing factor (CRF), Science, 218 (1982) 377-379. 39 Silverman, A.J. and Pickard, G.E., The hypothalamus. In Emson (Ed.), Chemical Neuroanatomy, Raven, New York, 1983, pp. 295-336. 40 Smyth, D.G., fl-Endorphin and related peptide in pituitary, brain, pancreas and antrum, Br. Med. Bull., 39 (1983) 25-30. 41 Thomas, W.E., Studies of neurotransmitter chemistry of central nervous system neurons in primary tissue culture, Life Sci., 38 (1986) 297-308. 42 Tiligada, E. and Wilson, J.E, Dopaminergic inhibition of alpha-melanocyte-stimulating hormone release from superfused slices of rat hypothalamus, Brain Res., 457 (1988) 379-382. 43 Vale, W., Spiess, J., Rivier, C. and Rivier, J., Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and fl-endorphin, Science, 213 (1981) 1394-1397. 44 Vermes, I., Tilders, F.J.H. and Stoof, J.C., Dopamine inhibits the release of immunoreactive fl-endorphin from rat hypothalamus in vitro, Brain Res., 326 (1985) 41-46. 45 Weber, E., Evans, C.V. and Barchas, J.D., Predominance of the
46
47
48 49
50
amino-terminal octapeptide fragment of dynorphin in rat brain regions, Nature, 299 (1982) 77-79. Weber, E., Sonders, M., Quarum, M., McLeans, S., Pou, S. and Keana, J.F., 1,3-di(2-[5-3H]tolyl) guanidine: a selective ligand that labels sigma-type receptors for psychotomimetic opiates and antipsychotic drugs, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 8784-8788. White, B.A. and Carter-Bancroft, F., Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples, J. Biol. Chem., 257 (1982) 8569-8572. Yasuda, N., Greer, M.A., Greer, S.E. and Panton, P., Studies on the site of action of vasopressin in inducing adrenocorticotropin secretion, Endocrinology, 103 (1978) 906. Zhang, R., Hisano, S., Chikamori-Aoyama, M. and Daikokv, S., Synaptic association between enkephalin-containing axon terminals and proopiomelanocortin-containing neurons in the arcuate nucleus of rat hypothalamus, Neurosci. Lett., 82 (1987) 151-156. Zis, A.P., Opioidergic regulation of hypothalamo-pituitaryadrenal function in depression and Cushing's disease: an interim report, Psychoendocrinology, 13 (1988) 419-430.
115
BRESM 7 ~
Regulation of proopiomelanocortin messenger RNA concentrations by opioid peptides in primary cell cultures of rat hypothalamus Sylvie l'H6reault and Nicholas Barden Molecular Psychogenetics Laboratory, CHUL Research Centre and Laval University, Ste Foy, Que. (Canada)
(Accepted 11 December 1990) Key words: fl-Endorphin; mRNA; Opioid peptide receptor; Corticotropin-releasing factor
The effects of opioid peptides on a 1.1-kb long proopiomelanocortin messenger RNA (POMC mRNA) have been investigated in rat hypothalamic cells maintained in culture. Most opioid peptides exerted an inhibitory control on POMC mRNA steady-state concentrations. fl-Endorphin1_31 caused a 65% maximal inhibitory effect (IC50 = 6.1 x 10-9 M) while slightly less inhibition was caused by Met- and Leu-enkephalin, dynorphin AI_8 and DADLE ([D-AIa2,D-Leus] enkephalin). The effects of fl-endorphinl_3~ and of Met-enkephalin were completely reversed by the t$ opioid antagonist ICI 174,864 while the r-receptor specific antagonist binaltorphimine or the o-receptor specific antagonist DTG (1,3-di(2-tolyl) guanidine) respectively blocked the inhibitory actions of dynorphin A~_8 and of DADLE. The #-receptor specific agonist DAGO ([D-Aia2,N-Me-Phe4,Glys-OL]enkephalin)did not affect POMC mRNA levels. The failure of the dopaminergic D 2 antagonist haloperidol to modify the inhibitory effects of opioid peptides argues for a direct inhibitory opioid peptide modulation of hypothalamic POMC mRNA levels mediated by the 6-, r- and o- (but not #-) receptors in vivo.
INTRODUCTION fl-Endorphin, the most potent known naturally occuring analgesic agent, was first identified and isolated from porcine pituitary 5'6. After the discovery of the opioid properties of fl-endorphin 7'28, this 31-residue peptide was found to originate from a multifunctional prohormone, proopiomelanocortin (POMC), a 31 kDa peptide which is also the precursor of adrenocorticotrophic hormone (ACTH), a- and fl-melanocyte-stimulating hormones (a-MSH and fl-MSH) and fl-lipotropin. POMC m R N A and POMC-derived peptides have been found in various tissues including brain (hypothalamus), pancreas, placenta and testis 2,11,34. However, the regulation of POMC gene transcription, as well as the nature and release of the peptides formed from the precursor have several tissue-specific characteristics 2'16"4°. POMC gene regulation in the pituitary gland has been extensively investigated. The release of a-MSH and fl-endorphin from rat neurointermediate lobe as well as the control of POMC m R N A levels in this tissue have been shown to be under direct tonic inhibition by dopamine 27,32. In contrast with neurointermediate lobe melanotropes, anterior pituitary gland corticotropes are not directly innervated by hypo-
thalamic neurons. Circulating glucocorticoids and hypothalamic factors, which reach the anterior pituitary via the portal capillary system, are thus the major factors regulating the anterior pituitary POMC system. A C T H release is stimulated by corticotropin-releasing factor and inhibited by glucocorticoid feedback action 15'43"4s. Although corticotropin-releasing factor plays a pivotal role in the control of the hypothalamic-pituitary-adrenal axis 1'18'38, endogenous opioids are also involved in this process either directly or via effects on monoaminergic neurotransmitters 2. Buckingham 8 has shown that hypothalamic corticotropin-releasing factor secretion is stimulated by low fl-endorphin concentrations while it is reduced by high concentrations of fl-endorphin. It has also been shown that corticotropin-releasing factorimmunoreactive perikarya and fl-endorphin-immunoreactive fibers are co-localized in the paraventricular nucleus of the hypothalamus 36 and that this hypothalamic fl-endorphin originates from the POMC-containing neurons in the mediobasal hypothalamus 2°. The predominant pathway for processing of hypothalamic POMC leads to the formation of fl-endorphinl_31 and a-MSH. Large amounts of these POMC-derived peptides are found in the hypothalamus 33 and, to a lesser extent, in limbic and
Correspondence: N. Barden, Molecular Psychogenetics Laboratory, CHUL Research Centre and Laval University, 2705 Blvd. Laurier, Ste Foy, Qu6., Canada GIV4G2.
116 brainstem regions 36,39. These regions are innervated by P O M C - c o n t a i n i n g fibres originating from the arcuate nucleus of the hypothalamus and may be involved in control of behavior and nociception as well as hormonal and autonomic functions23"36. In view of the fact that endogenous opioid peptides are present in the hypothalamus t3 and are involved in n e u r o h u m o r a l regulation 1,18,3°, it is important to study the regulation of hypothalamic P O M C m R N A concentrations. Some studies have reported that P O M C m R N A and POMC-related peptides are under an inhibitory dopaminergic 24"25'42"44 and glucocorticoid4 control and under a stimulatory control by serotonin 24'25. Z h a n g et al. found evidence for synaptic association between Met-enkephalin-containing axon terminals and POMCcontaining n e u r o n s in the medio-basal hypothalamus 49 and autoregulation of the P O M C system by the precursor-derived products has been assessed since the discovery of interconnections between P O M C neurons in the medio-basal hypothalamus 1°. We have investigated the possibility that naturally occurring endogenous opioid peptides including flendorphin1_31, Met- and Leu-enkephalins and dynorphin A1 8, a m a j o r dynorphin product in brain regions 45, could regulate the P O M C m R N A levels in primary cultures of rat hypothalamic neurons. In addition, specific opioid receptor agonists and antagonists have been used to characterize the type of opioid receptors involved in the control of the hypothalamic P O M C m R N A levels. Although corticotropin-releasing factor-containing n e u r o n s are often intimately associated with P O M C peptides throughout the brain 49, there is but scant
24 well plates previously treated with gelatin, poly-L-lysine(mol. wt. 90,000) and PBS+ 10% fetal calf serum and maintained in the same chemically defined medium, but without serum, at 37 °C in a humidified CO2:air, 5%:95% atmosphere. The medium was changed 5 days later and then each 2 days thereafter. After 15 days of culture, cells were incubated with test substances dissolved in the incubation medium containing 10-5 M ascorbic acid and bacitracin 30/~g/ml. Control incubations contained medium, ascorbic acid and bacitracin only. Drugs Drugs and suppliers were as follows: fl-endorphin (rat 1-31, Peninsula Lab.); dynorphin A (1-8 porcine, Peninsula Lab.); Met-enkephalin (Sigma); Leu-enkephalin (Peninsula Lab.); DAGO ([D-Ala2,N-Me-Phe4,Glys-0L]-enkephalin acetate, Sigma); DADLE ([D-Ala2,D-Leus]-enkephalin, Peninsula Lab.); ICI 174, 864 (N,N-diallyl-Tyr-Aib(a-iso-butyric acid)-Aib-Phe-Leu, Cambridge Res. Biochem.); Binaltorphimine (Binaltorphimine HCi, RBI); DTG (1,3-di(2-tolyl) guanidine, RBI); haioperidol (Sigma); bacitracin (Sigma). Haloperidol was dissolved in ethanol/HCl 0.01 N and diluted with the medium (DMEM-F12 containing supplements, ascorbic acid and bacitracin). In these experiments, control incubations also contained the same final concentration of ethanol (0.01%). RNA isolation For Northern blot analysis, total RNA was extracted using guanidinium isothiocyanate. RNA was separated on 1% agaroseformaldehyde denaturing gels and blotted onto a nylon filter. For dot blot analysis, cells were scraped from the dishes after 48 h of incubation with the test substance and cytoplasmic RNA was extracted according to the procedure of White and CarterBancroft47. Each cytoplasmic RNA preparation was serially diluted in 10 × standard saline citrate (SSC) and dot blotted onto duplicate nylon filters. RNA was fixed to the membrane by a 2 min UV exposure.
1
knowledge about interactions between these two peptidergic systems. For this reason, we have also studied the effect of corticotropin-releasing factor on the P O M C m R N A level content of rat hypothalamic cells. O u r results show an inhibitory peptidergic control of the P O M C m R N A levels by both opioid peptides, mediated by ~c-, 6- and o-receptors (but not by /~receptors) and also by corticotropin-releasing factor.
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MATERIALS AND METHODS Hypothalamic cell culture Female Sprague-Dawley rats were mated during a two day period and embryos, taken between days 16 and 18 of gestation, placed in sterile phosphate-buffered saline (PBS) containing glucose, 0.6%, pH 7.4, before excision of the hypothalamus under a dissecting microscope. Cell cultures were prepared using a modified technique of Faivre-Bauman et a1.17. Hypothalami were collected in antibioticfree Dulbecco's modified Eagle's medium (DMEM:F12, 50:50, pH 7.0), supplemented with insulin (5 ~g/ml), selenium (15 nM), human transferrin (50/tg/ml), glutamine (50 mg/ml), putrescine (50/~M), progesterone (10 nM) and 10% fetal calf serum. The tissue was minced and then mechanically dissociated by passages through an elongated Pasteur pipette. Cells were seeded (1 million cells/well) in
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Fig. 1. POMC mRNA in hypothalamic cells and tissue. Total RNA was extracted from rat hypothalamic tissue (lane 1, 30 pg RNA) or cells maintained in culture during 16 days (lane 2, 20 ag RNA), separated on 1% agarose-formaldehyde denaturing gel, and blotted onto a nylon filter. The northern blot was hybridized with a POMC cRNA probe. Under our hybridization conditions, a single strong band corresponding to the 1.1 kb POMC mRNA, is identified in each lane.
117 cRNA probes Filters were hybridized with a [32p]UTP-labelled cRNA probes prepared by run-off transcription, using T7 RNA polymerase, of a 750 bp cDNA corresponding to most of exon 3 of the rat POMC gene (kindly provided by Dr. J. Drouin) inserted in the vector pGEM-1. Control of the total amount of RNA on blots was achieved by hybridization of duplicate filters with a fl-actin cRNA probe produced from a 1500 bp fl-actin PstI fragment inserted in pGEM-1.
Hybridization Filters were prehybridized for 4 h at 42 °C in the following buffer: 50% (v/v) formamide, 5 x SSC, 8 x Denhardt's, 0.1% SDS, 50 mM phosphate, 200 pg/ml yeast tRNA and 0.025% (W/V) denatured salmon sperm DNA. Hybridization took place in the same buffer containing 2 x 106 cpm/ml of 32p-labeUed cRNA, at 65 °C for 16-20 h. Filters were washed twice (20 min each) in 1 x SSC, 0.1% SDS
at room temperature and twice (1 h each) in 0.1 x SSC, 0.1% SDS at 70 °C. Filters were wrapped in Saran Wrap and exposed to Kodak X-OMAT A R films with (POMC) or without (fl-actin) intensifying screens.
Data analysis Films were analyzed with a Ras Image analysis system (Amersham) and the slope of the linear regression of the optical density of four or more serial dilutions was calculated. Only dilutions which were in the linear range of analysis were used for slope calculation. The ratio of the slope for POMC mRNA relative to the slope of fl-actin mRNA in the same sample is expressed as a percentage of the slope ratio found in the control incubation. Statistical analysis was performed with the Duncan-Kramer test23 after analysis of variance.
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On Northern blot analysis, POMC mRNA isolated from cultured hypothalamic cells was identical to that isolated from hypothalamic tissue (Fig. 1) and corresponds to the approximately 1.1 kb POMC mRNA found in pituitary gland n. Several opioid peptides markedly inhibited the accumulation of POMC mRNA in hypothalamic cells. These include fl-endorphin1_31, Met- and Leu-enkephalins, dynorphin AI_ 8 and the o-receptorspecific agonist DADLE but not the/~-receptor-specific agonist DAGO (Fig. 2). fl-Endorphinl_31 appeared to be the most potent of these inhibitory opioid peptides and caused a 65% maximal inhibition of POMC mRNA concentrations with an IC50 of 6.1 × 10-9 M (Fig. 3). Further investigation of receptor-specific actions showed
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118 that the inhibitory effects of enkephalins and of flendorphinl_3] were reversed by the 6-receptor antagonist ICI 174,864 while those of dynorphin and of D A D L E were, respectively, reversed by addition of the K-receptor antagonist, binaltorphimine or of the o-receptor antagonist D T G (Fig. 4). Incubation of hypothalamic cells with receptor-specific antagonists alone did not result in any change in P O M C m R N A concentrations (Fig. 4). We have previously shown 24 that dopamine is a potent inhibitor of P O M C m R N A concentrations in hypothaiamic cells. H o w e v e r , while the effect of dopamine is completely reversed by addition of the dopaminergic antagonist haloperidol, this drug had no effect on the inhibitory action of fl-endorphin]_3~ , dynorphin AI_s or Met-enkephalin (Fig. 5). Incubation of hypothalamic cells with 10 -7 M corticotropin-releasing factor caused a 30.2 + 2.8% (P < 0.05) reduction in P O M C m R N A concentrations while dexamethasone, 10 -7 M, inhibited P O M C m R N A steady-state concentrations by 29.3 +
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Fig. 4. Pharmacological analysis of opioidergic effects on POMC mRNA levels in hypothalamic cells. Specific opioid-receptor antagonists (ICI 174,864, Binaltorphimine or DTG, 10-5 M) were used in the presence or absence of an opioid agonist (fl-endorphin I 3], dynorphin Al_8, Met-enkephalin or DADLE, 10 7 M) for 48 h. Total RNA was dot blot analyzed as previously described. The results shown are the mean + S.E.M. of at least two separate experiments, each of which consisted of three replicate incubations. The significance of differences between means was evaluated by the Duncan-Kramer test after analysis of variance. **P < 0.01.
As shown by Northern blot analysis of total R N A (Fig. 1), the 1.1 kb P O M C m R N A present in both hypothalamic tissue and cultured neurons is similar to that found in pituitary gland 11. The probe used here is specific for rat P O M C m R N A and the lack of any cross hybridization with 28S or 18S ribosomal R N A , permitted analysis of P O M C m R N A concentrations in cell cultures by dot blot hybridization techniques. Primary cultures of hypothalamic neurons have been well characterized 17 and shown to contain numerous peptide and aminergic neurotransmitters including a - M S H and dopamine 41. On the basis of morphological and electrical properties, these cultured neurons resemble cells in vivo. The apparent decreased level of P O M C gene expression in cultured cells as compared to intact tissue (Fig. 1) may be due either to lower basal levels of expression in vitro compared to in vivo, or decreased viability of neurons derived specifically from the arcuate nucleus compared to other hypothalamic regions. We have shown that opioid peptides exert an inhibitory control over P O M C m R N A levels. In a dose
119 dependent manner, fl-endorphinl_31 appeared to be by far the most potent opioid peptide, followed by enkephalins and dynorphin AI_ s. D A D L E , a o-opiate receptor agonist 3 had a moderate effect while D A G O , a ~-opiate receptor agonist 21 had no significant effect. Although a peptidase inhibitor was present in all incubations, we cannot exclude the possibility that differences in apparent potency stem from different rates of degradation. However, in this respect, it is interesting to note that the more stable analogs containing D-amino acids are the peptides which show the least or no activity. The inhibitory effects of D A D L E , dynorphin and Met-enkephalin were respectively reversed by opioid receptor antagonists specific for o-, r- and 6-receptors TM 37,46. Although fl-endorphinl_31 has been found to be potent at both 6 and/~ opioid receptors, the fact that fl-endorphinl_a:induced inhibition could be totally reversed by a 6-opioid receptor-specific antagonist suggests that POMC mRNA levels in hypothalamic cells are predominantly controlled via 6, r- and o-opioid receptors. This is further supported by the fact that the /~-opioid receptor-specific agonist D A G O did not decrease POMC mRNA concentrations. Thus ~ opioid receptors do not appear to be involved in the opioid peptide control of POMC mRNA concentrations. Although endogenous opioid peptides may be synthesized by hypothalamic cells, they do not appear to be released into the culture medium, since the addition of opioid receptor antagonists alone did not cause any significant change in POMC mRNA levels. The differences in the potency of each opioid peptide to induce an inhibitory effect may be explained by the nature and concentration of the opioid receptor involved, the affinity of the agonist for the receptor as well as, as mentioned above, by the stability of the opioid peptide under assay conditions. Because of this multiplicity of variables, it is difficult to make any direct comparisons of the relative potencies of the opioid peptides used in this study. It is, nevertheless, interesting to compare the effect of Met-enkephalin, which is mediated by the 6-opioid receptor, and the effect of fl-endorphin1_31 which can act at both 6- and/~-receptors but only appears to act through the 6-receptor to modify POMC mRNA levels. The greater apparent potency of fl-endorphinl_31 to decrease POMC mRNA concentrations, in comparison to Met-enkephalin, cannot therefore be ascribed to synergistic actions on both 6- and ~-receptors. This conclusion is further supported by the failure of D A G O , a ~-receptor-specific agonist, to further increase the inhibitory effects of Met-enkephalin on POMC mRNA concentrations (results not shown). Previous studies have shown that acute morphine (a /~-opioid receptor agonist 35) administration does not affect levels of immuno-
reactive fl-endorphin in the hypothalamus 26, although, in a different in vivo study it was found that, during morphine tolerance, the hypothalamic POMC system was down regulated 29. We found no effect of a p-receptorspecific agonist (DAGO) on POMC mRNA levels, which may be compared to the lack of effect of acute morphine on fl-endorphin concentrations. While hypotheses of a down regulation of the hypothalamic POMC system by opioid peptides have been formulated 1°'49, it seems unlikely, from our results, that the chronic morphine effect could be produced by direct action on p-opioid receptors. There remains the possibility that acute and chronic effects of opiate receptor agonists may differ and that chronic stimulation may lead to variations in the expression of opiate receptors themselves. In in vivo studies opioid peptide agonists could act either directly or indirectly and, in this context, it is known that opioid peptides can suppress L H R H , TRH, oxytoxin and corticotropin-releasing factor under certain conditions, and can stimulate prolactin and arginine-vasopressin ls'3°. It appears that a number of the hormonal effects produced by opioid peptides in vivo are mediated through dopaminergic, serotonergic and/or noradrenergic mechanisms 18'19'3°. A previous study 24 has shown that dopamine exerts inhibitory control over POMC mRNA concentrations, while serotonergic action was slightly stimulatory. However, the inhibitory effects of opioid peptides seen in this study could not be reversed by a dopamine receptor blocker, indicating that, in hypothalamic cells in culture, inhibitory opioid peptide actions on POMC mRNA levels are not mediated by the dopaminergic system. The fact that 6-, r- and a-opioid receptors 14 have been detected in the arcuate nucleus of the hypothalamus argues strongly for a direct action of opioid peptides on POMC mRNA levels. Because of the known involvement of hypothalamic POMC in the control of the hypothalamic-pituitaryadrenal axis 8"9'5°, an action which could be exerted via effects on corticotropin-releasing factor, and of the colocalization of corticotropin-releasing factor neurons with fl-endorphin-containing neuron terminals 36, it is important to know if certain components of the hypothalamic-pituitary-adrenal axis exert a regulatory feedback action on hypothalamic POMC mRNA levels. Nikolarakis et al. 33 found that the release of fl-endorphin was maximal 1 h after direct application of corticotropinreleasing factor into the arcuate nucleus of the rat and returned to basal levels after 2 h. We found that corticotropin-releasing factor could decrease POMC mRNA levels after 48 h of incubation with hypothalamic neurons in culture. Dexamethasone, a glucocorticoid analogue, also caused a down regulation of POMC
120 m R N A levels, in a g r e e m e n t with results from in vivo studies of h y p o t h a l a m i c P O M C m R N A c o n c e n t r a t i o n s 4. In s u m m a r y , o u r results d e m o n s t r a t e a negative r e g u l a t i o n of h y p o t h a l a m i c P O M C m R N A c o n c e n t r a tions by o p i o i d peptides m e d i a t e d t h r o u g h 6-, ~c- a n d or-
opioid r e c e p t o r subtypes. This indicates that h y p o t h a lamic P O M C - c o n t a i n i n g n e u r o n s m a y have the ability to a u t o r e g u l a t e their levels of P O M C m R N A b y f o r m a t i o n of its t r a n s l a t i o n p r o d u c t , f l - e n d o r p h i n .
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