DETECTION OF 5-HYDROXYTRYPTAMINE(2) RECEPTORS BY RADIOLIGAND BINDING,NORTHERN BLOT ANALYSIS, AND CA2+ RESPONSES IN RAT PRIMARY ASTROCYTE CULTURES

Radioligand binding, Northern blot analysis, and changes in [Ca2+]i we re used to study serotonin [5-hydroxytryptamine (5HT)] receptor subtyp es in primary cultures of astrocytes from neonatal rat cerebral cortex . Radioligand binding studies revealed the presence of 5HT2, but not t he 5HT1 or 5HT3 receptor subtypes. Radioligand binding was also used t o show the presence of serotonin uptake sites, which had previously be en shown to be present by [H-3]-5HT uptake, and also alpha1-adrenergic receptors as has previously been reported by binding studies. Norther n blot analysis of cortical astrocyte mRNA demonstrated the presence o f transcripts for 5HT2 receptors, but failed to identify mRNA for 5HT1 a or 5HT1c receptors. Thus, results from Northern blot analysis correl ated with the radioligand binding data which showed only 5HT2 receptor s. Equilibrium saturation studies, using 125[I]-LSD to label 5HT2 rece ptors, yielded a K(D) of 9 nM and a B(max) of 177 fmol/mg protein. Rad ioligand binding studies or primary astrocyte cultures prepared from o ther brain regions also showed the presence of alpha1-adrenergic, 5HT2 receptor, and 5HT-uptake sites, but no detectable 5HT1a receptors, wh ich were the only 5HT1 receptors studied. Studies demonstrating 5HT-in duced, spiperone- and ketanserin-sensitive increases in free [Ca2+]i a s measured by FURA-2, showed that the 5HT2 receptors were functional i n 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 eviden ce of 5HT1a or 5HT1c subtypes.