Neuropeptides 5: 567-570. 1985
INFLUENCE OF ACUTE.
SUBCHRONIC AND
CHRONIC TREAThENT WITI+ NEJJROLEPTIC (BALOPERIUOL) ON ENEEPEALINS AND TEEIR PRECURSORS IN THE STRIA’J’U$j OF RAT BRAIN.D. Blauc, #. Cupo. E. Castanas*, N. Bourhim*. P. Giraud’ , M.J. Bannon f
and
L.E. Eiden .Centre d’Imnunologie
INSERM-CNRS de Marseille-Luminy, Case 906, 13288
Marseflle Cedex 9, France. *
Facultede Sdecine Nord, boulevard P. Dramard, 13326
Marseille Cedex 15, France. # Laboratory of Cell Biology, NIMR,Bethesda. Maryland 20205, USA. (Reprint requests and correspondence to A.
Cupo) .
ABSTRACT
we examined
the effects of chronic, subchronlc and acute treatment with haloperidol on the ME, the MERGL and enkephalin precursor Concentrations in rat brain. The changes affected primarily the striatum. The ME content was greatly increased by the treatment , the precursor level was decreased by the haloperidol treatment. The specific mRNA for proenkephalin A increased. For these reasons, we conclude that the effect of haloperldol Increase both the biosynthesis and the processing of precursors of enkephalins In the striatum.INTRODUCTION
The enkephalina
(Met-enkephalin (HE), Met-enkephalin-Arg-Phe (MRRF) , Met-enkephalln-Arg-Gly-Leu (MERGL) and Leu-enkephalln (LE) may actas
either neurotransmltters or neuromodulators in the nervous system. These molecules occur at high concentration la the etriatum. The peptldes are contained in the sequence of the preproenkephalin A molecule (1, 2). Little Is known about the factors that regulate eakepballn biosynthesis or metabolism in the striatum.Bong et al. (3, 4) reported that Ealoperldol, a dopamlne receptor blocker, increased the ~t-enkephalln content in the atriaturn, region richly innervated by dopamlue-contalning neurons. Recently a correlation between the Increase in ME content and of the mRNA for proenkzphalin A has been reported (5, 6).
However discrepancies vere observed between the two rates of increase.
Probably the augnumtation of preproenkephalln
A
biosynthesis may not be the sole me&mime responsible for the elevated ME content. We described recently a nev fmumloglcal method for the quantification of the protein precursor of MB In individual brain areas of the rat (7). We apply the method to the investigation of the regulation of enkephalln biosynthesis in the brain of rats which are treated by au acute , subchronic and chronic treatment with halopertdol. The effects of haloperidol on the HE, MBRGL and HE precursor levels are examined by tadlo%mnmoassay in the strfatum, hypothalamusand hypophysie. In
the utriatum, a koncomlttant increase of biosynthesis and of processing may explain the great %ncreaseof the HE level in
rats treated chronically ; but WC cannot exclude the degradation of ME. Studies on the proenkephalfn mRNA detected by RNA blot analysis (“Northern”)reinforce
thisconclusion.
567
MATERIALS AND METHODS
Treatment of animals : Lou rats (150-200 g body weight) were housed in a constant temperature room (Zl-23'C) with a 12 hr light-dark cycle, with food and drink ad libitum. Haloperidol (5 mg/ml) was purchased from Laboratoires Janssen-Lebrun. The animals were injected intraperitoneally with 2 mgfkgfday ; control rats received the same volume of 0.9% NaCl solution. The animals were sacrified by decapitation at the same time of day (9 am to 12 am) and the brain regions were dissected out and frozen in liquid nitrogen until use.
Determination of opioid peptides and precursor contents : Each part of the brain was homogeneized in 500 01 of O.lN KC1 by sonication, then heated at 95°C for 15 min to destroy proteolytic and processing enzymes. The opioid peptides (ME and MERGL) and precursor concentrations were determined by specific radioimmunoassays, described previously (7, 8, 9).
Preparation of mRNA and RNA blot analysis : Striatum mRNA was extracted according to the procedure of Rowe et al. as modified by Eiden et al. (10).
RESULTS Acute treatment (Fig. 1).
Control and treated rats were killed at 5, 30 min and 1, 2, 4 and 6 hours after the haloperidol NaCl or injection. No differences
and the precursor contents was observed in the striatum during the period of observation following treatment.
ACUTE TREATMENT
STRATUM HYF’OTWAWUS HYFWHYSIS
in the ME, the MERGL and hypothalamus
IL--____
E 0123456 0123456 0123456
Figure 1 : The effect of acute treatment with haloperidol on the ME MERGL and precursor distribution in the rat brain (n = 6). The results are expressed as pmole per mg of wet tissue. Closed symbols are control rats, and open symbols
treated rats ( 0 ME , A MERGL, n precursors) Subchronic and chronic treatment
The effects of haloperidol treatment were observed in the striatum and hypophysis but not in hypothalamus.
568
SUBCHRDNlC AND CtRQNlC TREATMEt&
WITH HbLOPERlDOL
STRIATUM IffPOTHAmm )_c4 tmIPHYSlS
P A. d’
..__ *..-A-..* sL*
L_:i.._*
b.:;1 2 3 4 18 21 1 2 3 A 18 21 1 2 3 4 18 21 c TIME (d-1
Figure 2 : The effects of subchronic and chronic treatment with haloperidol on the ME, the MERGL and the precursor distribution in the rat brain (n = 6). The results are expressed as pmole/mg of wet tissue. Closed symbols : control rat ; open symbols : treated rats ( l ME ;
AMERGL ;
n precursor).
Chronic treatment
The animals were injected daily for 18 and 21 days. In the striatum, we observed an increase of ME content -(44 and 64% respectively). The MERGL content was not affected by the treatment. Contrarily, the protein precursors were reduced by 40 and 52% by the treatment. No increase was observed in the hypothalamus.
Subchronic treatment
In order to understand how the phenomenon take place in striatum, subchronic treatment was instituted. After the first day of treatment, we observed an increase of ME content (50%) which reached a maximum the second day (65%), then tended toward a plateau the thirth and fourth days of treatment, the plateau values corresponded to the values measured in
chronically treated rats. The first day the precursor content, decreased in a first step (42%). We observed then the second day an increase of the precursor concomittant with the increase of ME. Nevertheless, the precursor content of treated rats was inferior to that of the control values, the precursor level tended toward the values of chronically treated rats since the third day of treatment.
Preliminary studies on the mRNA for proenkephalin A were reported in the following table; The results are expressed as arbitrary units.
Control
7.5
Treated rats
1D 2D 3D 4D 21D 50 60 6 10 18.5
We observed a great increase of biosynthesis on the first treatment which reached a maximum the second day then decrease In chronically treated rats, the rate of increase (2,5) was in with those reported by (5, 6).
569
day of
the third day,
good agreement
DISCUSSION
We examined the effects of haloperidol on the ME, the MERGL, and the precursor contents in the striatum of rats which were treated by an acute, subchronic and chronic treatment with haloperidol. In acute treatment no difference was observed. During subchronic treatment the ME content increased, on the first day of treatement, it reached a maximum the second day
concomittantly we observed an increase of proenkephalin mRNA and a decrease of protein precursors. Probably we deal here with a juxtaposition of several phenomena : the simultaneous increase of biosynthesis and acceleration of processing of the precursor which may account for the elevation of ME concentration but the discrepancies observed between the rate of the
biosynthesis (mRNA) and the increase of ME allow us to suppose an augmentation of ME degradation. The effects of haloperidol on the enkephalin biosynthetic pathway was established by the fourth day of treatment : the respective concentration of ME, MERGL and precursor content were quite similar in rats treated chronically. We examined already the effects of others drugs, which block the dopaminergic transmission, on the enkephalin biosynthesis (opioid peptides and precursors), we observe the same phenomena as those described for the haloperidol effects (preliminary results).
Acknowledgements
The authors express their sincere thanks to Dr. H. Rickenberg for carefully reading of the manuscript and to Th. Jarry and M.C Barry for technical assistance.
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