- Email: [email protected]
Upregulation of delta opioid receptor gene expression in NG108-15 cells by naloxone and ethanol
135
UPREGULATIONOFDELTAOPIOIDRECEPTORGENEEXPRESSIONIN NG108-15CELLS BY NALOXONEAND ETHANOL. S. Jenab and C. Inturrisi, Department of Pharmacology, Cornell University Medical College, New York, NY 10021. Pharmacological studies have shown homologous and heterologous upregulation of the delta opioid receptor (DOR) in NG108-15 cells by opioid antagonists or ethanol (1, 2, 3). This cell line expresses the DOR but not the mu or kappa opioid receptors permitting the study of DOR receptor regulation without the possible influence of coordinate changes in other opioid receptors. We have used solution hybridization and Northern blot analysis to measure homologous and heterologous upregulation of DOR mRNAby naloxone and/or ethanol. We show by Northern blot that six DORtranscripts hybridize specifically to a DORcDNAderived riboprobe. Each of the six transcripts increased proportionately with either treatment so that no difference was observed in the fraction of the total hybridization signal produced by each band. In addition, we found that the largest of the DOR transcripts in NG108-15 cell extracts is equivalent in size to a single DORtranscript present in mousewhole brain extracts. NG108-15 cells were grown as described by Jenab and Inturrisi (4). Semi-confluent cells were treated with naloxone (1 #M), ethanol (200 mM) or both naloxone (1 uM) and ethanol (200 mM) for selected time points from 6 to 168 hours. Total and polyA+RNA isolation, Northern blot analysis and the solution hybridization procedure were as described by Jenab and Inturrisi (4). To eliminate nonspecific hybridization, each Northern f i l t e r was incubated with ribonuclease (4). The 32p labelled DOR antisense riboprobe was a 1074 base long transcript containing all of the coding region of the DORcDNAof Evans et al. (5). An unlabelled 1082 base long sense transcript was used as the calibration standard. The standard curve for the DORsense standard is linear from 2 to 500 pg of sense transcript. Total RNA concentrations were determined by a solution hybridization assay using an 18S rRNAriboprobe and a standard calibration curve established from rat liver total RNA (6). T h i s combination of solution hybridization assays allows the measurement of DORtranscript levels to be expressed as pg DOR transcripts/ug total RNA (4). Fig. 1. shows that poly A+ RNA from NG108-15 cells contains 6 transcripts that survived ribonuclease treatment ranging in size from 9.1 to 2.1 kb. The presence of multiple DORtranscripts in NG108-15 cell extracts is in agreement with other reports (3, 5). Each of the transcripts contains the DOR coding sequence and preliminary sequencing studies (7) indicate that different poly A addition sites may account for the observed differences in transcript size. In contrast, we find that mouse brain poly A+ RNA contains a single hybridization band of approximately 9.1 kb (Fig. I). The use of ribonuclease may have eliminated cross-hybridizing bands responsible for the multiple Northern bands reported for mouse brain RNAby others (5). The DORcDNA was isolated from the mouse-rat hybrid NG108-15 cell line but appears to originate from the mouse sequences (8). Importantly, this single DOR transcript corresponds to the largest of the six DOR transcripts found in NG108-15 cells. This 9.1 kb transcript represents about 36% of the total band intensity signal as measured by phosphoimager analysis (4). It is not known to what degree each of the six DOR transcripts from NG108-15 cells are translated to yield receptor protein. However, our results suggest that the largest of the DORtranscripts, commonto both NG108-15 cells and mouse brain may be of particular interest for further study. Incubation of NG108-15 cells with 200 mM ethanol or I pM naloxone, treatments that have reviously been shown to upregulate DORbinding (1, 2, 3), doubled DORmRNAtranscript evels (Table 1). DOR mRNAlevels peaked at 24 to 48 hrs after exposure to either ethanol or naloxone. At 168 hrs DORmRNAlevels in NG108-15 cells exposed to naloxone had returned to control (untreated) levels while the levels in ethanol treated cells remained nearly equal to peak values. Thus, the time-course of the inductive effect of ethanol and naloxone on DORmRNAin NG108-15 cells differs. Furthermore, exposure to a combination of ethanol plus naloxone for 24 hrs produced an additive effect, so that DORmRNAtranscripts were increased 3 fold (Table 1). Northern blot analysis
~
36 Table I The Effect of a 24 hr Exposure to Ethanol and/or Naloxone on the Level of DORmRNA Transcripts in NG108-15 cells. Mean DORTranscripts Treatment (oqlua RNA) untreated 0.11 ethanol (200 mM) 0.20 * naloxone (1 uM) 0.21 * ethanol plus naloxone 0.31 ** • significantly different (p<.05) from untreated • * significantly different (p<.05) from ethanol or naloxone treatment
0--.
a b 6
c q'
~D, e ~ .... ....
d •
~
~;ii~ •
A~,
kb i_-~I I-~.8
i ° '
-4.2 -3.5 ~ ~i~~ i!
Fig. 1. A Northern blot analysis of mouse and NG108-15 cell RNA extracts. PolyA+ RNA from mouse brain (lanes a and b) and from NG108-15 cells (lane d) was separated on a 1.2% agarose gel, transferred to nitrocellulose and hybridized with the DOR riboprobe. Note, the single ribonuclease resistant hybridization band of approximately 9.1 kb in length in the mouse extract (arrow) while 6 separate hybridization bands ranging from 9.1 to 2.1 kb were observed with the NG108-15 extract. The size of the hybridization bands was estimated by use of labelled (lane c) and unlabelled DNA markers. 0 marks the origin of the electrophoresis. identified six DOR transcript bands ranging in size from 9.1 to 2.1 kb. The above treatments increased each ofthe six bands proportionately, so that no difference was observed in the fraction of the total hybridization signal produced by each band of the Northern blot. Opioid agonists desensitize and downregulate DOR (2, 3) and homologous upregulation of DORby opioid antagonists such as naloxone may be due in part to the ability of the antagonist to block endogenous opioid peptide (agonist) induced downregulation of DOR (2). Since the opioid antagonist induced increase in DOR is not blocked by cycloheximide and is associated with a loss of lysosomal activity, i t may also involve the processing of latent receptors and/or an inhibition of receptor degradation. NG108-15cells express opioid peptides and a compensatory increase in opioid peptide biosynthesis and release could occur with long term (i.e., >24 hr) exposure to naloxone resulting in a decrease of the induction of DOR mRNA. This compensatory mechanism would be expected to have l i t t l e effect on ethanol induction of DOR mRNAsince ethanol does not appear to produce its induction via the blockade of opioid peptide induced downregulation of DOR (3) Our results demonstrate that each of the DOR transcripts in NG108-15 cells are subject to homologous (naloxone) as well as heterologous (ethanol) upregulation. I t remains to be determined what mechanisms are responsible for these two types of DOR mRNAand receptor upregulation. Supported by NIDA Grants DA01457 and DA00198 (CEI) and Training Grant DA07274 (SJ). 1. J. Barg, R. Levy and R. Simantov (1984) Neurosci. Lett. 40, 133-137 2. M.M. Belcheva, J. Barg, R.J. McHale, X.M. Gao, D.M. Chuang and C.J. Coscia (1991) J. Pharmacol. Exp. Ther. 259, 302-309 3. M.E. Charness, G. Hu, R.H. Edwards and L.A. Querimit (1993) Mol. Pharmacol. 44, 1119-1127 4. S. Jenab and C.E. Inturrisi (1994) Mol. Brain Res. in press 5. C.J. Evans, D.E. Keith, Jr., H. Morrison, K. Magendzo and R.H. Edwards (1992) Science 258, 1952-1955 6. Y-S. Zhu, A.D. Branch, H.D. Robertson, T.H. Huang, S.O. Franklin and C.E. Inturrisi (1992) Mol. Brain Res. 12, 173-180 7. K. Magendzo, A. Merickel, D. Keith, Jr. and C.J. Evans (1993) Soc. Neurosci. Abstr. 23, 72 8. B.L. Kieffer, K. Befort, C. Gaveriaux-Ruff and C.J. Hirth (1992) Proc. Natl. Acad. Sci. USA 89, 12048-12052
UPREGULATIONOFDELTAOPIOIDRECEPTORGENEEXPRESSIONIN NG108-15CELLS BY NALOXONEAND ETHANOL. S. Jenab and C. Inturrisi, Department of Pharmacology, Cornell University Medical College, New York, NY 10021. Pharmacological studies have shown homologous and heterologous upregulation of the delta opioid receptor (DOR) in NG108-15 cells by opioid antagonists or ethanol (1, 2, 3). This cell line expresses the DOR but not the mu or kappa opioid receptors permitting the study of DOR receptor regulation without the possible influence of coordinate changes in other opioid receptors. We have used solution hybridization and Northern blot analysis to measure homologous and heterologous upregulation of DOR mRNAby naloxone and/or ethanol. We show by Northern blot that six DORtranscripts hybridize specifically to a DORcDNAderived riboprobe. Each of the six transcripts increased proportionately with either treatment so that no difference was observed in the fraction of the total hybridization signal produced by each band. In addition, we found that the largest of the DOR transcripts in NG108-15 cell extracts is equivalent in size to a single DORtranscript present in mousewhole brain extracts. NG108-15 cells were grown as described by Jenab and Inturrisi (4). Semi-confluent cells were treated with naloxone (1 #M), ethanol (200 mM) or both naloxone (1 uM) and ethanol (200 mM) for selected time points from 6 to 168 hours. Total and polyA+RNA isolation, Northern blot analysis and the solution hybridization procedure were as described by Jenab and Inturrisi (4). To eliminate nonspecific hybridization, each Northern f i l t e r was incubated with ribonuclease (4). The 32p labelled DOR antisense riboprobe was a 1074 base long transcript containing all of the coding region of the DORcDNAof Evans et al. (5). An unlabelled 1082 base long sense transcript was used as the calibration standard. The standard curve for the DORsense standard is linear from 2 to 500 pg of sense transcript. Total RNA concentrations were determined by a solution hybridization assay using an 18S rRNAriboprobe and a standard calibration curve established from rat liver total RNA (6). T h i s combination of solution hybridization assays allows the measurement of DORtranscript levels to be expressed as pg DOR transcripts/ug total RNA (4). Fig. 1. shows that poly A+ RNA from NG108-15 cells contains 6 transcripts that survived ribonuclease treatment ranging in size from 9.1 to 2.1 kb. The presence of multiple DORtranscripts in NG108-15 cell extracts is in agreement with other reports (3, 5). Each of the transcripts contains the DOR coding sequence and preliminary sequencing studies (7) indicate that different poly A addition sites may account for the observed differences in transcript size. In contrast, we find that mouse brain poly A+ RNA contains a single hybridization band of approximately 9.1 kb (Fig. I). The use of ribonuclease may have eliminated cross-hybridizing bands responsible for the multiple Northern bands reported for mouse brain RNAby others (5). The DORcDNA was isolated from the mouse-rat hybrid NG108-15 cell line but appears to originate from the mouse sequences (8). Importantly, this single DOR transcript corresponds to the largest of the six DOR transcripts found in NG108-15 cells. This 9.1 kb transcript represents about 36% of the total band intensity signal as measured by phosphoimager analysis (4). It is not known to what degree each of the six DOR transcripts from NG108-15 cells are translated to yield receptor protein. However, our results suggest that the largest of the DORtranscripts, commonto both NG108-15 cells and mouse brain may be of particular interest for further study. Incubation of NG108-15 cells with 200 mM ethanol or I pM naloxone, treatments that have reviously been shown to upregulate DORbinding (1, 2, 3), doubled DORmRNAtranscript evels (Table 1). DOR mRNAlevels peaked at 24 to 48 hrs after exposure to either ethanol or naloxone. At 168 hrs DORmRNAlevels in NG108-15 cells exposed to naloxone had returned to control (untreated) levels while the levels in ethanol treated cells remained nearly equal to peak values. Thus, the time-course of the inductive effect of ethanol and naloxone on DORmRNAin NG108-15 cells differs. Furthermore, exposure to a combination of ethanol plus naloxone for 24 hrs produced an additive effect, so that DORmRNAtranscripts were increased 3 fold (Table 1). Northern blot analysis
~
36 Table I The Effect of a 24 hr Exposure to Ethanol and/or Naloxone on the Level of DORmRNA Transcripts in NG108-15 cells. Mean DORTranscripts Treatment (oqlua RNA) untreated 0.11 ethanol (200 mM) 0.20 * naloxone (1 uM) 0.21 * ethanol plus naloxone 0.31 ** • significantly different (p<.05) from untreated • * significantly different (p<.05) from ethanol or naloxone treatment
0--.
a b 6
c q'
~D, e ~ .... ....
d •
~
~;ii~ •
A~,
kb i_-~I I-~.8
i ° '
-4.2 -3.5 ~ ~i~~ i!
Fig. 1. A Northern blot analysis of mouse and NG108-15 cell RNA extracts. PolyA+ RNA from mouse brain (lanes a and b) and from NG108-15 cells (lane d) was separated on a 1.2% agarose gel, transferred to nitrocellulose and hybridized with the DOR riboprobe. Note, the single ribonuclease resistant hybridization band of approximately 9.1 kb in length in the mouse extract (arrow) while 6 separate hybridization bands ranging from 9.1 to 2.1 kb were observed with the NG108-15 extract. The size of the hybridization bands was estimated by use of labelled (lane c) and unlabelled DNA markers. 0 marks the origin of the electrophoresis. identified six DOR transcript bands ranging in size from 9.1 to 2.1 kb. The above treatments increased each ofthe six bands proportionately, so that no difference was observed in the fraction of the total hybridization signal produced by each band of the Northern blot. Opioid agonists desensitize and downregulate DOR (2, 3) and homologous upregulation of DORby opioid antagonists such as naloxone may be due in part to the ability of the antagonist to block endogenous opioid peptide (agonist) induced downregulation of DOR (2). Since the opioid antagonist induced increase in DOR is not blocked by cycloheximide and is associated with a loss of lysosomal activity, i t may also involve the processing of latent receptors and/or an inhibition of receptor degradation. NG108-15cells express opioid peptides and a compensatory increase in opioid peptide biosynthesis and release could occur with long term (i.e., >24 hr) exposure to naloxone resulting in a decrease of the induction of DOR mRNA. This compensatory mechanism would be expected to have l i t t l e effect on ethanol induction of DOR mRNAsince ethanol does not appear to produce its induction via the blockade of opioid peptide induced downregulation of DOR (3) Our results demonstrate that each of the DOR transcripts in NG108-15 cells are subject to homologous (naloxone) as well as heterologous (ethanol) upregulation. I t remains to be determined what mechanisms are responsible for these two types of DOR mRNAand receptor upregulation. Supported by NIDA Grants DA01457 and DA00198 (CEI) and Training Grant DA07274 (SJ). 1. J. Barg, R. Levy and R. Simantov (1984) Neurosci. Lett. 40, 133-137 2. M.M. Belcheva, J. Barg, R.J. McHale, X.M. Gao, D.M. Chuang and C.J. Coscia (1991) J. Pharmacol. Exp. Ther. 259, 302-309 3. M.E. Charness, G. Hu, R.H. Edwards and L.A. Querimit (1993) Mol. Pharmacol. 44, 1119-1127 4. S. Jenab and C.E. Inturrisi (1994) Mol. Brain Res. in press 5. C.J. Evans, D.E. Keith, Jr., H. Morrison, K. Magendzo and R.H. Edwards (1992) Science 258, 1952-1955 6. Y-S. Zhu, A.D. Branch, H.D. Robertson, T.H. Huang, S.O. Franklin and C.E. Inturrisi (1992) Mol. Brain Res. 12, 173-180 7. K. Magendzo, A. Merickel, D. Keith, Jr. and C.J. Evans (1993) Soc. Neurosci. Abstr. 23, 72 8. B.L. Kieffer, K. Befort, C. Gaveriaux-Ruff and C.J. Hirth (1992) Proc. Natl. Acad. Sci. USA 89, 12048-12052