Clozapine and the mitogen-activated protein kinase signal transduction pathway: implications for antipsychotic actions.

BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathways respond to dopaminergic and serotonergic agents and mediate short- and long-term effects of intracellular signaling in neurons. Here we show that the antipsychotic agent, clozapine, selectively activates the MEK/ERK MAPK pathway, and inhibition of this pathway reverses clozapine's actions in the conditioned avoidance response (CAR) paradigm, a rodent behavioral assay of antipsychotic activity. METHODS: Phosphorylation patterns of MAPK pathway enzymes were determined by quantitative immunoblot analysis and immunohistochemistry of rat prefrontal cortex. Kinase inhibitors were used to assess the role of MAPK signaling pathways in mediating clozapine-induced suppression of CAR. RESULTS: Clozapine administration selectively increased phosphorylation of MEK1/2 but had no effect on p38 or JNK phosphorylation. Pretreatment with the 5-HT2A agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride blocked the clozapine-induced increase in MEK1/2 phosphorylation. Immunohistochemistry revealed that clozapine treatment elevated the number of cells in the prefrontal cortex positive for phosphoERK, the downstream substrate of MEK1/2. Prior administration of MEK1/2 inhibitors U0126 or Sl327, or ERK inhibitor 5-iodotubercidin, reversed suppression of CAR induced by clozapine, whereas administration of vehicle, JNK or p38 inhibitors (L-JNK-1 and SB203580, respectively) had no effect. Inhibition of kinases upstream to MEK1/2 (PI-3K, PKC, and CaMKII) by administration of LY294002, bisindolylmaleimide, or KN-62, respectively, also reversed clozapine-induced suppression of CAR. CONCLUSIONS: These data support the hypothesis that the MEK/ERK signal transduction cascade participates in clozapine's antipsychotic actions.

Studies of calmodulin-dependent regulation.

Methods are presented for purifying bovine testes calmodulin and the calmodulin-regulated plasma-membrane calcium ATPase from human erythrocytes by calcium dependent affinity chromatography. The assay of CaM Kinase II using a synthetic peptide substrate is also described.

Induction of nitric oxide synthase and over-production of nitric oxide by interleukin-1beta in cultured lacrimal gland acinar cells.

PURPOSE: Inflammation of the lacrimal gland is one of the major causative factors in aqueous tear-deficient dry eye syndrome. Pro-inflammatory cytokine production is upregulated in lacrimal gland autoimmune disease (i.e. Sjögren's syndrome) and is associated with cell death. The expression of inducible nitric oxide synthase (iNOS/NOS-2) is known to be induced in the presence of pro-inflammatory cytokines in several secretory epithelial cell types. We hypothesize that pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta), cause a marked increase in nitric oxide (NO) production via induction of iNOS in lacrimal gland epithelial cells and that this may be a significant pathophysiological pathway of dry eye syndrome. METHODS: Cultured immortalized rabbit lacrimal gland acinar cells were incubated with IL-1beta, iNOS inhibitor, or IL-1 receptor antagonist (IL-1ra). Colorimetric detection of NO(2)(-) and NO(3)(-) in the media, measured by the Griess reaction, was used as an index of NO production. Expression of iNOS was determined by SDS-PAGE and Western blot. RESULTS: IL-1beta stimulated a concentration-dependent and time-dependent increase in NO production. IL-1beta-induced NO production was significantly antagonized by co-incubation with IL-1ra or the iNOS-specific inhibitor, 1400W. Expression of iNOS protein was greatest at 4hr after addition of IL-1beta, and was nearly undetectable at 12hr. IL-1ra greatly reduced IL-1beta-induced iNOS expression. CONCLUSIONS: Lacrimal gland acinar cells are able to produce iNOS in response to the pro-inflammatory cytokine IL-1beta. The amount of iNOS expressed and the subsequent levels of NO that are produced by lacrimal cells are far lower than those seen in macrophages, but are consistent with those reported for other cell types in the literature. This pathway of iNOS induction and overproduction of NO may be a factor in lacrimal gland cell death in dry eye syndrome. Inhibitors of iNOS or IL-1 receptor may be beneficial for controlling lacrimal gland inflammation.

Peroxisome proliferator-activated receptor agonists inhibit interleukin-1beta-mediated nitric oxide production in cultured lacrimal gland acinar cells.

Development of dry eye disease often occurs in individuals with autoimmune disorders such as Sjögren's syndrome. The cause of dry eye in these patients is thought to be due, at least in part, to lymphocytic infiltration of the lacrimal glands, with subsequent loss of secretion of the aqueous component of tear film. How this lymphocytic infiltration leads to loss of secretion is not fully understood. We have previously shown that the proinflammatory cytokine, interleukin-1beta (IL-1beta), can stimulate the production of nitric oxide (NO) in cultured lacrimal gland acinar cells. It is possible that IL-1beta, produced by the infiltrating macrophages, stimulates production of inducible nitric oxide synthase (iNOS), and subsequently excessive production of NO. Peroxynitrate and other radical byproducts associated with excessive synthesis of NO may be detrimental to normal function of the lacrimal gland. Here we show that the peroxisome proliferator-activated receptor (PPAR)alpha and gamma agonists can inhibit NO production in cultured lacrimal gland acinar cells. Further, this is accomplished without loss of iNOS expression or tetrahydrobiopterin. These data suggest that the use of ointments or eye drops containing these PPAR agonists may provide an effective therapeutic intervention for the prevention of dry eye in Sjögren's syndrome patients.

Nitric oxide and cyclic GMP-mediated protein secretion from cultured lacrimal gland acinar cells.

PURPOSE: Nitric oxide (NO) donors and NO synthase (NOS) substrates were tested for their use to stimulate protein secretion from cultured lacrimal gland acinar cells, through activation of guanylate cyclase. METHOD: Rabbit lacrimal gland epithelial cells (RLG cells) were incubated with NO donors and/or NOS substrates and the protein released into culture medium was determined with bicinchoninic acid assay. Guanylate cyclase activation by NO precursors was determined by measurement of c-GMP produced. RESULTS: Both NO donors and NOS substrates were able to stimulate protein release from RLG cells. Among 6 compounds studied, sodium nitroprusside, isosorbide dinitrate and N(a)-benzoyl L-arginine ethyl ester (BAEE) were most potent to release protein over 100% of the basal release. The guanylate cyclase activity was stimulated by these NO precursors and was inhibited by guanylate cyclase inhibitor, [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). CONCLUSION: NO donors and NOS substrates were able to stimulate protein release from RLG cells via activation of guanylate cyclase and c-GMP release, which was blocked by guanylate cyclase inhibitor, ODQ. It indicates that NO donors and NOS substrates could be used for the treatment of dry eye syndrome if the same holds true in dry eye animal models.