cAMP-dependent phosphorylation of soluble and crude microtubule fractions of rat cerebral cortex after prolonged desmethylimipramine treatment

European Journal of Pharmacology - Molecular Pharmacology' Section, 172 (1989) 305-316

305

Elsevier EJPMOL 90029

cAMP-dependent phosphorylation of soluble and crude microtubule fractions of rat cerebral cortex after prolonged desmethylimipramine treatment J o r g e P e r e z , D a n i e l a T i n e l l i , N i c o l e t t a B r u n e l l o a n d G i o r g i o R a c a g n i 1., Center of Neuropharmacology, Institute of Pharmacological Sciences, University of Milan, Milan, Italy, and i Institute of Pharmacology, University of Pavia, Pavia, Italy

Received 2 May 1989, accepted 16 May 1989

We have analyzed the cAMP-dependent phosphorylation system in the cerebral cortex and hippocampus of rats after acute and chronic administration of desmethylimipramine. Prolonged desmethylimipramine administration modified the cAMP-dependent endogenous phosphorylation of a protein band with apparent molecular weight 280 kDa from the cerebrocortical-soluble fraction. The effect appeared to be specific and associated with the chronic but not the acute administration of desmethylimipramine since we did not obtain any modification in other endogenous cAMP phosphoproteins of either the particulate or soluble fraction of the cerebral cortex. 280 kDa was identified as the soluble microtubule associated protein 2 on the basis of molecular weight, endogenous phosphorylation and immunological recognition. Prolonged desmethylimipramine administration did not induce any modification in the soluble cAMP-dependent endogenous phosphorylation of 280 kDa in other brain areas such as hippocampus, striatum or cerebellum, suggesting a region-specific effect of chronic desmethylimipramine treatment. Microtubule-associated protein 2 is a neuronal protein highly enriched in the dendritic portion of neurons and represents one of the major substrates in the cell for the type II cAMP protein kinase. Since the type II cAMP protein kinase that catalyzes the phosphorylation of microtubule-associated protein 2 copurifies with microtubules, we performed endogenous phosphorylation using increasing concentrations of cAMP in a crude microtubule preparation where microtubule-associated protein 2 appeared to be more concentrated. Under our conditions the maximal effect occurred at 1 ~M cAMP, revealing increased 32p incorporation in microtubule-associated protein 2 from a crude microtubule preparation obtained from the cerebral cortex of rats treated with desmethylimipramine. Photoaffinity labelling with 8-azido[32p]cAMP of the various fractions obtained during the preparation of crude microtubules (S 1, S2 and crude microtubules) revealed an increase in the labelling of a protein band with apparent molecular weight of 52 kDa after desmethylimipramine treatment. The labelling of a 47 kDa protein band, which is also present in S 1 and S2 fractions was, however, not altered by drug treatment. In conclusion, our studies demonstrated that prolonged desmethylimipramine treatment elicited specific changes in the phosphorylation system associated with a crude microtubule fraction. Antidepressants; cAMP-dependent phosphorylation; Microtubule-associated protein

1. Introduction Various lines of evidence have clearly d e m o n strated that the a d m i n i s t r a t i o n of d e s m e t h y l -

* To whom all correspondence should be addressed: Center of Neuropharmacology, Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milan, Italy.

i m i p r a m i n e ( D M I ) , a tricyclic a n t i d e p r e s s a n t drug, is a s s o c i a t e d with a d a p t i v e changes in the central m o n o a m i n e r g i c system ( R a c a g n i a n d Brunello, 1984). Since there is a lag p h a s e of 15-20 d a y s before the start of beneficial activity of a n t i d e p r e s s a n t t h e r a p y ( H e n n i n g e r a n d C h a r n e y , 1987), the n e u r o c h e m i c a l basis of the p h a r m a c o l o g i c a l effects of D M I s h o u l d b e studied after its prolonged a d m i n i s t r a t i o n . C h r o n i c a l l y a d m i n i s t e r e d

0922-4106/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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antidepressants elicit down-regulation of several neurotransmitter receptor systems. One of the most significant modifications produced by prolonged DMI treatment is the desensitization of the norepinephrine (NE)-sensitive adenylate cyclase (Vetulani and Sulser, 1975), often paralleled by a reduction in the number of fl-receptors (Banerjee et al., 1977). Stimulation of fl-receptors induces biological responses by regulating the concentration of the second messenger, cAMP, and the diverse effects of cAMP on cell functions are mediated through the activation of cAMP-dependent protein kinase (cAMP-PK), the enzyme responsible for the phosphorylation of endogenous substrate proteins (Kuo and Greengard, 1969; Walter et al., 1978; Beavo and Mumby, 1982). Regulation of the activity or the amount of cAMP-PK may modulate important physiological processes involving the phosphorylation of endogenous proteins. Among the neuronal phosphoproteins, synapsin I and high molecular weight microtubule-associated protein 2 (MAP-2) (De Camilli, 1984) are of particular interest because of their different subcellular localization. Synapsin I is present in presynaptic terminals associated with synaptic vesicles and its phosphorylation might play a role in neurotransmitter release (Greengard, 1987). On the other hand the phosphorylation of MAP-2, which is specifically concentrated in dendrites and perikarya, could be involved in the regulation of microtubule function (Vallee, 1980; Matus et al., 1981; De Camilli et al., 1984). Since antidepressants have been shown to affect neurotransmission pre- and postsynaptically, we studied whether the cAMP-dependent phosphorylation system could be an intracellular target for the action of antidepressants. We now report on the cAMP-dependent phosphorylation system in the soluble and crude microtubule fractions of rat brain after acute and prolonged DMI treatment.

2. M a t e r i a l s a n d m e t h o d s

2.1. Materials DMI was a generous gift of Chiesi Farmaceutici (Parma, Italy). EGTA, EDTA, morpholin ethane-

sulfonic acid (MES), piperazine-N,N'-bis(2ethanesulfonic acid) (PIPES), 3-isobutyl-l-methyl xanthine (IBMX), aprotinin, pepstatin A, phenylmethylsulfonyl fluoride (PMS-F), RII-bovine heart regulatory subunit and cAMP were purchased from Sigma Chemical Co. Molecular weight standards and all other materials for gel electrophoresis were obtained from Bio-Rad. Dithiothreitol (DTT) was obtained from Boehringer Mannheim GmbH, Mannheim, FRG. "y-[32p]ATP (10 Ci/mmole), 125I-streptavidin (40/~ Ci//~ g), MAP-2, a- and fl-tubulin monoclonal antibodies and biotinylated antimouse antibody were obtained from Amersham Corp. 8-Azido-[32p]cAMP (8-N 3[32p]cAMP) (66.7 Ci/mmol) was obtained from ICN (Irvine, CA, USA). 2.2. Treatment schedule Male Sprague-Dawley rats (125-150 g, Charles River, Calco, Italy) were housed under standard laboratory conditions with water and food ad libitum and 14 h light-dark cycles (6 : 00 a.m.-8 : 00 p.m.). The animals received injections of saline or DMI (10 mg/kg) intraperitoneally acutely and twice daily for 1, 4, 8 and 15 days where not otherwise stated. The animals were killed by decapitation 1 h after the last injection. The brains were rapidly dissected on ice and then kept at - 80 ° C until assayed. 2.3. Endogenous phosphorylation The assays were performed as previously described (Walaas et al., 1983a,b). Cerebral cortex and hippocampus were homogenized in a glassteflon homogenizer in cold buffer containing 10 mM Tris-HC1 (pH 7.4), 2 mM EDTA, 1 mM DTT, 50 U / m l aprotinin, 2 /~g/ml pepstatin A, 0.1 mM PMS-F. After centrifugation, aliquots of particulate or soluble fraction (2 mg protein/ml) were preincubated at 30 °C for 90 s in the reaction mixture (final vol. 100 /~l) containing 25 mM Tris-HC1 (pH 7.4), 6 mM MgSO4, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, in the absence or presence of 5 #M cAMP plus 1 mM IBMX. The reaction was initiated by the addition of 2 /~M "y[32p]ATP.

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Fig. 1. Crude microtubule preparation from rat cerebral cortex. 10% SDS-PAGE of pellet (P) after the first centrifugation (100000x g for 1 h at 4 ° C ) , and of S2 and crude microtubule (M) fractions after the second centrifugation on sucrose 20% (100000 x g for 1 h at 37 o C). See Materials and methods for details.

Incubation periods of 15 and 60 s were used for particulate and soluble fractions, respectively. The reaction was terminated by the addition of 100/zl of 'stop solution' (62.5 mM Tris-HC1 (pH 6.8), 10% glycerol, 2.3% sodium dodecyl sulfate (SDS w / v ) , 5% 2 - f l - m e r c a p t o e t h a n o l , 0.001% Bromophenol Blue (w/v)). The samples were mixed, boiled for 2 min and a 15 /~g aliquot of protein was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described earlier (Laemmli, 1970) using 6% and 10% of acrylamide. The gels were stained, destained, dried and subjected to autoradiography. The molecular weight standards used were: myosin (200000 Da), flgalactosidase (116250 Da), phosphorylase b (97400 Da), albumin (66200 Da), Ovalbumin (42699 Da), carbonic anhydrase (31000 Da), trypsin inhibitor (21500 Da), lysozyme (14400 Da). 32p incorporation into protein bands on different gels was demonstrated by cutting the bands out from the dried gels and quantifying the 32p content by liquid scintillation counting.

2.4. Crude microtubule preparation A crude microtubule fraction was obtained from rat cerebral cortex by a modification of the microtubule assembly procedure (Shelanski et al., 1973). The tissues were homogenized (1 g/ml) in a Polytron at slow speed in polymerization buffer containing 50 mM PIPES (pH 6.8), 0.1 mM MgC12, 0.1 mM EGTA, 0.5 mM 2-/~-mercaptoethanol, 2 /~g/ml pepstatin, 50 U / e l aprotinin, 1 mM PMSo F. The homogenate was centrifuged at 100 000 x g for 1 h at 4 ° C. For polymerization, the supernatant S 1 (fig. 1) was incubated at 37°C for 30 min under agitation in a Dubnoff water-bath, then was stratified on an equal volume of sucrose 20% in the polymerization buffer and centrifuged at 100000 × g for 1 h at 37°C. The supernatant S2 (fig. 1) was collected and dialyzed before use. The translucent pellet, M (fig. 1), was gently washed with water then resuspended in the appropriate buffer according to the experimental procedure. SDS-PAGE was performed on each preparation in order to verify the purification procedure (fig. 1).

308

2.5. Western blotting Following SDS-PAGE, the gels were either stained or transferred to nitrocellulose for subsequent immunoblot analysis (Towbin et al., 1979). The blotted nitrocellulose sheets were blocked by gentle agitation with 3% bovine serum albumin (BSA) in buffer A (pH 7.3) containing: 137 mM NaC1, 2.7 mM KC1, 1.5 mM K 2 H P O 4, 8 mM N a 2 H P O 4. Incubation with primary antibodies (1:1000 dilution) was performed at room temperature for 1 h in 50 mM K H : P O 4 / K 2 H P O 4 (pH 7.4), 0.15 mM NaC1 for MAP-2 and in buffer A-0.25% BSA for tubulin. After incubation the blots were washed in buffer A-0.25% BSA 3 times and then placed in the same solution containing a 1/250 dilution of biotinylated antimouse antibody for 1 h at room temperature. The blots were again washed 3-4 times in buffer A-0.25% BSA and incubated with 125Istreptavidin at the appropriate dilution in the same buffer. The blots were again washed 3-4 times, dried and autoradiographed.

with minor modifications. The reaction mixture (final volume, 100 ~1) contained 50 mM MES (pH 6.2), 10 mM MgCI2, 1 mM IBMX, 0.5 mM 2-/3mercaptoethanol, 1 /~M 8-N3-[32p]cAMP and aliquots of 50 #1 (200 #g proteins) of S 1, S2 or crude microtubule fraction, in the absence or presence of 100/~M unlabelled cAMP. Incubation was carried out for 60 min in the dark at 4 ° C and the samples were then irradiated for 10 min at 254 nm with a u.v. lamp (Spectroline, mod. E N F / 2 4 F ) . The reaction was blocked by the addition of 100 /~1 stop solution. The samples were mixed, heated at 100 ° C for 1 min. and subjected to SDS-PAGE. The radioactive bands localized by autoradiography were cut out from gel and the 32p content was quantified by liquid scintillation counting. The standard photoactivated incorporation procedure was performed using purified RII of bovine heart.

2.8. Protein determination Protein was determined by the method of Bradford (1976) using BSA as standard.

2.6. Phosphorylation of crude microtubules Endogenous phosphorylation was performed as previously described (Sloboda et al., 1975). Briefly, crude microtubule fractions obtained from rat cerebral cortex were resuspended in buffer containing 50 mM PIPES (pH 6.8), 0.1 mM MgC12, 0.1 mM 2-]3-mercaptoethanol, 50 U / m l aprotinin, 2 /~g/ml pepstatin, 0.1 mM PMS-F. Aliquots of 50 ~al (100 /~g of proteins) were preincubated at 30 ° C for 90 s without addition or in the presence of 0.5, 1, 2.5, 5, 10/~M cAMP plus IBMX (1 mM). The reaction was initiated by the addition of 7132p]ATP (final concentration 2 /~M). After 1 min of incubation at 30 ° C, phosphorylation was blocked by adding 50 /~1 of stop solution. The samples were then subjected to SDS-PAGE. Radioactive bands were localized by autoradiography, cut out from the dried gel and the 32p content was quantified by liquid scintillation counting.

2.7. 8-N3-[3:P]cAMP incorporation Photoaffinity labelling experiments were performed as described previously (Walter et al., 1977)

2.9. Statistical analyses All statistical analyses were done with either Student's t-test or Dunnett's t-test for multiple comparisons.

3. Results

3.1. cAMP-dependent endogenous phosphorylation in various rat brain areas The endogenous substrate proteins for cAMPPK were evaluated in both the soluble and the particulate fraction of rat cerebral cortex and hippocampus. Repeated administration of DMI modified the pattern of phosphorylation of a soluble cAMP-dependent phosphoprotein as shown by the autoradiograph in fig. 2 (upper panel). DMI treatment for 8 and 15 days induced an increase of endogenous phosphorylation in the protein band of apparent molecular weight 280 kDa under basal and stimulated (5 /~M cAMP) conditions. The

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endogenous phosphorylation of two other bands with apparent molecular weight between 97 and 116 kDa appeared to be enhanced after repeated DMI treatment under basal conditions. However, in contrast with the 280 kDa band, the addition of cAMP did not induce any modification in the phosphorylation of the latter bands (fig. 2, upper panel). The amount of radioactive phosphate incorporated into 280 kDa after 8 and 15 days of DMI treatment was compared with that after saline only (fig. 2 lower panel). The increase in 32p incorporation induced by DMI was 100% and 140% respectively under basal conditions vs. that of saline-treated rats. Moreover, an increase of approximately 120% in the incorporation of 32p

was observed under stimulated conditions after prolonged DMI treatment when compared to the controls. In contrast, acute and in vitro (10 -5 M) DMI administration failed to produce these effects (data not shown). The electrophoretic mobility and the increased 32p incorporation seen in the presence of cAMP suggested that the 280 kDa band could represent the microtubule-associated protein, MAP-2. This possibility was tested by means of Western Blotting using the monoclonal antibody, anti-MAP-2, providing further evidence that 280 kDa phosphoprotein is identified with soluble MAP-2. Figure 3 (panel A) shows the pattern of cerebrocortical soluble proteins separated on 6% SDS-PAGE (lane 1) and the polypeptide recognized by the antibody, anti-MAP-2 (lane 2). Interestingly, 15 days of treatment had no effect on the hippocampus phosphoprotein that migrated at the position of MAP-2 under both basal and stimulated conditions (fig. 4). Similar experiments performed with the particulate fraction evidenced no difference in the cAMP-dependent endogenous phosphorylation of both cerebral cortex and hippocampus in control and DMI-treated rats (data not shown).

3.2. Endogenous phosphorylation in the crude microtubule fraction Partial characterization of the crude microtubule fraction (fig. 1) was obtained by Western Blotting. Figure 3 (panel B) shows the pattern of cerebrocortical crude microtubule proteins, (lane 1) characterized by two bands at 280 kDa and 50 kDa, corresponding to MAP-2 and c~-/? tubulin respectively, and recognized by the antibodies anti-MAP-2 (lane 2) and anti-a-/? tubulin (lane 3). Previous studies showing that cAMP-PK is associated with a crude microtubule fraction (Vallee et al., 1981: Theurkauf and Vallee, 1982) prompted us to determine whether prolonged DMI treatment would affect the phosphorylation system associated with this fraction. In order to check this hypothesis, crude microtubule fractions from control and DMI-treated rats were subjected to endogenous phosphorylation using various concentrations of cAMP as described in Materials and methods. Maximal stimulation was obtained

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1 and 2) and DMI-treated rats (lanes 3,4). Autoradiography of the same gel (Fig. 6A) showed three protein bands in the control and the DMI-treated S 1 fraction with apparent molecular weight 52, 47 and 45 kDa which incorporated 8-N3-[32p]cAMP (lanes 5 and 7). Non-specific binding in the control and DMI-treated S 1 was determined in the presence of 100 ~tM cAMP (lanes 6 and 8). The analysis of the amount of photoactivated incorporation into these bands revealed an increase in the 52 kDa band after DMI treatment (fig. 6B). Photoaffinity labelling of the S2 fraction (fig. 7) showed a high percentage of binding in a band of 47 kDa. However, there were no differences in the covalent binding of 8-N3-[32p]cAMP between saline (lanes 1,2)- and DMI (lanes 3,4)-treated animals. The SDS-PAGE (fig. 8, panel A) of the crude microtubule fraction from cerebral cortex of

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Fig. 4. Upper panel. Autoradiography showing endogenous phosphorylation of soluble fraction from hippocampus of control and DMI-treated rats (10 mg/kg twice daily for 15 days). Phosphorylation was performed under basal and stimulated conditions (5 /~M c A M P + I mM IBMX) and proteins were separated by 6% SDS-PAGE. Lower panel. Histograms showing the 32p incorporation in the 280 kDa band localized by autoradiography as described in Materials and methods. The values are the means of three experiments.

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control (lanes 1,2) and DMI-treated rats (lanes 3,4) showed a similar pattern of protein, where MAP-2 (280 kDa) and tubulin (50 kDa) are present. Autoradiography (fig. 8, lanes 5 and 7) of the same gel showed an increase in the photoactivated incorporation of the 52 kDa after repeated DMI administration. Lanes 6 and 8 show the nonspecific binding obtained after the addition of 100 /~M cold cAMP. DMI treatment (fig. 8, panel B) elicited an increase of 187% in the amount of photoactivated 32p incorporation in the 52 kDa protein when compared to control animals.

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4. Discussion 47

Extensive experimental evidence indicates that antidepressant drugs can act on different neuronal systems at pre- and postsynaptic sites. Changes in receptor sensitivity of monoamine or other neurotransmitter systems have been reported following the prolonged administration of tricyclic antidepressants. The time course of these receptor mod-

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ifications closely approximates the delay in clinical antidepressant effects observed in patients. However the intracellular events related to these receptor modifications are still partially unclear. Since chronic treatment with antidepressants is known to produce changes in those receptor systems coupled to the regulation of second messengers, which in turn modulate the phosphorylation status of neuronal proteins, it was of interest to test whether the phosphorylation system might be an intracellular target of antidepressants. Accordingly, we analysed the cAMP-dependent phosphorylation system after acute and chronic DMI administration. Various endogenous substrates for cAMP-dependent PK have been identi-

fied in the CNS (Lohmann et al., 1980). These phosphoproteins can differ as to cellular and subcellular localization and regional distribution (Walaas et al., 1983a,b; Greengard, 1987). The cAMP-dependent phosphorylation of a protein band from the cerebrocortical soluble fraction, with apparent molecular weight 280 kDa, was found to be modified after prolonged DMI administration (fig. 2). This effect appeared to be specific and associated with the chronic administration of DMI since we observed no modification in the other endogenous cAMP phosphoproteins of both particulate and soluble fractions. Moreover, acute treatment or the in vitro addition of DMI (10 -5 M) directly to endogenous phosphorylation assay mixtures, failed to modify the cAMP-dependent phosphorylation system (data not shown). The fact that acute or in vitro treatment with DMI is devoid of any effect on cAMP-dependent phosphorylation suggests strongly that, similarly to antidepressant-induced receptor modification, this is a long-term effect which might require several days to develop. Molecular weight, endogenous phosphorylation and antibody recognition indicate that 280 kDa could be the soluble microtubule-associated protein, MAP-2. It has been demonstrated that MAP-2 is a neuronal protein highly concentrated in the dendritic portion of neurons (Matus et al., 1981; Caceres et al., 1983; De Camilli et al., 1984). In addition, the state of its phosphorylation can be modulated by cAMP-PK (Sloboda et al., 1975; Vallee, 1980; Vallee et al., 1981; Goldenring et al., 1985). This observation could indicate that MAP-2 is a postsynaptic neuronal target for the action of neurotransmitters coupled to the second messenger-phosphorylation system. On this basis, the modification in the endogenous phosphorylation of MAP-2 may indicate that prolonged DMI administration produces changes in postsynaptic intracellular pathways. It has been demonstrated that pre- and postsynaptic changes after DMI administration can occur in different regions of the brain (Korf, 1983). Since MAP-2 was found to be present in the great majority of neurons (De Camilli et al., 1984), we also analyzed cAMP-dependent endogenous phosphorylation in other brain areas. Prolonged DMI administration did not induce any modification in

313 the soluble cAMP-dependent endogenous phosphorylation of 280 kDa in hippocampus (fig. 4), striatum or cerebellum (data not shown). These findings suggest a region-specific effect of chronic DMI treatment. This apparent discrepancy could be explained by taking several factors into consideration, e.g. the different time course of antidepressant effects in various brain areas (Kinnier et al., 1980), the modulation by antidepressants of a single protein of the phosphorylation system and different subcellular localization of the phosphorylation system components (Lohmann et al., 1984; De Camilli et al., 1986). Moreover, even if it is not yet possible to correlate these changes with a modification in the function of specific adrenoceptor populations, the differential effects in the brain areas could be ascribed to the relative distribution of B-1 adrenoceptors, down-regulated by chronic antidepressant treatment and more concentrated in the cerebral cortex (Minneman et al., 1979), and j~-2 adrenoceptors (relatively insensitive to antidepressants and more abundant in the cerebellum (Minneman et al., 1979; Frazer et al., 1988)); the absence of a noradrenergic input from the locus coeruleus to the striatum (Lindvall and By~Srklund, 1978). It should be noted that other bands from the soluble fraction of both cerebral cortex and hippocampus were modified after chronic DMI treatment under either basal or stimulated conditions. However these bands are not endogenous substrates for cAMP-PK since the addition of cAMP did not induce any modification. It has been demonstrated that soluble MAP-2 is one of the more prominent proteins that copurify with tubulin in the microtubule fraction of the brain (Sloboda et al., 1975; Kim et al., 1979). Several studies have demonstrated that cAMP-PK can be associated to the microtubule fraction (Vallee et al., 1981; Theurkauf et al,, 1982; Lohmann et al., 1984; De Camilli et al., 1986). These findings support the concept that endogenous substrate proteins and protein kinase have a similar subcellular distribution. We have therefore analyzed both the endogenous phosphorylation of MAP-2 in the cerebrocortical crude microtubule fraction and the photoaffinity labelling of cAMP-PK after chronic DMI treatment. Under our experimental conditions, an

increase in 32p incorporation was found in the MAP-2 of the crude microtubule fraction after DMI treatment (fig. 5) without changes in the concentration of MAP-2 as revealed by SDSPAGE. The above-mentioned results suggest that the endogenous phosphorylation of MAP-2 in the soluble and crude microtubule fractions of rat cerebral cortex appears to be modified after repeated DMI administration. It is generally accepted that the increase of 32p in endogenous substrate protein after a pharmacological treatment could be due either to a change in substrate concentration (Nestler et al., 1981; 1982), to a modified activity of protein kinase or phosphatase (Zatz and O'Dea, 1976; Johansson and Anderson, 1981) or to a modification in the concentration of kinase or phosphatase modulators (Kuo et al., 1979; Beavo and Mumby, 1982; Hemmings et al., 1984). There are several reports of changes in the regulation of protein kinases in a variety of systems after acute and chronic administration of drugs, and such modifications may reflect important short- and long-term control of physiological processes (Soderling et al., 1973; Liu et al., 1981: Lohmann and Walter, 1984). It has been shown, for example, that chronic, but not acute, morphine treatment modifies the activity of the cAMP-dependent PK in specific regions of the brain (Nestler and Tallman, 1988) and that DMI can alter the activity of the cAMP-dependent PK in the pineal gland (Moyer et al., 1986). Since we have observed changes in endogenous phosphorylation in the soluble and microtubule fractions of rat cortical tissues following DMI treatment, we investigated the effect of chronic DMI administration on the cAMP-PK associated with these fractions. We have therefore analyzed the subcellular distribution of the cAMP-PK in the S 1, S2 and crude microtubule fraction (M). Since the regulatory subunit of PK is the only well-characterized receptor for cAMP (Walter and Greengard, 1982), photoaffinity labelling has been used to study the regulation of the amount of cAMP-dependent PK. Under our experimental conditions, three photoactivated bands were found in the S 1 from control and DMI-treated rats (fig. 6). cAMP displaced labelled photoactivated incor-

314 p o r a t i o n only in 52 a n d 47 k D a i n d i c a t i n g that there are two c A M P b i n d i n g p r o t e i n s in this fraction. A c c o r d i n g to the m o l e c u l a r weight, these p r o t e i n b a n d s could represent the r e g u l a t o r y subunit of c A M P - P K I a n d c A M P - P K II, R I a n d R I I respectively. D M I t r e a t m e n t specifically affected the covalent b i n d i n g of c A M P to 52 k D a , suggesting an increase in the a m o u n t of R I I which rem a i n e d u n d e t e c t a b l e in the S D S - P A G E because of the presence of other p r o t e i n s m i g r a t i n g at the same m o l e c u l a r weight. T w o p h o t o a c t i v a t e d b a n d s with a p p a r e n t m o l e c u l a r weight 52 a n d 47 k D a were also d e t e c t e d in the S2 fraction b u t D M I t r e a t m e n t d i d not i n d u c e any m o d i f i c a t i o n (fig. 7) since most of the 52 k D a c o p u r i f i e d with the crude m i c r o t u b u l e fraction (fig. 8). In fact, an increase in the p h o t o a c t i v a t e d i n c o r p o r a t i o n in 52 k D a was f o u n d after chronic D M I in the crude m i c r o t u b u l e fraction, suggesting that the increase in the r e g u l a t o r y s u b u n i t of P K II could be associated with the m i c r o t u b u l e fraction. It is interesting to note that the p a t t e r n of p r o t e o l y t i c f r a g m e n t s d i d not differ between control a n d D M I - t r e a t e d rats, suggesting that the increase o b s e r v e d in R I I was not d u e to a change in p r o t e i n d e g r a d a t i o n b u t could have been elicited either b y a m o d i f i c a tion in the t r a n s c r i p t i o n a l or t r a n s l a t i o n a l activity or b y a different subcellular redistribution. In conclusion, our d a t a d e m o n s t r a t e that p r o longed D M I t r e a t m e n t elicited changes in the e n d o g e n o u s p h o s p h o r y l a t i o n of M A P - 2 in the soluble and crude m i c r o t u b u l e fractions a n d that this effect a p p e a r s to be m e d i a t e d b y a m o d ulation of c A M P - d e p e n d e n t PK. The lack of effect of acute or in vitro D M I a d m i n i s t r a t i o n suggests that this m o d i f i c a t i o n m a y be related to the a d a p t i v e changes elicited b y p r o l o n g e d antid e p r e s s a n t t r e a t m e n t at the s y n a p t i c level. H o w ever, we d o not yet k n o w the b i o c h e m i c a l mechanisms u n d e r l y i n g this effect which could elicit changes in the functional state of m i c r o t u b u l e p o l y m e r i z a t i o n or d e p o l y m e r i z a t i o n .

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