Paul C. Brandt

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 clozapines 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 clozapines antipsychotic actions.

Anesthetic-induced alteration of Ca2+ homeostasis in neural cells: a temperature-sensitive process that is enhanced by blockade of plasma membrane Ca2+-ATPase isoforms.

Background Many inhalation anesthetics at clinically relevant concentrations inhibit plasma membrane Ca2+‐adenosine triphosphatase (PMCA) ion pumping in brain synaptic membranes and in cultured cells of neural origin. In this study, the authors investigated the effect of inhalation anesthetics on cytosolic calcium homeostasis in cortical neurons maintained at physiologic and room temperatures and on cortical neurons and pheochromocytoma cells with antisense blockade of specific PMCA isoforms. Methods Using Ca2+‐specific confocal microfluorimetry, the anesthetic effects on Ca2+ dynamics were examined in mouse embryonic cortical neurons in association with ligand‐stimulated Ca2+ influx. Studies were done at 21 [degree sign]C and 37 [degree sign]C. Mouse embryonic cortical neurons with oligodeoxyribonucleotide blockade of PMCA2 expression and transfected rat pheochromocytoma cells with blocked expression of PMCA1 were also examined. Results Baseline and poststimulation peak cytosolic calcium concentrations ([Ca2+]i) were increased, and Ca2+ clearance was delayed in cells exposed at 37 [degree sign]C, but not at 21 [degree sign]C, to concentrations <or= to 1 minimum alveolar concentration (MAC)‐equivalent of halothane, isoflurane, and sevoflurane. Neurons exposed to xenon solutions <or= to 0.4, 0.6, and 0.8 MAC showed dose‐related perturbations of cytosolic Ca2+. Calcium dynamics were altered in neural cells with blocked PMCA isoform production, but at much lower halothane concentrations: 0.5 MAC for cortical neurons and 0.1 MAC for pheochromocytoma cells. Conclusions By extruding Ca2+ through the plasma membrane, PMCA maintains resting neuronal [Ca2+]i at low levels and clears physiologic loads of Ca2+ after influx through calcium channels. Inhalation anesthetics perturb this process and thus may interfere with neurotransmitter release, altering interneuronal signaling.

Induction of nitric oxide synthase and over-production of nitric oxide by interleukin-1β 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. Sjögrens 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.

Splicing of the Muscle-Specific Plasma Membrane Ca2+-ATPase Isoform PMCA1c Is Associated with Cell Fusion in C2 Myocytes

Abstract: The regulation of intracellular calcium is essential for proper muscle function. Muscle cells have several mechanisms for dealing with the rapid and large changes in cytosolic calcium level that occur during contraction. Among these is the plasma membrane Ca2+‐ATPase (PMCA), which pumps calcium from the cytosol to the extracellular space. We have previously shown that in human fetal muscle the PMCA1 isoforms present are PMCA1a‐d, with PMCA1b and c predominating. Alternative splicing of mRNAs encoding proteins involved in muscle contraction is common in developing muscle. Therefore, we examined the expression of muscle‐specific PMCA mRNAs in pre‐ and postfusion mouse C2 myoblasts. The housekeeping form of the Ca2+‐ATPase, PMCA1b, was found at all times and under all conditions. However, the other predominating isoform found in muscle, PMCA1c, was expressed on myotube formation. Simple cell‐cell contact was not sufficient to induce PMCA1c expression, as cells plated at confluence but harvested before myotube formation did not express PMCA1c. The induction of this muscle‐specific Ca2+‐ATPase at myotube formation suggests that it may play an important role in muscle function.

Down regulation of interleukin-1β-induced nitric oxide production in lacrimal gland acinar cells by sex steroids

Purpose. Because the ocular surface is constantly exposed to allergens and irritants, it was reasoned that one cause of dry eye might be damage from inflammatory responses normally regulated by sex steroids. To test this hypothesis, we determined if sex steroids could down regulate nitric oxide (NO) production induced by interleukin-1β (IL-1β) in cultured rabbit lacrimal gland acinar cells. Methods. Cultured rabbit lacrimal gland acinar cells were exposed to IL-1β to stimulate NO production. Stimulated cells were treated with different sex steroids and expression of iNOS protein determined by Western blotting and NO production by a nitrate/nitrite colorimetric assay. Results. It was found that the androgens testosterone, dihydrotestosterone, dehydroepiandrosterone and dehydroepiandrosterone-sulfate and 17β-estradiol were able to inhibit interleukin-1β-induced NO production in rabbit lacrimal gland acinar cells at physiological concentrations, while progesterone was not able to inhibit NO production. Sex steroid inhibition of NO production was not due to down regulation of iNOS protein production nor was it due to down regulation of GTP cyclohydrolase I with consequent loss of tetrahydrobiopterin production. Conclusions. The results reported here show that androgens and estrogens can down regulate cytokine-mediated responses in cells that are part of the ocular surface protection system and thereby may have an important role in regulating inflammatory responses in the eye. Deficiencies in these steroids, as occurs in postmenopausal women, may lead to damage of the cells responsible for producing the fluids that protect the ocular surface and subsequently to dry eye disease.

Peroxisome Proliferator-Activated Receptor Agonists Inhibit Interleukin-1β-Mediated Nitric Oxide Production in Cultured Lacrimal Gland Acinar Cells

Development of dry eye disease often occurs in individuals with autoimmune disorders such as Sjögrens 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 Sjögrens 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.

Elevated Glucocorticoid Receptor Transactivation and Down-regulation of α1 Integrin Are Associated with Loss of Plasma Membrane Ca2+-ATPase Isoform 1

We have previously shown that inhibition of expression of the plasma membrane Ca2+-ATPase isoform 1 in PC6 cells leads to loss of nerve growth factor-mediated neurite extension (Brandt, P. C., Sisken, J. E., Neve, R. L., and Vanaman, T. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13843–13848). Cells lacking plasma membrane Ca2+-ATPase 1 did not attach to collagen-coated plates as tightly as controls, suggesting that a defect in adhesion might be underlying the inability to extend neurites. We report here that cell lines lacking plasma membrane Ca2+-ATPase 1 do not produce α1 integrin, which is required for both collagen adherence and neurite extension. Because α1 integrin gene transcription can be down-regulated by glucocorticoids, the response of cells to glucocorticoids was investigated. Cortisol-dependent transactivation from the mouse mammary tumor virus promoter in cells lacking plasma membrane Ca2+-ATPase 1 was stimulated 145–216-fold over untreated cells compared with 15–26-fold for controls. This increase was not due to increased binding affinity of the receptor for cortisol, an increased number of cortisol-binding sites, or increased translocation of the receptor to the nucleus. Expression of additional glucocorticoid receptor-dependent genes required for neurite extension must also be altered in cells missing the plasma membrane Ca2+-ATPase 1 because constitutive expression of α1 integrin did not restore their nerve growth factor-mediated neurite extension capability. The impact of plasma membrane Ca2+-ATPase isoform 1 on other signaling systems and the resultant profound yet subtle effects on PC6 cells strongly suggests that it plays an important role in modulating signal transduction pathways downstream of Ca2+-mediated signals.

Long-lasting distortion of GABA signaling in MS/DB neurons after binge-like ethanol exposure during initial synaptogenesis

Using a well-established model of binge-like ethanol treatment of rat pups on postnatal days (PD) 4-9, we found that maturation of GABAA receptor (GABAAR) miniature postsynaptic currents (mPSCs) was substantially blunted for medial septum/diagonal band (MS/DB) neurons in brain slices on PD 11-16. Ethanol reduced mPSC amplitude, frequency, and decay kinetics, while attenuating or exaggerating allosteric actions of zolpidem and allopregnanolone, respectively. The impact of ethanol in vivo was long lasting as most changes in MS/DB GABAAR mPSCs were still observed as late as PD 60-85. Maturing MS/DB neurons in naïve brain slices PD 4-16 showed increasing mPSC frequency, decay kinetics, and zolpidem sensitivity that were nearly identical to our earlier findings in cultured septal neurons (DuBois et al., 2004, 2006). These rapidly developing mPSC parameters continued to mature through the first month of life then stabilized throughout the remainder of the lifespan. Finally, equivalent ethanol-induced alterations in GABAAR mPSC signaling were present in MS/DB neurons from both male and female animals. Previously, we showed ethanol treatment of cultured embryonic day 20 septal neurons distorts the maturation of GABAAR mPSCs predicting that early stages of GABAergic transmission in MS/DB neurons are vulnerable to intoxication injury (DuBois et al., 2004, 2006). Since the overall character, timing, and magnitude of GABAergic mPSC developmental- and ethanol-induced changes in the in vivo model so closely mirror chronologically equivalent adaptations in cultured septal neurons, this suggests that such parallel models of ethanol impairment of GABAergic synaptic development in vivo and in vitro should be useful for translational studies exploring the efficacy and mechanism of action of potential therapeutic interventions from the cellular to whole animal level.

Identification of a Synaptic Membrane-Localized Isoform of the Calcium-Pumping ATPase

Calcium-pumping membrane ATPases play the essential function of terminating Ca2+-dependent responses. Two broad classes of Ca2+-pumping ATPases are now known to exist in animal cells, those of internal membranes (100 kDa) and the plasma membrane enzymes (120–140 kDa). These two families of Ca2+-pumping ATPases differ substantially in molecular, enzymatic and regulatory properties commensurate with their differiential localization and physiological roles. Recent studies in a number of laboratories have provided evidence that multiple isoforms of each type of Ca2+ -ATPase are produced in different cell types as products of either differential splicing events or different genes. These sets of isoforms are almost certain to have subtle differences in functional properties related to the requirements of a particular biological setting.