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Regulation of both preproenkephalin mRNA and its derived opioids by haloperidol — a method for measurement of peptides and mRNA in the same tissue extract
Molecular Brain Research, 8 (1990) 243-248 Elsevier
243
BRESM 70231
Regulation of both preproenkephalin mRNA and its derived opioids by haloperidol a method for measurement of peptides and mRNA in the same tissue extract Mary E. Abood 1, James H. Eberwine 2, Elizabeth Erdelyi 1 and Christopher J. Evans 1 t Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University Medical Center, Stanford CA 94305 (U.S.A.) and 2Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104 (U.S.A.) (Accepted 3 April 1990)
Key words: Opioid peptide; Preproenkephalin mRNA; Haloperidol; Neuroleptic; Radioimmunoassay; Reverse-phase HPLC
The goal of this study was to delineate the effects of dopamine antagonists on the regulation of preproenkephalin mRNA and opioid peptides in the rat brain. We have developed a method whereby both mRNA and peptides can be efficiently measured in the same tissue extract, thus reducing the effects of intraspecies variation, differences in dissection and the number of animals required for statistical significance. A sub-chronic dose of haloperidol (3 mg/kg given i.p. in 100/~1 DMSO daily for 5 days) produced a 1.8-fold increase (P < 0.001) in striatal preproenkephalin mRNA levels when compared to animals injected with vehicle dimethyl sulfoxide (DMSO) employing the same schedule. Total opioid peptides as measured by a radioimmunoassay directed to the N-terminus of enkephalins and endorphins were elevated 1.6 fold (P < 0.001) in the rat striatum. However in other brain regions examined no increases were observed either in preproenkephalin mRNA or the tissue levels of opioid peptides. Analysis of the opioid-like immunoreactive peptides by reverse-phase HPLC analysis showed no dramatic changes in the ratios of the various opioid peptides between haloperidol and vehicle injected animals. Naive animals showed no statistical differences in opioid peptide levels compared to the haloperidol treated animals. There was a statistically significant decrease (30%) in the opioid peptide content of the animals injected with vehicle daily for 5 days when compared with the animals merely sacrificed, or those given acute injections (either with haloperidol or vehicle) the day of sacrifice. We have demonstrated that opioid peptides and mRNA can be measured in the same tissue extract and the data are suggestive that increases in stored opioid peptides may not directly parallel increases in mRNA. INTRODUCTION T h e levels of p r o e n k e p h a l i n - d e r i v e d peptides in the rat brain striatum are p o s t u l a t e d to be u n d e r tonic inhibitory control by d o p a m i n e 15 in a similar fashion to that of p r o o p i o m e l a n o c o r t i n in cells of the i n t e r m e d i a t e lobe of the rat pituitary 2. D o p a m i n e r e c e p t o r antagonists, such as h a l o p e r i d o l , have been shown to increase both p r e p r o e n k e p h a l i n m R N A levels and e n k e p h a l i n p e p t i d e levels in the nucleus accumbens and caudate p u t a m e n , but not in o t h e r brain areas 12-14. It appears that the regulation m a y occur at all levels of e n k e p h a l i n gene expression, i.e., from transcription through processing of the p r o e n k e p h a l i n precursor 12-15. In o r d e r to examine the time course of d o p a m i n e r g i c regulation of opioid p e p t i d e and m R N A levels concurrently, we have develo p e d a m e t h o d for extracting peptides and m R N A from the same tissue extracts. This a p p r o a c h has the a d d e d a d v a n t a g e of reducing the n u m b e r of animals required to o b t a i n statistically significant d a t a as well as the effects of intraspecies variation and differences in dissection. By
interfacing r a d i o i m m u n o a s s a y with reverse-phase highp e r f o r m a n c e liquid c h r o m a t o g r a p h y ( R P - H P L C ) we d e t e r m i n e d the identity of the o p i o i d p e p t i d e s p r e s e n t in the co-extracted tissues. MATERIALS AND METHODS
Treatment paradigm Male Sprague-Dawley rats from Bantin and Kingman (200-250 g) were injected intraperitoneally with a 0.1 mi injection volume of haloperidol (3 mg/kg) or bromocriptine (3 mg/kg) or 100% DMSO (vehicle) daily for the time period (1-5 days). On the last day of treatment all animals received an injection 4 h prior to sacrifice. Rats were sacrificed by decapitation and the brains dissected by the Glowinski-Iversen procedure7 and immediately frozen in liquid nitrogen. Tissue preparation Tissues were homogenized with a polytron in 5 M guanidinium thiocyanate (GTC), 10 mM EDTA, 50 mM Tris-HCI, pH 7.5 and 8% fl-mercaptoethanol using 7 vols. per wet weight GTC extract. RNA was obtained in a modification of the procedure originally described by Cathala 1. Extracts were precipitated overnight at 4 °C with 7 vols. of 4 M LiC1, and the RNA pelleted by centrifugation at 12,000 g for 90 min. The pellet was resuspended in 3 M LiC1 (4
Correspondence: M.E. Abood, Department of Pharmacology, Box 524, Medical College of Virginia, Richmond, VA 23298, U.S.A. 0169-328X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
244 ml/0.5 g tissue), spun at 12,000 g for 60 min and resuspended in solubilization buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), using 4 ml/0.5 g tissue). The RNA was then extracted by a 1:1 phenol/chloroform extraction, followed by a 1:1 chloroform extraction. Following addition of 0.1 vol. of 3 M NaOAc, pH 5.5 to the aqueous phase, RNA was precipitated overnight a t - 2 0 °C by addition of 2.5 vols. of ethanol. (See also Fig. 1). Alternatively, RNA was isolated by centrifugation through a CsC! gradient 3. Peptides were extracted from the GTC supernatants using reverse phase C18 Sep-Pak cartridges. The extract was acidified with acetic acid (to 5%) and adsorbed onto Sep-Paks, pre-activated with 6 ml methanol then equilibrated with 10 ml 5% acetic acid. After loading the sample, cartridges were washed twice with 5 ml 5% acetic acid then peptides were eluted with acetic acid-acetone (17 M acetic acid:H20:acetone, 2:5:20, v:v). Following elution the acetic acid and acetone were removed by centrifugal evaporation using a speed vac concentrator at room temperature and the residue redissolved in 5% acetic acid. Alternatively, peptides were extracted directly from tissues by sonication in 5 vols. per gram wet weight acid-acetone (12 N HChH20:acetone, 1:6:40, v:v). Insoluble material was pelleted by centrifugation, the acid-acetone was removed by evaporation and the residue was redissolved in 5% acetic acid 17.
N-terminus and has been described in great detail elsewhere tt. For RP-HPLC analysis of the immunoreactive material 2 × 100 mm columns packed with Hypersil WP300 C8 resin were employed. For details of the tandem column system see Maidment et a1.11. Peptides were eluted at 0.5 ml/min using an acetonitrile gradient (see Fig. 4 for gradient details) in 50 mM sodium phosphate pH 2.8.
Preparation of preproenkephalin probes and hybridization conditions Equivalent amounts of total RNA (20 #g as measured by Comparisonof totalopioidpeptldL~sextractedfromaci(I-acehmeversus(;TC-I,iCI A striatum 3000 2500 hypothalamus
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Radioimmunoassay and RP-HPLC Total opioid peptides were measured by a chemical/immunoassay directed to the N-terminus of enkephalins and endorphins 17. For samples redissolved in acetic acid following Sep Pak or acid-acetone extraction an aliquot (5-25 #1) was removed, evaporated to dryness and the residue redissolved in 100 #1 RIA buffer (see below) diluted 1:10. The sample was then acetylated by addition of 5 #1 acetic anhydride dissolved in 10 #1 DMSO. The sample was dried and 100 #1 6% ammonium hydroxide added which hydrolyses O-acetylated phenolic esters and reacts with the excess of acetic anhydride. Following removal of the ammonium hydroxide by centrifugal evaporation, the sample was redissolved in RIA buffer consisting of 0.15 M potassium phosphate, 0.1% gelatin, 0.2% Tween 20, pH 7.5. For the acetylation of the HPLC fractions, aliquots were evaporated to dryness, redissoived in RIA buffer, diluted 1:10 and acetylated as described above. The RIA employed was a solid phase assay utilizing antisera generated to a-N-acetyl-fl-endorphin. The assay cross-reacts with all peptides with a-N-acetyl-YGGF-X at the
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Fig. 1. Procedure for isolating peptides and mRNA from the same tissue extract. The steps for isolating peptides and mRNA from the same tissue extract are illustrated here.
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Fig. 2. Comparison of tissue extraction in acid:acetone vs GTC and the effect of haloperidol treatment on opioid peptides and preproenkephalin mRNA levels. A: tissues were extracted in 5 vols. acid:acetone or 5 M GTC, absorbed to SepPak C18 and eluted in acid:acetone (as described in Methods). Following chemical acetylation, the ~-N-acetyl-YGGF-X were measured by RIA. B: total RNA was prepared from the GTC-LiCI pellet as described in the Methods and slot blots prepared from the individual tissues. Shown is the analysis of the resulting autoradiograms. The optical density units (O.D. Units, vertical axis) are relative values and can only be compared within tissues and not between tissues. The empty bars represent the tissues from control rats and the solid from the haloperidol treated. The error bars represent standard errors of the mean. The * indicates statistical significance at the P < 0.001 level according to Student's t-test. They represent averages from 5 animals per treatment group.
245 acetone in several rat tissues we found the total concentration of opioid-like immunoreactive peptides was equivalent for the two procedures (Fig. 2A). As shown by Northern blot analysis, a 1.5 kb message corresponding to rat preproenkephalin was detected with both the synthetic oligonucleotide probe and the cDNA probe (data not shown). Thereafter, we measured preproenkephalin mRNA with the oligonucleotide probe exclusively. We examined several rat brain regions as well as adrenals for haloperidol- or bromocriptine-induced alterations in preproenkephalin mRNA or total opioid peptide levels. All the areas examined are known to contain preproenkephalin products TM. The only consistent change was a haloperidol-induced increase (1.5-2 fold) in both mRNA and peptide levels in the striatum compared to the vehicle-injected controls (Fig. 2), as had been previously reported 12. There were no consistent changes in the bromocriptine-treated animals (data not shown). In order to address the question of whether enkephalin mRNA and peptide levels are coordinately or differentially regulated by haioperidol, we carried out a time course of drug treatment. After 1 day of haloperidol treatment, (i.e. injections 2 and 24 h prior to sacrifice), both peptide and mRNA levels were elevated compared with the animals injected for 5 days with DMSO (controls) (Fig. 3). Peptide and mRNA levels remained elevated for the remainder of the treatment period, with mRNA levels reaching maximum levels at day 2 (Fig. 3). An acute injection of haloperidol did not increase preproenkephalin mRNA or total opioid peptide levels (Fig. 3). Interestingly, the peptide levels in the naive (uninjected control) animals were higher than that of the
absorbance at 260 nm) were loaded on formaldehyde-agarose gels and transferred to nitrocellulose (Northern analysis) 16. Alternatively, aliquots of RNA (approximately 2 #g) were denatured and dotted onto nitrocellulose using a Schleicher and Schuell slot blot apparatus 16. Preproenkephalin mRNA was detected with both a synthetic oligonucleotide to the rat cDNA (nt 400-436) and a rat cDNA probe obtained from S Sabol (Lab. Biochem. Gen., NIH) TM. The oligonucleotide was synthesized by phosphoramidite chemistry on a Model 8600 Biosearch DNA synthesizer and labeled with 32p by a 5"-kinase reaction 16. The cDNA clone was 32p-labeled by random priming using hexanucleotide primers 6. Blots were prehybridized in 5x Denhardt's (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, fraction V), 0.1 mg/ml salmon sperm DNA, 30% formamide, 6x SSC (20x SSC is 3 M NaC1, 0.3 M sodium citrate, pH 7.0) and 50 mM Tris-HCl, pH 8 for 2-12 h at 37 °C. Then probe (0.5-1 x 107 dpm) was added in the same buffer and hybridization allowed to proceed overnight at 37 °C. Filters were washed in 0.5x SSC, 0.01% SDS for 1 h at 37 °C and exposed to film. The resulting autoradiograms were scanned using a Loats image analysis system and the optical density units recorded. In addition, the blots were stripped and re-hybridized with a fl-actin cDNA probe (obtained from L. Kedes) to correct for slight differences in loading.
RESULTS
There are several effective procedures for isolating RNA from brain tissues involving GTC, including one using a CsC1 gradient through which to centrifuge the extract, and another involving LiCI precipitation of the RNA 1'3. When we compared the extraction in G T C CsC1 vs GTC-LiC1, we found the recovery of RNA similar with both procedures. However, the opioid peptides recovered from the GTC-CsCI extracts appeared to be more variable (data not shown). We thus chose to use the LiCI procedure as shown in Fig. 1. When we compared tissue extraction in GTC-LiC1 and acid-
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Fig. 3. Time course of haloperidol regulation of enkephalin peptides and preproenkephalin mRNA. Total opioid peptides and preproenkephalin mRNA from 6 striata from 3 animals per treatment group were extracted as described in Methods. Animals were treated daily (i.e.) injections 2 and 24 h prior to sacrifice) with haloperidol (haidol) as indicated in the figure. The 'acute' treatment represents animals given a single injection of haloperidol in DMSO (acute haidol) or DMSO alone (acute control) 2 h prior to sacrifice. The analysis of preproenkephalin mRNA is from autoradiograms from duplicate slot blots and thus the O.D. units can be directly compared. The error bars represent standard errors of the mean. The * indicates significance at the P < 0.001 level relative to the 5 day control using Student's t-test.
246 RP-HPLC OF STRIATAL OPIOID-LIKE IMMUNOREACTIVE MATERIAL F R O M H A L O P E R I D O L T R E A T E D AND C O N T R O L RATS
treated animals versus vehicle-treated animals (Fig. 5 and data not shown).
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Fig. 4. RP-HPLC analysis of striatal total opioid peptide immunoreactivity from control and haloperidol-treated rats. Extracts from the control and haloperidol treated rats (used for Fig. 3) were separated on RP-HPLC (using a sodium phosphate:acetonitrile gradient) followed by RIA. The closed circles represent the data from control animals and the open circles represent data from haloperidol-treated rats.
5 day injected control. The data indicate that repeated injection with the vehicle, DMSO, leads to a decrease in total opioid peptide levels, although m R N A levels are unchanged from uninjected animals. Haloperidol clearly increases preproenkephalin m R N A levels, whereas the increase in peptide levels seems to be influenced by at least two factors, the increase in m R N A and the injection paradigm. In order to address whether the increase in peptide levels (relative to the 5 day injected control) seen after 1 day of haloperidol treatment was due to an effect of the drug, or was a consequence of the injection, we modified the injection paradigm such that each animal was injected with either DMSO vehicle or haloperidol each day for the treatment period. Thus, for example, the animals that were treated with haloperidol for 1 day had received 4 daily DMSO injections prior to haloperidol. We observed the same results as obtained in Fig. 3A, i.e., total opioid peptide levels were elevated after 1 day of haloperidol treatment as compared with 5 day injected controls. We examined the effect of haloperidol treatment on the different opioid peptides by interfacing the RIA with R P - H P L C . Fig. 4 shows the R P - H P L C analysis of opioid-like immunoreactive material from striata of rats that had received 5 daily injections of haloperidol or DMSO ('control'). The amount of immunoreactivity co-chromatographing on the R P - H P L C with the opioid peptide standards represented greater than 80% of the striatal opioid-like immunoreactive material measured in the crude extracts. In addition, we also determined the opioid peptide profiles after 1 and 2 days of haloperidol treatment, and saw no significant changes in the ratios of the various opioid peptides in 1, 2 and 5 day haloperidol-
DISCUSSION We have shown that opioid peptides can be efficiently extracted from a GTC-LiCI extract. Thus, peptides and m R N A can be isolated from the same tissue extract. During the course of our experiments, a method whereby peptides could be isolated from G T C - C s C I extracts was reported 9. In our hands the peptide recovery was more variable when extracted from a CsCI gradient than from the LiC1 supernatant. This may be due to the more complete separation of large proteins from peptides in the LiC1 extract. In the LiCI method proteins precipitate with the RNA, whereas in the CsCI gradient ultracentrifugation, large proteins are predominantly in a plug at the top of the centrifuge tube and this plug may trap some peptides. Since the recovery of m R N A was equivalent in both methods, we chose to use the GTC-LiCI extraction protocol for our regulation studies. We examined the effects of sub-chronic haloperidol treatment (3 mg/kg, i.p. daily) on total opioid peptides and preproenkephalin m R N A in several rat brain regions. Previously, Mocchetti et al. had reported that haloperidol had no effect on preproenkephalin m R N A levels in cortex, hypothalamus and brainstem ~2. Similarly, we found that total opioid peptide and preproenkephalin m R N A levels were increased only in striatum and not in other brain areas. The subtype of dopamine receptor responsible for these effects remains to be clearly established. Antipsychotic agents are thought to ameliorate schizophrenic hallucinations via their antagonism at D 2 receptors 4. Haloperidol has a higher affinity for D 2 than for D 1 receptors, however, at the doses used for these and previous studies on enkephalin gene expression, both receptors would be blocked 4. Two separate studies using selective D 1 and O 2 antagonists yielded conflicting results; one group found chronic administration of SCH23390, a selective D 1 antagonist, elevated preproenkephalin m R N A levels, while another found a decrease after treatment with SCH2339013"14. In a pilot experiment, we also found a slight increase with SCH23390, but it remains uncertain as to which subtype of receptor controls enkephalin gene expression. Haloperidol treatment results in increases in preproenkephalin m R N A , translatable precursor and stored opioid peptides lz-15. A time course of haloperidol treatment is one way to estimate whether the regulation of peptides and m R N A levels is coordinately regulated. When we injected animals for one day with haloperidol, we saw an increase in both m R N A and total opioid
247 peptide levels relative to a 5 day injected control animal. However, whereas the mRNA levels were increased within 24 h when compared to naive (uninjected animals), peptide levels following 1 day of haloperidol treatment were not significantly elevated above the levels in the naive animals. The only significant alteration in opioid peptide levels was a 30% decrease in the opioid peptide content in animals given 5 daily injections of DMSO vehicle. Opioid peptides are thought to be released during stress 1°, and our results suggest that the stress associated with daily injections may alter stored opioid peptide levels. If this is indeed the case it is unclear whether the increase in opioid peptides in haloperidol treated rats is due to the drug-induced increase in mRNA levels or interference with the stress effect. In order to determine whether the increase in peptide levels was due to an effect of the drug or a result of the injection, we modified the injection paradigm such that each animal received an injection with either haloperidol or the DMSO vehicle each day for the treatment period. Using this paradigm, there was still a significant increase in the animals which received 1 day of haloperidol (and 4 days of DMSO) when compared to animals which received DMSO for 5 days. However, the peptide levels in the vehicle injected animals were still lower than in the naive animals. It is still unclear whether the decrease in opioid peptide levels observed is due to injection per se or injection with DMSO. In order to address this, another series of experiments using injection paradigms of DMSO vs saline vs needle sticks would be required. Nevertheless, these data imply that due to potential stress effects related to injecting the animals, experiments relating to the measurements of opioid peptide levels must be very carefully controlled. Perhaps administration of haloperidol via minipumps may eliminate the stress due to injections. One of the advantages of using the chemical acetylation protocol followed by radioimmunoassay for quantitation of total opioid peptides is the ability to then separate the various opioid-active components by HPLC. Analysis of the opioid-like immunoreactive peptides by
R P - H P L C revealed no dramatic changes in the ratios of various opioid peptides in 1, 2 and 5 day haloperidoltreated versus vehicle-treated animals. Although there was some fluctuation in the various peptide levels from day to day with haloperidol, there was approximately a 2 fold increase of immunoreactivity eluting at the position of Met-enkephalin, Leu-enkephalin, Met-enkephalinRGL, Met-enkephalin-RF and BAM18. BAM18 had not previously been examined with respect to a potential role in haloperidol's effect on brain opioid systems, yet as shown here, is a major processing product in the striatum. BAM18 has a higher affinity for p and r receptors than for 6, whereas Met-enkephalin is thought to have specificity for t~ or/~ receptors s. Since the ratios of peptides with respect to one another were not altered by haloperidol treatment, this implies that the drug treatment itself does not alter proenkephalin processing and there would be no dramatic change in opioid receptor selectivity of the stored proenkephalin products. In this study, the total tissue content of opioid peptides has been measured. It remains to be determined whether the released opioid peptide profile is altered by haloperidol treatment. In vivo dialysis studies on basal and potassium-stimulated release of opioid peptides indicate that Met-enkephalin, Leu-enkephalin, Met-enkephalinRGL and Met-enkephalin-RF are released in naive animals 11. Additionally, it has been reported that on death there is considerable release of opioid peptides into the extracellular space 5. The contribution of this release upon death to tissue content of opioid peptides is unknown and this may further complicate the interpretation of peptide measurements following drug treatments. In conclusion, we have demonstrated that opioid peptides and mRNA can be measured in the same tissue extract, and the data are suggestive that increases in stored opioid peptides are not precisely linked to increases in mRNA.
ABBREVIATIONS
ME-RF ME-RGL RIA RP-HPLC
DMSO GTC LE ME
dimethyl sulfoxide guanidinium thiocyanate leucine-enkephalin methionine enkephalin
REFERENCES 1 Cathala, G., Savouret, J.-F., Mendez, B., West, B.L., Karin, M., Martial, J.A. and Baxter, J.D., Laboratory methods: A method for isolation of intact, translationally active ribonucleic acid, DNA, 2 (1983) 329-335.
Acknowledgements. This work was supported by NIDA Grant DA-05010, and by individual post-doctoral fellowship MH-09577 and a Peter Pande fellowship (M.E.A.).
methionine-enkephalin-arginine6-phenylalanine7 methionine-arginine6-glycineT-leucine8 radioimmunoassay reverse-phase high-performance liquid chromatography
2 Chen, C.L.C., Dionne, ET., Roberts, J.L., Regulation of pro-opioimelanocortin mRNA levels in rat pituitary by dopaminergic compounds, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 2211-2215. 3 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of biologically active ribonucleic acid from
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sources enriched in ribonuclease, Biochemistry, 18 (1979) 52945299. Creese, I., Sibley, D.R., Hamblin, H.W. and Left, S.E., The classification of dopamine receptors: relationship to radioligand binding, Annu. Rev. Neurosci., 6 (1983) 43-71. Evans, C.J., Brumbaugh, D.R., Erdelyi, E., Rudolph, V.D., Barchas, J.D. and Maidment, N.T., In vivo microdialysis of extracellular opioid peptides in the rat brain, INRC abstracts, 1989. Feinberg, A.P. and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific acitivity, Analyt. Biochem., 132 (1983) 6-13. Glowinski, J. and Iversen, L.L., Regional studies of catecholamines in rat brain. I. The disposition of 3H-norepinephrine, 3H-dopamine and 3H-DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. Hurlbut, D.E., Evans, C.J., Barchas, J.D. and Leslie, EM., Pharmacological properties of a proenkephalin-A derived opioid peptide: BAM 18, Eur. J. PharmacoL, 138 (1987) 359-366. Iadarola, M.J. and Naranjo, J.R., Extraction of peptides from guanidine thiocyanate homogenates used to isolate RNA, Peptides, 9 (1988) 669-671. Madden, J., Akil, H., Patrick, R.L. and Barchas, J.D., Stress-induced parallel changes in central opioid levels and pain responsiveness in the rat, Nature, 265 (1977) 358-360. Maidment, N.T., Brumbaugh, D.R., Rudolph, V.D., Erdelyi, E. and Evans, C.J., Microdialysis of extracellular endogenous opioid peptides from rat brain in vivo, Neuroscience, 33 (1989) 549-557.
12 Mocchetti, I., Schwartz, J.P. and Costa, E., Use of mRNA hybridization and radioimmunoassay to study mechanisms of drug-induced accumulation of enkephalins in rat brain structures, Mol. Pharmacol., 28 (1985) 86-91. 13 Mocchetti, I., Naranjo, J.R. and Costa, E., Regulation of striatat enkephalin turnover in rats receiving antagonists of specific dopamine receptors subtypes, J. Pharmacol. Exp. Ther., 241 (1987) 1120-1124. 14 Morris, B.J., Hollt, V. and Herz, A., Dopaminergic regulation of striatal proenkephalin mRNA and prodynorphin mRNA: contrasting effects of D1 and D2 antagonists, Neuroscience, 25 (1988) 525-532. 15 Sabol, S.L., Yoshikawa, K. and Hong, J.-S., Regulation of methionine enkephalin precursor messenger RNA in rat striatum by haloperidol and lithium, Biochem. Biophys. Res. Commun., 113 (1983) 391-399. 16 Sambrook, J., Fritsch, E.E and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, 1989, pp. 7.31-32; 11.31-32. 17 Weber, E., Truscott, R.J.W., Evans, C.J., Sullivan, S., Angwin, P. and Barchas, J.D., Alpha-N-acetyl fl-endorphins in the pituitary: Immunohistochemical localization using antibodies raised against dynorphin (1-13), J. Neurochem., 36 (1981) 1977-1985. 18 Yoshikawa, K., Williams, C. and Sabol, S.L., Rat brain preproenkephalin mRNA: cDNA cloning, primary structure and distribution in the central nervous system, J. Biol. Chem., 259 (1984) 14301-14306.
243
BRESM 70231
Regulation of both preproenkephalin mRNA and its derived opioids by haloperidol a method for measurement of peptides and mRNA in the same tissue extract Mary E. Abood 1, James H. Eberwine 2, Elizabeth Erdelyi 1 and Christopher J. Evans 1 t Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University Medical Center, Stanford CA 94305 (U.S.A.) and 2Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104 (U.S.A.) (Accepted 3 April 1990)
Key words: Opioid peptide; Preproenkephalin mRNA; Haloperidol; Neuroleptic; Radioimmunoassay; Reverse-phase HPLC
The goal of this study was to delineate the effects of dopamine antagonists on the regulation of preproenkephalin mRNA and opioid peptides in the rat brain. We have developed a method whereby both mRNA and peptides can be efficiently measured in the same tissue extract, thus reducing the effects of intraspecies variation, differences in dissection and the number of animals required for statistical significance. A sub-chronic dose of haloperidol (3 mg/kg given i.p. in 100/~1 DMSO daily for 5 days) produced a 1.8-fold increase (P < 0.001) in striatal preproenkephalin mRNA levels when compared to animals injected with vehicle dimethyl sulfoxide (DMSO) employing the same schedule. Total opioid peptides as measured by a radioimmunoassay directed to the N-terminus of enkephalins and endorphins were elevated 1.6 fold (P < 0.001) in the rat striatum. However in other brain regions examined no increases were observed either in preproenkephalin mRNA or the tissue levels of opioid peptides. Analysis of the opioid-like immunoreactive peptides by reverse-phase HPLC analysis showed no dramatic changes in the ratios of the various opioid peptides between haloperidol and vehicle injected animals. Naive animals showed no statistical differences in opioid peptide levels compared to the haloperidol treated animals. There was a statistically significant decrease (30%) in the opioid peptide content of the animals injected with vehicle daily for 5 days when compared with the animals merely sacrificed, or those given acute injections (either with haloperidol or vehicle) the day of sacrifice. We have demonstrated that opioid peptides and mRNA can be measured in the same tissue extract and the data are suggestive that increases in stored opioid peptides may not directly parallel increases in mRNA. INTRODUCTION T h e levels of p r o e n k e p h a l i n - d e r i v e d peptides in the rat brain striatum are p o s t u l a t e d to be u n d e r tonic inhibitory control by d o p a m i n e 15 in a similar fashion to that of p r o o p i o m e l a n o c o r t i n in cells of the i n t e r m e d i a t e lobe of the rat pituitary 2. D o p a m i n e r e c e p t o r antagonists, such as h a l o p e r i d o l , have been shown to increase both p r e p r o e n k e p h a l i n m R N A levels and e n k e p h a l i n p e p t i d e levels in the nucleus accumbens and caudate p u t a m e n , but not in o t h e r brain areas 12-14. It appears that the regulation m a y occur at all levels of e n k e p h a l i n gene expression, i.e., from transcription through processing of the p r o e n k e p h a l i n precursor 12-15. In o r d e r to examine the time course of d o p a m i n e r g i c regulation of opioid p e p t i d e and m R N A levels concurrently, we have develo p e d a m e t h o d for extracting peptides and m R N A from the same tissue extracts. This a p p r o a c h has the a d d e d a d v a n t a g e of reducing the n u m b e r of animals required to o b t a i n statistically significant d a t a as well as the effects of intraspecies variation and differences in dissection. By
interfacing r a d i o i m m u n o a s s a y with reverse-phase highp e r f o r m a n c e liquid c h r o m a t o g r a p h y ( R P - H P L C ) we d e t e r m i n e d the identity of the o p i o i d p e p t i d e s p r e s e n t in the co-extracted tissues. MATERIALS AND METHODS
Treatment paradigm Male Sprague-Dawley rats from Bantin and Kingman (200-250 g) were injected intraperitoneally with a 0.1 mi injection volume of haloperidol (3 mg/kg) or bromocriptine (3 mg/kg) or 100% DMSO (vehicle) daily for the time period (1-5 days). On the last day of treatment all animals received an injection 4 h prior to sacrifice. Rats were sacrificed by decapitation and the brains dissected by the Glowinski-Iversen procedure7 and immediately frozen in liquid nitrogen. Tissue preparation Tissues were homogenized with a polytron in 5 M guanidinium thiocyanate (GTC), 10 mM EDTA, 50 mM Tris-HCI, pH 7.5 and 8% fl-mercaptoethanol using 7 vols. per wet weight GTC extract. RNA was obtained in a modification of the procedure originally described by Cathala 1. Extracts were precipitated overnight at 4 °C with 7 vols. of 4 M LiC1, and the RNA pelleted by centrifugation at 12,000 g for 90 min. The pellet was resuspended in 3 M LiC1 (4
Correspondence: M.E. Abood, Department of Pharmacology, Box 524, Medical College of Virginia, Richmond, VA 23298, U.S.A. 0169-328X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
244 ml/0.5 g tissue), spun at 12,000 g for 60 min and resuspended in solubilization buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), using 4 ml/0.5 g tissue). The RNA was then extracted by a 1:1 phenol/chloroform extraction, followed by a 1:1 chloroform extraction. Following addition of 0.1 vol. of 3 M NaOAc, pH 5.5 to the aqueous phase, RNA was precipitated overnight a t - 2 0 °C by addition of 2.5 vols. of ethanol. (See also Fig. 1). Alternatively, RNA was isolated by centrifugation through a CsC! gradient 3. Peptides were extracted from the GTC supernatants using reverse phase C18 Sep-Pak cartridges. The extract was acidified with acetic acid (to 5%) and adsorbed onto Sep-Paks, pre-activated with 6 ml methanol then equilibrated with 10 ml 5% acetic acid. After loading the sample, cartridges were washed twice with 5 ml 5% acetic acid then peptides were eluted with acetic acid-acetone (17 M acetic acid:H20:acetone, 2:5:20, v:v). Following elution the acetic acid and acetone were removed by centrifugal evaporation using a speed vac concentrator at room temperature and the residue redissolved in 5% acetic acid. Alternatively, peptides were extracted directly from tissues by sonication in 5 vols. per gram wet weight acid-acetone (12 N HChH20:acetone, 1:6:40, v:v). Insoluble material was pelleted by centrifugation, the acid-acetone was removed by evaporation and the residue was redissolved in 5% acetic acid 17.
N-terminus and has been described in great detail elsewhere tt. For RP-HPLC analysis of the immunoreactive material 2 × 100 mm columns packed with Hypersil WP300 C8 resin were employed. For details of the tandem column system see Maidment et a1.11. Peptides were eluted at 0.5 ml/min using an acetonitrile gradient (see Fig. 4 for gradient details) in 50 mM sodium phosphate pH 2.8.
Preparation of preproenkephalin probes and hybridization conditions Equivalent amounts of total RNA (20 #g as measured by Comparisonof totalopioidpeptldL~sextractedfromaci(I-acehmeversus(;TC-I,iCI A striatum 3000 2500 hypothalamus
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Radioimmunoassay and RP-HPLC Total opioid peptides were measured by a chemical/immunoassay directed to the N-terminus of enkephalins and endorphins 17. For samples redissolved in acetic acid following Sep Pak or acid-acetone extraction an aliquot (5-25 #1) was removed, evaporated to dryness and the residue redissolved in 100 #1 RIA buffer (see below) diluted 1:10. The sample was then acetylated by addition of 5 #1 acetic anhydride dissolved in 10 #1 DMSO. The sample was dried and 100 #1 6% ammonium hydroxide added which hydrolyses O-acetylated phenolic esters and reacts with the excess of acetic anhydride. Following removal of the ammonium hydroxide by centrifugal evaporation, the sample was redissolved in RIA buffer consisting of 0.15 M potassium phosphate, 0.1% gelatin, 0.2% Tween 20, pH 7.5. For the acetylation of the HPLC fractions, aliquots were evaporated to dryness, redissoived in RIA buffer, diluted 1:10 and acetylated as described above. The RIA employed was a solid phase assay utilizing antisera generated to a-N-acetyl-fl-endorphin. The assay cross-reacts with all peptides with a-N-acetyl-YGGF-X at the
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Fig. 1. Procedure for isolating peptides and mRNA from the same tissue extract. The steps for isolating peptides and mRNA from the same tissue extract are illustrated here.
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Fig. 2. Comparison of tissue extraction in acid:acetone vs GTC and the effect of haloperidol treatment on opioid peptides and preproenkephalin mRNA levels. A: tissues were extracted in 5 vols. acid:acetone or 5 M GTC, absorbed to SepPak C18 and eluted in acid:acetone (as described in Methods). Following chemical acetylation, the ~-N-acetyl-YGGF-X were measured by RIA. B: total RNA was prepared from the GTC-LiCI pellet as described in the Methods and slot blots prepared from the individual tissues. Shown is the analysis of the resulting autoradiograms. The optical density units (O.D. Units, vertical axis) are relative values and can only be compared within tissues and not between tissues. The empty bars represent the tissues from control rats and the solid from the haloperidol treated. The error bars represent standard errors of the mean. The * indicates statistical significance at the P < 0.001 level according to Student's t-test. They represent averages from 5 animals per treatment group.
245 acetone in several rat tissues we found the total concentration of opioid-like immunoreactive peptides was equivalent for the two procedures (Fig. 2A). As shown by Northern blot analysis, a 1.5 kb message corresponding to rat preproenkephalin was detected with both the synthetic oligonucleotide probe and the cDNA probe (data not shown). Thereafter, we measured preproenkephalin mRNA with the oligonucleotide probe exclusively. We examined several rat brain regions as well as adrenals for haloperidol- or bromocriptine-induced alterations in preproenkephalin mRNA or total opioid peptide levels. All the areas examined are known to contain preproenkephalin products TM. The only consistent change was a haloperidol-induced increase (1.5-2 fold) in both mRNA and peptide levels in the striatum compared to the vehicle-injected controls (Fig. 2), as had been previously reported 12. There were no consistent changes in the bromocriptine-treated animals (data not shown). In order to address the question of whether enkephalin mRNA and peptide levels are coordinately or differentially regulated by haioperidol, we carried out a time course of drug treatment. After 1 day of haloperidol treatment, (i.e. injections 2 and 24 h prior to sacrifice), both peptide and mRNA levels were elevated compared with the animals injected for 5 days with DMSO (controls) (Fig. 3). Peptide and mRNA levels remained elevated for the remainder of the treatment period, with mRNA levels reaching maximum levels at day 2 (Fig. 3). An acute injection of haloperidol did not increase preproenkephalin mRNA or total opioid peptide levels (Fig. 3). Interestingly, the peptide levels in the naive (uninjected control) animals were higher than that of the
absorbance at 260 nm) were loaded on formaldehyde-agarose gels and transferred to nitrocellulose (Northern analysis) 16. Alternatively, aliquots of RNA (approximately 2 #g) were denatured and dotted onto nitrocellulose using a Schleicher and Schuell slot blot apparatus 16. Preproenkephalin mRNA was detected with both a synthetic oligonucleotide to the rat cDNA (nt 400-436) and a rat cDNA probe obtained from S Sabol (Lab. Biochem. Gen., NIH) TM. The oligonucleotide was synthesized by phosphoramidite chemistry on a Model 8600 Biosearch DNA synthesizer and labeled with 32p by a 5"-kinase reaction 16. The cDNA clone was 32p-labeled by random priming using hexanucleotide primers 6. Blots were prehybridized in 5x Denhardt's (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, fraction V), 0.1 mg/ml salmon sperm DNA, 30% formamide, 6x SSC (20x SSC is 3 M NaC1, 0.3 M sodium citrate, pH 7.0) and 50 mM Tris-HCl, pH 8 for 2-12 h at 37 °C. Then probe (0.5-1 x 107 dpm) was added in the same buffer and hybridization allowed to proceed overnight at 37 °C. Filters were washed in 0.5x SSC, 0.01% SDS for 1 h at 37 °C and exposed to film. The resulting autoradiograms were scanned using a Loats image analysis system and the optical density units recorded. In addition, the blots were stripped and re-hybridized with a fl-actin cDNA probe (obtained from L. Kedes) to correct for slight differences in loading.
RESULTS
There are several effective procedures for isolating RNA from brain tissues involving GTC, including one using a CsC1 gradient through which to centrifuge the extract, and another involving LiCI precipitation of the RNA 1'3. When we compared the extraction in G T C CsC1 vs GTC-LiC1, we found the recovery of RNA similar with both procedures. However, the opioid peptides recovered from the GTC-CsCI extracts appeared to be more variable (data not shown). We thus chose to use the LiCI procedure as shown in Fig. 1. When we compared tissue extraction in GTC-LiC1 and acid-
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Fig. 3. Time course of haloperidol regulation of enkephalin peptides and preproenkephalin mRNA. Total opioid peptides and preproenkephalin mRNA from 6 striata from 3 animals per treatment group were extracted as described in Methods. Animals were treated daily (i.e.) injections 2 and 24 h prior to sacrifice) with haloperidol (haidol) as indicated in the figure. The 'acute' treatment represents animals given a single injection of haloperidol in DMSO (acute haidol) or DMSO alone (acute control) 2 h prior to sacrifice. The analysis of preproenkephalin mRNA is from autoradiograms from duplicate slot blots and thus the O.D. units can be directly compared. The error bars represent standard errors of the mean. The * indicates significance at the P < 0.001 level relative to the 5 day control using Student's t-test.
246 RP-HPLC OF STRIATAL OPIOID-LIKE IMMUNOREACTIVE MATERIAL F R O M H A L O P E R I D O L T R E A T E D AND C O N T R O L RATS
treated animals versus vehicle-treated animals (Fig. 5 and data not shown).
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Fig. 4. RP-HPLC analysis of striatal total opioid peptide immunoreactivity from control and haloperidol-treated rats. Extracts from the control and haloperidol treated rats (used for Fig. 3) were separated on RP-HPLC (using a sodium phosphate:acetonitrile gradient) followed by RIA. The closed circles represent the data from control animals and the open circles represent data from haloperidol-treated rats.
5 day injected control. The data indicate that repeated injection with the vehicle, DMSO, leads to a decrease in total opioid peptide levels, although m R N A levels are unchanged from uninjected animals. Haloperidol clearly increases preproenkephalin m R N A levels, whereas the increase in peptide levels seems to be influenced by at least two factors, the increase in m R N A and the injection paradigm. In order to address whether the increase in peptide levels (relative to the 5 day injected control) seen after 1 day of haloperidol treatment was due to an effect of the drug, or was a consequence of the injection, we modified the injection paradigm such that each animal was injected with either DMSO vehicle or haloperidol each day for the treatment period. Thus, for example, the animals that were treated with haloperidol for 1 day had received 4 daily DMSO injections prior to haloperidol. We observed the same results as obtained in Fig. 3A, i.e., total opioid peptide levels were elevated after 1 day of haloperidol treatment as compared with 5 day injected controls. We examined the effect of haloperidol treatment on the different opioid peptides by interfacing the RIA with R P - H P L C . Fig. 4 shows the R P - H P L C analysis of opioid-like immunoreactive material from striata of rats that had received 5 daily injections of haloperidol or DMSO ('control'). The amount of immunoreactivity co-chromatographing on the R P - H P L C with the opioid peptide standards represented greater than 80% of the striatal opioid-like immunoreactive material measured in the crude extracts. In addition, we also determined the opioid peptide profiles after 1 and 2 days of haloperidol treatment, and saw no significant changes in the ratios of the various opioid peptides in 1, 2 and 5 day haloperidol-
DISCUSSION We have shown that opioid peptides can be efficiently extracted from a GTC-LiCI extract. Thus, peptides and m R N A can be isolated from the same tissue extract. During the course of our experiments, a method whereby peptides could be isolated from G T C - C s C I extracts was reported 9. In our hands the peptide recovery was more variable when extracted from a CsCI gradient than from the LiC1 supernatant. This may be due to the more complete separation of large proteins from peptides in the LiC1 extract. In the LiCI method proteins precipitate with the RNA, whereas in the CsCI gradient ultracentrifugation, large proteins are predominantly in a plug at the top of the centrifuge tube and this plug may trap some peptides. Since the recovery of m R N A was equivalent in both methods, we chose to use the GTC-LiCI extraction protocol for our regulation studies. We examined the effects of sub-chronic haloperidol treatment (3 mg/kg, i.p. daily) on total opioid peptides and preproenkephalin m R N A in several rat brain regions. Previously, Mocchetti et al. had reported that haloperidol had no effect on preproenkephalin m R N A levels in cortex, hypothalamus and brainstem ~2. Similarly, we found that total opioid peptide and preproenkephalin m R N A levels were increased only in striatum and not in other brain areas. The subtype of dopamine receptor responsible for these effects remains to be clearly established. Antipsychotic agents are thought to ameliorate schizophrenic hallucinations via their antagonism at D 2 receptors 4. Haloperidol has a higher affinity for D 2 than for D 1 receptors, however, at the doses used for these and previous studies on enkephalin gene expression, both receptors would be blocked 4. Two separate studies using selective D 1 and O 2 antagonists yielded conflicting results; one group found chronic administration of SCH23390, a selective D 1 antagonist, elevated preproenkephalin m R N A levels, while another found a decrease after treatment with SCH2339013"14. In a pilot experiment, we also found a slight increase with SCH23390, but it remains uncertain as to which subtype of receptor controls enkephalin gene expression. Haloperidol treatment results in increases in preproenkephalin m R N A , translatable precursor and stored opioid peptides lz-15. A time course of haloperidol treatment is one way to estimate whether the regulation of peptides and m R N A levels is coordinately regulated. When we injected animals for one day with haloperidol, we saw an increase in both m R N A and total opioid
247 peptide levels relative to a 5 day injected control animal. However, whereas the mRNA levels were increased within 24 h when compared to naive (uninjected animals), peptide levels following 1 day of haloperidol treatment were not significantly elevated above the levels in the naive animals. The only significant alteration in opioid peptide levels was a 30% decrease in the opioid peptide content in animals given 5 daily injections of DMSO vehicle. Opioid peptides are thought to be released during stress 1°, and our results suggest that the stress associated with daily injections may alter stored opioid peptide levels. If this is indeed the case it is unclear whether the increase in opioid peptides in haloperidol treated rats is due to the drug-induced increase in mRNA levels or interference with the stress effect. In order to determine whether the increase in peptide levels was due to an effect of the drug or a result of the injection, we modified the injection paradigm such that each animal received an injection with either haloperidol or the DMSO vehicle each day for the treatment period. Using this paradigm, there was still a significant increase in the animals which received 1 day of haloperidol (and 4 days of DMSO) when compared to animals which received DMSO for 5 days. However, the peptide levels in the vehicle injected animals were still lower than in the naive animals. It is still unclear whether the decrease in opioid peptide levels observed is due to injection per se or injection with DMSO. In order to address this, another series of experiments using injection paradigms of DMSO vs saline vs needle sticks would be required. Nevertheless, these data imply that due to potential stress effects related to injecting the animals, experiments relating to the measurements of opioid peptide levels must be very carefully controlled. Perhaps administration of haloperidol via minipumps may eliminate the stress due to injections. One of the advantages of using the chemical acetylation protocol followed by radioimmunoassay for quantitation of total opioid peptides is the ability to then separate the various opioid-active components by HPLC. Analysis of the opioid-like immunoreactive peptides by
R P - H P L C revealed no dramatic changes in the ratios of various opioid peptides in 1, 2 and 5 day haloperidoltreated versus vehicle-treated animals. Although there was some fluctuation in the various peptide levels from day to day with haloperidol, there was approximately a 2 fold increase of immunoreactivity eluting at the position of Met-enkephalin, Leu-enkephalin, Met-enkephalinRGL, Met-enkephalin-RF and BAM18. BAM18 had not previously been examined with respect to a potential role in haloperidol's effect on brain opioid systems, yet as shown here, is a major processing product in the striatum. BAM18 has a higher affinity for p and r receptors than for 6, whereas Met-enkephalin is thought to have specificity for t~ or/~ receptors s. Since the ratios of peptides with respect to one another were not altered by haloperidol treatment, this implies that the drug treatment itself does not alter proenkephalin processing and there would be no dramatic change in opioid receptor selectivity of the stored proenkephalin products. In this study, the total tissue content of opioid peptides has been measured. It remains to be determined whether the released opioid peptide profile is altered by haloperidol treatment. In vivo dialysis studies on basal and potassium-stimulated release of opioid peptides indicate that Met-enkephalin, Leu-enkephalin, Met-enkephalinRGL and Met-enkephalin-RF are released in naive animals 11. Additionally, it has been reported that on death there is considerable release of opioid peptides into the extracellular space 5. The contribution of this release upon death to tissue content of opioid peptides is unknown and this may further complicate the interpretation of peptide measurements following drug treatments. In conclusion, we have demonstrated that opioid peptides and mRNA can be measured in the same tissue extract, and the data are suggestive that increases in stored opioid peptides are not precisely linked to increases in mRNA.
ABBREVIATIONS
ME-RF ME-RGL RIA RP-HPLC
DMSO GTC LE ME
dimethyl sulfoxide guanidinium thiocyanate leucine-enkephalin methionine enkephalin
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Acknowledgements. This work was supported by NIDA Grant DA-05010, and by individual post-doctoral fellowship MH-09577 and a Peter Pande fellowship (M.E.A.).
methionine-enkephalin-arginine6-phenylalanine7 methionine-arginine6-glycineT-leucine8 radioimmunoassay reverse-phase high-performance liquid chromatography
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