Identification of 5-hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes

1 5-Hydroxytryptamine (5-HT) elicited a dose-dependent stimulation of intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in cultured astrocytes derived from neonatal rat (Sprague Dawley) thalamic/hypothalamic area with a potency (pEC(50)) of 6.68 +/- 0.08 (mean +/- s.e.mean). 2 In order to characterize the 5-HT receptor responsible for the cyclic AMP accumulation the effects of a variety of compounds were investigated on basal cyclic AMP levels (agonists) and 5-carboxamidotryptamine (5-CT) stimulated cyclic AMP levels (antagonists). The rank order of potency for the agonists investigated was 5-CT (pEC(50) = 7.81+/-0.09) > 5-methoxytryptamine (5-MeOT) (pEC(50) = 6.86 +/- 0.36) > 5-HT (pEC(50) = 6.68 +/- 0.08). The following compounds, at concentrations up to 10 mu M, did not affect basal cyclic AMP levels 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), cisapride, sumatriptan, DOI and RU 24969. The rank order of potency of antagonists was methiothepin (pK(i) = 7.98 +/- 0.25) > mesulergine (pK(i) = 7.58 +/- 0.18) > ritanserin (pK(i) = 7.20 +/- 0.24) > clozapine (pK(i) = 7.03 +/- 0.19) > mianserin (pK(i) = 6.41 +/- 0.19). The following compounds, at concentrations up to 10 mu M, were inactive: ketanserin, WAY100635, GR127935. This pharmacological profile is consistent with that of 5-HT7 receptor subtype-mediated effects. 3 The cultured astrocytes exhibited regional heterogeneity in the magnitude of cyclic AMP accumulation (E(max)). Cells cultured from the thalamic/hypothalamic area had significantly higher E(max) values (588 +/- 75% and 572 +/- 63% of basal levels for 5-CT and 5-HT, respectively) compared to brainstem (274 +/- 51% and 318 +/- 46%, respectively) and colliculus astrocytes (244 +/- 15% and 301 +/- 24%, respectively). No significant differences in pEC(50) (for either 5-HT or 5-CT) values were observed. 4 Reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for the 5-HT7 receptor confirmed expression of messenger RNA for this receptor subtype by the cultured astrocytes derived from all regions investigated. Primers specific for the 5-HT6 receptor also amplified a cDNA fragment from the same samples. 5 From these findings, we conclude that astrocytes cultured from a number of brain regions express functional 5-HT receptors positively coupled to adenylyl cyclase and that the level of receptor expression or the efficiency of receptor coupling is regionally-dependent. The pharmacological profile of the receptor on thalamic/hypothalamic astrocytes suggests that the 5-HT7 receptor is the dominant receptor that is functionally expressed even though astrocyte cultures have the capacity to express both 5-HT6 and 5-HT7 receptor messenger RNA.