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Actions of lithium on brain adenylyl cyclase: Role of magnesium
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S-33 John Cade Symposium on Lithium - Part 2 total number of spikes. to increase c-fos expression in DA target areas. Thus 5-HT2-receptor antagonism should facilitate information processing through frontal DA innervated circuits. In contrast. a l-adrenoceptor antagonism by prazosin was found to reduce phasi, but not tonic activity in mesolimbic DA neurons and to effectively antagonize the MK-80l evoked DA release in nucleus accumbens, without changing basal release. Although blockade of DA receptors appears critical for antipsychotic activity. antagonism of 5-HT2A-receptors and a l-adrenoceptors may help to normalize central DA system perturbations in psychosis at a presynaptic level.
IS-33 I John Cade Symposium on Lithium - Part 2 18-33-1 I Actions of.Lithium on Brain Adenylyl Cyclase: Role of MagneSium
A. M erk, Department of Neurobiology. Pharmacological Research. H. Lundbeck AlS. Copenhagen-Valby. Denmar k
Lithium affects cAMP formation in the brain [1]. There is now evidence of 9 different types of adenylyl cyclase. having distinct regulatory characteristics [2]. Type 1, III and VIII adenylyl cyclases are sensitive to calcium-calmodulin (Ca2+ -CaM) and are thus stimulated by physiological concentrations of Ca2+. Magnesium (Mg2+) regulates adenylyl cyclases throughtwo independent cation sites; one Mg2+ site modulates the activity of G proteins. and a second Mg2+ site on the catalytic proteins regulates the catalytic activities. The in vitro and chronic effects of lithium on brain adenylyl cyclase have been studied. Chronic lithium was studied by treating rats for 4 weeks. yielding serum-lithium of 0.7-0. 8 rnmol/l. Lithium inhibited Ca2+ -CaM-stimulated adenylyl cyclase activity, indicating altered activity of the catalytic protein independent of the protein kinase C arm. The inhibitory effect of lithium in vitro on cAMP formation stimulated by noradrenaline, Gpp(NH)p. forskolin, or Ca2+ -CaM was found to be counterac ted by increasing concentrations of Mg2+ , indicating that lithium in situ inhibits adenylyl cyclases by displacing Mg2+ from its regulatory ion site. However, following chronic treatment. Mg2+ did not antagonize the inhibitory effect of lithium. suggesting that the acute and chronic effects of lithium on adenylyl cyclases are exerted by different mechanisms. [1] Moerk A. Geisler A,1. Neurochern. 65(1995) 134-139. [2] Cooper DMF, Mons N,Karpen lW, Nature 374 (1 995) 421 -424.
18-33-21 Marcks: A Key to the long-TermTherapeutic Action of Mood Stabilizers in the Brain? R.H. Lenox, D.G. Watson, R. K. McNamara. Departments of Psychiatry, Pharmacology and Neuroscience, University of Florida College of Medicine, Gainesville, Florida, U.S.A.
Ongoing studies in our laboratory and others have provided evidence for a role of PKC in mediating the effects of chronic lithium in the brain (Manji and Lenox, 1994). We have reported that chronic lithium at clinically relevant concentrations significantly reduces the expression of a major PKC substrate, MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate), in rat hippocampus (Lenox et al, 1992). This protein has been implicated in synaptic signaling and cytoskeletal remodeling. and recent studies in our laboratory have mapped the constitutive MARCKS gene expression in the hippocampal formation. Exposure of immortalized hippocampal cells (HN33) in culture to phorbol esters induces a down-regulation of MARCKS protein via a PKC-dependent mechanism (Watson et al, 1994). Similar studies in HN33 cell exposed to lithium results in a time-dependent reduction in MARCKS expression (Lenox et al, 1993). which we have now shown to be dependent upon the relative concentrations of lithium and inositol, as well as the level of muscarinic receptor activation. We have compared the effects of chronic exposure to lithium, valproate and carbamazepine, on the expression of MARCKS protein in HN33 cells. Sodium valproate exposure produced a significant dose-dependent but inositol-independent reduction in MARCKS protein
in both the soluble and membrane fractions following long-term exposure (3- 7 days). In contrast. no changes in MARCKS protein levels were detected following long-term exposure to carbamazepine or following acute exposure to any of these three agents. Regulation of MARCKS protein may represent a pharmacological property shared by selective mood stabilizers with therapeutic efficacy in the prophylactic treatment of manic-depressive illness and may provide an important avenue for elucidating the pathobiology of this illness • Supported by NIMH Grant # MH50105.
I8-33-31by Regulation of Transmembrane Signalling Systems Mood Stabilizing Agents: Therapeutic Implications Husseini K. Manji . Molecular Pathophysiology Program . Wayne State University School of Medicine
Lithium. valproic acid (VPA), and Carbamazepine (CBZ) are widely used in the treatment for bipolar affective disorder, but despite their efficacy. the molecular mechanisms underlying their therapeutic actions have not fully been elucidated. In recent years it has become increasingly clear that rather than any single neurotransmitter system being responsible for depression or mania, multiple interacting and overlapping systems are involved in regulating mood, and that most effective drugs may exert their therapeutic efficacy by affecting the functional balance between interacting systems. In this context. signal transduction pathways are in a pivotal position in the CNS and represent attractive targets to explain the efficacy of these mood-stabilizing agents in treating multiple aspects oft he illness. We have found that in both platelets from healthy volunteers, and rat prefrontal cortex. chronic lithium affects both basal and post-receptor adenylyl cyclase activity, and significantly increases pertussis-catalyzed 32 [ p]ADP-ribosylation. Preliminary studies from our laboratory suggest that chronic lithium administration impairs the ability of GTPyS to dissociate Gi, suggesting a post-translational process stabilizing the inactive beterotrimeric (apy ) form of the protein. Chronic lithium also produces a significant decrease in f3HjPDBU binding in several hippocampal structures (most notably subiculum and CAI region), effects which are due to isozyme-specific decrease in levels of membrane-associated PKC a and {3 ; in view of lithium's significant effects on PKC outlined above, we have also investigated the effecrs of VPA on various aspects of this family of enzymes. Chronic VPA (0.5 mM) produces a significant decrease in PKC activity in both the membrane and cytosolic fractions of C6 glioma cells; these effects are accompanied by isozyme-selective decreases in the levelsofPK C Cl' and p. Consistent with its effects on PKC isozyrnes, we findthat VPAexerts major effects on a major PKC substrate (MARCKS), and on the activity of AP-l (a transcriptional factor known to be regulated by PKC). We have also investigated CBZ's effects 0 0 signal transduction pathways and have found a concentration-dependent attenuation of both basal and post-receptor (Forskolin, cholera toxin or pertussis toxin) stimulated cyclic AMP accumulation. Using in vivo microdialysis, we have found that similar to what was observed in vitro. CBZ concentration-dependently reduced FSK stimulated cAMP levels in rat prefrontal cortex. We have used a FSK affinity column to purify ACs from rat brain. and findthat CBZ also exerts a significant inhibitory effect on the purified ACs. Overall, the results suggest that signal transduction pathways are targets for the actions of mood stabilizing agents; given their key roles in the amplification and integration of signals in the central nervous system, these findings have clear implications not only for research into the etiology/pathophysiology of manic-depressive illness. but also for thc development of innovative treatment strategies.
18-33-4 1The Inositol Depletion Hypothesis: Promise and Problems R.H. Belmaker. Division ofPsychiatry. Ben Gurian University Beersheva, Israel
Lithium (Li) inhibits inositol monophosphatase and reduces brain inositol. This effect was proposed as a mechanism of Li action. since reduced inositol could compromise resynthesis of phosphatidylinositol (PI) and thus moderate the second messenger response to agonists that work by breakdown of PI to IP3. However, many authors found that
S-33 John Cade Symposium on Lithium - Part 2 total number of spikes. to increase c-fos expression in DA target areas. Thus 5-HT2-receptor antagonism should facilitate information processing through frontal DA innervated circuits. In contrast. a l-adrenoceptor antagonism by prazosin was found to reduce phasi, but not tonic activity in mesolimbic DA neurons and to effectively antagonize the MK-80l evoked DA release in nucleus accumbens, without changing basal release. Although blockade of DA receptors appears critical for antipsychotic activity. antagonism of 5-HT2A-receptors and a l-adrenoceptors may help to normalize central DA system perturbations in psychosis at a presynaptic level.
IS-33 I John Cade Symposium on Lithium - Part 2 18-33-1 I Actions of.Lithium on Brain Adenylyl Cyclase: Role of MagneSium
A. M erk, Department of Neurobiology. Pharmacological Research. H. Lundbeck AlS. Copenhagen-Valby. Denmar k
Lithium affects cAMP formation in the brain [1]. There is now evidence of 9 different types of adenylyl cyclase. having distinct regulatory characteristics [2]. Type 1, III and VIII adenylyl cyclases are sensitive to calcium-calmodulin (Ca2+ -CaM) and are thus stimulated by physiological concentrations of Ca2+. Magnesium (Mg2+) regulates adenylyl cyclases throughtwo independent cation sites; one Mg2+ site modulates the activity of G proteins. and a second Mg2+ site on the catalytic proteins regulates the catalytic activities. The in vitro and chronic effects of lithium on brain adenylyl cyclase have been studied. Chronic lithium was studied by treating rats for 4 weeks. yielding serum-lithium of 0.7-0. 8 rnmol/l. Lithium inhibited Ca2+ -CaM-stimulated adenylyl cyclase activity, indicating altered activity of the catalytic protein independent of the protein kinase C arm. The inhibitory effect of lithium in vitro on cAMP formation stimulated by noradrenaline, Gpp(NH)p. forskolin, or Ca2+ -CaM was found to be counterac ted by increasing concentrations of Mg2+ , indicating that lithium in situ inhibits adenylyl cyclases by displacing Mg2+ from its regulatory ion site. However, following chronic treatment. Mg2+ did not antagonize the inhibitory effect of lithium. suggesting that the acute and chronic effects of lithium on adenylyl cyclases are exerted by different mechanisms. [1] Moerk A. Geisler A,1. Neurochern. 65(1995) 134-139. [2] Cooper DMF, Mons N,Karpen lW, Nature 374 (1 995) 421 -424.
18-33-21 Marcks: A Key to the long-TermTherapeutic Action of Mood Stabilizers in the Brain? R.H. Lenox, D.G. Watson, R. K. McNamara. Departments of Psychiatry, Pharmacology and Neuroscience, University of Florida College of Medicine, Gainesville, Florida, U.S.A.
Ongoing studies in our laboratory and others have provided evidence for a role of PKC in mediating the effects of chronic lithium in the brain (Manji and Lenox, 1994). We have reported that chronic lithium at clinically relevant concentrations significantly reduces the expression of a major PKC substrate, MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate), in rat hippocampus (Lenox et al, 1992). This protein has been implicated in synaptic signaling and cytoskeletal remodeling. and recent studies in our laboratory have mapped the constitutive MARCKS gene expression in the hippocampal formation. Exposure of immortalized hippocampal cells (HN33) in culture to phorbol esters induces a down-regulation of MARCKS protein via a PKC-dependent mechanism (Watson et al, 1994). Similar studies in HN33 cell exposed to lithium results in a time-dependent reduction in MARCKS expression (Lenox et al, 1993). which we have now shown to be dependent upon the relative concentrations of lithium and inositol, as well as the level of muscarinic receptor activation. We have compared the effects of chronic exposure to lithium, valproate and carbamazepine, on the expression of MARCKS protein in HN33 cells. Sodium valproate exposure produced a significant dose-dependent but inositol-independent reduction in MARCKS protein
in both the soluble and membrane fractions following long-term exposure (3- 7 days). In contrast. no changes in MARCKS protein levels were detected following long-term exposure to carbamazepine or following acute exposure to any of these three agents. Regulation of MARCKS protein may represent a pharmacological property shared by selective mood stabilizers with therapeutic efficacy in the prophylactic treatment of manic-depressive illness and may provide an important avenue for elucidating the pathobiology of this illness • Supported by NIMH Grant # MH50105.
I8-33-31by Regulation of Transmembrane Signalling Systems Mood Stabilizing Agents: Therapeutic Implications Husseini K. Manji . Molecular Pathophysiology Program . Wayne State University School of Medicine
Lithium. valproic acid (VPA), and Carbamazepine (CBZ) are widely used in the treatment for bipolar affective disorder, but despite their efficacy. the molecular mechanisms underlying their therapeutic actions have not fully been elucidated. In recent years it has become increasingly clear that rather than any single neurotransmitter system being responsible for depression or mania, multiple interacting and overlapping systems are involved in regulating mood, and that most effective drugs may exert their therapeutic efficacy by affecting the functional balance between interacting systems. In this context. signal transduction pathways are in a pivotal position in the CNS and represent attractive targets to explain the efficacy of these mood-stabilizing agents in treating multiple aspects oft he illness. We have found that in both platelets from healthy volunteers, and rat prefrontal cortex. chronic lithium affects both basal and post-receptor adenylyl cyclase activity, and significantly increases pertussis-catalyzed 32 [ p]ADP-ribosylation. Preliminary studies from our laboratory suggest that chronic lithium administration impairs the ability of GTPyS to dissociate Gi, suggesting a post-translational process stabilizing the inactive beterotrimeric (apy ) form of the protein. Chronic lithium also produces a significant decrease in f3HjPDBU binding in several hippocampal structures (most notably subiculum and CAI region), effects which are due to isozyme-specific decrease in levels of membrane-associated PKC a and {3 ; in view of lithium's significant effects on PKC outlined above, we have also investigated the effecrs of VPA on various aspects of this family of enzymes. Chronic VPA (0.5 mM) produces a significant decrease in PKC activity in both the membrane and cytosolic fractions of C6 glioma cells; these effects are accompanied by isozyme-selective decreases in the levelsofPK C Cl' and p. Consistent with its effects on PKC isozyrnes, we findthat VPAexerts major effects on a major PKC substrate (MARCKS), and on the activity of AP-l (a transcriptional factor known to be regulated by PKC). We have also investigated CBZ's effects 0 0 signal transduction pathways and have found a concentration-dependent attenuation of both basal and post-receptor (Forskolin, cholera toxin or pertussis toxin) stimulated cyclic AMP accumulation. Using in vivo microdialysis, we have found that similar to what was observed in vitro. CBZ concentration-dependently reduced FSK stimulated cAMP levels in rat prefrontal cortex. We have used a FSK affinity column to purify ACs from rat brain. and findthat CBZ also exerts a significant inhibitory effect on the purified ACs. Overall, the results suggest that signal transduction pathways are targets for the actions of mood stabilizing agents; given their key roles in the amplification and integration of signals in the central nervous system, these findings have clear implications not only for research into the etiology/pathophysiology of manic-depressive illness. but also for thc development of innovative treatment strategies.
18-33-4 1The Inositol Depletion Hypothesis: Promise and Problems R.H. Belmaker. Division ofPsychiatry. Ben Gurian University Beersheva, Israel
Lithium (Li) inhibits inositol monophosphatase and reduces brain inositol. This effect was proposed as a mechanism of Li action. since reduced inositol could compromise resynthesis of phosphatidylinositol (PI) and thus moderate the second messenger response to agonists that work by breakdown of PI to IP3. However, many authors found that