Detection of 5‐hydroxytryptamine2 receptors by radioligand binding, northern blot analysis, and Ca2+ responses in rat primary astrocyte cultures

Abstract

Radioligand binding, Northern blot analysis, and changes in [Ca²⁺]i were used to study serotonin [5‐hydroxytryptamine (5HT)] receptor subtypes in primary cultures of astrocytes from neonatal rat cerebral cortex. Radioligand binding studies revealed the presence of 5HT₂, but not the 5HT₁ or 5HT₃ receptor subtypes. Radioligand binding was also used to show the presence of serotonin uptake sites, which had previously been shown to be present by [³H]‐5HT uptake, and also α₁‐adrenergic receptors as has previously been reported by binding studies. Northern blot analysis of cortical astrocyte mRNA demonstrated the presence of transcripts for 5HT₂ receptors, but failed to identify mRNA for 5HT_1α or 5HT_1c receptors. Thus, results from Northern blot analysis correlated with the radioligand binding data which showed only 5HT₂ receptors. Equilibrium saturation studies, using ¹²⁵[I]‐LSD to label 5HT₂ receptors, yielded a K_D of 9 nM and a B_max of 177 fmol/mg protein. Radioligand binding studies or primary astrocyte cultures prepared from other brain regions also showed the presence of α₁‐ adrenergic, 5HT₂ receptor, and 5HT‐uptake sites, but no detectable 5HT_1a receptors, which were the only 5HT₁ receptors studied. Studies demonstrating 5HT‐induced, spiperone‐ and ketanserin‐sensitive increases in free [Ca²⁺]i as measured by FURA‐2, showed that the 5HT₂ receptors were functional in these cells. These data provide clear evidence for the existence of both 5HT₂ receptors and 5HT‐uptake sites in the same primary astrocyte cultures from neonatal rat cerebral cortex, with no detectable evidence of 5HT_1a or 5HT_1c subtypes.