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The delta2-opioid receptor subtype stimulates phosphoinositide metabolism in mouse periaqueductal gray matter
ELSEVIER
PI1 SOO243205(98)00083-6
Life Sciences, Vol. 62, No. 16, pp. PL 255258, 1998 Copyright 0 1998 Eaevier science Inc. Printed in the USA. All rights resewed 0024-3205/98 $19.00 + .oo
PHARMACOLOGY LETTERS Accelerated Communication
THE DELTA,-OPIOID RECEPTOR SUBTYPE STIMULATES PHOSPHOINOSITIDE METABOLISM IN MOUSE PERIAQUEDUCTAL GRAY MATTER Marta Rodriguez-Diaz, Neurofarmacologia,
Javier Garz6n and Pilar Stichez-Bltiquez
Instituto Cajal C.S.I.C., 28002 Madrid, Spain
(Submitted November 3, 1997, accepted December 8, 1997; received in final form January 29, 1998)
Abstract. The delta@)-opioid agonists [D-Pen2J]enkephalin (DPDPE) and [D-Ala*]deltorphin Il increased the formation of inositol phosphates (IPs) in mice periaqueductal gray matter (PAG) slices pre-labeled with myo-[3H]inositol. Both s-agonists caused an increase in IP accumulation in a dosedependent manner (l-100 FM) and which was pertussis toxin (0.5 &mouse, icv) sensitive. This effect was blocked by the &antagonist ICI-174.864 (10 PM). The prese?ce of subtypes of the &opioid receptor (6, and 6,) in PAG has been suggested by pharmacological studies. In this brain structure, naltrindrole S-isothiocyanate (5’-NTII), but not 7-benzylidenenaltrexone (BNTX), antagonized the effects of DPDPE and [D-Ala*]deltorphin II, suggesting the involvement of a population of delta receptors sensitive to the a,-antagonist NT II on this effect. To further investigate the participation of &receptor subtypes in the stimulation of IPs formation, mice were injected with antisense oligodeoxynucleotides (ODNs) directed to nucleotides 7-26 or 29-46 of the cloned h-receptor mRNA, and PAG slices from these animals were used in in vitro assays. The results demonstrate that the reported increase of phosphoinositide (PI) hydrolysis depends on the agonist activation of the 6,-opioid receptor subtype in the PAG. Q 1998 Elsevier Science Inc. ?ky Word:
delta opioid receptor subtypes, periaqueductal gray matter, phosphoinositide hydrolysis, antisense oligodeoxynucleotides, delta opioid agonists, delta opioid antagonists
Introduction Pharmacological studies in vivo (1,2) and in vitro (3) have suggested the existence of subtypes of the &opioid receptor, which have been termed 6, and 6, (see 4 for review). The &,-receptor has been defined as the site activated by DPDPE, and which is sensitive to the antagonist BNTX. [DAla*]deltorphin II and DSLET have been defined as agonists at the 6,-receptor, which is sensitive to antagonism by NT II. Despite the functional evidence of &receptor heterogeneity, only one &opioid receptor has been obtained by molecular cloning (5). The mRNA encodes a protein of 372 amino acids which shows structural homology to G protein-coupled receptors. The dissimilar effects observed in the antinociceptive potency of h-agonists after in viva administrationof ODNs to the cloned &receptor (6-9), also support the existence of subtypes of the delta-opioid receptor. In mice undergoing treatment with ODNs to h-receptor mRNA, a reduction in the levels of the encoded protein has also been demonstrated following icv injection of ‘*‘I-IgGs directed to the s-receptor (9). Correspondence should he addressed to: Dr Pilar S&nchez-Blkquez. 28002 Madrid, Espaiia., (e-mail:[email protected])
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Classically it is assumed that opioids are able to inhibit adenylyl cyclase, close voltage-sensitive Ca*’ channels and hyperpolarize the cell by opening K’channels (10). Recent studies have revealed that delta-opioids also mobilize Ca*’ from intracellular stores in several cell types (11-13). They also stimulate inositol 1,4,5triphosphate formation in cells transfected with the cloned S-receptor (14,15), and in undifferentiated NG108-15 cells (13). ln the present study, an investigation was made into the effect of &opioid receptor stimulation on phosphoinositide (PI) metabolism by the pharmacologically defined 6,- (DPDPE) or 6,- ([D-Ala2]deltorfln ll) agonists. The results indicate that &agonists stimulate the formation of lPs in periaqueductal gray matter slices pre-labelled with myo-[3H]inositol. Further, the data revealed that a population of delta receptors sensitive to S-NT II@, subtype?) is involved in the regulation of this effector system via pertussis toxin-sensitive G proteins.
Materials and Methods Animals. The experimental animals were albino male mice CD- 1 (Charles River, Barcelona, Spain) weighing 22 to 25 g. These were kept in groups of ten at 22°C under conditions of a 12 h light/dark cycle (8 a.m./p.m.). Food and water were provided ad libitum. Mice were housed and used strictly in accordance with the guidelines of the European Community about Care and Use of Laboratory Animals. Synthesis of UDNs. End-capped phosphorothioate ODNs were synthesized on a CODER 300 DNA synthesizer by phosphoramidite chemistry (16). Crude ODNs were purified by reverse-phase chromatography with COP cartridges (Cruachem, Great Britain). The eluted ODNs in 50% acetonitrile-water were then lyophilized (Rouan RC 1009/RCT 90, France). Sequences were as follows : ODN6,, 5’-G*C*ACGGGCAGAG GGCACC*A*G-3’ corresponding to nucleotides 7 to 26 of the murine &receptor gene, and ODN6,, 5’-A*G*AGGGCACCAGCTCC*A*T-3’ corresponding to nucleotides 29 to 46 of the same gene sequence (5). The random oligo (ODN-RD) with the sequence 5’-C*C*CTTAT’lTACTACTC*G*C-3’, was used as a control. These ODNs have been previously characterized in antinociceptive studies (9). Administration of ODNs. lntracerebroventricular (icv) injections were made directly into the lateral ventricle. Mice were lightly anaesthetized with ether. Injection was made manually 2 mm lateral and 2 mm caudal to the bregma, at a depth of 3 mm, using a 10 pl Hamilton microliter syringe with a 26 gauge needle. Injection volumes were 4 ~1. ODNs were reconstituted in sterile water immediately prior to use. To assess the specificity of treatments three separate control groups of mice were employed: non-injected animals (naive), those which received the vehicle (saline), and animals injected with a random sequence (ODN-RD). Each ODN treatment was performed on a distinct group of 16 to 24 mice by the following schedule: on days 1 and 2 with lnmol, on days 3 and 4 with 2 nmol, and on day 5 with 3 nmol (9). On day 6 mice were decapitated and inositol phospholipid breakdown in periaqueductal gray slices was assayed. Znosifol phospholipid breakdown assay. Agonist stimulation of the formation of [3H]inositol phosphates in myo-[3H]inositol-labelled periaqueductal gray slices was measured by the method of Berridge et al. (17) with some modifications. Cross-chopped tissue slices were washed three times in warm buffer A [millimolar composition: NaCl, 142.0, KCI, 5.6, MgCl,, 1, CaCl,, 2.2, NaHCO,,3.6, HEPES, 30, and glucose, 5.6; bubbled with O,-CO, (95%-5%) and the pH adjusted to 7.41. Slices (approximately 400 yg of protein) were dispersed in buffer A containing 5 uWml(O.30 pM) of myo[3H]inositol and 10 mM lithium chloride in a final volume of 200 ~1. After the pre-incubation period, 50 pl of agonist was added and reactions allowed to proceed for a further 60 min with gentle shaking. Basal [3H]inositol phosphates accumulation was determined by incubating parallel samples in absence
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of agonist. Reactions were terminated by the addition of 0.94 ml chloroform/methanol (1:2). allowed to stand for 15 min, and sonicated for 10 s to disrupt the slices. Following the addition of 0.31 ml chloroform and 0.3 1 ml water, the tubes were vortexed and phases were separated by centrifugation at 3500 rpm for 5 min. Samples (0.75 ml) of the aqueous phases were diluted to 3 ml with water and 0.5 ml of Dowex-1X8 resin (formate form). Tubes were then vigorously mixed for 30 min and centrifuged. The supematant was aspirated and the resin washed twice with 2.5 ml 5 mM myo-inositol. Labeled inositol phosphates were elute from the resin with 0.5 ml 1M ammonium formate/O.l M formic acid. A 0.4 ml aliquot was removed for counting (Beckman LS-5801) after the addition of 2 ml ECOLUME scintillation fluid. Chemicals. Myo-2-[3H]inositol (lo-25 Ci/mmol) in an ethanol:water solution, 9: 1 was obtained from New England Nuclear. DPDPE and [D-Ala2]deltorphin II were purchased from Peninsula Laboratories (San Carlos, CA), ICI-174,864 from CRB (Cambridge, UK), BNTX and NT II came from RBI (Natick, MA.). Pertussis toxin was obtained from LIST Biological Labs (Campbell, CA)
Results Cross-chopped pre-labeled PAG slices responded to DPDPE and [D-Ala2]deltorphin II with an increase in IPs formation, although the magnitude of the response varied among animals. The results show that the stimulation was dose-dependent from 100 nM to 100 pM. Maximal stimulation occurred at 100 uM, with average percentage increases of 161i35 and 189&39 over basal values (2.641to.09 dpm/pg protein) for DPDPE and [D-Ala2]deltorphin II respectively (fig.lA). Various receptor antagonists were tested against the action of a fixed dose (100 uM) of both agonists. The effect of DPDPE and [D-Ala2]deltorphin II on total IP accumulation was blocked by ICI-174.864 (10 PM), indicating that the response was &opioid receptor mediated (fig. 1B). The participation of different &receptor subtypes in the regulation of phospholipase C activity in the mouse PAG was also investigated. The ability of &agonists to induce stimulation of PI hydrolysis was not altered by BNTX (lo-100 uM). In contrast, NT II (10 PM) significantly antagonized the stimulatory effects of DPDPE and [D-Ala’ldeltorphin II (fig. 1B). The involvement of receptor-regulated G proteins on this stimulatory effect was also examined. Pre-treatment of the slices with pertussis toxin (25 pg/ml) for 2 h only slightly reduced IP formation when in the presence of 100 uM DPDPE (fig. 1C). After increasing the incubation period to 4 h, the effect of DPDPE on IP hydrolysis diminished in both vehicle and pertussis toxin treated slices. Interestingly, in cross-chopped PAG slices obtained from in viva pertussis toxin pre-treated mice to.5 ug/mouse, icv; 6 days before sacrifice (18)] the complete blockage of DPDPE-induced stimulation of PI hydrolysis could be observed (fig. 1D). The [DAla2]deltorphin B-stimulated accumulation of IPs was even more sensitive to the impairing effect of the toxin. The effect of the agonist on IP formation was significantly reduced in mice pre-treated with 0.1 and 0.5 pg/mouse, icv (fig. 1 D). Administration of either pertussis toxin or the vehicle alone had not effect on basal IP formation (not shown). The observed efficacy of in viva administration of pertussis toxin to impair receptor-activated G protein-induced effects, allowed the extension of the study to mice subchronically treated with ODNs known to inhibit the synthesis of the cloned 8-opioid receptor (9). In these animals, the stimulation of IPs formation induced by DPDPE and [D-Ala2]deltorphin II in periaqueductal gray slices was completely blocked (fig. 2). The stimulation of IP formation over basal values for DPDPE was 161%&35, 149%&7,23%*8 and lO%ti respectively for non-injected control animals, mice treated with the random oligo, and those with the ODN directed to nucleotides 7-26 and 29-46 of the mRNA coding for the cloned &opioid receptor. Also, subchronic administration of these ODNS significantly imoaired the increase in IPs accumulation induced by [D-Ala2]deltorphin II, values were 189%&39,
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177%*42, 3%&l and 2%*1, respectively. The slight reduction in PI metabolism observed in mice subchronically injected with the ODN-RD. was also observed in PAG slices from mice receiving the vehicle (not shown). This demonstrated the efficacy and selectivity of the ODNs that target the 6receptor.
B
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Fig. 1 Delta-opioid receptor-mediated accumulation of inositi phosphates in mouse PAG. Panel A: slices were pre-incubated with myo-[‘Hlinositol for 30 min and then incubated with agonists (100 nM-100 pM) for 60 min. The results, expressed as radioactivity in the total IP fraction, are presented as percentage stimulation of IP formation over basal activity (2.649Ll.09dpn#g protein). Bach point is the mean+S.E.M. of 8-12 determinations. Panel E: antagonists ICI-174.864, NT II and BNTX (10 pM) were added at the beginning of the incubation period, 30 min before DPDPE or [DAla2]deltorphin II (100 pM). Slices were incubated for an additional period of 60 min. and total [3H]IPrecorded Panel C: slices were preincubated in the presence or absence of activated pertussis toxin 5 or 25 pg/ml for 120 min and subsequently assayed for the response to DPDPE (100 pM). Panel D: PAG from pertussis toxin-treated mice (0.1 or 0.5 @mouse icv. 6 days before sacrifice) were used to test their responses to delta agonists at 100 pM. Values are the mean~.E.M. from triplicate determinations of at least 5 separate assays.* Significantly different from the control group receiving saline, and ** from the group receiving the agonist. Analysis of variance, StudentNewman-Keuls test. P < 0.05.
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[D-Ala2]deltorphin II
r 250 200
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Control
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150 100 50
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Fig.2 Effect of subchronic icv administration of ODNs to &opioid receptors on the IPs formation induced by DPDPE and [D-Ala’~eltorphin Il. Stimulation of inositol phospholipid hydrolysis by the agonists (100 pM) was determined in periaqueductal gray slices from control (non-injected mice) and mice receiving the random oligo or the ODNs directed to nucleotides 7-26 or 2946 of the cloned &receptor mRNA (see methods). The results, expressed as radioactivity in the total IP fraction, are presented as percentage stimulation of IP formation over basal activity. Values are the mean&.E.M. from at least 6 determinations. * Significantly different from the control group receiving agonist. Analysis of variance, Student-Newman-Keuls test, P < 0.05.
Discussion The present study provides the first direct evidence that mouse brain 6-opioid receptors stimulate phosphoinositide hydrolysis. The coupling of b-receptors to this second messenger pathway has previously been described for the a-agonist DPDPE in neuroblastoma x glioma NG 108- 15 cells ( 13), and in Xenopus oocytes (14) and in Ltk- fibroblast (15) expressing cloned S-opioid receptors. These effects were mediated by delta receptor activation of G proteins sensitive to pertussis toxin. The present study also indicates that pertussis toxin-sensitive G proteins are regulated by delta-opioid receptors in the stimulation of PI formation in mouse PAG. Notwithstanding, the regulation of Gz proteins by 6 receptors in PI formation in L&cells (15), plus data from in vitro assays, suggest that participation of pertussis toxin-resistant Gz or Gq proteins in this opioid effect cannot be disregarded. In the current investigation, the pharmacologically defined &-agonist DPDPE and the selective agonist at the &-receptors [D-Ala*]deltorphin II, were equally effective in increasing the formation of inositol phosphates in slices of mouse periaqueductal gray matter. Both agonists caused an ICI- 174,864 reversible stimulation of IP accumulation, indicating the involvement of the &opioid receptor in this effect. In addition, the data show that only NT II, the selective antagonist at the 6,receptor, blocked the actions of DPDPE and [D-Ala’ldeltorphin Il. BNTX @,-antagonist) was without effect, suggesting the predominance of b,-opioid receptor subtype in the stimulation of IP formation in the mouse PAG.Moreover, in this brain structure the data revealed functional binding of DPDPE to the &,-subtype to increase PI metabolism, regardless the limited efficacy of this compound to produce effective antinociception through 6,-receptors following icv administration (4). This apparent discrepancy can be explained if the b,-receptor mediates the production of antinociception and the regulation of phosphoinositide metabolism via different coupling mechanisms. Thus, the relative efficacy of an agonist might depend on the nature of the G proteins coupled to the receptor, favoring the possibility that the coupling of Sopioid receptor with different G proteins/signaling pathways could determine receptor function. This would then explain the
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agonist/antagonist profiles for the delta ligands. In this respect, and under appropriate conditions, the antagonism of DPDPE on [D-Ala’ldeltorphin II-evoked effects has been documented in antinociceptive studies (19). Further, DPDPE antagonized the activating effect exerted by [DAla2]deltorphin II on Gi2 and G, proteins in mouse periaqueductal gray matter (20). As stated in the introduction, previous experiments with ODNs to mRNA coding for the &opioid receptor (6-9) have shown differences in the antinociceptive profiles of DPDPE and [DAla2]deltotphin lI. By using antisense oligodeoxynucleotides directed to nucleotides 7-26 or 29-46 of the cloned a-receptor mRNA [which respectively blocked 6,,- and Qinduced antinociception (9)], it was hoped that further investigation of the participation of S-receptor subtypes on IP accumulation in this brain area would be possible. The data obtained confirm the conclusions drawn from the use of selective antagonists, and further support the idea that stimulation of PI hydrolysis within the PAG is mediated through a population of &receptors, whose characteristics correlate with the 6,-receptor subtype. In summary, this study indicates that in addition to adenylyl cyclase inhibition, the closing of voltage-sensitive Ca2’ channels and the opening of K’ channels, 6-opioid receptors regulate phospholipase C activity in areas of the mouse brain. Acknowledgements This work was supported by funds provided by Comisidn Inter-ministerial Ciencia y Tecnologfa (EspaAa) I SAF95-0003. References 1. Q. JIANG, A.E. TAKEMORI, M. SULTANA, P.S. PORTOGHESE, W.D. BOWEN, H.I. MOSBERG and
R. PORRECA. J. Pharmacol. Exp. Ther. 257 1069-1075 (1991). 2. A. MATITA, T. VANDERAH, H.I. MOSBERG and F. PGRRECA. J. Pharmacol. Exp. Ther. 258 583-587 (1991). 3. B. BUZAS, S. IZENWASSER, P.S. PORTOGHESE and B.M. COX. Life Sci. &l 101-106 (1994). 4. P.A. ZAKI, E.J. BILSKY, T.W. VANDERAH, J. LAI, C.J. EVANS and F. PORRECA. Annu. Rev. Pharmacol. Toxicol. 36 379-401 (1996). 5. K. YASUDA, K. RAYNOR, H. KONG, C. BREDER, J. TAKEDA, T. REISINE and G.I. BELL. Proc. Natl. Acad. Sci. USA 90 6736-6740 (1993). 6. J. LAI, E.J. BILSKY, R.B. ROTHMAN and F. PORRECA. Nemo Report. 5 1049-1052 (1994). 7. E.J. BILSKY, R.N. BERNSTEIN, V.J. HRUBY, R.B. ROTHMAN, J. LA1 and F. PORRECA. J. Pharmacol. Exp. Ther. 277 491-501 (1996). 8. G.C. ROSSI, W. SU, L. LEVENTHAL, H. SU and G.W. PASTERNAK. Brain Res. 275 176-179 (1997). 9. P. SANCHEZBLAZQUEZ, A. GARCkESPANA and J. GARZ6N. J. Pharmacol. Exp. Ther. 280 1423-1431( 1997). 10. S.R. CHILDERS. Life Sci. &3 1991-2003 (1991). 1 I. W. JING, N.M. LEE, H.H. LOH and S.A. THAYER. Mol. Pharmacol. 42 1083-1089 (1992). 12. M.A. CONNOR, A. PLANNER and G. HENDERSON. Regu. Pept. 54 65-66 (1994). 13. D. SMART and D.G. LAMBERT. J. Neurochem. &i 1462-1466 (1996). 14. T. MIYAMAE, N. FUKUSHIMA, Y. MISU and H. UEDA. FEBS Lett. 333 31 l-314 (1993). 15. R.C. TSU, J.S.C. CHAN and Y .H. WONG. J. Neurochem. 64 2700-2707 ( 1995). 16. M.D. MATTEUCCI and M.H. CARUTHERS. J. Am. Chem. Sot. j_@ 31853191 (1981). 17. M.J. BERRIDGE, C.P. DOWNES and M.E. HANLEY. B&hem. J. 206 587-595 (1982). 18. P. SANCHEZ-BLAZQUEZ and J. GARZGN. Eur. J. Pharmacol. 152 357-361 (1988). 19. T. VANDERAH, A.E. TAKEMORI, M. SULTANA, P.S. PORTOGHESE, H.I. MOSBERG, V.J. HRUBY, R.C. HAASETH, T. MATSUNAGA and F. PORRECA European J. Pharmacol. 252 133-137 (1994). 20. J. GARZGN, A. GARCkESPANA and P. SANCHEZ-BLAZQUEZ. J. Pharmacol. Exp. Ther. m 549-557 (1997).
PI1 SOO243205(98)00083-6
Life Sciences, Vol. 62, No. 16, pp. PL 255258, 1998 Copyright 0 1998 Eaevier science Inc. Printed in the USA. All rights resewed 0024-3205/98 $19.00 + .oo
PHARMACOLOGY LETTERS Accelerated Communication
THE DELTA,-OPIOID RECEPTOR SUBTYPE STIMULATES PHOSPHOINOSITIDE METABOLISM IN MOUSE PERIAQUEDUCTAL GRAY MATTER Marta Rodriguez-Diaz, Neurofarmacologia,
Javier Garz6n and Pilar Stichez-Bltiquez
Instituto Cajal C.S.I.C., 28002 Madrid, Spain
(Submitted November 3, 1997, accepted December 8, 1997; received in final form January 29, 1998)
Abstract. The delta@)-opioid agonists [D-Pen2J]enkephalin (DPDPE) and [D-Ala*]deltorphin Il increased the formation of inositol phosphates (IPs) in mice periaqueductal gray matter (PAG) slices pre-labeled with myo-[3H]inositol. Both s-agonists caused an increase in IP accumulation in a dosedependent manner (l-100 FM) and which was pertussis toxin (0.5 &mouse, icv) sensitive. This effect was blocked by the &antagonist ICI-174.864 (10 PM). The prese?ce of subtypes of the &opioid receptor (6, and 6,) in PAG has been suggested by pharmacological studies. In this brain structure, naltrindrole S-isothiocyanate (5’-NTII), but not 7-benzylidenenaltrexone (BNTX), antagonized the effects of DPDPE and [D-Ala*]deltorphin II, suggesting the involvement of a population of delta receptors sensitive to the a,-antagonist NT II on this effect. To further investigate the participation of &receptor subtypes in the stimulation of IPs formation, mice were injected with antisense oligodeoxynucleotides (ODNs) directed to nucleotides 7-26 or 29-46 of the cloned h-receptor mRNA, and PAG slices from these animals were used in in vitro assays. The results demonstrate that the reported increase of phosphoinositide (PI) hydrolysis depends on the agonist activation of the 6,-opioid receptor subtype in the PAG. Q 1998 Elsevier Science Inc. ?ky Word:
delta opioid receptor subtypes, periaqueductal gray matter, phosphoinositide hydrolysis, antisense oligodeoxynucleotides, delta opioid agonists, delta opioid antagonists
Introduction Pharmacological studies in vivo (1,2) and in vitro (3) have suggested the existence of subtypes of the &opioid receptor, which have been termed 6, and 6, (see 4 for review). The &,-receptor has been defined as the site activated by DPDPE, and which is sensitive to the antagonist BNTX. [DAla*]deltorphin II and DSLET have been defined as agonists at the 6,-receptor, which is sensitive to antagonism by NT II. Despite the functional evidence of &receptor heterogeneity, only one &opioid receptor has been obtained by molecular cloning (5). The mRNA encodes a protein of 372 amino acids which shows structural homology to G protein-coupled receptors. The dissimilar effects observed in the antinociceptive potency of h-agonists after in viva administrationof ODNs to the cloned &receptor (6-9), also support the existence of subtypes of the delta-opioid receptor. In mice undergoing treatment with ODNs to h-receptor mRNA, a reduction in the levels of the encoded protein has also been demonstrated following icv injection of ‘*‘I-IgGs directed to the s-receptor (9). Correspondence should he addressed to: Dr Pilar S&nchez-Blkquez. 28002 Madrid, Espaiia., (e-mail:[email protected])
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Classically it is assumed that opioids are able to inhibit adenylyl cyclase, close voltage-sensitive Ca*’ channels and hyperpolarize the cell by opening K’channels (10). Recent studies have revealed that delta-opioids also mobilize Ca*’ from intracellular stores in several cell types (11-13). They also stimulate inositol 1,4,5triphosphate formation in cells transfected with the cloned S-receptor (14,15), and in undifferentiated NG108-15 cells (13). ln the present study, an investigation was made into the effect of &opioid receptor stimulation on phosphoinositide (PI) metabolism by the pharmacologically defined 6,- (DPDPE) or 6,- ([D-Ala2]deltorfln ll) agonists. The results indicate that &agonists stimulate the formation of lPs in periaqueductal gray matter slices pre-labelled with myo-[3H]inositol. Further, the data revealed that a population of delta receptors sensitive to S-NT II@, subtype?) is involved in the regulation of this effector system via pertussis toxin-sensitive G proteins.
Materials and Methods Animals. The experimental animals were albino male mice CD- 1 (Charles River, Barcelona, Spain) weighing 22 to 25 g. These were kept in groups of ten at 22°C under conditions of a 12 h light/dark cycle (8 a.m./p.m.). Food and water were provided ad libitum. Mice were housed and used strictly in accordance with the guidelines of the European Community about Care and Use of Laboratory Animals. Synthesis of UDNs. End-capped phosphorothioate ODNs were synthesized on a CODER 300 DNA synthesizer by phosphoramidite chemistry (16). Crude ODNs were purified by reverse-phase chromatography with COP cartridges (Cruachem, Great Britain). The eluted ODNs in 50% acetonitrile-water were then lyophilized (Rouan RC 1009/RCT 90, France). Sequences were as follows : ODN6,, 5’-G*C*ACGGGCAGAG GGCACC*A*G-3’ corresponding to nucleotides 7 to 26 of the murine &receptor gene, and ODN6,, 5’-A*G*AGGGCACCAGCTCC*A*T-3’ corresponding to nucleotides 29 to 46 of the same gene sequence (5). The random oligo (ODN-RD) with the sequence 5’-C*C*CTTAT’lTACTACTC*G*C-3’, was used as a control. These ODNs have been previously characterized in antinociceptive studies (9). Administration of ODNs. lntracerebroventricular (icv) injections were made directly into the lateral ventricle. Mice were lightly anaesthetized with ether. Injection was made manually 2 mm lateral and 2 mm caudal to the bregma, at a depth of 3 mm, using a 10 pl Hamilton microliter syringe with a 26 gauge needle. Injection volumes were 4 ~1. ODNs were reconstituted in sterile water immediately prior to use. To assess the specificity of treatments three separate control groups of mice were employed: non-injected animals (naive), those which received the vehicle (saline), and animals injected with a random sequence (ODN-RD). Each ODN treatment was performed on a distinct group of 16 to 24 mice by the following schedule: on days 1 and 2 with lnmol, on days 3 and 4 with 2 nmol, and on day 5 with 3 nmol (9). On day 6 mice were decapitated and inositol phospholipid breakdown in periaqueductal gray slices was assayed. Znosifol phospholipid breakdown assay. Agonist stimulation of the formation of [3H]inositol phosphates in myo-[3H]inositol-labelled periaqueductal gray slices was measured by the method of Berridge et al. (17) with some modifications. Cross-chopped tissue slices were washed three times in warm buffer A [millimolar composition: NaCl, 142.0, KCI, 5.6, MgCl,, 1, CaCl,, 2.2, NaHCO,,3.6, HEPES, 30, and glucose, 5.6; bubbled with O,-CO, (95%-5%) and the pH adjusted to 7.41. Slices (approximately 400 yg of protein) were dispersed in buffer A containing 5 uWml(O.30 pM) of myo[3H]inositol and 10 mM lithium chloride in a final volume of 200 ~1. After the pre-incubation period, 50 pl of agonist was added and reactions allowed to proceed for a further 60 min with gentle shaking. Basal [3H]inositol phosphates accumulation was determined by incubating parallel samples in absence
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of agonist. Reactions were terminated by the addition of 0.94 ml chloroform/methanol (1:2). allowed to stand for 15 min, and sonicated for 10 s to disrupt the slices. Following the addition of 0.31 ml chloroform and 0.3 1 ml water, the tubes were vortexed and phases were separated by centrifugation at 3500 rpm for 5 min. Samples (0.75 ml) of the aqueous phases were diluted to 3 ml with water and 0.5 ml of Dowex-1X8 resin (formate form). Tubes were then vigorously mixed for 30 min and centrifuged. The supematant was aspirated and the resin washed twice with 2.5 ml 5 mM myo-inositol. Labeled inositol phosphates were elute from the resin with 0.5 ml 1M ammonium formate/O.l M formic acid. A 0.4 ml aliquot was removed for counting (Beckman LS-5801) after the addition of 2 ml ECOLUME scintillation fluid. Chemicals. Myo-2-[3H]inositol (lo-25 Ci/mmol) in an ethanol:water solution, 9: 1 was obtained from New England Nuclear. DPDPE and [D-Ala2]deltorphin II were purchased from Peninsula Laboratories (San Carlos, CA), ICI-174,864 from CRB (Cambridge, UK), BNTX and NT II came from RBI (Natick, MA.). Pertussis toxin was obtained from LIST Biological Labs (Campbell, CA)
Results Cross-chopped pre-labeled PAG slices responded to DPDPE and [D-Ala2]deltorphin II with an increase in IPs formation, although the magnitude of the response varied among animals. The results show that the stimulation was dose-dependent from 100 nM to 100 pM. Maximal stimulation occurred at 100 uM, with average percentage increases of 161i35 and 189&39 over basal values (2.641to.09 dpm/pg protein) for DPDPE and [D-Ala2]deltorphin II respectively (fig.lA). Various receptor antagonists were tested against the action of a fixed dose (100 uM) of both agonists. The effect of DPDPE and [D-Ala2]deltorphin II on total IP accumulation was blocked by ICI-174.864 (10 PM), indicating that the response was &opioid receptor mediated (fig. 1B). The participation of different &receptor subtypes in the regulation of phospholipase C activity in the mouse PAG was also investigated. The ability of &agonists to induce stimulation of PI hydrolysis was not altered by BNTX (lo-100 uM). In contrast, NT II (10 PM) significantly antagonized the stimulatory effects of DPDPE and [D-Ala’ldeltorphin II (fig. 1B). The involvement of receptor-regulated G proteins on this stimulatory effect was also examined. Pre-treatment of the slices with pertussis toxin (25 pg/ml) for 2 h only slightly reduced IP formation when in the presence of 100 uM DPDPE (fig. 1C). After increasing the incubation period to 4 h, the effect of DPDPE on IP hydrolysis diminished in both vehicle and pertussis toxin treated slices. Interestingly, in cross-chopped PAG slices obtained from in viva pertussis toxin pre-treated mice to.5 ug/mouse, icv; 6 days before sacrifice (18)] the complete blockage of DPDPE-induced stimulation of PI hydrolysis could be observed (fig. 1D). The [DAla2]deltorphin B-stimulated accumulation of IPs was even more sensitive to the impairing effect of the toxin. The effect of the agonist on IP formation was significantly reduced in mice pre-treated with 0.1 and 0.5 pg/mouse, icv (fig. 1 D). Administration of either pertussis toxin or the vehicle alone had not effect on basal IP formation (not shown). The observed efficacy of in viva administration of pertussis toxin to impair receptor-activated G protein-induced effects, allowed the extension of the study to mice subchronically treated with ODNs known to inhibit the synthesis of the cloned 8-opioid receptor (9). In these animals, the stimulation of IPs formation induced by DPDPE and [D-Ala2]deltorphin II in periaqueductal gray slices was completely blocked (fig. 2). The stimulation of IP formation over basal values for DPDPE was 161%&35, 149%&7,23%*8 and lO%ti respectively for non-injected control animals, mice treated with the random oligo, and those with the ODN directed to nucleotides 7-26 and 29-46 of the mRNA coding for the cloned &opioid receptor. Also, subchronic administration of these ODNS significantly imoaired the increase in IPs accumulation induced by [D-Ala2]deltorphin II, values were 189%&39,
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177%*42, 3%&l and 2%*1, respectively. The slight reduction in PI metabolism observed in mice subchronically injected with the ODN-RD. was also observed in PAG slices from mice receiving the vehicle (not shown). This demonstrated the efficacy and selectivity of the ODNs that target the 6receptor.
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Fig. 1 Delta-opioid receptor-mediated accumulation of inositi phosphates in mouse PAG. Panel A: slices were pre-incubated with myo-[‘Hlinositol for 30 min and then incubated with agonists (100 nM-100 pM) for 60 min. The results, expressed as radioactivity in the total IP fraction, are presented as percentage stimulation of IP formation over basal activity (2.649Ll.09dpn#g protein). Bach point is the mean+S.E.M. of 8-12 determinations. Panel E: antagonists ICI-174.864, NT II and BNTX (10 pM) were added at the beginning of the incubation period, 30 min before DPDPE or [DAla2]deltorphin II (100 pM). Slices were incubated for an additional period of 60 min. and total [3H]IPrecorded Panel C: slices were preincubated in the presence or absence of activated pertussis toxin 5 or 25 pg/ml for 120 min and subsequently assayed for the response to DPDPE (100 pM). Panel D: PAG from pertussis toxin-treated mice (0.1 or 0.5 @mouse icv. 6 days before sacrifice) were used to test their responses to delta agonists at 100 pM. Values are the mean~.E.M. from triplicate determinations of at least 5 separate assays.* Significantly different from the control group receiving saline, and ** from the group receiving the agonist. Analysis of variance, StudentNewman-Keuls test. P < 0.05.
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i$-OR Stimulates PI Metabolism
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Fig.2 Effect of subchronic icv administration of ODNs to &opioid receptors on the IPs formation induced by DPDPE and [D-Ala’~eltorphin Il. Stimulation of inositol phospholipid hydrolysis by the agonists (100 pM) was determined in periaqueductal gray slices from control (non-injected mice) and mice receiving the random oligo or the ODNs directed to nucleotides 7-26 or 2946 of the cloned &receptor mRNA (see methods). The results, expressed as radioactivity in the total IP fraction, are presented as percentage stimulation of IP formation over basal activity. Values are the mean&.E.M. from at least 6 determinations. * Significantly different from the control group receiving agonist. Analysis of variance, Student-Newman-Keuls test, P < 0.05.
Discussion The present study provides the first direct evidence that mouse brain 6-opioid receptors stimulate phosphoinositide hydrolysis. The coupling of b-receptors to this second messenger pathway has previously been described for the a-agonist DPDPE in neuroblastoma x glioma NG 108- 15 cells ( 13), and in Xenopus oocytes (14) and in Ltk- fibroblast (15) expressing cloned S-opioid receptors. These effects were mediated by delta receptor activation of G proteins sensitive to pertussis toxin. The present study also indicates that pertussis toxin-sensitive G proteins are regulated by delta-opioid receptors in the stimulation of PI formation in mouse PAG. Notwithstanding, the regulation of Gz proteins by 6 receptors in PI formation in L&cells (15), plus data from in vitro assays, suggest that participation of pertussis toxin-resistant Gz or Gq proteins in this opioid effect cannot be disregarded. In the current investigation, the pharmacologically defined &-agonist DPDPE and the selective agonist at the &-receptors [D-Ala*]deltorphin II, were equally effective in increasing the formation of inositol phosphates in slices of mouse periaqueductal gray matter. Both agonists caused an ICI- 174,864 reversible stimulation of IP accumulation, indicating the involvement of the &opioid receptor in this effect. In addition, the data show that only NT II, the selective antagonist at the 6,receptor, blocked the actions of DPDPE and [D-Ala’ldeltorphin Il. BNTX @,-antagonist) was without effect, suggesting the predominance of b,-opioid receptor subtype in the stimulation of IP formation in the mouse PAG.Moreover, in this brain structure the data revealed functional binding of DPDPE to the &,-subtype to increase PI metabolism, regardless the limited efficacy of this compound to produce effective antinociception through 6,-receptors following icv administration (4). This apparent discrepancy can be explained if the b,-receptor mediates the production of antinociception and the regulation of phosphoinositide metabolism via different coupling mechanisms. Thus, the relative efficacy of an agonist might depend on the nature of the G proteins coupled to the receptor, favoring the possibility that the coupling of Sopioid receptor with different G proteins/signaling pathways could determine receptor function. This would then explain the
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agonist/antagonist profiles for the delta ligands. In this respect, and under appropriate conditions, the antagonism of DPDPE on [D-Ala’ldeltorphin II-evoked effects has been documented in antinociceptive studies (19). Further, DPDPE antagonized the activating effect exerted by [DAla2]deltorphin II on Gi2 and G, proteins in mouse periaqueductal gray matter (20). As stated in the introduction, previous experiments with ODNs to mRNA coding for the &opioid receptor (6-9) have shown differences in the antinociceptive profiles of DPDPE and [DAla2]deltotphin lI. By using antisense oligodeoxynucleotides directed to nucleotides 7-26 or 29-46 of the cloned a-receptor mRNA [which respectively blocked 6,,- and Qinduced antinociception (9)], it was hoped that further investigation of the participation of S-receptor subtypes on IP accumulation in this brain area would be possible. The data obtained confirm the conclusions drawn from the use of selective antagonists, and further support the idea that stimulation of PI hydrolysis within the PAG is mediated through a population of &receptors, whose characteristics correlate with the 6,-receptor subtype. In summary, this study indicates that in addition to adenylyl cyclase inhibition, the closing of voltage-sensitive Ca2’ channels and the opening of K’ channels, 6-opioid receptors regulate phospholipase C activity in areas of the mouse brain. Acknowledgements This work was supported by funds provided by Comisidn Inter-ministerial Ciencia y Tecnologfa (EspaAa) I SAF95-0003. References 1. Q. JIANG, A.E. TAKEMORI, M. SULTANA, P.S. PORTOGHESE, W.D. BOWEN, H.I. MOSBERG and
R. PORRECA. J. Pharmacol. Exp. Ther. 257 1069-1075 (1991). 2. A. MATITA, T. VANDERAH, H.I. MOSBERG and F. PGRRECA. J. Pharmacol. Exp. Ther. 258 583-587 (1991). 3. B. BUZAS, S. IZENWASSER, P.S. PORTOGHESE and B.M. COX. Life Sci. &l 101-106 (1994). 4. P.A. ZAKI, E.J. BILSKY, T.W. VANDERAH, J. LAI, C.J. EVANS and F. PORRECA. Annu. Rev. Pharmacol. Toxicol. 36 379-401 (1996). 5. K. YASUDA, K. RAYNOR, H. KONG, C. BREDER, J. TAKEDA, T. REISINE and G.I. BELL. Proc. Natl. Acad. Sci. USA 90 6736-6740 (1993). 6. J. LAI, E.J. BILSKY, R.B. ROTHMAN and F. PORRECA. Nemo Report. 5 1049-1052 (1994). 7. E.J. BILSKY, R.N. BERNSTEIN, V.J. HRUBY, R.B. ROTHMAN, J. LA1 and F. PORRECA. J. Pharmacol. Exp. Ther. 277 491-501 (1996). 8. G.C. ROSSI, W. SU, L. LEVENTHAL, H. SU and G.W. PASTERNAK. Brain Res. 275 176-179 (1997). 9. P. SANCHEZBLAZQUEZ, A. GARCkESPANA and J. GARZ6N. J. Pharmacol. Exp. Ther. 280 1423-1431( 1997). 10. S.R. CHILDERS. Life Sci. &3 1991-2003 (1991). 1 I. W. JING, N.M. LEE, H.H. LOH and S.A. THAYER. Mol. Pharmacol. 42 1083-1089 (1992). 12. M.A. CONNOR, A. PLANNER and G. HENDERSON. Regu. Pept. 54 65-66 (1994). 13. D. SMART and D.G. LAMBERT. J. Neurochem. &i 1462-1466 (1996). 14. T. MIYAMAE, N. FUKUSHIMA, Y. MISU and H. UEDA. FEBS Lett. 333 31 l-314 (1993). 15. R.C. TSU, J.S.C. CHAN and Y .H. WONG. J. Neurochem. 64 2700-2707 ( 1995). 16. M.D. MATTEUCCI and M.H. CARUTHERS. J. Am. Chem. Sot. j_@ 31853191 (1981). 17. M.J. BERRIDGE, C.P. DOWNES and M.E. HANLEY. B&hem. J. 206 587-595 (1982). 18. P. SANCHEZ-BLAZQUEZ and J. GARZGN. Eur. J. Pharmacol. 152 357-361 (1988). 19. T. VANDERAH, A.E. TAKEMORI, M. SULTANA, P.S. PORTOGHESE, H.I. MOSBERG, V.J. HRUBY, R.C. HAASETH, T. MATSUNAGA and F. PORRECA European J. Pharmacol. 252 133-137 (1994). 20. J. GARZGN, A. GARCkESPANA and P. SANCHEZ-BLAZQUEZ. J. Pharmacol. Exp. Ther. m 549-557 (1997).