Activation of Astroglial Connexin Is Involved in Concentration-Dependent Double-Edged Sword Clinical Action of Clozapine

Abstract

Clozapine (CLZ) is a gold-standard antipsychotic against treatment-refractory schizophrenia, but is one of the most toxic antipsychotic agents. Pharmacological mechanisms of the double-edged sword clinical action of CLZ remain to be clarified. To explore the mechanisms of CLZ, the present study determined the astroglial transmission associated with connexin43 (Cx43), which is the most principal expression in astrocytes and myocardial cells, and expression of Cx43 in primary cultured astrocytes. Both acute and subchronic administrations of CLZ concentration-dependently increased Cx43-associated astroglial release of l-glutamate and d-serine, whereas therapeutic-relevant concentration of CLZ acutely did not affect but subchronically increased astroglial release. In contrast, after the subchronic administration of therapeutic-relevant concentration of valproate (VPA), acute administration of therapeutic-relevant concentration of CLZ drastically increased Cx43-associated astroglial releases. VPA increased Cx43 expression in cytosol fraction without affecting plasma membrane fraction, whereas CLZ increased Cx43 expression in both fractions. Acute administration of therapeutic-relevant concentration of CLZ drastically increased Cx43 expression in the plasma membrane fraction of astrocytes subchronically treated with VPA. The present findings suggest that CLZ-induced the activation of Cx43-associated channel activity and transported Cx43 to plasma membrane, probably contribute to the double-edged sword clinical action of CLZ, such as improvement of cognitive dysfunction and CLZ-induced myocarditis.

Upregulated Connexin 43 Induced by Loss-of-Functional S284L-Mutant α4 Subunit of Nicotinic ACh Receptor Contributes to Pathomechanisms of Autosomal Dominant Sleep-Related Hypermotor Epilepsy

“…All experiments in this study were designed with equally sized animal groups (N = 6) without carrying out a formal power analysis, in keeping with previous studies [21][22][23]33,[50][51][52]. All values are expressed as mean ± standard deviation (SD) and P < 0.05 (two-tailed) was considered statistically significant for all tests.…”

“…Amino-3-(3-hydroxy-5-methyl -isoxazol-4-yl)propanoic acid (AMPA) and nicotine ditartrate [35] were obtained from Wako Chemicals (Osaka, Japan). The selective Cx43 inhibitor, GAP19 [33,36] was obtained from Funakoshi (Tokyo, Japan). Zonisamide sodium salt (ZNS) was provided by Dainippon-Sumitomo Pharma (Osaka, Japan).…”

“…Total plasma membrane proteins of rat frontal cortex and primary cultured astrocytes were extracted using Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA) [33]. Simple Western analyses were performed using Wes (ProteinSimple, Santa Clara, CA, USA) according to the ProteinSimple user manual [21,33].…”

“…Total plasma membrane proteins of rat frontal cortex and primary cultured astrocytes were extracted using Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA) [33]. Simple Western analyses were performed using Wes (ProteinSimple, Santa Clara, CA, USA) according to the ProteinSimple user manual [21,33]. Lysate of primary cultured astrocytes were mixed with a master mix (ProteinSimple, San Jose, CA, USA) to a final concentration of 1 × sample buffer, 1 × fluorescent molecular weight marker and 40 mM dithiothreitol, then heated at 95 • C for 5 min.…”

“…In particular, inhibitors of connexin channels can prevent the onset of epileptic seizures [26,31,32]. Recently, we demonstrated that an atypical antipsychotic, clozapine, was able to increase hemichannel activity as well as Cx43 expression in a concentration dependent manner in the plasma membrane of astrocytes [33,34]. The stimulatory effects of clozapine on Cx43 expression however plays a double-edged sword where it has demonstrated improvement in cognitive dysfunction in antipsychotic-refractory schizophrenia but has also resulted in clozapine-induced toxic myocarditis and convulsion via enhanced astroglial L-glutamate release [33,34].…”