Depressive disorder is one of the most important diseases influencing human health in the 21st century. Antidepressants can improve some depressive symptoms and signs of depressive disorder in patients. It is thought that neurotransmitters (especially serotonin and/or norepinephrine and/or dopamine) have important roles in antidepression effects, but their pharmacological effects on the intracellular signal transduction pathway remain unclear. The aim of this thesis is to explore the effect of the antidepressants paroxetine (a selective serotonin reuptake inhibitor) and nortriptyline (a tricyclic antidepressant) on the intracellular Ca2+-related signal transduction and apoptosis of human prostate cancer PC3 cells. By using the fura-2 method, in PC3 cell, we found paroxetine (at concentrations between 10-150 £gM) and nortriptyline (at concentrations between 50-500 £gM) increased [Ca2+]i of PC3 cells in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+ indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor nearly abolished paroxetine and nortriptyline-induced Ca2+ release. Conversely, pretreatment with nortriptyline greatly reduced the inhibitor-induced [Ca2+]i rise, suggesting that antidepressants released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C with U73122 inhibited paroxetine-induced [Ca2+]i rise by 80%, but did not change nortriptyline-induced [Ca2+]i rise. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. Nortriptyline-induced Ca2+ influx was inhibited by activation of protein kinase C. Nortriptyline at a concentration of 10 £gM increased the viability of the PC3 cells. At 50 £gM, nortriptyline killed 45% of the cells, and induced significant apoptosis, as measured by propidium iodide staining. Collectively, in PC3 cells, paroxetine induced [Ca2+]i rise is caused by phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Nortriptyline increased [Ca2+]i via phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx from the protein kinase C-sensitive pathway. Nortriptyline also induced apoptosis at a higher level. The results of this thesis may be helpful for understanding the effects of antidepressants on the intracellular signaling of cultured cells, and might illustrate a new possible mechanism concerning antidepressants¡¦ therapeutic effects or clinical side effects.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Carbon skeleton of polysubstituted pyroglutamates with three contiguous asymmetric centers was built up in one base-induced coupling/cyclization reaction of £\-sulfonylacetamide with 2-bromo-2-propenoates and has been used as the key step in the formal synthesis of isoguvacine paroxetine, and psudoheliotridane.

Neuropathic pain (NPP) is a chronic syndrome suffered by patients with multiple sclerosis (MS), for which there is no cure. Underlying cellular mechanisms involved in its pathogenesis are multifaceted, resulting in significant challenges in its management. In addition to its complex pathophysiology, the clinical management of MS-induced NPP is further complicated by the lack of clinical therapeutics trials specific to this population. The primary aim of the work underlying this thesis was to contribute to the evidence-based management of individuals with MS-induced NPP through the completion of two clinical therapeutics trials in this population. A secondary aim was to describe pain variability in this patient population through the development and validation of a pain variability algorithm tool. Resulting from this work, we demonstrated that nabilone – a synthetic oral cannabinoid – represents an effective, well-tolerated and novel treatment for MS-induced NPP. Additionally, we show that the SSRI paroxetine was poorly tolerated in our patient population, with a correspondingly high attrition rate. As a result, we were unable to determine any treatment effect in this trial due to insufficient recruitment due to drop-out. Lastly, we were able to define and describe pain instability in this cohort, noting that approximately 30% of individuals with MS-induced NPP experiencing highly variable daily pain. The results of these projects provide novel information for this patient population. Patients currently living with the daily burden of MS-induced NPP would benefit from additional trials ensuing from this, and other, research in order to initiate a momentum for much-needed clinical research in this complicated patient cohort.

Neuropathic pain (NPP) is a chronic syndrome suffered by patients with multiple sclerosis (MS), for which there is no cure. Underlying cellular mechanisms involved in its pathogenesis are multifaceted, resulting in significant challenges in its management. In addition to its complex pathophysiology, the clinical management of MS-induced NPP is further complicated by the lack of clinical therapeutics trials specific to this population. The primary aim of the work underlying this thesis was to contribute to the evidence-based management of individuals with MS-induced NPP through the completion of two clinical therapeutics trials in this population. A secondary aim was to describe pain variability in this patient population through the development and validation of a pain variability algorithm tool. Resulting from this work, we demonstrated that nabilone – a synthetic oral cannabinoid – represents an effective, well-tolerated and novel treatment for MS-induced NPP. Additionally, we show that the SSRI paroxetine was poorly tolerated in our patient population, with a correspondingly high attrition rate. As a result, we were unable to determine any treatment effect in this trial due to insufficient recruitment due to drop-out. Lastly, we were able to define and describe pain instability in this cohort, noting that approximately 30% of individuals with MS-induced NPP experiencing highly variable daily pain. The results of these projects provide novel information for this patient population. Patients currently living with the daily burden of MS-induced NPP would benefit from additional trials ensuing from this, and other, research in order to initiate a momentum for much-needed clinical research in this complicated patient cohort.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 195-231). Also available in print.

Orientador: Carlos Roque Duarte Correia / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-06T00:38:56Z (GMT). No. of bitstreams: 1 Pastre_JulioCezar_M.pdf: 2844371 bytes, checksum: 6abfb0e87d60113816e3b01d9a02d6b9 (MD5) Previous issue date: 2005 / Mestrado / Quimica Organica / Mestre em Química

No / The effect of the selective serotonin reuptake inhibitor fluoxetine was examined on the 5-HT4 receptor-mediated relaxation in the rat isolated ileum. Fluoxetine unsurmountably antagonized the relaxation to exogenous 5-HT with abolition of the response at 10 ¿M. Fluoxetine (10 ¿M) also caused a gradual loss of the resting tension. These effects of fluoxetine were prevented by a prior addition of the 5-HT4 receptor selective antagonist GR113808 (100 nM), which itself caused a contraction of the tissues when administered alone. Fluoxetine (10 ¿M) also failed to prevent the relaxation due to exogenous 5-HT and the 5-HT4 receptor agonist 5-methoxytryptamine in tissues taken from the rats treated with para-chlorophenylalanine (300 mg kg¿1) for 3 and 6 days, which reduced the 5-HT level in the mucosa by 88 and 97.5% respectively. The contraction of the tissues with GR113808 indicates the presence of an endogenous 5-HT tone at the 5-HT4 receptor in the rat ileum. It is hypothesized that in the presence of fluoxetine, the concentration of endogenous 5-HT at the receptor was increased sufficiently to reduce or abolish the relaxation to 5-HT added exogenously. The inability of fluoxetine to prevent the relaxation to 5-HT in the presence of GR113808 or after the p-CPA treatment supports this hypothesis. The effect of the selective serotonin reuptake inhibitor fluoxetine was examined on the 5-HT4 receptor-mediated relaxation in the rat isolated ileum. Fluoxetine unsurmountably antagonized the relaxation to exogenous 5-HT with abolition of the response at 10 ¿M. Fluoxetine (10 ¿M) also caused a gradual loss of the resting tension. These effects of fluoxetine were prevented by a prior addition of the 5-HT4 receptor selective antagonist GR113808 (100 nM), which itself caused a contraction of the tissues when administered alone. Fluoxetine (10 ¿M) also failed to prevent the relaxation due to exogenous 5-HT and the 5-HT4 receptor agonist 5-methoxytryptamine in tissues taken from the rats treated with para-chlorophenylalanine (300 mg kg¿1) for 3 and 6 days, which reduced the 5-HT level in the mucosa by 88 and 97.5% respectively. The contraction of the tissues with GR113808 indicates the presence of an endogenous 5-HT tone at the 5-HT4 receptor in the rat ileum. It is hypothesized that in the presence of fluoxetine, the concentration of endogenous 5-HT at the receptor was increased sufficiently to reduce or abolish the relaxation to 5-HT added exogenously. The inability of fluoxetine to prevent the relaxation to 5-HT in the presence of GR113808 or after the p-CPA treatment supports this hypothesis. The effect of the selective serotonin reuptake inhibitor fluoxetine was examined on the 5-HT4 receptor-mediated relaxation in the rat isolated ileum. Fluoxetine unsurmountably antagonized the relaxation to exogenous 5-HT with abolition of the response at 10 ¿M. Fluoxetine (10 ¿M) also caused a gradual loss of the resting tension. These effects of fluoxetine were prevented by a prior addition of the 5-HT4 receptor selective antagonist GR113808 (100 nM), which itself caused a contraction of the tissues when administered alone. Fluoxetine (10 ¿M) also failed to prevent the relaxation due to exogenous 5-HT and the 5-HT4 receptor agonist 5-methoxytryptamine in tissues taken from the rats treated with para-chlorophenylalanine (300 mg kg¿1) for 3 and 6 days, which reduced the 5-HT level in the mucosa by 88 and 97.5% respectively. The contraction of the tissues with GR113808 indicates the presence of an endogenous 5-HT tone at the 5-HT4 receptor in the rat ileum. It is hypothesized that in the presence of fluoxetine, the concentration of endogenous 5-HT at the receptor was increased sufficiently to reduce or abolish the relaxation to 5-HT added exogenously. The inability of fluoxetine to prevent the relaxation to 5-HT in the presence of GR113808 or after the p-CPA treatment supports this hypothesis.

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)- benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells and OC2 human oral cancer cells was unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended MDCK and OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. m-3M3FBS at concentrations between 0.1-20 £gM increased [Ca2+]i in a concentration-dependent manner in MDCK cells, however in OC2 cells, m-3M3FBS at concentrations between 10-60 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signals were reduced partly by removing extracellular Ca2+ in the two cell types. m-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, m-3M3FBS pretreatment abolished the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin, cyclopiazonic acid or 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, cyclopiazonic acid or BHQ partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. Collectively, in MDCK and OC2 cells, m-3M3FBS induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels and other unidentified Ca2+ channels. Additionally, 5-100 £gM of m-3M3FBS killed cells in a concentration-dependent manner in OC2 cells. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 £gM) induced apoptosis in a Ca2+-independent manner. We were also interested in knowing whether BAPTA suppressed cell death during oxidative stress in MDCK cells. BAPTA loading altered tBHP (tert-butyl hydroperoxide) and H2O2-induced cell death in a concentration-dependent manner. This suggests that the cell death induced by tBHP and H2O2 appears to be Ca2+-dependent in MDCK cells. The tBHP and H2O2-induced cell death was not suppressed by 2 £gM U73122 (PLC inhibitor), 50 £gM zVAD-fmk (caspase inhibitor), 2 £gM cyclosporin A (a potent inhibitor of the MPTP), 20 £gM PD98059 (ERK inhibitor) or 2 £gM SP600125 (JNK inhibitor). This suggests that the tBHP and H2O2-induced MDCK cells death was not via the PLC, MPTP, caspase, ERK or JNK pathways. Propidium iodide staining, caspase-3 activity assay and cell morphology data suggest that tBHP and H2O2-induced cell death was necrosis, not via apoptosis, and the cell death appears to be caspase-independent and Ca2+-dependent. The effect of the antidepressant paroxetine on [Ca2+]i in OC2 human oral cancer cells is unclear. This study also explored whether paroxetine changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Paroxetine at concentrations between 100-1000 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 50% by removing extracellular Ca2+. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished paroxetine¡Vinduced [Ca2+]i rise. Inhibition of PLC with U73122 did not alter paroxetine-induced [Ca2+]i rise. Paroxetine at 10-50 £gM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca2+ was chelated with BAPTA. Propidium iodide staining suggests that apoptosis played a role in the death. Collectively, in OC2 cells, paroxetine induced [Ca2+]i rise by causing PLC-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Paroxetine also induced cell death in a Ca2+-independent manner.

Depression is a physiological disorder that may be treated by increasing the body¡¦s amount of one or a few of the following neurotransmitters: serotonin, dopamine and norepinephrine. Although there are seven distinct classes of antidepressants, selective serotonin reuptake inhibitors (SSRIs) and tetracyclic antidepressants are widely prescribed and generally regarded as the first-line drugs in the treatment of depression. However, many physiological roles of some SSRIs appear to be dissociated with the inhibition of serotonin reuptake. For instance, paroxetine, a member of SSRIs and maprotiline, a member of tetracyclic antidepressant, have been shown to induce apoptosis or to prevent other agents from inducing apoptosis in several cell lines. Thus the effects of these two drugs on the apoptosis are still controversial. The aim of this study is to investigate the molecular mechanisms of paroxetine and maprotiline in induction of cell death in human osteosarcoma and murine neuroblastoma cells. First, WST-1 reduction assays and propidium iodide-staining assays were used to determine cell viability and apoptosis in the presence of paroxetine and maprotiline. Then immunoblotting was used to measure the activity of apoptotic markers caspase-3 and mitogen-activated protein kinases (MAPKs) to survey the apoptotic pathways induced by these two antidepressants. The experimental results may be helpful to understand the pharmacological and toxicological effects of these two antidepressants in cells from important organs. Results showed that paroxetine caused apoptosis via the activation of caspase-3 in cultured human osteosarcoma cells (MG63). Although paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine was also found to induce [Ca2+]i increases but pretreatment with BAPTA/AM, a Ca2+ chelator, prevented paroxetine-induced [Ca2+]i increases, and thus did not protect cells from death. These results suggest that paroxetine caused Ca2+-independent apoptosis via the activation of p38 MAPK-associated caspase-3 in MG63 cells. Maprotiline was also found to induce apoptosis through increased caspase-3 activation in murine neuroblastoma Neuro-2a cells. Induction of JNK phosphorylation contributed to the activation of caspase-3 resulting in maprotiline-induced Neuro-2a cell apoptosis. Thus, it appears that maprotiline induced apoptosis via JNK/caspase-3-dependent signaling pathways. Blockage of activation of ERK was found to increase the activation of caspase-3 leading to an enhancement of maprotiline-induced apoptosis. These data suggest that ERK was a survival signal to oppose maprotiline-caused apoptotic effect in Neuro-2a cells. Thus the activation of caspase-3 by maprotiline appears to depend on the activation of JNK and the inactivation of ERK. [Ca2+]i measurement in the presence of maprotiline showed that the antidepressant induced [Ca2+]i increases. Interestingly, pretreatment with BAPTA/AM could suppress maprotiline-induced ERK phosphorylation, enhance caspase-3 activation and increase maprotiline-induced apoptosis. In conclusion, this study demonstrates that maprotiline induced apoptosis in murine neuroblastoma cells through activation of JNK-associated caspase-3 pathways. Maprotiline also evoked an anti-apoptotic response that was both Ca2+- and ERK-dependent. This thesis contains some published data in the journal of Toxicology and Applied Pharmacology and some data were submitted in the journal of Toxicology Letters.