Inhibition of specific adenylyl cyclase isoforms by lithium and carbamazepine, but not valproate, may be related to their antidepressant effect.

OBJECTIVES: Lithium, valproate, and carbamazepine decrease stimulated brain cyclic-AMP (cAMP) levels. Adenylyl cyclase (AC), of which there are nine membrane-bound isoforms (AC1-AC9), catalyzes the formation of cAMP. We have recently demonstrated preferential inhibition of AC5 by lithium. We now sought to determine whether carbamazepine and valproate also preferentially inhibit specific AC isoforms or decrease cAMP levels via different mechanisms. METHODS: COS7 cells were transfected with one of AC1-AC9, with or without D1-dopamine receptors. Carbamazepine's and valproate's effect on forskolin- or D1 agonist-stimulated ACs was studied. The effect of Mg(2+) on lithium's inhibition was studied in membrane-enriched fraction from COS7 cells co-expressing AC5 and D1 receptors. AC5 knockout mice were tested for a behavioral phenotype similar to that of lithium treatment. RESULTS: Carbamazepine preferentially inhibited forskolin-stimulated AC5 and AC1 and all D1 agonist-stimulated ACs, with AC5 and AC7 being the most sensitive. When compared to 1 or 3 mM Mg(2+), 10 mM Mg(2+) reduced lithium-induced AC5 inhibition by 70%. In silico modeling suggests that among AC isoforms carbamazepine preferentially affects AC1 and AC5 by interacting with the catechol-estrogen site. Valproate did not affect any forskolin- or D1 receptor-stimulated AC. AC5 knockout mice responded similarly to antidepressant- or lithium-treated wild-types in the forced-swim test but not in the amphetamine-induced hyperactivity mania model. CONCLUSIONS: Lithium and carbamazepine preferentially inhibit AC5, albeit via different mechanisms. Lithium competes with Mg(2+), which is essential for AC activity; carbamazepine competes for AC's catechol-estrogen site. Antidepressant-like behavior of AC5 knockout mice in the forced-swim test supports the notion that AC5 inhibition is involved in the antidepressant effect of lithium and carbamazepine. The effect of lithium and carbamazepine to lower cAMP formation in AC5-rich dopaminergic brain regions suggests that D1-dopamine receptors in these regions are involved in the antidepressant effect of mood stabilizers.

Lithium preferentially inhibits adenylyl cyclase V and VII isoforms.

Lithium ions' inhibition of adenylyl cyclase (AC) has not been previously studied for the newly discovered AC isoforms. COS7 cells were transfected with each of the nine membrane-bound AC isoforms cDNAs with or without D1- or D2-dopamine receptor cDNA. AC activity was measured as [3H]cAMP accumulation in cells pre-incubated with [3H]adenine followed by incubation with phosphodiesterase inhibitors together with either the D1 agonist SKF-82958 alone, or forskolin, in the presence or absence of the D2 agonist quinpirole. At 1 mm or 2 mm lithium inhibited only AC-V activity when the enzyme was stimulated by forskolin, a direct activator of AC. Lithium inhibited AC-V (by 50%), AC-VII (by 40%) and AC-II (by 25%) when stimulated via the D1 receptors, but did not affect the Ca2+-activated isoforms when stimulated by the Ca2+ ionophore A23187. Quinpirole inhibits AC via the Gi protein. Lithium did not affect quinpirole-inhibited FSK-activated AC-V activity nor did it affect superactivated AC-V or AC-I following the removal of quinpirole. The data suggest interference of lithium with transduction pathways mediated via AC-V or AC-VII; only the active conformation of these AC isoforms is inhibited by lithium; the inhibitory effect of lithium is abolished when the enzyme is superactivated. The marked inhibition of AC-V and AC-VII by lithium suggests that these two isoforms may be involved in mediating the mood-stabilizing effect of lithium.

Possible involvement of post-dopamine D2 receptor signalling components in the pathophysiology of schizophrenia.

Par-4 has been suggested to mediate dopamine neurotransmission. Dopamine D2 receptor (DRD2) activation induces a signalling complex of AKT1, PP2A and beta-arrestin2 which dephosphorylates/inactivates AKT1 thereby activating GSK-3beta, transducing dopamine-dependent behaviour. DRD2 activation also results in down-regulation of PKA activity. Among other substrates PKA phosphorylates GSK-3beta. Prolonged DRD2 activation leads to its 'desensitization' which involves GRKs and beta-arrestins. beta-arrestin1 binds to phosphorylated receptors preventing further G-protein stimulation. This study examined whether Par-4, beta-arrestin1, AKT1 and GSK-3beta are involved in the pathophysiology of schizophrenia. Lymphocytes obtained from schizophrenia and bipolar patients and healthy controls recruited from the Beer-Sheva Mental Health Center were transformed by Epstein-Barr virus (EBV) into lymphocyte-derived cell lines (LDCL). Post-mortem brain samples were obtained from the Rebecca L. Cooper Brain Bank, Parkville, Australia. The study was approved by the IRB committees of Beer-Sheva, Israel and Parkville, Australia. Levels of the specific proteins were assayed by Western blotting. beta-arrestin1 protein levels were significantly ~2-fold increased in LDCL from schizophrenia patients while Par-4 protein levels were unaltered. A 63% significant decrease was found in frontal cortex phospho-Ser9-GSK-3beta protein levels in schizophrenia but not in those of AKT1, Par-4 or beta-arrestin1. Elevated beta-arrestin1 protein levels in LDCL and decreased phospho-Ser9-GSK-3beta protein levels in post-mortem frontal cortex of schizophrenia patients vs. control groups support the possible involvement of these proteins in the pathophysiology of schizophrenia. However, since we did not find differences in beta-arrestin1, AKT1 and Par-4 protein levels in post-mortem frontal cortex of schizophrenia patients and although GSK-3beta participates in other signalling cascades we can not rule out the possibility that the differences found reflect deviation in DRD2 signalling.