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Chronic mianserin treatment decreases 5-HT2 receptor binding without altering 5-HT2 receptor mRNA levels
European Journal of Pharmacology - Molecular Pharmacology Section, 207 ( 1'091) 169-172 g, 1991 Elsevier Science Publishers B.V. 0922-41(t6/91/$03.50 ADONIS 092241069100123X
169
EJPMOL 80050
Short communication
Chronic mianserin treatment decreases 5-HT z receptor binding without altering 5-HT~ receptor mRNA levels Bryan L. R o t h and R o l a n d D. C i a r a n e l l o Nancy Pritzker LaboratoD' oJ"Molecular and Decelopmental Neurobiolol,% Department of Psvchiuto' and Bekat'ioral Sciences, StanJord Unicersity Medical Center, StanJbrd, CA 94305, U.S.A. Received 2 April 1991, accepted 9 April 1991
Rats were injected with 15 mg/kg (i.p.) mianserin or vehicle (saline) for 4~ 10 or 21 days and 5-HT 2 receptor binding and mRNA levels measured. Treatment with mianserin induced a substantial decrease in 5-HT~ radioligand binding (~4-59~ decrease; P < 0.05 vs. control). No changes in the amount of 5-HT2 or, as a control,/3-actin mRNAs were found (P > 0.05 vs. control). These results indicate that mianserin decreases 5-HT~ radioligand binding without altering steady-state 5-HT, mRNA levels. 5-HT 2 receptor; mRNA; Gene expression
1. Introduction
Serotonin 2 (5-HT,) receptors are regulated by many exogenous and endogenous substances including pharmacologic agents (e.g. mianserin, clozapine), protein kinases, and developmentally- and genetically-induced factors (see Roth et al., 1990 for a recent review). The effect of mianserin is perhaps the most interesting since it decreases 5-HT 2 receptor binding after a single injection (Blaekshear and Sanders-Bush, 1982; Blackshear et al., 1983). This effect of mianserin is maintained for at least 48 h, and is apparently not caused by persistent occupation of the receptor by reversiblybound ligand (Sanders-Bush et al., 1987). The mechanism(s) by'which mianserin and other agents (clozapine, loxapine) alter 5-HT 2 binding is unknown. The 5-HT 2 receptor e D N A has recently been cloned and its complete sequence published (Pritchett et al., 1988). This has provided us with an opportunity to re-address the problem of 5-HT 2 receptor regulation. In particular, we are now able to determine whether the decrease in receptor binding induced by mianserin is accompanied by 5-HT, receptor m K N A alterations. In this paper we show that mianserin alters 5-HT,
Correspondence to: Bryan L. Roth. MD, PhD. Nancy Pritzker Laboratory of Developmental and Molecular Neurobiology, Beckman Center. B-002. Department of Psychiatry. Stanford University Medical Center, Stanford. CA 94305, U.S.A.
radioligand binding without changing the steady-state levels of 5-I-tT. receptors mRNAs.
2. MateriaIs and methods
2.1. An#rials and their treatment Male Sprague-Dawley rats (150-200 g) were obtained from Simonson and allowed to acclimate to the animal care facility for 1 week prior to the experiment. Rats were injected intraperitoneally with either vehicle (saline) or mianserin (15 m g / k g ) for 4, i0 or 21 days. At 24 h after the last i~ection the rats were decap!tated, brains rapidly removed, forebrains dissected out, hemisected, frozen and stored at - 8 0 ° C until use. No effect was noted on levels of 5-HT 2 receptors or m R N A with - 80 ° C storage. Z Z RNA preparation and Northern blot analysis F~r m R N A analysis the ~3rocedure of Davis (Davis et al., 1986) was followed. 13r!efly, one-half of a forebrain was homogenized in guanidinium-isothiocyanate and RNA purified by CsCI gradient ultracentrifugation. Atter equalization by OD measurements (260/280 rim), pellets were dissolved in R N A loading buffer (1 × MOPS, 50% fom~amide, 8% formaldehyde, 5% glycerol, 0.2% bromophenol blue) and run at 300 V for 2 h on a 1% agarose-formaldehyde gel in 1 × MOPS buffer.. R N A was transferred to Duralon-UV mem-
170
branes (Stratagene, San Diego, CA, U.S.A.) in 10 × SSC, crosslinked by UV irradiation (attto-crosslink settings: Stratalinker: Stratagene) and hybridized with either oligonucleotide or plasmid-derived probes (see below). For both types of probes the following hyt~ridization mixture was used: 50Of deionized formamide, 5 × SSC, 2 × Denhardt's, 1":~ sodium dodecyl sulfate (SDS), 10% dextran sulfate, 250 /.tg/ml yeast tRNA, 50(1 ,ag/rnl sheared, denatured herring sperm DNA. For oligonucleotide probes incubation was carried out at 37 ° C while 42 ° C was used for plasmid-derived probes. After overnight incubation, membranes were washed lor 1 h in 2 × SSC/0.1% SDS at room temperature and then washed for 40 rain at 55°C in either 0.2 × SSC/0.1q;~ SDS (for plasmid-derived probes) or 1 × SSC/0.1c:~ SDS (oligonucleotidc probes). Membranes were then autoradiographed on Kodak X-Omat fihn and Cronex intensifying screens at - 8 0 ° C . Bands were quantified by scanning laser densitomctry (Zeineh, Los Angeles, CA, U.S.A.) with various exposures to ensure linearity of film response. Blots were re-probed with /3-actin after washing and overnight exposure to determine loss of 5-HT, mRNA signal.
0
28S 18S
F-'I
1
2. 4. Receptor binding assays One-half of a forebrain (corresponding to the half not used for mRNA studies) was homogenized in binding buffer (50 mM Tris-CI; pH 7.40) and centrifuged at 20,000 × g for 30 min. The brain membranes were then resuspended in 10 ml of binding buffer and re-centrifuged (2(I,000 × g; 30 rain) and the resulting membrane pellets used for radioligand binding assays. 5-HT~ receptors were labeled with [~H]ketanserin (70 Ci/mmol; New England Nuclear) as previously detailed (Roth et al., t987) using mianserin to distinguish between tetrabenazine-sensitive sites and 5-HT 2 receptors in a total volume of 0.5 ml. Mianserin (1 ,aM) was used to determine non-specific binding. Specific binding was 70-80% of total binding. Saturation binding studies
5HT2 mRNA ACTIN mRNA
P:~ 5HT2 BINDING 125
=p(O.O5
100
g, N Z
75
O U
N
50 25
4-DAY
Ii
IO-DAY
21-DAY
MIANSERINTREATMENT
2.3. Probes For measurement of /3-actin mRNA, an 1821 bp Bgll (bp 2201-4022) fragment was used (kindly provided by J. Eberwine). For 5-HT, mRNA measurement, a synthetic antisense DNA oligonucleotide probe complementary to bases 730-781 (+72 to + 1231 was synthesized (Applied Biosystems) and labeled with terminal deoxynucleotidyl transferase and [a-32p]dATP to a final specific activity of 0.8-1.0 × 10'~ c p m / u g DNA. Plasmid-derived probes were labeled using the random-primer technklue and [a-32p]dCTP to a specific activity of 1-1.2 × 19~ c p m / g g DNA. These probes and specificity of hybridiz~tion have been previously described (Roth ct al., 199!).
,.
Fig. I. Effect of 4, 10 and 21 days of mianserin treatment on rat forebrain 5-HT e receptors and mRNA levels. 1A shows a typical autoradiogram for forebrain 5-HT: mRNA levels from control (CONT) rats. rats treated for 10 days (10) or 21 days (211 with mianserin (15 mg/kg). 1B shows the same blot re-probed for/3-actin mRNA. 1C shows data averaged from four different experiments (8-16 animals/data p,)int) for 5-HT 2 mRNA (open columns), 5-HT 2 receptor binding (cross-hatched columns) and /3-actin mRNA (filled columns) all expressed as percent of control (saline-injected) aninlals. Con',rol specific cpm bound/mg protein (1 nM [3H]ketanserin) was 23.473_+ 1043. * P < 0.05 vs. control it-test).
were performed using 11 concentrations of unlabeled ligand over a 1000-fold concentration range. Following incubation at 25 °C for 90 min, the membranes were harvested onto 0.1% polyethyleneimine pre-treated G F / C glass-fiber filters (Whatmar,, Boston, MA, U.S.A.) using a Brandel Cell Harvester. Radioactivity bound to filters was determined by liquid scintillation spectrometry. Data were analyzed as previously detailed (Roth et al., 19871 using the LIGAND problem (Munson and Rodbard, 19801. Protein was determined using the Bio-Rad (Richmond, CA, U.S.A.) procedure.
3. Results
Similar to results reported by other investigators (Blackshear et al., 1983, Sanders-Bush et al., 1987), we found that mianserin treatment for 4, 10 or 21 days decreased 5-HT 2 radioligand binding (fig. IC). No significant change was found in the equilibrium dissocia-
17] TABLE 1 Effect of mi-mse,in on 5-HT~ receptor binding parameters. Data represent mean_+S.D. of computer-derived estimates for a typical experiment in which rats were injected with saline (control) or mianserin (35 mg/kg; i.p.) for 4 days and [~H]ketanserin binding measured as detailed in Materiab, and melhods Control K,j(nM) Bma~ (fmol/mg)
2.3-+ !.1 128 -+30
Mianserintreated 1.3-+ 0.9"* 56 -+ 13 *
• P < 0.05 vs. control (F-test): * * P > [L{}5vs. control.
tion constant (K,~) for [3H]ketanserin for the 5-1-1T, receptor (table 1) for a typica! exr~eriment of 4 days duration. Similar results were noted at 14 and 21 days of mianserin treatment (not shown). The K j values are similar to those previously published (Roth et al., 19871. Figure 1A and B show representative results obtained after 10 and 21 days of continuous mianserin treatment for the 5-HT 2 m R N A (fig. 1A). The same blot was re-probed to measure/3-actin (fig. I B) m R N A , No significant change is seen in the levels of these m R N A s following mianserin treatment. Figure 1C shows a summary, of four separate experiments in which 8-16 animals were analyzed at each time point and m R N A s were quantified by scanning laser densitometry. Again, no significant (P > 0.(151 changes were noted for any m R N A species following mianserin treatment though 5-HT 2 receptor binding is significantly diminished at each time point (P < 0.05).
4. Discussion
O u r major finding is that mianserin decreases 5-HT~ radioligand binding without changing the steady-state levels of 5-HT, m R N A . O u r results suggest that mianserin alters radioligand binding to 5-HT= receptors by an undefined transcriptional or post-transcriptional process (e.g. m R N A stability, m R N A transiation, post-translational modifications). The precise mechanism by which this occurs is unknown and will be the subject of further study. O u r data indicate that mianserin probably does not alter 5-H'I'= gene transcription. Previous investigators have amply demonstrated that mianserin (Sanders-Bush et al., 1987), as well as loxapine and clozapine (Matsubara and Meltzer, 1989) can specifically decrease 5-HT e receptor binding. Hove this occurs is unkno~vn and has been the subject of intense study. Thus, for instance, Sanders-Bush and colleagues (1987) showed that mianserin did not a p p e a r to bind reversibly er irreversibly to the 5-HT, receptor. Matsubara and M e h z e r (19891 found that a single dose of clozapine decreased 5-HT z binding for 4 days and
suggested that the re-appearance of 5-HT~ receptors d e p e n d e d upon the re~synthcsis of 5-HT~ receptors. Several mechanisms can be invoked to describe these effects of mianserin on 5-HT, receptor binding including transcriptional, translational and post-transiationa[ events (see Roth et ai., 1990 for review). These will be discussed in turn. The most obvious possibility is that mianserin alters 5-HT, receptor gene transcription and thereby levels of receptor mRNAs. As we have shown, 4, 10 or 21 days of mianserin treatment does not significantly change 5-HT 2 m R N A levels. It is possible that mianserin alters receptor or receptor m R N A turnover (e.g. changes in receptor m R N A or protein synthesis and degradation), but not steady-state receptor m R N A levels. This possibility is not addressed by these studies, but could be studied with specific anti-receptor antibodies or intron-specific probes. Conceivably, mianserin could affect the ability of 5-HT~ receptor m R N A to be translated into protein. The means by which this could occur is unknown, but could, conceivably, be mediated by altering m R N A stability or processing. This possibility can be investigated using techniques of in vitro translation, though the extremely low abundance of 5-!-IT, and receptor m R N A makes this approach technically challenging. Finally, mianserin could alter 5-HT, receptors by post-translational processing. This could occur by at least two potential mechanisms. First, mianserin, or a metabotite could irreversibly bind to the ligand recognition site on the 5-HT~ receptor, Our findings, as well as those of other investigators imply that the effect of mianserin is not simply due to residual drug binding to the receptor. Such a reversibly bound ligand would be expected to cause an ir~crease in K a and no change in Bm~,~. We measured instead a decrease in Bm~,~with no significant change in K d (table 1). Second, mianserin could induce a rapid internalization and degradation of the 5-HT, receptors. Eurther investigations of these possibilities will be facilitated by the use of anti-receptor antibodies which are currently being prepared in our laboratoly. In conclusion we demonstrate that mianserin decreases 5-HT, receptor binding without altering receptor m R N A levels. These results imply that mianserin does not decrease 5-HT, receptor levels by diminishing the steady-state levels of 5-HT, m R N A .
References
I31ackshear, M. and E. Sanders-Bush, 1982. Seroloni,1receptor sensitivity after acute and chronic trcatmcn{ with mianscrin. J. Pharmaco!. Exp. Ther. 221, 3{)3. Btackshear, M.. R. Friedman and E. Sanders-Bush. 19S3. Acute and chronic effects of serotonin antagonists on serotonin binding sites, N. S. Arch. Pharmac~d. 324. 125.
]
I/c'tl ilt'~l)l~;ii:h ti~t c h d l . i t l t ' l t / . i l i O l ] i!] I~lt.tllil !~iiltlll! 7 ",'.'.',uIl!'.. .\i];~l. I~h,chc'tn. 1il7. '.]tl i'rilc'!lcil. I).l~> A \t. It.~ch kl \~.~.'n~. () t.dc't, bt. l)nt 1<~>. !.( Sillh ,..ncl t ' i l <.~ ,'bl!i~ i~s~, ~illtlCltllt" ,1I]~f ilil)t'lilill,il g\t~lc".'.tiilt ~! cl<~nmd I,Ii .
Nctilo~'~i~'in
4~, I>;. :~,,
'ltr! ~Ilw~ A,II¢'I II}!;VI~Utl~I~ WiHIlp~tll~,Oll Ol I0~.~ Of ~il~."~ ;tltCl br;lilt I.*~.~.". ,, ~,I dr;i~.,. I l~i~K~,*,~n J l~'h.~m,~.'ol. I'~:L I~,L
169
EJPMOL 80050
Short communication
Chronic mianserin treatment decreases 5-HT z receptor binding without altering 5-HT~ receptor mRNA levels Bryan L. R o t h and R o l a n d D. C i a r a n e l l o Nancy Pritzker LaboratoD' oJ"Molecular and Decelopmental Neurobiolol,% Department of Psvchiuto' and Bekat'ioral Sciences, StanJord Unicersity Medical Center, StanJbrd, CA 94305, U.S.A. Received 2 April 1991, accepted 9 April 1991
Rats were injected with 15 mg/kg (i.p.) mianserin or vehicle (saline) for 4~ 10 or 21 days and 5-HT 2 receptor binding and mRNA levels measured. Treatment with mianserin induced a substantial decrease in 5-HT~ radioligand binding (~4-59~ decrease; P < 0.05 vs. control). No changes in the amount of 5-HT2 or, as a control,/3-actin mRNAs were found (P > 0.05 vs. control). These results indicate that mianserin decreases 5-HT~ radioligand binding without altering steady-state 5-HT, mRNA levels. 5-HT 2 receptor; mRNA; Gene expression
1. Introduction
Serotonin 2 (5-HT,) receptors are regulated by many exogenous and endogenous substances including pharmacologic agents (e.g. mianserin, clozapine), protein kinases, and developmentally- and genetically-induced factors (see Roth et al., 1990 for a recent review). The effect of mianserin is perhaps the most interesting since it decreases 5-HT 2 receptor binding after a single injection (Blaekshear and Sanders-Bush, 1982; Blackshear et al., 1983). This effect of mianserin is maintained for at least 48 h, and is apparently not caused by persistent occupation of the receptor by reversiblybound ligand (Sanders-Bush et al., 1987). The mechanism(s) by'which mianserin and other agents (clozapine, loxapine) alter 5-HT 2 binding is unknown. The 5-HT 2 receptor e D N A has recently been cloned and its complete sequence published (Pritchett et al., 1988). This has provided us with an opportunity to re-address the problem of 5-HT 2 receptor regulation. In particular, we are now able to determine whether the decrease in receptor binding induced by mianserin is accompanied by 5-HT, receptor m K N A alterations. In this paper we show that mianserin alters 5-HT,
Correspondence to: Bryan L. Roth. MD, PhD. Nancy Pritzker Laboratory of Developmental and Molecular Neurobiology, Beckman Center. B-002. Department of Psychiatry. Stanford University Medical Center, Stanford. CA 94305, U.S.A.
radioligand binding without changing the steady-state levels of 5-I-tT. receptors mRNAs.
2. MateriaIs and methods
2.1. An#rials and their treatment Male Sprague-Dawley rats (150-200 g) were obtained from Simonson and allowed to acclimate to the animal care facility for 1 week prior to the experiment. Rats were injected intraperitoneally with either vehicle (saline) or mianserin (15 m g / k g ) for 4, i0 or 21 days. At 24 h after the last i~ection the rats were decap!tated, brains rapidly removed, forebrains dissected out, hemisected, frozen and stored at - 8 0 ° C until use. No effect was noted on levels of 5-HT 2 receptors or m R N A with - 80 ° C storage. Z Z RNA preparation and Northern blot analysis F~r m R N A analysis the ~3rocedure of Davis (Davis et al., 1986) was followed. 13r!efly, one-half of a forebrain was homogenized in guanidinium-isothiocyanate and RNA purified by CsCI gradient ultracentrifugation. Atter equalization by OD measurements (260/280 rim), pellets were dissolved in R N A loading buffer (1 × MOPS, 50% fom~amide, 8% formaldehyde, 5% glycerol, 0.2% bromophenol blue) and run at 300 V for 2 h on a 1% agarose-formaldehyde gel in 1 × MOPS buffer.. R N A was transferred to Duralon-UV mem-
170
branes (Stratagene, San Diego, CA, U.S.A.) in 10 × SSC, crosslinked by UV irradiation (attto-crosslink settings: Stratalinker: Stratagene) and hybridized with either oligonucleotide or plasmid-derived probes (see below). For both types of probes the following hyt~ridization mixture was used: 50Of deionized formamide, 5 × SSC, 2 × Denhardt's, 1":~ sodium dodecyl sulfate (SDS), 10% dextran sulfate, 250 /.tg/ml yeast tRNA, 50(1 ,ag/rnl sheared, denatured herring sperm DNA. For oligonucleotide probes incubation was carried out at 37 ° C while 42 ° C was used for plasmid-derived probes. After overnight incubation, membranes were washed lor 1 h in 2 × SSC/0.1% SDS at room temperature and then washed for 40 rain at 55°C in either 0.2 × SSC/0.1q;~ SDS (for plasmid-derived probes) or 1 × SSC/0.1c:~ SDS (oligonucleotidc probes). Membranes were then autoradiographed on Kodak X-Omat fihn and Cronex intensifying screens at - 8 0 ° C . Bands were quantified by scanning laser densitomctry (Zeineh, Los Angeles, CA, U.S.A.) with various exposures to ensure linearity of film response. Blots were re-probed with /3-actin after washing and overnight exposure to determine loss of 5-HT, mRNA signal.
0
28S 18S
F-'I
1
2. 4. Receptor binding assays One-half of a forebrain (corresponding to the half not used for mRNA studies) was homogenized in binding buffer (50 mM Tris-CI; pH 7.40) and centrifuged at 20,000 × g for 30 min. The brain membranes were then resuspended in 10 ml of binding buffer and re-centrifuged (2(I,000 × g; 30 rain) and the resulting membrane pellets used for radioligand binding assays. 5-HT~ receptors were labeled with [~H]ketanserin (70 Ci/mmol; New England Nuclear) as previously detailed (Roth et al., t987) using mianserin to distinguish between tetrabenazine-sensitive sites and 5-HT 2 receptors in a total volume of 0.5 ml. Mianserin (1 ,aM) was used to determine non-specific binding. Specific binding was 70-80% of total binding. Saturation binding studies
5HT2 mRNA ACTIN mRNA
P:~ 5HT2 BINDING 125
=p(O.O5
100
g, N Z
75
O U
N
50 25
4-DAY
Ii
IO-DAY
21-DAY
MIANSERINTREATMENT
2.3. Probes For measurement of /3-actin mRNA, an 1821 bp Bgll (bp 2201-4022) fragment was used (kindly provided by J. Eberwine). For 5-HT, mRNA measurement, a synthetic antisense DNA oligonucleotide probe complementary to bases 730-781 (+72 to + 1231 was synthesized (Applied Biosystems) and labeled with terminal deoxynucleotidyl transferase and [a-32p]dATP to a final specific activity of 0.8-1.0 × 10'~ c p m / u g DNA. Plasmid-derived probes were labeled using the random-primer technklue and [a-32p]dCTP to a specific activity of 1-1.2 × 19~ c p m / g g DNA. These probes and specificity of hybridiz~tion have been previously described (Roth ct al., 199!).
,.
Fig. I. Effect of 4, 10 and 21 days of mianserin treatment on rat forebrain 5-HT e receptors and mRNA levels. 1A shows a typical autoradiogram for forebrain 5-HT: mRNA levels from control (CONT) rats. rats treated for 10 days (10) or 21 days (211 with mianserin (15 mg/kg). 1B shows the same blot re-probed for/3-actin mRNA. 1C shows data averaged from four different experiments (8-16 animals/data p,)int) for 5-HT 2 mRNA (open columns), 5-HT 2 receptor binding (cross-hatched columns) and /3-actin mRNA (filled columns) all expressed as percent of control (saline-injected) aninlals. Con',rol specific cpm bound/mg protein (1 nM [3H]ketanserin) was 23.473_+ 1043. * P < 0.05 vs. control it-test).
were performed using 11 concentrations of unlabeled ligand over a 1000-fold concentration range. Following incubation at 25 °C for 90 min, the membranes were harvested onto 0.1% polyethyleneimine pre-treated G F / C glass-fiber filters (Whatmar,, Boston, MA, U.S.A.) using a Brandel Cell Harvester. Radioactivity bound to filters was determined by liquid scintillation spectrometry. Data were analyzed as previously detailed (Roth et al., 19871 using the LIGAND problem (Munson and Rodbard, 19801. Protein was determined using the Bio-Rad (Richmond, CA, U.S.A.) procedure.
3. Results
Similar to results reported by other investigators (Blackshear et al., 1983, Sanders-Bush et al., 1987), we found that mianserin treatment for 4, 10 or 21 days decreased 5-HT 2 radioligand binding (fig. IC). No significant change was found in the equilibrium dissocia-
17] TABLE 1 Effect of mi-mse,in on 5-HT~ receptor binding parameters. Data represent mean_+S.D. of computer-derived estimates for a typical experiment in which rats were injected with saline (control) or mianserin (35 mg/kg; i.p.) for 4 days and [~H]ketanserin binding measured as detailed in Materiab, and melhods Control K,j(nM) Bma~ (fmol/mg)
2.3-+ !.1 128 -+30
Mianserintreated 1.3-+ 0.9"* 56 -+ 13 *
• P < 0.05 vs. control (F-test): * * P > [L{}5vs. control.
tion constant (K,~) for [3H]ketanserin for the 5-1-1T, receptor (table 1) for a typica! exr~eriment of 4 days duration. Similar results were noted at 14 and 21 days of mianserin treatment (not shown). The K j values are similar to those previously published (Roth et al., 19871. Figure 1A and B show representative results obtained after 10 and 21 days of continuous mianserin treatment for the 5-HT 2 m R N A (fig. 1A). The same blot was re-probed to measure/3-actin (fig. I B) m R N A , No significant change is seen in the levels of these m R N A s following mianserin treatment. Figure 1C shows a summary, of four separate experiments in which 8-16 animals were analyzed at each time point and m R N A s were quantified by scanning laser densitometry. Again, no significant (P > 0.(151 changes were noted for any m R N A species following mianserin treatment though 5-HT 2 receptor binding is significantly diminished at each time point (P < 0.05).
4. Discussion
O u r major finding is that mianserin decreases 5-HT~ radioligand binding without changing the steady-state levels of 5-HT, m R N A . O u r results suggest that mianserin alters radioligand binding to 5-HT= receptors by an undefined transcriptional or post-transcriptional process (e.g. m R N A stability, m R N A transiation, post-translational modifications). The precise mechanism by which this occurs is unknown and will be the subject of further study. O u r data indicate that mianserin probably does not alter 5-H'I'= gene transcription. Previous investigators have amply demonstrated that mianserin (Sanders-Bush et al., 1987), as well as loxapine and clozapine (Matsubara and Meltzer, 1989) can specifically decrease 5-HT e receptor binding. Hove this occurs is unkno~vn and has been the subject of intense study. Thus, for instance, Sanders-Bush and colleagues (1987) showed that mianserin did not a p p e a r to bind reversibly er irreversibly to the 5-HT, receptor. Matsubara and M e h z e r (19891 found that a single dose of clozapine decreased 5-HT z binding for 4 days and
suggested that the re-appearance of 5-HT~ receptors d e p e n d e d upon the re~synthcsis of 5-HT~ receptors. Several mechanisms can be invoked to describe these effects of mianserin on 5-HT, receptor binding including transcriptional, translational and post-transiationa[ events (see Roth et ai., 1990 for review). These will be discussed in turn. The most obvious possibility is that mianserin alters 5-HT, receptor gene transcription and thereby levels of receptor mRNAs. As we have shown, 4, 10 or 21 days of mianserin treatment does not significantly change 5-HT 2 m R N A levels. It is possible that mianserin alters receptor or receptor m R N A turnover (e.g. changes in receptor m R N A or protein synthesis and degradation), but not steady-state receptor m R N A levels. This possibility is not addressed by these studies, but could be studied with specific anti-receptor antibodies or intron-specific probes. Conceivably, mianserin could affect the ability of 5-HT~ receptor m R N A to be translated into protein. The means by which this could occur is unknown, but could, conceivably, be mediated by altering m R N A stability or processing. This possibility can be investigated using techniques of in vitro translation, though the extremely low abundance of 5-!-IT, and receptor m R N A makes this approach technically challenging. Finally, mianserin could alter 5-HT, receptors by post-translational processing. This could occur by at least two potential mechanisms. First, mianserin, or a metabotite could irreversibly bind to the ligand recognition site on the 5-HT~ receptor, Our findings, as well as those of other investigators imply that the effect of mianserin is not simply due to residual drug binding to the receptor. Such a reversibly bound ligand would be expected to cause an ir~crease in K a and no change in Bm~,~. We measured instead a decrease in Bm~,~with no significant change in K d (table 1). Second, mianserin could induce a rapid internalization and degradation of the 5-HT, receptors. Eurther investigations of these possibilities will be facilitated by the use of anti-receptor antibodies which are currently being prepared in our laboratoly. In conclusion we demonstrate that mianserin decreases 5-HT, receptor binding without altering receptor m R N A levels. These results imply that mianserin does not decrease 5-HT, receptor levels by diminishing the steady-state levels of 5-HT, m R N A .
References
I31ackshear, M. and E. Sanders-Bush, 1982. Seroloni,1receptor sensitivity after acute and chronic trcatmcn{ with mianscrin. J. Pharmaco!. Exp. Ther. 221, 3{)3. Btackshear, M.. R. Friedman and E. Sanders-Bush. 19S3. Acute and chronic effects of serotonin antagonists on serotonin binding sites, N. S. Arch. Pharmac~d. 324. 125.
]
I/c'tl ilt'~l)l~;ii:h ti~t c h d l . i t l t ' l t / . i l i O l ] i!] I~lt.tllil !~iiltlll! 7 ",'.'.',uIl!'.. .\i];~l. I~h,chc'tn. 1il7. '.]tl i'rilc'!lcil. I).l~> A \t. It.~ch kl \~.~.'n~. () t.dc't, bt. l)nt 1<~>. !.( Sillh ,..ncl t ' i l <.~ ,'bl!i~ i~s~, ~illtlCltllt" ,1I]~f ilil)t'lilill,il g\t~lc".'.tiilt ~! cl<~nmd I,Ii .
Nctilo~'~i~'in
4~, I>;. :~,,
'ltr! ~Ilw~ A,II¢'I II}!;VI~Utl~I~ WiHIlp~tll~,Oll Ol I0~.~ Of ~il~."~ ;tltCl br;lilt I.*~.~.". ,, ~,I dr;i~.,. I l~i~K~,*,~n J l~'h.~m,~.'ol. I'~:L I~,L