5-Hydroxytryptamine_2B receptors stimulate Ca²⁺ increases in cultured astrocytes from three different brain regions

Abstract

The expression of 5-hydroxytryptamine-2B (5-HT_2B) receptor mRNA has recently been shown in cultured astrocytes. Here the expression of functional 5-HT_2B receptors has been studied in cultured astrocytes from rat cerebral cortex, hippocampus, and brain stem. Fluo-3- and fura-2-based microspectrofluorometry was used for measuring changes in intracellular free calcium concentrations ([Ca²⁺]_i). The 5-HT_2B agonist α-methyl 5-HT (40 nM) produced rapid transient increases in [Ca²⁺]_i in astrocytes from all three brain regions studied, and these responses were blocked by the selective 5-HT_2B antagonist rauwolscine (1 μM). The specificity of the responses to α-methyl 5-HT was further demonstrated by the failure of 4-(4-fluorobenzoyl)-1-(4-phenylbutyl)-piperidine oxalate (1 μM), a specific 5-HT_2A/5-HT_2C antagonist, to block these responses. The 5-HT_2B-induced increases in [Ca²⁺]_i persisted in Ca²⁺-free buffer, indicating that the increase in [Ca²⁺]_i results from mobilization of intracellular Ca²⁺ stores. The expression of 5-HT_2B receptors on astroglial cells was further verified immunohistochemically and by Western blot analysis. These results provide evidence of the existence of 5-HT_2B receptors on astrocytes in primary culture.

Introduction

Serotonin [5-hydroxytryptamine (5-HT)] is an important neurotransmitter in the mammalian central nervous system (CNS), and disturbances in the 5-HT system have been demonstrated in several mental disorders, such as depression, anxiety, schizophrenia, and migraine. The 5-HT receptor family consists at present of seven major classes based on structural and pharmacological properties, the 5-HT_1–7 receptors, which are further divided into several subtypes (Hoyer and Martin, 1997). Many of the receptors have been cloned and, with the exception of the 5-HT₃ receptor, found to belong to the superfamily of G protein-coupled receptors with the predicted seven transmembrane-spanning structure (Saudou and Hen, 1994). The 5-HT_2B receptor (previously named 5-HT_2F) was originally cloned from the rat stomach fundus, where it is most abundant and causes smooth muscle contraction (Cohen and Fludzinski, 1987). 5-HT_2B-receptor mRNA has been detected at low levels in the human brain (Kursar et al., 1994), and the 5-HT_2B receptor has been shown in neurons from human cerebral cortex (Bonhaus et al., 1995). In rodent studies, stimulation of 5-HT_2B receptors has been seen to induce anxiolysis, hyperphagia, and reduced grooming (Duxon et al., 1997a, Kennett et al., 1997, Kennett et al., 1996). However, some studies have failed to detect 5-HT_2B mRNA in the rat CNS (Foguet et al., 1992, Kursar et al., 1992, Pompeiano et al., 1994), whereas others have found the receptor mRNA in the rat brain (Flanigan et al., 1995), in the spinal cord of rat (Helton et al., 1994), and the receptor protein in the cerebellum, septum, hypothalamus, and amygdala from rat (Duxon et al., 1997b). In cultured astrocytes the serotonin receptors were originally detected by Hertz and colleagues (Hertz et al., 1979), and to date six out of 14 currently known 5-HT receptor subtypes have been found in rat and mice. These are the 5-HT_1A (Azmitia et al., 1996, Whitaker-Azmitia et al., 1993), the 5-HT_2A (Deecher et al., 1993, Hagberg et al., 1998, Nilsson et al., 1991), the 5-HT_5A (Carson et al., 1996), and the 5-HT_6,7 receptors (Hirst et al., 1997). Recently, mRNA for the 5-HT_2B receptor was shown in cultured rat astrocytes (Hirst et al., 1998). However, functional 5-HT_2B receptors have hitherto not been reported on astrocytes.

The 5-HT₂ receptors are thought to be linked to the release of intracellular Ca²⁺ induced by inositol 1,3,4-trisphosphate (IP₃) (Kursar et al., 1992, Wainscott et al., 1993), and we have previously shown that 5-HT_2A receptors on cultured astrocytes activate the phosphoinositol hydrolysis/Ca²⁺ signal transduction system (Nilsson et al., 1991) and open voltage-independent Ca²⁺ channels (Hagberg et al., 1998). The purpose of the present study was to investigate whether cultured astrocytes from different brain regions also express functional 5-HT_2B receptors coupled to the intracellular Ca²⁺ homeostasis.

We here report 5-HT_2B immunoreactivity on astrocytes in primary cultures from rat cerebral cortex, hippocampus, and brain stem. Microspectrofluorometric measurements of increases in [Ca²⁺]_i after application of a selective 5-HT_2B ligand indicate the expression of functional 5-HT_2B receptors in these brain regions.