Antipsychotics, chlorpromazine and haloperidol inhibit voltage-gated proton currents in BV2 microglial cells

Abstract

Microglial dysfunction and neuroinflammation are thought to contribute to the pathogenesis of schizophrenia. Some antipsychotic drugs have anti-inflammatory activity and can reduce the secretion of pro-inflammatory cytokines and reactive oxygen species from activated microglial cells. Voltage-gated proton channels on the microglial cells participate in the generation of reactive oxygen species and neuronal toxicity by supporting NADPH oxidase activity. In the present study, we examined the effects of two typical antipsychotics, chlorpromazine and haloperidol, on proton currents in microglial BV2 cells using the whole-cell patch clamp method. Chlorpromazine and haloperidol potently inhibited proton currents with IC₅₀ values of 2.2 μM and 8.4 μM, respectively. Chlorpromazine and haloperidol are weak bases that can increase the intracellular pH, whereby they reduce the proton gradient and affect channel gating. Although the drugs caused a marginal positive shift of the activation voltage, they did not change the reversal potential. This suggested that proton current inhibition was not due to an alteration of the intracellular pH. Chlorpromazine and haloperidol are strong blockers of dopamine receptors. While dopamine itself did not affect proton currents, it also did not alter proton current inhibition by the two antipsychotics, indicating dopamine receptors are not likely to mediate the proton current inhibition. Given that proton channels are important for the production of reactive oxygen species and possibly pro-inflammatory cytokines, the anti-inflammatory and antipsychotic activities of chlorpromazine and haloperidol may be partly derived from their ability to inhibit microglial proton currents.

Introduction

Schizophrenia is a debilitating mental disorder that affects 0.30–0.66% of the population (McGrath et al., 2008, Van Os and Kapur, 2009). Schizophrenia is characterized by positive symptoms (hallucinations and delusions), negative symptoms (socially withdrawn behavior), cognitive dysfunction, and affective dysregulation (Van Os and Kapur, 2009). The current antipsychotic drugs used to treat schizophrenic patients are all dopaminergic D₂ receptor antagonists to some degree, and effective for treating hallucinations and delusions (Coyle et al., 2010). Thus, schizophrenia is thought to be associated with dysregulated dopaminergic neurotransmission.

Recent studies also point to an association between neuroinflammation and free radicals and the pathogenesis of schizophrenia (Monji et al., 2013, Reddy and Yao, 1996). Schizophrenic patients have increased levels of lipid peroxidation and altered antioxidant enzyme activities. For example, elevated plasma lipid peroxides in schizophrenic patients were positively associated with the severity of negative symptoms but negatively with glutathione peroxidase in red blood cells (Mahadik et al., 1998). In addition, schizophrenic patients had elevated levels of blood superoxide dismutase, a key enzyme to detoxify superoxide radicals, compared to normal individuals, and the degree of elevation was correlated with positive symptoms (Zhang et al., 2003).

In rat, chronic administration of chlorpromazine, a typical antipsychotic, increased the activities of antioxidant enzymes and inhibited lipid peroxidation in the brain (Roy et al., 1984). In vitro, chlorpromazine has been shown to be a powerful scavenger of hydroxyl radicals, organic peroxyl radicals, and hypochlorous acid. It also inhibited hydroxyl radical formation and peroxidation of liposomal lipid and arachidonic acid (Jeding et al., 1995). Similarly, haloperidol, which belongs to a different chemical class of antipsychotic drugs from chlorpromazine, was also able to scavenge hypochlorous acid (Jeding et al., 1995).

Microglia are major sources of pro-inflammatory cytokines and reactive oxygen species such as superoxide and nitric oxide in the central nervous system (Block et al., 2007). Chlorpromazine and haloperidol have been shown to reduce the production of nitric oxide and pro-inflammatory cytokines from activated microglial cells (Kato et al., 2007, Labuzek et al., 2005).

Voltage-gated proton channels (Hv1) are expressed predominantly in immune cells such as microglia, where they mediate the rapid movement of protons across the cell membrane (DeCoursey, 2003, DeCoursey, 2008, Okochi et al., 2009, Ramsey et al., 2006, Sasaki et al., 2006, Wu et al., 2012). Proton channels are often closely associated with NADPH oxidase. NADPH oxidase transports electrons across the plasma membrane to reduce molecular oxygen into superoxide and to generate other downstream reactive oxygen species, leaving protons in the cytosol (DeCoursey, 2003, DeCoursey, 2008). In parallel, intracellular reactive oxygen species are generated, which stimulate the production of pro-inflammatory cytokines such as tumor necrosis factor-α (Kim et al., 2010). The NADPH oxidase inhibitor, diphenyliodonium, suppressed microglial over-activation, and inhibited the production of reactive oxygen species and tumor necrosis factor-α (Qian et al., 2007). To function optimally, NADPH oxidase requires an intracellular pH of 7.5 and a polarized membrane potential (DeCoursey et al., 2003, Morgan et al., 2005). Proton channels are suitable for both to remove intracellular protons and to maintain membrane potential, which are vital to keeping NADPH oxidase continuously active. In the present study, we hypothesized that the ability to suppress microglial activation and the anti-inflammatory properties of chlorpromazine and haloperidol would be related to the ability to inhibit proton currents.