Journal of Neuroscience Research 28:4048 (1991)
Differential Effect of Lithium on fos Protooncogene Expression Mediated by Receptor and Postreceptor Activators of Protein Kinase C and Cyclic Adenosine Monophosphate: Model for Its Antimanic Action M.M. Divish, G. Sheftel, A. Boyle, V.D. Kalasapudi, D.F. Papolos, and H.M. Lachman Department of Psychiatry, Program of Behavioral Genetics (M.M.D., G.S., A.B., V.D.K., D.F.P., H.M.L.) and Department of Medicine (H.M.L.), Albert Einstein College of Medicine, Bronx, New York
Lithium salts are the most effective agents used in Key words: muscarinic receptor, manic depression, treating manic-depressive illness. It has been sug- down regulation, adenylate cyclase, lithium gested that lithium’s therapeutic efficacy could be due to an inhibitory effect on either inositol phospholipid (IP) and/or cyclic nucleotide metabolism. We have INTRODUCTION investigated the effect of lithium on these two signal Lithium salts have been used in the treatment of transduction pathways in PC12 pheochromocytoma manic depression and recurrent unipolar depression for a cells by studying a common effector target, expres- number of years, yet the specific target of its therapeutic sion of the fos protooncogene. We find that lithium, action remains obscure. Several investigators have sugat therapeutic doses, has an augmenting effect on gested that lithium influences inositol phospholipid (IP) phosphatidylinositol (PI)-mediated fos expression in- pathways by interfering with inositol synthesis, through duced by activating a muscarinic cholinergic path- its inhibition of the enzyme myoinositol 1-phosphatase way, whereas it has no effect, a t tenfold the thera- (Hallcher and Sherman, 1980; Berridge and Irvine, peutic dose, on fos expression induced by receptor or 1989). Depletion of inositol would be expected to lead to postreceptor activators of cyclic adenosine mono- a decrease in phosphatidylinositol 4,5-bisphosphate phosphate (CAMP). The lithium augmenting effect is (PIP,), which is hydrolyzed into diacylglycerol (DAG) also observed when the cells are treated with phorbol and inositol 1,4,5-trisphosphate (IP,) by ligand-activated esters, which directly activate protein kinase C phospholipase C (PLC). IP, mobilizes intracellular (PKC), suggesting that the level of lithium’s interac- Ca2+, and DAG activates protein kinase C (PKC), a tion with the IP pathway is at the postreceptor level. central serine and threonine kinase that modulates a numWe also show that phorbol esters induce extensive ber of effector targets, including ion channels and neudown regulation of subsequent cholinergic and phor- rotransmitter receptors (Berridge and Irvine, 1989; Nishbol ester responsiveness as well as heterologous down izuka, 1986; Kikkawa and Nishizuka, 1986; Sigel and regulation of CAMP responses. Treatment of down- Roland, 1988). Following activation by agonists or phorregulated cells with lithium leads to an enhanced re- bol esters, PKC binds to the membrane in conjunction sponsiveness when cells are rechallenged with ago- with DAG, Ca2+ , and phosphatidylserine. nists that activate PKC but not by agonists that stimulate CAMP. We also show that carbamazepine, another antimanic agent, has an inhibitory effect on Received March 9, 1990; revised May 31, 1990; accepted June 5 , CAMP-mediated fos but no effect on the IP pathway. 1990. The opposite effects of lithium and carbamazepine on Address reprint requests to Herbert M. Lachman, Department of Medtwo critical transducing systems suggest a model for icine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, the antimanic action of these agents. 0 1991 Wiley-Liss, Inc.
Lithium and fos Protooncogene Expression
It has been suggested that an influence on IP metabolism could be a mechanism for the therapeutic efficacy of lithium in manic depression (Berridge and Irvine, 1989; Lachman and Papolos, 1989; Sherman et al., 1986). Supporting this hypothesis are the reports that lithium inhibits PLC-mediated events in the brain. For example, cortical slices derived from rats treated with lithium exhibit reduced agonist stimulated inositol phosphate production (Godfrey et al., 1989). Also, lithium inhibits muscarinic cholinergic responses in the hippocampus, which is mediated by PLC (Worley et al., 1988). Other investigators have suggested that the target of lithium’s effect on cells may be cyclic nucleotide metabolism (Avissar et al., 1988; Newman and Belmaker, 1987). For example, it has been reported that therapeutic concentrations of lithium inhibit agonist stimulated adenylate cyclase activity in rats (Mork and Geisler, 1989). Avissar et al. (1988) have shown that lithium blocks agaonist-induced guanosine triphosphate (GTP) binding in membranes, an effect that is a measure of guanine nucleotide regulatory protein (G-protein) activation. They postulated that lithium reduces cyclic adenosine monophosphak (CAMP) responsiveness by inhibiting G,, the G-protein that regulates adenylate cyclase. TG understand further lithium’s influence on second messenger pathways, it would be useful to study an effector system that is influenced by both the IP and cyclic nucleotide pathways. Activation of fos protooncogene expression is an ideal target for such studies, since fos gene transcription is inhitiated by both PKCand CAMP-dependent pathways (Curran and Morgan, 1985; Rauscher et al., 1988; Mellon et al., 1989). fos protein dimerizes with the jun protooncogene product to form the AP- 1 transcription factor, which regulates the expression of a number of AP-l-sensitive genes (Rauscher et al., 1988). We have previously demonstrated that PC12 rat pheochromocytoma cells, treated with agonists that stimulate PKC, rapidly accumulate large quantities of fos mRNA. When lithium is added to the culture medium, PKC-mediated activation of fos expression is augmented severalfold (Kalasapudi et al., in press). In this report, we have extended our original studies and compared the effect of various concentrations of lithium on fos expression induced by receptor and postreceptor activators of PKC and cAMP in PC12 cells. In addition, we have explored the interaction between the cAMP and IP pathways by studying the effect of muscarinic cholinergic agonists and phorbol esters on CAMPmediated fos expression. The data show that, in PC12 cells, lithium has an enhancing effect on fos expression induced by receptor and postreceptor activators of PKC, whereas it has no direct effect on agonists that stimulate CAMP. By contrast, the antiepileptic and antimanic agent
41
carbamazepine has an inhibitory effect on CAMP-activated fos but no effect on the IP pathway. Furthermore, we show that treatment with phorbol esters leads to extensive down regulation of receptor systems that activate both PKC and adenylate cyclase. Whereas lithium increases the responsiveness of down-regulated PLC/PKC pathways, it has no effect on down-regulated cAMP responses. The differential effect of lithium on down-regulated receptor systems coupled to PKC and cAMP may be important in understanding its therapeutic efficacy.
MATERIALS AND METHODS Cell Cultures PC 12 cells were maintained in tissue culture flasks in RPMI supplemented with 10% horse serum and 5% fetal calf serum, both heat inactivated at 56°C. Fortyeight hours before RNA extraction, the cells were trypsinized from a nearly confluent T- 125 flask, plated on ten 100 mm Falcon tissue culture plates, and placed in a 5% CO, incubator at 37°C and 100% relative humidity. Under these conditions, each plate contained approximately 5 x lo6-] X lo7 cells after 48 hr. The clone of PC12 cells used in these experiments attaches to plastic surfaces without requiring a protein matrix. Drugs were added from 1,OOOX and 10,OOOX stocks, with the exception of lithium, which is a 400X aqueous solution before dilution to a final concentration of 10 mM. The vehicles used for dissolving the drugs included water, dimethylsulfoxide (DMSO), and 70% ethanol. In control experiments, neither of these agents had an effect on fos gene expression at the concentrations used in these studies (data not shown). In the challenge experiments, cell were treated with agonists for 4 hr, after which the drugs were removed by washing the cells three times in complete medium over a 15 min period. The cells were then rechallenged 4-48 hr later, and total cellular RNA was extracted 30 min after the rechallenge. RNA Analysis Cells were trypsinized and harvested by vigorous pipetting and centrifugation at 1,000 rpm for 5 min. Total cellular RNA was extracted using the hot phenol technique as previously described (Lachman and Skoultchi, 1984). Ten micrograms of total cellular RNA was separated by electrophoresis through 0.9% agarose containing 3% formaldehyde. The samples were denatured prior to loading by adding 4 volumes of loading buffer containing 60% formamide and 20% formaldehyde and heating to 65°C for 5 min. Following electrophoresis, the RNA was transferred to nylon filters (Nytran, Schleichter and Schuel) through 20 X SSC (1 X SSC is 0.15 M sodium chloride and 0 .O 15 M sodium citrate). The filters were baked at 80°C in vacuuo for 2 hr and prehybridized
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A
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"3 Fig. 1. A: Effect of lithium on carbamylcholine-mediated fos challenge (lanes 4, 6, and 8, respectively) or were challenged expression. Total cellular RNA was extracted from PC 12 cells and rechallenged under the same conditions but in the presence following treatment with carbamylcholine (CB) for 30 min of 10 mM lithium (lane 5, 4 hr rechallenge; lane 7, 24 hr with and without preincubation with lithium chloride for 16 hr rechallenge; and lane 9, 48 hr rechallenge). B: Effect of acti(Li). RNA was analyzed for fos and histone H3 mRNAs by nomycin D on fos mRNA. Control and lithium-treated (10 mM Northern analysis as described in Materials and Methods. for 16 hr) PC12 cells were treated with CB for 15 min, after Lane 1, untreated control; lane 2, 1 mM CB; lane 3, I mM CB which actinomycin D ( 5 pgiml) was added. Total cellular RNA plus 10 mM Li. Down regulation with cholinergic stimulation was extracted after an additional 15 rnin (lane 1, no lithium; was induced by challenging the cells for 4 hr with 1 mM CB. lane 3, plus lithium) and 30 rnin (lane 2, no lithium; lane 4, After washing with complete medium, the cells were rechal- plus lithium). Lane 5 shows RNA extracted from control cells lenged for 30 min with 1 mM CB at 4, 24, and 48 hr after the treated with CB for 45 min, without actinomycin D.
in 5 x SSC, 10 X Denhardt's solution, 50 mM phosphate buffer, pH 6.8, 0.1% sodium dodecyl sulfate (SDS) and 0.25 mg/ml denatured sheared salmon sperm DNA. The filters were hybridized for 16 hr at 65°C with denatured DNA probes that were radiolabeled with 32P by nick translation or by the random primer technique. The probes used in these experiments included pMMMfos, a mouse genomic fos plasmid (Miller et al., 1984); pGAPDHl3, a rat glyceraldehyde 3-phosphate dehydrogenase cDNA clone (Piechaczyk et al., 1984); and pRAH3-2, a mouse histone H3 cDNA clone (Alterman et al., 1984).
RESULTS Effect of Lithium on fos Expression During Carbamylcholine-Mediated Down Regulation Total cellular RNA was extracted from PC12 cells and analyzed for fos mRNA by Northern filter analysis. The autoradiogram in Figure 1A shows that fos mRNA is not detected in untreated PC12 cells (lane 1). However, upon treatment of the cells for 30 min with carbamylcholine, a stable cholinergic agonist, a large induction of fos mRNA occurs (lane 2). Carbamylcholine is a nonspecific cholinergic agonist. However, previous experiments indicate that, in this clone of PC12 cells, the carbamylcholine-mediated induction of fos mRNA is due to the activation of an M1 muscarinic receptor subtype, which is coupled to PLC, since induction is blocked by low concentrations of pirenzepine (Kalasapudi et al., in
press). Nicotinic cholinergic receptor stimulation does not induce fos expression in the clone of PC12 used in these experiments, in contrast to the findings reported by Greenberg et al. (1986). Following treatment of the cells with 10 mM lithium chloride for 16 hr, there is an augmentation of the carbamylcholine-mediated induction of fos mRNA (lane 3), similar to the results we previously described. Since down-regulated muscarinic responses are thought to play a role in the development of mania (Janowsky et al., 1980; Dilsaver, 1986), we investigated the effect of lithium on cells that were overstimulated with carbamylcholine which induces cholinergic down regulation. Cells were challenged with carbamylcholine for 4 hr then rechallenged with carbamylcholine, 4 hr (lane 4), 24 hr (lane 6), and 48 hr (lane 8) after the challenge. Total cellular RNA was extracted from the cells 30 min after rechallenge and analyzed for fos RNA. The autoradiogram in Figure 1A shows that the induction of fos mRNA, following a previous cholinergic challenge, is markedly attenuated. However, if the cells are challenged and rechallenged with carbamylcholine in the presence of 10 mM lithium, fos RNA levels increase at each time point (lanes 5 , 7, and 9) compared with cells that were not treated with lithium. Densitometric analysis revealed that the lithium-mediated increase in fos mRNA ranged from twofold to 3.5-fold (data not shown). The lithium-mediated increase in fos mRNA seen in down-regulated cells is not due to its accumulation during the challenge period since residual fos
Lithium and fos Protooncogene Expression
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fos
Fig. 2. A: Lithium and carbamylcholine dose response. Cells were treated with 0.05, 0.25, and 1.0 mM CB for 30 min and analyzed for fos and H3 mRNA (lanes 1-3, respectively). Cells were challenged with 0.05, 0.25, and 1.0 mM CB for 4 hr and rechallenged for 30 min with the same concentration of CB after 24 hr (lanes 4-6, respectively). Control cells (lane 7) and cells treated with 1, 5 , and 10 mM Li for 16 hr (lanes 8-10), were induced with 1 mM CB for 30 min. Cells were challenged with CB as in Figure 1 and rechallenged 24 hr later in the absence (lane 11) or presence of 1 , 5 , or 10 mM Li (lanes 12-14, respectively). B: Effect of PMA on fos expres-
sion. Cells were analyzed for fos and GAPDH mRNAs following treatment with the phorbol ester PMA (20 ngiml) for 30 rnin in the absence of Li (lane 1) or with 1, 5 , and I0 mM Li (lanes 2 4 ) . Cells were also challenged with PMA for 4 hr and rechallenged 24 hr later with PMA in the absence (lane 5) or presence of 10 mM Li (lane 6). Lane 7, Fos and GAPDH mRNA following 30 rnin with I mM CB. Lane 8, 1 mM CB 24 hr following a challenge with PMA. Note the RNA in lanes 7 and 8 are derived from an experiment separate from the one shown in lanes 1-6.
mRNA is not detected at the indicated time points unless cells are rechallenged (data not shown). Thus, in agreement with our previous observations, lithium can increase fos expression in cells that display a down-regulated muscarinic cholinergic receptor system. To control for the equal loading and transfer of the RNA, the filters were rehybridized with a histone H3 probe. Since H3 expression is cell cycle dependent, it also serves as a control for cell replication during the rechallenge and lithium treatment periods. A reduction in replicating cells should be reflected by a decrease in the level of H3 mRNA (Alterman et al., 1984). As can be seen in Figure l A , the level of H3 RNA in this experiment is not significantly altered by cholinergic challenge or lithium treatment. One possible explanation for the augmenting effect is that the half life of fos mRNA is increased by lithium. A number of studies have demonstrated that mRNAs with relatively short half-lives, such as fos, can be stabilized by protein synthesis inhibitors, which presumably inhibit the synthesis of nucleases that degrade mRNAs (Stimac et al., 1984). A similar effect on the addition of relatively high concentrations of lithium could conceivably lead to enhanced fos mRNA accumulation and would likely represent a toxic effect of the drug rather than a relevant physiological action. The data in Figure 1B argue against this possibility. Cells were treated with carbamylcholine in the presence and absence of 10 mM lithium. After 15 min, the transcription inhibitor actinomycin D was added, and total cellular RNA was extracted after an additional 15 and 30 min. When transcription is inhibited, mRNA levels decrease at a rate proportional to its half-life (Nepveu et al., 1987). As can
be seen in Figure l B , the level of fos mRNA rapidly declines following the addition of actinomycin D, both in the absence (lanes 1, 2) and in the presence (lanes 3 , 4) of 10 mM lithium. Densitometric analysis did not reveal a significant difference in decline of fos mRNA in the presence or absence of lithium. The level of the control histone H3 mRNA is relatively unchanged by actinomycin D. Since fos mRNA accumulation is transient, we had to establish that the actinomycin D-mediated decrease in fos mRNA was not due to its natural disappearance from the cells following agonist stimulation. Therefore, RNA was also extracted from cells that were treated with carbamylcholine for 45 rnin but not with actinomycin D. As can be seen in lane 5 , fos mRNA is easily detected in cells that are not treated with actinomycin D. We conclude that fos mRNA is rapidly degraded in PC 12 cells in the absence and presence of lithium and that its increased accumulation in cells treated with agonists plus lithium is not due to RNA stabilization.
Dose Response of Carbamylcholine and Lithium on Cholinergic- and Phorbol Ester-Mediated Down Regulation We next determined the dose response for carbamylcholine-mediated down regulation. PC 12 cells were treated for 30 rnin with SO, 250, and 1 , 0 0 0 pM carbamylcholine, and RNA was extracted and analyzed for fos mRNA. As can be seen in Figure 2A (lanes 1-3), fos mRNA is induced by carbamylcholine over a 20-fold concentration range. The cells were also challenged with 50, 250, and 1,000 pM carbamylcholine for 4 hr, washed vigorously with complete medium, then rechallenged 24 hr later with the same dose of carbamylcho-
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line, Thirty minutes after rechallenge, total cellular RNA was extracted. Similar to the data shown in Figure 1, the induction of fos mRNA is markedly attenuated at each concentration of carbamylcholine (lanes 4-6). Thus muscarinic cholinergic responsiveness can be effectively down regulated over a wide carbamylcholine concentration range. To determine the dose response of lithium, cells were treated with carbamylcholine in the absence (lane 7) and presence of 1, 5, and 10 mM lithium (lanes 8-10). The effective therapeutic range of lithium is 0.5-1.2 mM (Braastroup et al., 1970). As can be seen in a comparison between lane 7 and lanes 8-10, fos mRNA accumulation is augmented at each concentration of lithium. In this experiment, the level of fos mRNA was increased by 50% at 1 mM lithium and by twofold at 5 and 10 mM. The cells were also challenged with carbamylcholine in the absence (lane 1 1) and presence of 1, 5, and 10 mM lithium (lanes 12-14), then rechallenged 24 hr later. The level of fos mRNA is increased when down-regulated cells are rechallenged with carbamylcholine in the presence of lithium. Densitometric analysis revealed that lithium increased the level of fos mRNA by a factor of two- to threefold at 1 and 5 mM concentrations and fivefold at 10 mM. The level of control histone H3 mRNA is essentially unchanged for each experimental set (lanes 1-3, 4-6, 7-10, 11-14). Thus the changes in fos mRNA mediated by lithium are not due to artifacts in the transfer and loading of RNA. To determine whether the effects observed with carbamycholine are mimicked by agents that directly activate PKC, cells were treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) for 30 rnin in the absence (Fig. 2B, lane 1) and presence of 1, 5, and 10 mM lithium (Fig. 2B, lanes 2-4) and analyzed for fos RNA. The level of fos mRNA is not significantly increased at lower concentrations of lithium. However, at 10 mM, there is a fourfold increase compared to the PMA sample without lithium. As a control in this experiment, we used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, which is constitutively expressed in many cell types (Piechaczyk et al., 1984). Prolonged treatment with phorbol esters is associated with PKC down regulation (Nishizuka, 1986). To determine whether a similar effect is observed in this system, cells were treated with 20 ng/ml PMA for 4 hr and were rechallenged with PMA 24 hr later. Thirty minutes following rechallenge, RNA was extracted. As can be seen in a comparison between lanes 1 and 5 in Figure 2B, challenge with PMA leads to down regulation of the fos mRNA signal upon subsequent rechallenge. However, similar to the results shown in Figure 1, when the cells are challenged and rechallenged in the presence
of 10 mM lithium, an augmentation of the fos signal is observed (lane 6). Thus the augmenting effect of lithium on fos expression, in both naive and down-regulated cells, appears to be mediated at a postreceptor level. Since carbamylcholine induces fos expression in our clone of PC12 cells by activating a muscarinic receptor linked to PKC, one would expect that challenge with PMA would lead to down regulation of carbamylcholine-activated fos mRNA. The experiment shown in lanes 7 and 8 indicates that the induction of fos mRNA by carbamylcholine (lane 7) is markedly attenuated if the cells were previously challenged with PMA and then rechallenged with carbamylcholine (lane 8). These data indicate that treatment with PMA leads to down regulation of subsequent muscarinic cholinergic responses.
Effect of Lithium and Carbamazepine on CAMP-Mediated fos Expression: Evidence for PMA-Mediated Heterologous Down Regulation The effect of lithium on CAMP-mediated fos expression was investigated. PC 12 cells were treated with prostaglandin E, (PGE,) for 30 min in the presence and absence of 1, 5 , and 10 mM lithium; forskolin alone, and forskolin with 10 mM lithium. The cells were also pretreated with the phosphodiesterase inhibitor 3-isobutyll-methyl xanthine (IBMX) for 5 min prior to adding the agonists. PGE, receptors are linked to the activation of adenylate cyclase throughout a G-regulatory protein (Bouzou et al., 1986). Forskolin directly activates adenylate cyclase. As can be seen in Figure 3A, activation of adenylate cyclase through the PGE, receptor (lane 1) or with forskolin (lane 5) leads to an increase in fos mRNA, presumably by increasing cAMP levels, which leads to activation of the CAMP-responsive DNA element known to be located on the fos gene (Mellon et al., 1989). However, in contrast to the results obtained with carbamylcholine and PMA, the addition of lithium does not increase fos mRNA levels induced by either receptor or postreceptor activation of adenylate cyclase (lanes 24 and 6). Therefore, the lithium-augmenting effect on fos expression we observe with carbamylcholine and PMA appears to be specific for the PLC/PKC pathway. Since down regulation of major neurotransmitter systems is thought to be involved in the pathogenesis of both mania (Dilsaver, 1986; Janowsky et al., 1980) and depression (van Praag, 1979), we were interested in determining whether lithium had an effect on down-regulation cAMP responses. Since PKC has been shown to inhibit the cAMP pathway in other systems (Kassis et al., 1985; Kelleher et al., 1984; Johnson et al., 1986), we induced down regulation with PMA and then rechallenged the cells 24 hr later with either PGE, or forskolin. As can be seen in lane 7, PMA markedly attenuates the capacity of PGE, to induce fos mRNA accumulation.
Lithium and fos Protooncogene Expression
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B
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GAPDH Fig. 3. A: Effect of lithium on CAMP-mediated fos expression. PC12 cells were pretreated with IBMX for 5 min and then treated with PGE, for 30 min in the absence (lane 1) and presence of 1, 5 , and 10 mM Li (lanes 2 4 ) , forskolin alone (lane 5), and forskolin with 10 mM Li (lane 6). Cells were challenged with PMA for 4 hr and then were rechallenged 24 hr later with PGE, (lane 7), PGE, plus 10 mM Li (lane 8), forskolin (lane 9), or forskolin plus 10 mM Li (lane 10). Cells
were challenged with CB for 4 hr and rechallenged 24 hr later with PGE, (lane 12) or PGE, plus 10 mM Li (lane 13). The control for this experiment is shown in lane 11, which is the level of fos and GAPDH mRNAs following the addition of PGE, to naive, unchallenged cells. B: Effect of carbamazepine on fos expression. Cells were treated with forskolin in the absence (lane 1) or presence (lane 2) of 0.05 mM carbamazepine (CZ), carbamylcholine alone (lane 3), or with CZ (lane 4).
Also, in contrast to down-regulated cells that are rechallenged with PMA or carbamylcholine, the addition of 10 mM lithium does not significantly affect the level of fos mRNA in cells rechallenged with PGE, (lane 8). A fos mRNA signal was not detected in down-regulated cells rechallenged with PGE, even with prolonged autoradiographic exposures. When PMA-down-regulated cells were rechallenged with forskolin in the absence and presence of lithium (lanes 9 and lo), a fos mRNA signal was easily detected, although both fos and GAPDH mRNAs were somewhat reduced in this experiment compared with similarly treated control cells (lanes 5 and 6 ) . We conclude that overstimulating the cells with PMA leads to heterologous down regulation of the PGE, receptor system, primarily at the receptor level. The data also show that the response of this down-regulated receptor system to rechallenge is not augumented by lithium, in marked contrast to our observations on down-regulated PLC/PKC pathways. We also investigated whether down regulation induced by carbamylcholine leads to heterologous downregulation of PGE, responsiveness. Cells were challenged with carbamylcholine in the presence and absence of lithium, then rechallenged 24 hr later with PGE,. In contrast to the case with cells challenged with PMA, challenge with carbamylcholine does not lead to down regulation of PGE, -mediated fos mRNA accumulation (compare lane 11, PGE, alone, with 12 and 13, carbamylcholine challenge and PGE, rechallenge, in the absence and presence of lithium). These data indicate that activation of the C-kinase linked to the MI muscarinic receptor in these cells does not induce heterologous down regulation of the PGE, receptor system. Further-
more, the finding that carbamycholine-challenged cells respond to PGE, indicates that prolonged cholinergic agonist stimulation does not lead to a nonspecific reduction in the capacity of cells to induce fos gene expression. Finally, we were interested in determining whether another antimanic agent has an effect on second messenger-mediated fos expression. The anticonvulsant carbamazepine (CZ) is an effective antimanic agent when used alone or in combination with lithium (Post, 1988). Although the exact target of its therapeutic effect in manic depression has not been established, CZ has been reported to have an inhibiting effect on adenylate cyclase (Palmer, 1979). To determine whether fos expression is affected by CZ, cells were pretreated with 50 pM CZ and then treated with either forskolin or carbamylcholine. As can be seen in Figure 3B, CZ reduces forskolinmediated fos mRNA accumulation (compare lanes 1 and 2), whereas no effect is observed when fos is induced with carbamylcholine (lanes 3 and 4). Thus, in contrast with lithium, CZ has a direct inhibitory effect on a CAMP response but no effect on PLC/PKC.
DISCUSSION It has been suggested by a number of investigators that lithium inhibits IP and/or cyclic nucleotide pathways (Avissar et al., 1988; Berridge and Irvine, 1989; Hallcher and Sherman, 1980; Newman and Belmaker, 1987; Worley et al., 1988). The putative actions of lithium on the IP cycle are thought to be secondary to the inhibition of two phosphatases that convert inositol phosphate intermediates into inositol, a precursor of PIP, (Berridge and Irvine, 1989). Reduced cyclic nucleotide
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Fig. 4. Hypothetical model for the antimanic action of lithium and carbamazepine. A: Neurotransmitter or neuropeptide receptor systems are coupled to the activation of phospholipase C (PLC) and protein kinase C (PKC) (left) or to cAMP (right), which have opposite effects on an effector target ( - and + , respectively). B: Reduced activation of PKC (thin-lined receptor system) or enhanced cAMP activation (bold lines) leads to a relative deficiency of (-) signals or a relative increase in ( + ) signals, resulting in excessive effector activation, abnormal neuronal responses, and mania. C: The Li-augmentation of PKC function or the CZ-mediated reduction in adenylate cyclase (AC) activity leads to the restoration of normal effector activation and an antimanic effect. The antimanic agent clonidine (CL) activates a,-adrenergic receptors linked to a G-regulatory protein (GJ, which inhibits AC (G, not shown). Other abbreviations include phosphatidylinositol 4,5-bisphosphate (PIP2),inositol 1,4,5-trisphosphate(IP3),cliacylglycerol (DG), phosphatidylserine (PS), and G-regulatory proteins that activate PLC and AC (G and G,, respectively). responsiveness is thought to be due to an effect on Gprotein activation (Avissar et al., 1988). To understand the effect of lithium on these critical signal transduction
pathways, we have examined a common target of phospholipid and cyclic nucleototide metabolism, activation of the fos protooncogene. The data clearly show that lithium augments fos expression induced by receptor and postreceptor activation of PKC but does not affect the cAMP pathway. Most strikingly, the augmenting effect is also observed when cholinergic or phorbol ester-mediated down-regulated cells are treated with activators of PKC. By contrast, down-regulated cAMP pathways are not affected by lithium. The muscarinic cholinergic system appears to be comparatively sensitive to the augmenting effect, since lithium concentrations within the therapeutic range are effective, whereas higher concentrations are required to augment phorbol ester-mediated fos accumulation. The increased sensitivity of the cholinergic pathway to lithium, compared with PMA, is not strictly due to differences between receptor and postreceptor activation of PKC, since we have previously found that bradykinin and nerve growth factor, agonists that stimulate PKC and fos expression in PC12 cells, are only augmented by 10 mM lithium (Kalasapudi et al., in press, and unpublished observations). According to the cholinergic hypothesis, susceptibility to manic episodes is due to reduced muscarinic cholinergic neurotransmission (Janowsky et al., 1980; Dilsaver, 1986). Thus the ability of therapeutic concentrations of lithium to augment a down-regulated muscarinic receptor system is consistent with its antimanic action according to this hypothesis. The lithium augmentation of fos expression is not consistant with its putative inhibition of the PLC pathway, since one would expect that fos expression would be inhibited, especially in cells that are challenged with carbamylcholine in the presence of lithium, a condition that may deplete cells of its pool of PIP, (Worley et al., 1988). Also, lithium should not influence phorbol estermediated events, since the direct activation of PKC by these agents bypasses the IP cycle that is thought to be the target of lithium. It has been shown that treatment with lithium leads to an increase in inositol phosphate intermediates in PC 12 cells stimulated with agonists that induce PIP, turnover (Van Calker et al., 1987). It is conceivable that this could lead to a disturbance in inosito1 phosphate-mediated Ca2+ mobilization that would effect the function of a Ca2+-dependent enzyme, such as PKC, in lithium-treated cells exposed to agonists that stimulate PIP, turnover. However, this is unlikely, since we observe a lithium-augmenting effect when PKC is directly activated with phorbol esters. This class of compounds does not stimulate PIP, turnover. Indeed, phorbol esters have been found to inhibit PIP, hydrolysis in PC12 cells (Vincentini et al., 1985). The data are more consistent with an enhancing effect of lithium on PKC function. It has been found that lithium has an affinity for
Lithium and fos Protooncogene Expression
phospholipids, in particular phosphotidylserine (PS) (Casal et al., 1987; Hauser and Shipley, 1981). Furthermore, it has been suggested that lithium could influence the function of membrane-bound proteins by altering their interaction with membrane phospholipids (Riddell and Arumugam, 1988). An effect of lithium at the membrane level leading to a change in the interaction between PKC with DAG and PS, and a subsequent increase in C-kinase activity, could account for the receptor- and postreceptor-mediated augmentation we observe. Alternatively, lithium may reduce PKC down regulation that ordinarily occurs following its activation. We also demonstrated that lithium neither inhibits nor enhances fos expression induced by receptor and postreceptor stimulation of adenylate cyclase and CAMP. If lithium inhibits this system by blocking G,, as has been suggested by Avissar et al. (1988), then one would expect fos mRNA induced by PGE, to be reduced by lithium, whereas no effect on forskolin induction should be observed. It is conceivable, however, that lithium could have an inhibitory effect on GJadenylate cyclase pathways linked to other receptor systems that may be more sensitive to lithium. It is also conceivable that lithium could have an indirect inhibitory effect on the cAMP pathway by enhancing PKC-mediated heterologous down regulation. However, we have not yet been able to demonstrate such an effect in PC12 cells (unpublished observation). In contrast to our results with lithium, we do find that CZ, another effective antimanic agent, does reduce fos mRNA levels induced by forskolin, whereas no effect on the PLC pathway is observed. Although we have not determined whether the inhibitory effect on CAMPmediated fos expression also occurs at the receptor level, our data are consistent with an inhibitory effect of CZ on adenylate cyclase and the absence of an effect on the IP pathway, consistent with data reported by other investigators (Palmer, 1979; Elphick et al., 1988). Thus two of the most commonly used antimanic agents appear to have an effect on different signal transduction pathways in PC12 cells. One possible mechanism to account for the antimanic properties of lithium and CZ, based on our experimental observations, is depicted in the model shown in Figure 4. In this model, PKC and cAMP have opposite effects on a common effector target, such as an ion channel (Fig. 4A). A manic episode is initiated because of a stressor overstimulating the cAMP arm, or because of an underactive PLCiPKC pathway, leading to excessive, signals (Fig. 4B). Presumably, the vulnerunopposed ability to these events would occur as a result of the mutant alleles thought to play a role in the pathogenesis of manic depression (Lachman and Papolos, 1989). An augmenting effect on PKC function by lithium, or an
+
47
inhibition of the cAMP arm by CZ, or perhaps by lithium-enhanced PKC heterologous down regulation, would lead to an antimanic effect (Fig. 4C). It is interesting to note that this model is also consistent with the mechanism of action of another antimanic agent, clonidine, which activates the a,-adrenergic receptor linked to Gi, the G-regulatory protein that inhibits adenylate cyclase (Hardy et al., 1986). Although we used fos expression as a convenient target activated by both the PLC/PKC and cAMP pathways, we have not yet addressed its augmentation by lithium as a potentially important aspect of the therapeutic response. The impact of increased fos expression on long-term neuronal responses in the treatment of mood disorders remains to be determined.
ACKNOWLEDGMENTS We thank Dr. Herman van Praag for his support during this study. The Program of Behavioral Genetics is supported by a grant from the Ruane Family Fund. H.M.L. is supported by NIH grant CA 4324601 and the Weill-Caulier Career Scientist Award. G.S. was supported by an NIH Cancer training grant (5R23CA47779). M.D. is supported by a Medical Student Research Training Fellowship from the Howard Hughes Medical Institute.
REFERENCES Alterman RB, Ganguly S , Schulze D, Marzluff W, Schildkraut C, Skoultchi A (1984): Cell cycle regulation of mouse histone H3 mRNA metabolism. Mol Cell Biol 4:123-132. Avissar S, Schreiber G, Danon A, Belmaker RH (1988): Lithium inhibits adrenergic and cholinergic increases in GTP binding in rat cortex. Nature 331:440-442. Berridge MJ, Irvine RF (1989): Inositol phosphates and cell signalling. Nature 341:197-205. Braastroup PC, Poulsen JC, Schou M, Thomsen K, Amidsen A ( 1970): Prophylactic lithium: Double-blind discontinuation in manic-depression and recurrent-depressive illness. Lancet 2: 326-330. Bouzou JC, Amar S , Vincent JP, Kitabgi P (1986): Neurotensinmediated inhibition of cyclic nucleotide formation in neuroblastoma NlEl15 cells. Involvement of the inhibitory GTPbinding component of adenylate cyclase. Mol Pharmacol 29: 489-496. Casal, HL, Mantsch HH, Paltauf F, Hauser H (1987): Infrared and 3'P-NMR studies of the effect of Li+ and Ca2+ on phosphatidylserines. Biochim Biophys Acta 919:275-286. Curran T, Morgan JI (1985): Superinduction of c-fos by nerve growth factor in the presence of peripherally active benzodiazepines. Science 229: 1265-1 268. Dilsaver SC (1986): Cholinergic mechanisms in depression. Brain Res Rev 11:285-316. Elphick M, Taghavi Z, Powell T, Godfrey PP (1988): Alteration of inositol phospholipid metabolism in rat cortex by lithium but not carbamazepine. Eur J Pharmacol 156:411-414. Godfrey PP, McClue SJ, White AM, Wood AM, Grahame-Smith DG (1989): Subacute and chronic in vivo lithium treatment inhibits
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agonist and sodium fluoride stimulated inositol phosphate production in rat cortex. J Neurochem 52:498-507. Greenberg ME, Ziff EB, Greene LA (1986): Stimulation of neuronal acetylcholine receptors induces rapid gene transcription. Science 234:SO-83. Hallcher L, Sherman WR (1980): The effects of lithium ion and other agents on the activity of myo-inositol-1-phosphatasefrom bovine brain. J Biol Chem 261:8100-8130. Hardy MC, Lecrubier Y, Widlocher D (1986): Efficacy of clonidine in 24 patients with acute mania. Am J Psychiatry 143: 1450-1453. Hauser H, Shipley GG (1981): Crystallization of the phosphotidylserine bilayers induced by lithium. J Biol Chem 256:1137711380. Janowsky DS, Risch SC, Parker D, Huey LY, Judd L (1980): Increased vulnerability to cholinergic stimulation in affective disorder patients. Psychopharmacol Bull 16:29-31. Johnson JA, Goka TJ, Clark RB (1986): Phorbol ester-induced augmentation and inhibition of epinephrine-stimulated adenylate cyclase in S49 lymphoma cells. J Cyclic Nucleotide Protein Phosphor Res 11:199-215. Kassis S , Zaremba T , Patel J, Fishman PH (1985): Phorbol esters and beta-adrenergic agonists mediate desensitization of adenylate cyclase in rat glioma C6 cells by distinct mechanisms. J Biol Chem 260:8911-8917. Kelleher DJ, Pessin JE, Ruoho AE, Johnson GI, (1984): Phorbol ester induces desensitization of adenylate cyclase and phosphorylation of the beta-adrenergic receptor in turkey erythrocytes. Proc Natl Acad Sci USA 81:4316-4320. Kikkawa U, Nishizuka Y (1986): The role of protein kinase C in transmembrane signalling. Annu Rev Cell Biol 2: 149-178. Lachman HM, Papolos DF (1989): Abnormal signal transduction: A hypothetical model for bipolar affective disorder. Life Sci 45: 1413-1426. Lachman HL, Skoultchi A1 (1984): Expression of c-myc changes during differentiation of mouse erythroleukemia cells. Nature 3 10: 592-594. Mellon PL, Clegg C, Correll LA, McKnight GS (1989): Regulation of transcription by CAMP-dependent protein kinase. Proc Natl Acad Sci USA 86:488-491. Miller AD, Curran T, Verma IM (1984): C-fos protooncogene can induce transformation: A novel mechanism of activation of a cellular oncogene. Cell 36:5 1-60. Mork A , Geisler A (1989): Effects of GTP on hormone-stimulated adenylate cyclase in cerebral cortex, striatum, and hippocampus from rats treated chronically with lithium. Biol Psychiatry 261279-288. Nepveu A, Marcu KB, Skoultchi AI, Lachman HM (1987): Contributions of transcriptional and post-transcriptional mechanisms to the regulation of c-myc expression in mouse erythroleukemia cells. Genes Dev 1:938-945.
Newman ME, Belmaker RH (1987): Effects of lithium in vitro and ex vivo on components of the adenylcyclase system in membranes from cerbral cortex of the rat. Neuropharmacology 26:211217. Nishizuka Y (1986): Studies and perspectives of protein kinase C. Science 233:305-312. Palmer C (1979): Interactions of antiepileptic drugs on adenylate cyclase and phosphodiesterases in rat and mouse cerebrum. Exp Neurol 63:322-335. Piechaczyk M, Blanchard JM, Marty L, Dani C, Panatieres F, ElSabouty S, Fort P, Jeanteur P (1984): Post-transcriptional regulation of glyceraldehyde-3-phosphatedehydrogenase gene expression in rat tissue. Nucleic Acids Res 12:6951-6963. Post RM (1988): Time course of clinical effects of carbamazepine: Implications for mechanisms of action. J Clin Psychiatry 49[SUPPI41135-46. Rauscher FJ, Sambucetti LC, Curran T, Distel RJ, Spiegalman BM (1988): Common DNA binding site for Fos protein complexes and transcription factor AP-I . Cell 52:471-480. Riddell FG, Arumugam S (1988): Surface charge effects upon membrane transport processes: The effects of surface charge on the monensin-mediated transport of lithium ions through phospholipid bilayers studied by ’Li-NMR spectroscopy. Biochim Biophys Acta 945:65-72. Sherman W, Gish B, Honchar M, Munsell LY (1986): Effects of lithium on phosphoinositide metabolism in vivo. Fed Proc 4.5: 2639-2646. Sigel E, Roland B (1988): Activation of protein kinase C differentially modulates neuronal Na+ , Ca2+ and gamma-aminobutyrate type A channels. Proc Natl Acad Sci USA 85:6192-6196. Stimac E, Groppi VE, Coffino P (1984): Inhibition of protein synthesis stabilizes histone mRNA. Mol Cell Biol 4:2082-2092. van Calker D, Assmann K, Greil W (1987): Stimulation by bradykinin, angiotensin 2 and carbachol of the accumulation of inositol phosphates in PC12 pheochromocytoma cells: Differential effects of lithium on mono and polyphosphates. J Neurochem 49: 1379-1384. van Praag HM (1979): Central Serotonin: Its relation to depression vulnerability and depressive prophylaxis. In Obiols J, Ballus C, Gonzales Manclus E, Pujol J (eds): “Biological Psychiatry Today.” New York: Elsever North Holland Biomedical Press. Vincentini LM, di Virgilio F, Ambrosini A, Pozzan T, Meldolesi (198s): Tumor promoter 12-myrisate, 13-acetate inhibits phosphoinositide hydrolysis and cytosolic Ca2+ rise induced by activation of muscarinic receptor in PC12 cells. Biochem Biophys Res Commun 127:310-317. Worley PF, Heller WA, Snyder SH, Baraban JM (1988): Lithium blocks a phosphoinositide-mediated cholinergic response in hippocampal slices. Science 239: 1428-1429.
Differential Effect of Lithium on fos Protooncogene Expression Mediated by Receptor and Postreceptor Activators of Protein Kinase C and Cyclic Adenosine Monophosphate: Model for Its Antimanic Action M.M. Divish, G. Sheftel, A. Boyle, V.D. Kalasapudi, D.F. Papolos, and H.M. Lachman Department of Psychiatry, Program of Behavioral Genetics (M.M.D., G.S., A.B., V.D.K., D.F.P., H.M.L.) and Department of Medicine (H.M.L.), Albert Einstein College of Medicine, Bronx, New York
Lithium salts are the most effective agents used in Key words: muscarinic receptor, manic depression, treating manic-depressive illness. It has been sug- down regulation, adenylate cyclase, lithium gested that lithium’s therapeutic efficacy could be due to an inhibitory effect on either inositol phospholipid (IP) and/or cyclic nucleotide metabolism. We have INTRODUCTION investigated the effect of lithium on these two signal Lithium salts have been used in the treatment of transduction pathways in PC12 pheochromocytoma manic depression and recurrent unipolar depression for a cells by studying a common effector target, expres- number of years, yet the specific target of its therapeutic sion of the fos protooncogene. We find that lithium, action remains obscure. Several investigators have sugat therapeutic doses, has an augmenting effect on gested that lithium influences inositol phospholipid (IP) phosphatidylinositol (PI)-mediated fos expression in- pathways by interfering with inositol synthesis, through duced by activating a muscarinic cholinergic path- its inhibition of the enzyme myoinositol 1-phosphatase way, whereas it has no effect, a t tenfold the thera- (Hallcher and Sherman, 1980; Berridge and Irvine, peutic dose, on fos expression induced by receptor or 1989). Depletion of inositol would be expected to lead to postreceptor activators of cyclic adenosine mono- a decrease in phosphatidylinositol 4,5-bisphosphate phosphate (CAMP). The lithium augmenting effect is (PIP,), which is hydrolyzed into diacylglycerol (DAG) also observed when the cells are treated with phorbol and inositol 1,4,5-trisphosphate (IP,) by ligand-activated esters, which directly activate protein kinase C phospholipase C (PLC). IP, mobilizes intracellular (PKC), suggesting that the level of lithium’s interac- Ca2+, and DAG activates protein kinase C (PKC), a tion with the IP pathway is at the postreceptor level. central serine and threonine kinase that modulates a numWe also show that phorbol esters induce extensive ber of effector targets, including ion channels and neudown regulation of subsequent cholinergic and phor- rotransmitter receptors (Berridge and Irvine, 1989; Nishbol ester responsiveness as well as heterologous down izuka, 1986; Kikkawa and Nishizuka, 1986; Sigel and regulation of CAMP responses. Treatment of down- Roland, 1988). Following activation by agonists or phorregulated cells with lithium leads to an enhanced re- bol esters, PKC binds to the membrane in conjunction sponsiveness when cells are rechallenged with ago- with DAG, Ca2+ , and phosphatidylserine. nists that activate PKC but not by agonists that stimulate CAMP. We also show that carbamazepine, another antimanic agent, has an inhibitory effect on Received March 9, 1990; revised May 31, 1990; accepted June 5 , CAMP-mediated fos but no effect on the IP pathway. 1990. The opposite effects of lithium and carbamazepine on Address reprint requests to Herbert M. Lachman, Department of Medtwo critical transducing systems suggest a model for icine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, the antimanic action of these agents. 0 1991 Wiley-Liss, Inc.
Lithium and fos Protooncogene Expression
It has been suggested that an influence on IP metabolism could be a mechanism for the therapeutic efficacy of lithium in manic depression (Berridge and Irvine, 1989; Lachman and Papolos, 1989; Sherman et al., 1986). Supporting this hypothesis are the reports that lithium inhibits PLC-mediated events in the brain. For example, cortical slices derived from rats treated with lithium exhibit reduced agonist stimulated inositol phosphate production (Godfrey et al., 1989). Also, lithium inhibits muscarinic cholinergic responses in the hippocampus, which is mediated by PLC (Worley et al., 1988). Other investigators have suggested that the target of lithium’s effect on cells may be cyclic nucleotide metabolism (Avissar et al., 1988; Newman and Belmaker, 1987). For example, it has been reported that therapeutic concentrations of lithium inhibit agonist stimulated adenylate cyclase activity in rats (Mork and Geisler, 1989). Avissar et al. (1988) have shown that lithium blocks agaonist-induced guanosine triphosphate (GTP) binding in membranes, an effect that is a measure of guanine nucleotide regulatory protein (G-protein) activation. They postulated that lithium reduces cyclic adenosine monophosphak (CAMP) responsiveness by inhibiting G,, the G-protein that regulates adenylate cyclase. TG understand further lithium’s influence on second messenger pathways, it would be useful to study an effector system that is influenced by both the IP and cyclic nucleotide pathways. Activation of fos protooncogene expression is an ideal target for such studies, since fos gene transcription is inhitiated by both PKCand CAMP-dependent pathways (Curran and Morgan, 1985; Rauscher et al., 1988; Mellon et al., 1989). fos protein dimerizes with the jun protooncogene product to form the AP- 1 transcription factor, which regulates the expression of a number of AP-l-sensitive genes (Rauscher et al., 1988). We have previously demonstrated that PC12 rat pheochromocytoma cells, treated with agonists that stimulate PKC, rapidly accumulate large quantities of fos mRNA. When lithium is added to the culture medium, PKC-mediated activation of fos expression is augmented severalfold (Kalasapudi et al., in press). In this report, we have extended our original studies and compared the effect of various concentrations of lithium on fos expression induced by receptor and postreceptor activators of PKC and cAMP in PC12 cells. In addition, we have explored the interaction between the cAMP and IP pathways by studying the effect of muscarinic cholinergic agonists and phorbol esters on CAMPmediated fos expression. The data show that, in PC12 cells, lithium has an enhancing effect on fos expression induced by receptor and postreceptor activators of PKC, whereas it has no direct effect on agonists that stimulate CAMP. By contrast, the antiepileptic and antimanic agent
41
carbamazepine has an inhibitory effect on CAMP-activated fos but no effect on the IP pathway. Furthermore, we show that treatment with phorbol esters leads to extensive down regulation of receptor systems that activate both PKC and adenylate cyclase. Whereas lithium increases the responsiveness of down-regulated PLC/PKC pathways, it has no effect on down-regulated cAMP responses. The differential effect of lithium on down-regulated receptor systems coupled to PKC and cAMP may be important in understanding its therapeutic efficacy.
MATERIALS AND METHODS Cell Cultures PC 12 cells were maintained in tissue culture flasks in RPMI supplemented with 10% horse serum and 5% fetal calf serum, both heat inactivated at 56°C. Fortyeight hours before RNA extraction, the cells were trypsinized from a nearly confluent T- 125 flask, plated on ten 100 mm Falcon tissue culture plates, and placed in a 5% CO, incubator at 37°C and 100% relative humidity. Under these conditions, each plate contained approximately 5 x lo6-] X lo7 cells after 48 hr. The clone of PC12 cells used in these experiments attaches to plastic surfaces without requiring a protein matrix. Drugs were added from 1,OOOX and 10,OOOX stocks, with the exception of lithium, which is a 400X aqueous solution before dilution to a final concentration of 10 mM. The vehicles used for dissolving the drugs included water, dimethylsulfoxide (DMSO), and 70% ethanol. In control experiments, neither of these agents had an effect on fos gene expression at the concentrations used in these studies (data not shown). In the challenge experiments, cell were treated with agonists for 4 hr, after which the drugs were removed by washing the cells three times in complete medium over a 15 min period. The cells were then rechallenged 4-48 hr later, and total cellular RNA was extracted 30 min after the rechallenge. RNA Analysis Cells were trypsinized and harvested by vigorous pipetting and centrifugation at 1,000 rpm for 5 min. Total cellular RNA was extracted using the hot phenol technique as previously described (Lachman and Skoultchi, 1984). Ten micrograms of total cellular RNA was separated by electrophoresis through 0.9% agarose containing 3% formaldehyde. The samples were denatured prior to loading by adding 4 volumes of loading buffer containing 60% formamide and 20% formaldehyde and heating to 65°C for 5 min. Following electrophoresis, the RNA was transferred to nylon filters (Nytran, Schleichter and Schuel) through 20 X SSC (1 X SSC is 0.15 M sodium chloride and 0 .O 15 M sodium citrate). The filters were baked at 80°C in vacuuo for 2 hr and prehybridized
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A
B 1 2 3 4 5 6 7 8 9
1 2 3 4 5
Fos
"3 Fig. 1. A: Effect of lithium on carbamylcholine-mediated fos challenge (lanes 4, 6, and 8, respectively) or were challenged expression. Total cellular RNA was extracted from PC 12 cells and rechallenged under the same conditions but in the presence following treatment with carbamylcholine (CB) for 30 min of 10 mM lithium (lane 5, 4 hr rechallenge; lane 7, 24 hr with and without preincubation with lithium chloride for 16 hr rechallenge; and lane 9, 48 hr rechallenge). B: Effect of acti(Li). RNA was analyzed for fos and histone H3 mRNAs by nomycin D on fos mRNA. Control and lithium-treated (10 mM Northern analysis as described in Materials and Methods. for 16 hr) PC12 cells were treated with CB for 15 min, after Lane 1, untreated control; lane 2, 1 mM CB; lane 3, I mM CB which actinomycin D ( 5 pgiml) was added. Total cellular RNA plus 10 mM Li. Down regulation with cholinergic stimulation was extracted after an additional 15 rnin (lane 1, no lithium; was induced by challenging the cells for 4 hr with 1 mM CB. lane 3, plus lithium) and 30 rnin (lane 2, no lithium; lane 4, After washing with complete medium, the cells were rechal- plus lithium). Lane 5 shows RNA extracted from control cells lenged for 30 min with 1 mM CB at 4, 24, and 48 hr after the treated with CB for 45 min, without actinomycin D.
in 5 x SSC, 10 X Denhardt's solution, 50 mM phosphate buffer, pH 6.8, 0.1% sodium dodecyl sulfate (SDS) and 0.25 mg/ml denatured sheared salmon sperm DNA. The filters were hybridized for 16 hr at 65°C with denatured DNA probes that were radiolabeled with 32P by nick translation or by the random primer technique. The probes used in these experiments included pMMMfos, a mouse genomic fos plasmid (Miller et al., 1984); pGAPDHl3, a rat glyceraldehyde 3-phosphate dehydrogenase cDNA clone (Piechaczyk et al., 1984); and pRAH3-2, a mouse histone H3 cDNA clone (Alterman et al., 1984).
RESULTS Effect of Lithium on fos Expression During Carbamylcholine-Mediated Down Regulation Total cellular RNA was extracted from PC12 cells and analyzed for fos mRNA by Northern filter analysis. The autoradiogram in Figure 1A shows that fos mRNA is not detected in untreated PC12 cells (lane 1). However, upon treatment of the cells for 30 min with carbamylcholine, a stable cholinergic agonist, a large induction of fos mRNA occurs (lane 2). Carbamylcholine is a nonspecific cholinergic agonist. However, previous experiments indicate that, in this clone of PC12 cells, the carbamylcholine-mediated induction of fos mRNA is due to the activation of an M1 muscarinic receptor subtype, which is coupled to PLC, since induction is blocked by low concentrations of pirenzepine (Kalasapudi et al., in
press). Nicotinic cholinergic receptor stimulation does not induce fos expression in the clone of PC12 used in these experiments, in contrast to the findings reported by Greenberg et al. (1986). Following treatment of the cells with 10 mM lithium chloride for 16 hr, there is an augmentation of the carbamylcholine-mediated induction of fos mRNA (lane 3), similar to the results we previously described. Since down-regulated muscarinic responses are thought to play a role in the development of mania (Janowsky et al., 1980; Dilsaver, 1986), we investigated the effect of lithium on cells that were overstimulated with carbamylcholine which induces cholinergic down regulation. Cells were challenged with carbamylcholine for 4 hr then rechallenged with carbamylcholine, 4 hr (lane 4), 24 hr (lane 6), and 48 hr (lane 8) after the challenge. Total cellular RNA was extracted from the cells 30 min after rechallenge and analyzed for fos RNA. The autoradiogram in Figure 1A shows that the induction of fos mRNA, following a previous cholinergic challenge, is markedly attenuated. However, if the cells are challenged and rechallenged with carbamylcholine in the presence of 10 mM lithium, fos RNA levels increase at each time point (lanes 5 , 7, and 9) compared with cells that were not treated with lithium. Densitometric analysis revealed that the lithium-mediated increase in fos mRNA ranged from twofold to 3.5-fold (data not shown). The lithium-mediated increase in fos mRNA seen in down-regulated cells is not due to its accumulation during the challenge period since residual fos
Lithium and fos Protooncogene Expression
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B.
A. I 2 3 4 5 6
7 8 910 1112 1314
1 2 3 4 5 6 7 8
fos
Fig. 2. A: Lithium and carbamylcholine dose response. Cells were treated with 0.05, 0.25, and 1.0 mM CB for 30 min and analyzed for fos and H3 mRNA (lanes 1-3, respectively). Cells were challenged with 0.05, 0.25, and 1.0 mM CB for 4 hr and rechallenged for 30 min with the same concentration of CB after 24 hr (lanes 4-6, respectively). Control cells (lane 7) and cells treated with 1, 5 , and 10 mM Li for 16 hr (lanes 8-10), were induced with 1 mM CB for 30 min. Cells were challenged with CB as in Figure 1 and rechallenged 24 hr later in the absence (lane 11) or presence of 1 , 5 , or 10 mM Li (lanes 12-14, respectively). B: Effect of PMA on fos expres-
sion. Cells were analyzed for fos and GAPDH mRNAs following treatment with the phorbol ester PMA (20 ngiml) for 30 rnin in the absence of Li (lane 1) or with 1, 5 , and I0 mM Li (lanes 2 4 ) . Cells were also challenged with PMA for 4 hr and rechallenged 24 hr later with PMA in the absence (lane 5) or presence of 10 mM Li (lane 6). Lane 7, Fos and GAPDH mRNA following 30 rnin with I mM CB. Lane 8, 1 mM CB 24 hr following a challenge with PMA. Note the RNA in lanes 7 and 8 are derived from an experiment separate from the one shown in lanes 1-6.
mRNA is not detected at the indicated time points unless cells are rechallenged (data not shown). Thus, in agreement with our previous observations, lithium can increase fos expression in cells that display a down-regulated muscarinic cholinergic receptor system. To control for the equal loading and transfer of the RNA, the filters were rehybridized with a histone H3 probe. Since H3 expression is cell cycle dependent, it also serves as a control for cell replication during the rechallenge and lithium treatment periods. A reduction in replicating cells should be reflected by a decrease in the level of H3 mRNA (Alterman et al., 1984). As can be seen in Figure l A , the level of H3 RNA in this experiment is not significantly altered by cholinergic challenge or lithium treatment. One possible explanation for the augmenting effect is that the half life of fos mRNA is increased by lithium. A number of studies have demonstrated that mRNAs with relatively short half-lives, such as fos, can be stabilized by protein synthesis inhibitors, which presumably inhibit the synthesis of nucleases that degrade mRNAs (Stimac et al., 1984). A similar effect on the addition of relatively high concentrations of lithium could conceivably lead to enhanced fos mRNA accumulation and would likely represent a toxic effect of the drug rather than a relevant physiological action. The data in Figure 1B argue against this possibility. Cells were treated with carbamylcholine in the presence and absence of 10 mM lithium. After 15 min, the transcription inhibitor actinomycin D was added, and total cellular RNA was extracted after an additional 15 and 30 min. When transcription is inhibited, mRNA levels decrease at a rate proportional to its half-life (Nepveu et al., 1987). As can
be seen in Figure l B , the level of fos mRNA rapidly declines following the addition of actinomycin D, both in the absence (lanes 1, 2) and in the presence (lanes 3 , 4) of 10 mM lithium. Densitometric analysis did not reveal a significant difference in decline of fos mRNA in the presence or absence of lithium. The level of the control histone H3 mRNA is relatively unchanged by actinomycin D. Since fos mRNA accumulation is transient, we had to establish that the actinomycin D-mediated decrease in fos mRNA was not due to its natural disappearance from the cells following agonist stimulation. Therefore, RNA was also extracted from cells that were treated with carbamylcholine for 45 rnin but not with actinomycin D. As can be seen in lane 5 , fos mRNA is easily detected in cells that are not treated with actinomycin D. We conclude that fos mRNA is rapidly degraded in PC 12 cells in the absence and presence of lithium and that its increased accumulation in cells treated with agonists plus lithium is not due to RNA stabilization.
Dose Response of Carbamylcholine and Lithium on Cholinergic- and Phorbol Ester-Mediated Down Regulation We next determined the dose response for carbamylcholine-mediated down regulation. PC 12 cells were treated for 30 rnin with SO, 250, and 1 , 0 0 0 pM carbamylcholine, and RNA was extracted and analyzed for fos mRNA. As can be seen in Figure 2A (lanes 1-3), fos mRNA is induced by carbamylcholine over a 20-fold concentration range. The cells were also challenged with 50, 250, and 1,000 pM carbamylcholine for 4 hr, washed vigorously with complete medium, then rechallenged 24 hr later with the same dose of carbamylcho-
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line, Thirty minutes after rechallenge, total cellular RNA was extracted. Similar to the data shown in Figure 1, the induction of fos mRNA is markedly attenuated at each concentration of carbamylcholine (lanes 4-6). Thus muscarinic cholinergic responsiveness can be effectively down regulated over a wide carbamylcholine concentration range. To determine the dose response of lithium, cells were treated with carbamylcholine in the absence (lane 7) and presence of 1, 5, and 10 mM lithium (lanes 8-10). The effective therapeutic range of lithium is 0.5-1.2 mM (Braastroup et al., 1970). As can be seen in a comparison between lane 7 and lanes 8-10, fos mRNA accumulation is augmented at each concentration of lithium. In this experiment, the level of fos mRNA was increased by 50% at 1 mM lithium and by twofold at 5 and 10 mM. The cells were also challenged with carbamylcholine in the absence (lane 1 1) and presence of 1, 5, and 10 mM lithium (lanes 12-14), then rechallenged 24 hr later. The level of fos mRNA is increased when down-regulated cells are rechallenged with carbamylcholine in the presence of lithium. Densitometric analysis revealed that lithium increased the level of fos mRNA by a factor of two- to threefold at 1 and 5 mM concentrations and fivefold at 10 mM. The level of control histone H3 mRNA is essentially unchanged for each experimental set (lanes 1-3, 4-6, 7-10, 11-14). Thus the changes in fos mRNA mediated by lithium are not due to artifacts in the transfer and loading of RNA. To determine whether the effects observed with carbamycholine are mimicked by agents that directly activate PKC, cells were treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) for 30 rnin in the absence (Fig. 2B, lane 1) and presence of 1, 5, and 10 mM lithium (Fig. 2B, lanes 2-4) and analyzed for fos RNA. The level of fos mRNA is not significantly increased at lower concentrations of lithium. However, at 10 mM, there is a fourfold increase compared to the PMA sample without lithium. As a control in this experiment, we used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, which is constitutively expressed in many cell types (Piechaczyk et al., 1984). Prolonged treatment with phorbol esters is associated with PKC down regulation (Nishizuka, 1986). To determine whether a similar effect is observed in this system, cells were treated with 20 ng/ml PMA for 4 hr and were rechallenged with PMA 24 hr later. Thirty minutes following rechallenge, RNA was extracted. As can be seen in a comparison between lanes 1 and 5 in Figure 2B, challenge with PMA leads to down regulation of the fos mRNA signal upon subsequent rechallenge. However, similar to the results shown in Figure 1, when the cells are challenged and rechallenged in the presence
of 10 mM lithium, an augmentation of the fos signal is observed (lane 6). Thus the augmenting effect of lithium on fos expression, in both naive and down-regulated cells, appears to be mediated at a postreceptor level. Since carbamylcholine induces fos expression in our clone of PC12 cells by activating a muscarinic receptor linked to PKC, one would expect that challenge with PMA would lead to down regulation of carbamylcholine-activated fos mRNA. The experiment shown in lanes 7 and 8 indicates that the induction of fos mRNA by carbamylcholine (lane 7) is markedly attenuated if the cells were previously challenged with PMA and then rechallenged with carbamylcholine (lane 8). These data indicate that treatment with PMA leads to down regulation of subsequent muscarinic cholinergic responses.
Effect of Lithium and Carbamazepine on CAMP-Mediated fos Expression: Evidence for PMA-Mediated Heterologous Down Regulation The effect of lithium on CAMP-mediated fos expression was investigated. PC 12 cells were treated with prostaglandin E, (PGE,) for 30 min in the presence and absence of 1, 5 , and 10 mM lithium; forskolin alone, and forskolin with 10 mM lithium. The cells were also pretreated with the phosphodiesterase inhibitor 3-isobutyll-methyl xanthine (IBMX) for 5 min prior to adding the agonists. PGE, receptors are linked to the activation of adenylate cyclase throughout a G-regulatory protein (Bouzou et al., 1986). Forskolin directly activates adenylate cyclase. As can be seen in Figure 3A, activation of adenylate cyclase through the PGE, receptor (lane 1) or with forskolin (lane 5) leads to an increase in fos mRNA, presumably by increasing cAMP levels, which leads to activation of the CAMP-responsive DNA element known to be located on the fos gene (Mellon et al., 1989). However, in contrast to the results obtained with carbamylcholine and PMA, the addition of lithium does not increase fos mRNA levels induced by either receptor or postreceptor activation of adenylate cyclase (lanes 24 and 6). Therefore, the lithium-augmenting effect on fos expression we observe with carbamylcholine and PMA appears to be specific for the PLC/PKC pathway. Since down regulation of major neurotransmitter systems is thought to be involved in the pathogenesis of both mania (Dilsaver, 1986; Janowsky et al., 1980) and depression (van Praag, 1979), we were interested in determining whether lithium had an effect on down-regulation cAMP responses. Since PKC has been shown to inhibit the cAMP pathway in other systems (Kassis et al., 1985; Kelleher et al., 1984; Johnson et al., 1986), we induced down regulation with PMA and then rechallenged the cells 24 hr later with either PGE, or forskolin. As can be seen in lane 7, PMA markedly attenuates the capacity of PGE, to induce fos mRNA accumulation.
Lithium and fos Protooncogene Expression
45
B
A I 2 3 4 5 6 7 8 9 10111213
I 2 3 4
Fos
GAPDH Fig. 3. A: Effect of lithium on CAMP-mediated fos expression. PC12 cells were pretreated with IBMX for 5 min and then treated with PGE, for 30 min in the absence (lane 1) and presence of 1, 5 , and 10 mM Li (lanes 2 4 ) , forskolin alone (lane 5), and forskolin with 10 mM Li (lane 6). Cells were challenged with PMA for 4 hr and then were rechallenged 24 hr later with PGE, (lane 7), PGE, plus 10 mM Li (lane 8), forskolin (lane 9), or forskolin plus 10 mM Li (lane 10). Cells
were challenged with CB for 4 hr and rechallenged 24 hr later with PGE, (lane 12) or PGE, plus 10 mM Li (lane 13). The control for this experiment is shown in lane 11, which is the level of fos and GAPDH mRNAs following the addition of PGE, to naive, unchallenged cells. B: Effect of carbamazepine on fos expression. Cells were treated with forskolin in the absence (lane 1) or presence (lane 2) of 0.05 mM carbamazepine (CZ), carbamylcholine alone (lane 3), or with CZ (lane 4).
Also, in contrast to down-regulated cells that are rechallenged with PMA or carbamylcholine, the addition of 10 mM lithium does not significantly affect the level of fos mRNA in cells rechallenged with PGE, (lane 8). A fos mRNA signal was not detected in down-regulated cells rechallenged with PGE, even with prolonged autoradiographic exposures. When PMA-down-regulated cells were rechallenged with forskolin in the absence and presence of lithium (lanes 9 and lo), a fos mRNA signal was easily detected, although both fos and GAPDH mRNAs were somewhat reduced in this experiment compared with similarly treated control cells (lanes 5 and 6 ) . We conclude that overstimulating the cells with PMA leads to heterologous down regulation of the PGE, receptor system, primarily at the receptor level. The data also show that the response of this down-regulated receptor system to rechallenge is not augumented by lithium, in marked contrast to our observations on down-regulated PLC/PKC pathways. We also investigated whether down regulation induced by carbamylcholine leads to heterologous downregulation of PGE, responsiveness. Cells were challenged with carbamylcholine in the presence and absence of lithium, then rechallenged 24 hr later with PGE,. In contrast to the case with cells challenged with PMA, challenge with carbamylcholine does not lead to down regulation of PGE, -mediated fos mRNA accumulation (compare lane 11, PGE, alone, with 12 and 13, carbamylcholine challenge and PGE, rechallenge, in the absence and presence of lithium). These data indicate that activation of the C-kinase linked to the MI muscarinic receptor in these cells does not induce heterologous down regulation of the PGE, receptor system. Further-
more, the finding that carbamycholine-challenged cells respond to PGE, indicates that prolonged cholinergic agonist stimulation does not lead to a nonspecific reduction in the capacity of cells to induce fos gene expression. Finally, we were interested in determining whether another antimanic agent has an effect on second messenger-mediated fos expression. The anticonvulsant carbamazepine (CZ) is an effective antimanic agent when used alone or in combination with lithium (Post, 1988). Although the exact target of its therapeutic effect in manic depression has not been established, CZ has been reported to have an inhibiting effect on adenylate cyclase (Palmer, 1979). To determine whether fos expression is affected by CZ, cells were pretreated with 50 pM CZ and then treated with either forskolin or carbamylcholine. As can be seen in Figure 3B, CZ reduces forskolinmediated fos mRNA accumulation (compare lanes 1 and 2), whereas no effect is observed when fos is induced with carbamylcholine (lanes 3 and 4). Thus, in contrast with lithium, CZ has a direct inhibitory effect on a CAMP response but no effect on PLC/PKC.
DISCUSSION It has been suggested by a number of investigators that lithium inhibits IP and/or cyclic nucleotide pathways (Avissar et al., 1988; Berridge and Irvine, 1989; Hallcher and Sherman, 1980; Newman and Belmaker, 1987; Worley et al., 1988). The putative actions of lithium on the IP cycle are thought to be secondary to the inhibition of two phosphatases that convert inositol phosphate intermediates into inositol, a precursor of PIP, (Berridge and Irvine, 1989). Reduced cyclic nucleotide
46
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Divish et al.
I
-
t EFFECTOR
8
cAMP
4
1
+
C Li+
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cAMP
-
1I
t
Fig. 4. Hypothetical model for the antimanic action of lithium and carbamazepine. A: Neurotransmitter or neuropeptide receptor systems are coupled to the activation of phospholipase C (PLC) and protein kinase C (PKC) (left) or to cAMP (right), which have opposite effects on an effector target ( - and + , respectively). B: Reduced activation of PKC (thin-lined receptor system) or enhanced cAMP activation (bold lines) leads to a relative deficiency of (-) signals or a relative increase in ( + ) signals, resulting in excessive effector activation, abnormal neuronal responses, and mania. C: The Li-augmentation of PKC function or the CZ-mediated reduction in adenylate cyclase (AC) activity leads to the restoration of normal effector activation and an antimanic effect. The antimanic agent clonidine (CL) activates a,-adrenergic receptors linked to a G-regulatory protein (GJ, which inhibits AC (G, not shown). Other abbreviations include phosphatidylinositol 4,5-bisphosphate (PIP2),inositol 1,4,5-trisphosphate(IP3),cliacylglycerol (DG), phosphatidylserine (PS), and G-regulatory proteins that activate PLC and AC (G and G,, respectively). responsiveness is thought to be due to an effect on Gprotein activation (Avissar et al., 1988). To understand the effect of lithium on these critical signal transduction
pathways, we have examined a common target of phospholipid and cyclic nucleototide metabolism, activation of the fos protooncogene. The data clearly show that lithium augments fos expression induced by receptor and postreceptor activation of PKC but does not affect the cAMP pathway. Most strikingly, the augmenting effect is also observed when cholinergic or phorbol ester-mediated down-regulated cells are treated with activators of PKC. By contrast, down-regulated cAMP pathways are not affected by lithium. The muscarinic cholinergic system appears to be comparatively sensitive to the augmenting effect, since lithium concentrations within the therapeutic range are effective, whereas higher concentrations are required to augment phorbol ester-mediated fos accumulation. The increased sensitivity of the cholinergic pathway to lithium, compared with PMA, is not strictly due to differences between receptor and postreceptor activation of PKC, since we have previously found that bradykinin and nerve growth factor, agonists that stimulate PKC and fos expression in PC12 cells, are only augmented by 10 mM lithium (Kalasapudi et al., in press, and unpublished observations). According to the cholinergic hypothesis, susceptibility to manic episodes is due to reduced muscarinic cholinergic neurotransmission (Janowsky et al., 1980; Dilsaver, 1986). Thus the ability of therapeutic concentrations of lithium to augment a down-regulated muscarinic receptor system is consistent with its antimanic action according to this hypothesis. The lithium augmentation of fos expression is not consistant with its putative inhibition of the PLC pathway, since one would expect that fos expression would be inhibited, especially in cells that are challenged with carbamylcholine in the presence of lithium, a condition that may deplete cells of its pool of PIP, (Worley et al., 1988). Also, lithium should not influence phorbol estermediated events, since the direct activation of PKC by these agents bypasses the IP cycle that is thought to be the target of lithium. It has been shown that treatment with lithium leads to an increase in inositol phosphate intermediates in PC 12 cells stimulated with agonists that induce PIP, turnover (Van Calker et al., 1987). It is conceivable that this could lead to a disturbance in inosito1 phosphate-mediated Ca2+ mobilization that would effect the function of a Ca2+-dependent enzyme, such as PKC, in lithium-treated cells exposed to agonists that stimulate PIP, turnover. However, this is unlikely, since we observe a lithium-augmenting effect when PKC is directly activated with phorbol esters. This class of compounds does not stimulate PIP, turnover. Indeed, phorbol esters have been found to inhibit PIP, hydrolysis in PC12 cells (Vincentini et al., 1985). The data are more consistent with an enhancing effect of lithium on PKC function. It has been found that lithium has an affinity for
Lithium and fos Protooncogene Expression
phospholipids, in particular phosphotidylserine (PS) (Casal et al., 1987; Hauser and Shipley, 1981). Furthermore, it has been suggested that lithium could influence the function of membrane-bound proteins by altering their interaction with membrane phospholipids (Riddell and Arumugam, 1988). An effect of lithium at the membrane level leading to a change in the interaction between PKC with DAG and PS, and a subsequent increase in C-kinase activity, could account for the receptor- and postreceptor-mediated augmentation we observe. Alternatively, lithium may reduce PKC down regulation that ordinarily occurs following its activation. We also demonstrated that lithium neither inhibits nor enhances fos expression induced by receptor and postreceptor stimulation of adenylate cyclase and CAMP. If lithium inhibits this system by blocking G,, as has been suggested by Avissar et al. (1988), then one would expect fos mRNA induced by PGE, to be reduced by lithium, whereas no effect on forskolin induction should be observed. It is conceivable, however, that lithium could have an inhibitory effect on GJadenylate cyclase pathways linked to other receptor systems that may be more sensitive to lithium. It is also conceivable that lithium could have an indirect inhibitory effect on the cAMP pathway by enhancing PKC-mediated heterologous down regulation. However, we have not yet been able to demonstrate such an effect in PC12 cells (unpublished observation). In contrast to our results with lithium, we do find that CZ, another effective antimanic agent, does reduce fos mRNA levels induced by forskolin, whereas no effect on the PLC pathway is observed. Although we have not determined whether the inhibitory effect on CAMPmediated fos expression also occurs at the receptor level, our data are consistent with an inhibitory effect of CZ on adenylate cyclase and the absence of an effect on the IP pathway, consistent with data reported by other investigators (Palmer, 1979; Elphick et al., 1988). Thus two of the most commonly used antimanic agents appear to have an effect on different signal transduction pathways in PC12 cells. One possible mechanism to account for the antimanic properties of lithium and CZ, based on our experimental observations, is depicted in the model shown in Figure 4. In this model, PKC and cAMP have opposite effects on a common effector target, such as an ion channel (Fig. 4A). A manic episode is initiated because of a stressor overstimulating the cAMP arm, or because of an underactive PLCiPKC pathway, leading to excessive, signals (Fig. 4B). Presumably, the vulnerunopposed ability to these events would occur as a result of the mutant alleles thought to play a role in the pathogenesis of manic depression (Lachman and Papolos, 1989). An augmenting effect on PKC function by lithium, or an
+
47
inhibition of the cAMP arm by CZ, or perhaps by lithium-enhanced PKC heterologous down regulation, would lead to an antimanic effect (Fig. 4C). It is interesting to note that this model is also consistent with the mechanism of action of another antimanic agent, clonidine, which activates the a,-adrenergic receptor linked to Gi, the G-regulatory protein that inhibits adenylate cyclase (Hardy et al., 1986). Although we used fos expression as a convenient target activated by both the PLC/PKC and cAMP pathways, we have not yet addressed its augmentation by lithium as a potentially important aspect of the therapeutic response. The impact of increased fos expression on long-term neuronal responses in the treatment of mood disorders remains to be determined.
ACKNOWLEDGMENTS We thank Dr. Herman van Praag for his support during this study. The Program of Behavioral Genetics is supported by a grant from the Ruane Family Fund. H.M.L. is supported by NIH grant CA 4324601 and the Weill-Caulier Career Scientist Award. G.S. was supported by an NIH Cancer training grant (5R23CA47779). M.D. is supported by a Medical Student Research Training Fellowship from the Howard Hughes Medical Institute.
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