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

Abstract

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 (pEC₅₀) 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 (pEC₅₀=7.81±0.09)>5-methoxytryptamine (5-MeOT) (pEC₅₀=6.86±0.36)>5-HT (pEC₅₀=6.68±0.08). The following compounds, at concentrations up to 10 μM, 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 meth- iothepin (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 μM, 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 pEC₅₀ (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.

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