PII S0024-3205(97)00405-0
PHARMACOLOGY LETTERS
Accelerated Communication
Departamento de Farmacologia, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain. 1Centro de Investigaciones Biomedicas, Consejo Superior de Investigaciones Científicas, Arturo Duperier 4, 28029 Madrid, Spain (Submitted January 31, 1997; accepted March 3, 1997; received in final form April 21, 1997)
Key Words: delta-9-tetrahydrocannabinol, proenkephalin, prodynorphin, spinal cord
Introduction
Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of the marihuana, is known to produce a variety of pharmacological effects including antinociception (1). In recent years, a great number of studies have attempted to elucidate the THC-mediated regulation of antinociception. Several lines of evidence indicate an interaction between cannabinoid and opioid systems. THC inhibits in a non competitive manner the binding of µ- and delta-, but not kappa-opioid receptor ligands to rat brain membranes (2). Welch and coworkers have found that the antinociceptive effects of intrathecal administration of morphine is enhanced by pretreatment of THC (3) and that the kappa-opioid receptor antagonist nor-binaltorphimine blocked THC-induced antinociception (4). In addition, pretreatment with an antisense oligodeoxynucleotide directed against the kappa-1receptor significantly reduced the antinociceptive effects of THC (5). Since kappa-opioid binding in the brain has been shown to remain unaltered by cannabinoids (2), and cannabinoid binding in the brain is not displaced by nor-binaltorphimine or the kappa-opioid agonist U-50,488, THC-induced antinociception may involve an increase in releasable kappa endogenous ligand dynorphin. Indeed, intrathecal administration of dynorphin1-8 or dynorphin 1-17 antisera partially blocked the THC-induced analgesia in the tail flick test (6,7), suggesting that spinal analgesia induced by THC may be mediated, at least in part, by an increase in dynorphin-related peptides in the spinal cord. However, the molecular mechanisms by which both the cannabinoid and opioid systems interact in the spinal cord remain to be determined. To this aim, the purpose of this study was to investigate the effects of five-days treatment with THC on prodynorphin (PDYN) and proenkephalin (PENK) gene expression in the spinal cord.
Methods
Animals: Adult male Sprague-Dawley rats, weighing 200-225 g, were obtained from Interfauna Iberica Laboratories (San Feliu de Codines, Barcelona, Spain) and maintained under conditions of controlled temperature (23 ± 1°C) and lighting (lights on 0800-2000 h), with food and water provided ad libitum.
Drug and treatment: THC (generously provided by the National Institute of Drug Abuse, NIDA, Baltimore, MD, USA) was dissolved in saline:ethanol:cremophor (18:1:1) and intraperitoneally administered (5 mg/kg;1 ml/kg) daily at 10:00 A.M. for five days. Five hours after the last injection rats were killed by decapitation. Spinal cord tissue from each animal was quickly removed and immediately frozen on dry ice and stored at -80º C until use.
Northern blot procedure: Total spinal cord RNA from vehicle or THC-treated group was isolated by the guanidinum thiocyanate method described by Chomczynski and Sacchi (8). Poly (A)+ RNA purification was performed through Amersham oligo(dT)-cellulose spin columns following the manufacturer instructions.
The probes used were restriction fragments prepared from plasmids containing cDNA for rat preprodynorphin, plasmid pSP64D1.7 (kindly provided by J. Douglass (9), insert: 1.7 kb digested with EcoRI and PstI); rat proenkephalin plasmid pSEAY1 (kindly provided by S. Sabol (10), insert: 0.97 kb, excised with SmaI-SacI) or human S26 ribosomal protein plasmid pHSS26 (kindly provided by S. Vincent (11); insert: 500 bp cleaved with HindIII-KpnI). The probes were labeled with alpha[32P] dCTP by random priming using the Megaprime DNA labeling system, and purified through Sephadex G-50 spin columns to specific activity of about 5 x 10 8 cpm/µg.
For Northern blot analysis, aliquots (20 µg) of Poly (A)+ RNA were denatured at 65ºC for 5 minutes in 5% formaldehyde, 50% formamide, 8% glycerol and then size-fractionated by gel electrophoresis (20 mA, 15 h) in a 0.9 % agarose gel under denaturing conditions. RNAs were then blotted and fixed to NYTRAN membranes. Prehybridization and hybridization were carried out as described by Thomas (12).
Quantitation analysis of autoradiograms was performed by using a Macintosh Power PC computer with the public domain NIH Image program (developed at U.S. National Institutes of Health and available from the internet by anonymous FTP from zippy.nimh.nih.gov). Densitometric analyses of autoradiograms were determined using Gel Plotting Macro provided by NIH Image program.
Results
RNA blots probed for prodynorphin, proenkephalin and S26 mRNAs are depicted in Figure 1. S26 is an abundant ribosomal protein and represents a suitable internal control for gene regulation experiments (11). S26 mRNA was used to standardize each lane for the amount of RNA applied. Data were expressed as the ratio of prodynorphin or proenkephalin optical density to that of S26 and as percent of control from vehicle-treated rats. The intensity of the hybridization signal of prodynorphin and proenkephalin is clearly more intense in the animals treated with THC than in vehicle-treated group (Figure 1A). Indeed, the densitometric computer analysis revealed that the i.p administration of THC (5 mg/kg; 5 days) increased prodynorphin (39%) and proenkephalin (34%) mRNA levels in the spinal cord when compared to the vehicle-treated group (Figure 1B).
Fig.1 Effects of THC and vehicle on proenkephalin, prodynorphin and S26 mRNA levels in the spinal cord. Rats receive either vehicle (saline:ethanol:cremophor, 18:1:1; 1 ml/kg; i.p., daily 5 days) or THC (5 mg/kg/day). Five hours after the last administration, animals were killed by decapitation and spinal cord was quickly removed and frozen over dry ice. Panel A represents mRNA blots probed for proenkephalin, prodynorphin and S26 from 20 µg mRNA Poly A prepared from pools of 6 rats per group. Panel B shows opioids mRNA/S26 mRNA levels expressed as percent of control. Bars represent the mean ±S.E.M. from 5 densitometric scans of autoradiograms made from the same blot probed successively for PDYN, PENK and S26 mRNA.
Discussion
The results of the present study clearly demonstrate that repeated administration of THC increases PDYN and PENK gene expression in the spinal cord. This enhancement of opioidergic activity induced by THC in the spinal cord may be related with the ability of this compound to induce analgesia. The observation that cannabinoids and opioids produce analgesia has sustained the idea that they may share a common mechanism of action. Studies in rats and mice have demonstrated that THC induced antinociception are mediated at both spinal and supraspinal analgesia (1,3). The localization of cannabinoids and opioid receptors in the periaqueductal gray of the brain and in the substantia gelatinosa in the spinal cord (13), areas closely related with the processing of pain signals, strengthens the hypothesis of an interaction between the cannabinoid and the opioid systems to produce analgesia. The increase in PENK and PDYN gene expression found in this study after repeated administration of THC may be a consequence of acute daily increase in release of the products cleaved from these opioid genes, that is, dynorphin A and methionine- or leucine-enkephalin. Consistent with this hypothesis, recently Mason and Welch (14) have shown that acute administration of THC in the subarachnoid space increases dynorphin A and leucine-enkephalin levels in the spinal cord, suggesting that THC induced antinociception mediated at this level involves the release of endogenous opioid ligands.
Whether the increase in PDYN and PENK gene expression in the spinal cord found in this study is mediated by opioid or cannabinoid receptors remains to be determined.
In summary, the results of the present study suggest that the effects of THC in the spinal cord involve an increase in opioid activity, and therefore sustain the hypothesis that an interaction between the cannabinoid and opioid systems in this region might play a role in cannabinoid induced analgesia.
Acknowledgements
This work was supported by the Universidad Complutense of Madrid grant PR294/95-6189, Concerted Action from the European Union grant BMH1-CT-94-1108 and Spanish Ministry of Education grant DGICYT UE95-0017. We thank the Research Technology Branch, NIDA, for the supplies of THC. J. Corchero is a Predoctoral Fellow supported by the "Comunidad Autonoma de Madrid" and J. Manzanares is supported by the Spanish Ministry of Education.
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Corresponding author: Jorge Manzanares, Ph.D., Instituto Pluridisciplinar,Universidad Complutense de Madrid,Paseo de Juan XXIII, 1, 28030 Madrid, SPAIN;(Phone)34-1-394-3271;(Fax)34-1-394-3264; (E-mail)jorgemr@eucmax.sim.ucm.es
