Identification of a new functional target of haloperidol metabolite: implications for a receptor-independent role of 3-(4-fluorobenzoyl) propionic acid.

Haloperidol, a dopamine D2 receptor blocker, is a classical neuroleptic drug that elicits extrapyramidal symptoms. Its metabolites include 3-(4-fluorobenzoyl) propionic acid (FBPA) and 4-(4-chlorophenyl)-4-piperidinol (CPHP). Until now, the biological significance of these metabolites has remained largely unknown. Here, we report that the administration of FBPA to mice effected a suppression of locomotor activity and induced catalepsy in a manner similar to that observed with haloperidol, whereas CPHP had no significant effects. Neither of these two metabolites, however, exhibited any ability to bind to the dopamine D2 receptor. FBPA blocked dopamine-induced extracellular signal-regulated kinase 1/2 phosphorylation, and it specifically affected mitogen-activated protein kinase kinase (MEK)1/2 activity in hippocampal HN33 cells. Moreover, FBPA was capable of direct interaction with MEK1/2, and inhibited its activity in vitro. We demonstrated the generation of haloperidol metabolites within haloperidol-treated cells by mass spectrometric analyses. Collectively, our results confirm the biological activity of FBPA, and provide initial clues as to the receptor-independent role of haloperidol.

Haloperidol induces calcium ion influx via L-type calcium channels in hippocampal HN33 cells and renders the neurons more susceptible to oxidative stress.

Haloperidol is a classical neuroleptic drug that is still in clinical use and can lead to abnormal motor activity following repeated administration. However, there is little knowledge of how it triggers neuronal impairment. In this study, we report that it induced calcium ion influx via L-type calcium channels and that the elevation of calcium ions induced by haloperidol appeared to render hippocampal cells more susceptible to oxidative stress. Indeed, the level of cytotoxic reactive oxygen species (ROS) and the expression of pro-apoptotic Bax increased in response to oxidative stress in haloperidol-treated cells, and these effects were inhibited by verapamil, a specific L-type calcium channel blocker, but not by the T-type calcium channel blocker, mibefradil. These findings indicate that haloperidol induces calcium ion influx via L-type calcium channels and that this calcium influx influences neuronal fate.

Serotonin stimulates GnRH secretion through the c-Src-PLC gamma1 pathway in GT1-7 hypothalamic cells.

Serotonin is a neurotransmitter that alters the hypothalamic-pituitary-adrenal axis. To date, however, the molecular mechanisms underlying the role of serotonin in hormone secretion have remained largely unclear. In this study, we report that serotonin activates phospholipase C (PLC) gamma1 in an Src-dependent manner in hypothalamic GT1-7 cells, and that pretreatment with either 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazole [3, 4-d] pyrimidine, an Src-kinase inhibitor, or U73122, a PLC inhibitor, attenuates the serotonin-induced increase in calcium levels. Also, PLC gamma1 binds to c-Src through the Src-homology (SH) 223 domain upon serotonin treatment. Moreover, calcium increase is alleviated in the cells transientlyexpressing SH223 domain-deleted PLC gamma1 or lipase inactive mutant PLC gamma1, as compared with cells transfected with wild-type PLC gamma1. Furthermore, the inhibition of the activities of either PLC or Src results in a significant diminution of the serotonin-induced release of gonadotropin-releasing hormone (GnRH). In addition, the results of our small-interfering RNA experiment confirm that endogenous PLC gamma1 is a prerequisite for serotonin-mediated signaling pathways. Taken together, our findings demonstrate that serotonin stimulates the release of GnRH through the Src-PLC gamma1 pathway, via the modulation of intracellular calcium levels.

Neurotensin enhances nitric oxide generation via the JAK2-STAT1 pathway in murine macrophage Raw264.7 cells during costimulation with LPS and IFNgamma.

Neurotensin has been known to be implicated in immune function, but its molecular mechanisms remain largely unclear. In this study, we report that neurotensin increased the intracellular calcium levels of murine macrophage Raw264.7 cells, and that this calcium increase disappeared in the presence of either U73122, a PLC inhibitor, or SR48692, a neurotensin receptor antagonist. Also, the production of nitric oxide (NO) during costimulation with lipopolysaccharide (LPS) and interferon gamma (IFNgamma) was potentiated by exposure to neurotensin, whereas neurotensin itself had no ability to induce NO generation. The up-regulation of NO generation was correlated with the induction of inducible NO synthase (iNOS). In addition, the activities of janus activated kinase 2 (JAK2)-signal transducer and activated transcription 1 (STAT1) and the migration capacity of macrophage were increased as the result of costimulation of neurotensin with LPS and IFNgamma, and pretreatment of either U73122 or SR48692 attenuated these phenomenon. Moreover, the neurotensin-mediated enhancement of NO generation and migration were observed in the wild-type JAK2 transfected cells, but not in the dominant negative JAK2 transfected cells. Together, these results demonstrate that neurotensin can effect enhancement in LPS/IFNgamma-induced NO generation and migration capacity, via the JAK2-STAT1 pathway.

Secretin induces neurite outgrowth of PC12 through cAMP-mitogen-activated protein kinase pathway.

The gastrointestinal functions of secretin have been fairly well established. However, its function and mode of action within the nervous system remain largely unclear. To gain insight into this area, we have attempted to determine the effects of secretin on neuronal differentiation. Here, we report that secretin induces the generation of neurite outgrowth in pheochromocytoma PC12 cells. The expressions of Tau and beta-tubulin, neuronal differentiation markers, are increased upon secretin stimulation. In addition, secretin induces sustained mitogen-activated protein kinase (MAPK) activation and also stimulates the cAMP secretion. Moreover, the neurite outgrowth elicited by secretin is suppressed to a marked degree in the presence of either PD98059, a specific MAPK/ERK kinase (MEK) inhibitor, or H89, a specific protein kinase A (PKA) inhibitor. Taken together, these observations demonstrate that secretin induces neurite outgrowth of PC12 cells through cAMP- MAPK pathway, and provide a novel insight into the manner in which secretin participates in neuritogenesis.

C-terminal part of AgRP stimulates insulin secretion through calcium release in pancreatic beta Rin5mf cells.

Agouti-related protein (AgRP) is an orexigenic peptide which is composed of three parts; the amino (N)-terminus, the middle part, and the carboxyl (C)-terminus. AgRP has been implicated in various cell signaling, but the precise role of each parts are currently unclear. In this study, we have attempted to determine which part of AgRP was critical for insulin secretion. We have found that the C-terminus of AgRP specifically increases the intracellular calcium concentration in pancreatic beta Rin5mf cells in a PLC-dependent manner, whereas the middle part and C-terminus have little effects on calcium release. This calcium response can be observed in the freshly isolated primary beta cells also. Moreover, amperometric measurement reveals that the C-terminus of AgRP increases the rate of exocytosis in Rin5mf cells. We further show that this region of AgRP is responsible for insulin secretion in a PLC-dependent manner. Taken together, these results indicate that the C-terminus of AgRP can participate in the insulin secretion in pancreatic beta cells, through the modulation of calcium release.