H1-antihistamines induce vacuolation in astrocytes through macroautophagy

Abstract

H1-antihistamines induce vacuolation in vascular smooth muscle cells, which may contribute to their cardiovascular toxicity. The CNS toxicity of H1-antihistamines may also be related to their non-receptor-mediated activity. The aim of this study was to investigate whether H1-antihistamines induce vacuolation in astrocytes and the mechanism involved. The H1-antihistamines induced large numbers of giant vacuoles in astrocytes. Such vacuoles were marked with both the lysosome marker Lysotracker Red and the alkalescent fluorescence dye monodansylcadaverine, which indicated that these vacuoles were lysosome-like acidic vesicles. Quantitative analysis of monodansylcadaverine fluorescence showed that the effect of H1-antihistamines on vacuolation in astrocytes was dose-dependent, and was alleviated by extracellular acidification, but aggravated by extracellular alkalization. The order of potency to induce vacuolation at high concentrations of H1-antihistamines (diphenhydramine > pyrilamine > astemizole > triprolidine) corresponded to their pKa ranking. Co-treatment with histamine and the histamine receptor-1 agonist trifluoromethyl toluidide did not inhibit the vacuolation. Bafilomycin A1, a vacuolar (V)-ATPase inhibitor, which inhibits intracellular vacuole or vesicle acidification, clearly reversed the vacuolation and intracellular accumulation of diphenhydramine. The macroautophagy inhibitor 3-methyladenine largely reversed the percentage of LC3-positive astrocytes induced by diphenhydramine, while only partly reversing the number of monodansylcadaverine-labeled vesicles. In Atg5^−/− mouse embryonic fibroblasts, which cannot form autophagosomes, the number of vacuoles induced by diphenhydramine was less than that in wild-type cells. These results indicated that H1-antihistamines induce V-ATPase-dependent acidic vacuole formation in astrocytes, and this is partly mediated by macroautophagy. The pKa and alkalescent characteristic of H1-antihistamines may be the major determinants of vacuolation, which may contribute to their CNS toxicity.

Introduction

The H1-antihistamines are among the most widely used medications in the world, most of which are available ‘over-the-counter’ for treating allergic reactions and motion sickness or alleviating cold symptoms. However, H1-antihistamines such as diphenhydramine, pyrilamine, and astemizole are known to have the CNS side-effects like convulsions, sedation, and hallucination. Diphenhydramine intoxication was reported to be the second leading cause of drug-induced seizures, which may be related to suicide or abuse (Thundiyil et al., 2007). However, the detailed mechanism is still unknown.

In the brain, astrocytes are the most numerous non-neuronal cell type and make up about 50% of human brain volume (Tower and Young, 1973). Astrocytes perform many functions, including biochemical support of endothelial cells that form the blood–brain barrier, provision of nutrients to nervous tissue, and maintenance of extracellular ion balance. Dysfunction of astrocytes may result in glutamate accumulation in the extracellular space, disturb GABA synthesis, and contribute to the generation of convulsions (Battaglioli and Martin, 1991, Binder and Steinhauser, 2006). Histamine elevates glutamine synthetase activity in cerebellar astrocytes, the key enzyme for glutamate metabolism and GABA synthesis in astrocytes (Rodriguez et al., 1989). And histamine H1 receptors are expressed in astrocytes (Hosli et al., 1984); however, whether the CNS toxicity of H1-antihistamines is related to astrocytes remains unclear.

H1-antihistamines induce vacuolation in vascular smooth muscle cells, and this may contribute to their cardiovascular toxicity (Morissette et al., 2008a). Recently, such vacuolar cytopathology has evoked much attention. It is known that the formation of numerous vacuoles visible under light microscopy is a response of various types of cells to basic compounds, such as procaine, procainamide, nicotine, atropine, and many others (Bawolak et al., 2010, Henics and Wheatley, 1999, Morissette et al., 2004, Morissette et al., 2008b). It is hypothesized that the sequestration of weak bases in the acidic vacuoles and then osmotic swelling is the reason for the vacuolation (Morissette et al., 2004). Moreover, macroautophagy is suggested to be involved in the vacuole formation, as the rim of a large fraction of vacuoles induced by H1-antihistamines is decorated by a specific marker of macroautophagy, LC3, in smooth muscle cells (Morissette et al., 2008a). Quinacrine, procaine, procainamide, and lidocaine, which also induced such vacuoles, increase the conversion from LC3-I to LC3-II, which indicates an elevated macroautophagy level (Bawolak et al., 2010). However, the detailed mechanism of vacuolation and the exact role of macroautophagy are still unclear. Consequently, we investigated the vacuolation in astrocytes induced by H1-antihistamines and the underlying mechanism, which may contribute to their CNS toxicity.