- Top of page
5-Hydroxytryptamine (5-HT) exerts a wide variety of behavioural and physiological effects through actions on multiple receptor subtypes. These receptors have been classified by operational, transductional and structural criteria into four distinct receptor classes (5-HT1, 5-HT2, 5-HT3 and 5-HT4), comprising ten receptor subtypes. Four additional re-combinant receptors (5-HT5a, 5-HT5b, 5-HT6 and 5-HT7) provide strong evidence for the existence of three additional receptor classes (Hoyer et al., 1994; Hoyer & Martin, 1996). Three of the receptor subtypes, namely: 5-HT4, 5-HT6 and 5-HT7 receptors are coupled to the stimulation of adenylyl cyclase. Despite sharing a common signal transduction mechanism these three receptors have unique and highly divergent amino acid sequences. The pharmacological profiles of these receptors are unique but consistent across species (Boess & Martin, 1994; Eglen et al., 1994).
The 5-HT7 receptor has been cloned from several species including the rat (Lovenberg et al., 1993; Meyerhof et al., 1993; Ruat et al., 1993; Shen et al., 1993), mouse (Plassat et al., 1993) and man (Bard et al., 1993). Functional assays measuring adenosine 3′: 5′-cyclic monophosphate (cyclic AMP) accumulation, have revealed the presence of receptors that may correspond to the 5-HT7 receptor subtype in guinea-pig brain (Shenker et al., 1985; Tsou et al., 1994) and rat brain (Markstein et al., 1986; Fayolle et al., 1988). Several pharmacological studies have suggested that functional 5-HT7 receptors are expressed by both intact peripheral tissues such as guinea-pig ileum (Carter et al., 1995), rabbit femoral vein (Martin & Wilson, 1994) and Cynomolgus monkey jugular vein (Leung et al., 1996) and by cultured cells derived from human vascular smooth muscle (Shoeffter et al., 1996).
Primary astrocyte cultures have been shown to express neurotransmitter receptors for amines, purines, amino acids and peptides (Kimelberg, 1995). There is evidence that stimulation of many of these receptors results in activation of second messenger systems affecting cyclic AMP, cyclic GMP, inositol phosphates and diacylglycerol levels (Kimbelberg, 1995). There is a controversey about the subtypes of 5-HT receptor expressed by astrocytes; this is largely due to the early studies on cultured astrocytes (Hertz et al., 1979; Tardy et al., 1982; Whitaker-Azmitia & Azmitia, 1986), preceding the development of selective 5-HT receptor ligands and the cloning of the multiple 5-HT receptor subtypes.
The aim of the present study was to investigate whether primary astrocyte cultures derived from different brain areas express 5-HT receptors which are positively coupled to ade-nylyl cyclase and to characterize them in terms of their pharmacology and molecular biology.
- Top of page
In the present study the expression of functional 5-HT receptors positively coupled to adenylyl cyclase in cultured astrocytes was investigated. Three 5-HT receptor subtypes have been shown to be positively linked to adenylyl cyclase; 5-HT4, 5-HT6 and 5-HT7 receptors. There is, however, some evidence for other receptor subtypes coupling to Gs and stimulating adenylyl cyclase. For example, cells transfected with 5-HT1D receptors expressed in mammalian cells have been shown to increase intracellular cyclic AMP levels in response to 5-HT (Maenhaut et al., 1991; Watson et al., 1994). Hence, it is possible that 5-HT receptor subtypes, other than 5-HT4, 5-HT6 or 5-HT7 receptors could elicit the increases in intracellular cyclic AMP levels observed. For these reasons, and because there are no selective agonists or antagonists currently available which distinguish between 5-HT6 and 5-HT7 receptors, an extensive characterization of the pharmacology of the receptors on the astrocytes was undertaken.
The classical, potent 5-HT1A receptor agonist 8-OH-DPAT, at concentrations up to 10 μM failed to stimulate cyclic AMP accumulation in cultured astrocytes. In binding studies on cloned rat, mouse, guinea-pig and human 5-HT7 receptors, 8-OH-DPAT has an affinity (p Ki of 6.3–7.5 (Shen et al., 1993; Ruat et al., 1993; Lovenberg et al., 1993; Plassat et al., 1993; Bard et al., 1993; Tsou et al., 1994). 8-OH-DPAT has a lower potency at stimulating cyclic AMP accumulation in cells transfected with cloned rat or mouse 5-HT7 receptors (Lovenberg et al., 1993; Plassat et al., 1993) or in guinea-pig hippocampal membranes (Tsou et al., 1994) (pEC50 values of 5.3-6). Interestingly, recent studies have shown that 8-OH-DPAT has either a low potency (pEC50 < 6) at putative 5-HT7 receptors expressed in Cynomolgus monkey jugular vein (Leung et al., 1996) or is completely inactive at stimulating cyclic AMP accumulation in human vascular smooth muscle cells which endogenously express 5-HT7 receptors (Schoeffter et al., 1996). In the present study the selective 5-HT1A receptor antagonist WAY 100635 (Fletcher et al., 1996) did not affect 5-CT stimulated cyclic AMP levels. Taken together, these results indicate that the 5-HT1A receptor is not involved in the stimulation of adenylyl cyclase in the cultured astrocytes.
Sumatriptan, a 5-HT1B and 5-HT1D receptor agonist and RU 24969, a 5-HT1A and 5-HT1B receptor agonist were inactive in the present study. GR127935, a 5-HT1B and 5-HT1D receptor antagonist (Skingle et al., 1996), had no effect on 5-CT stimulated cyclic AMP levels, excluding the involvement of these receptor subtypes. 5-HT1E and 5-HT1F receptors are not likely to be responsible for the increase in intracellular cyclic AMP observed, since there is no evidence that they are linked to adenylyl cyclase stimulation. Furthermore, 5-CT has low affinity for 5-HT1E and 5-HT1F receptors (p Ki 5.5–6.0) (Boess & Martin, 1994), whereas 5-CT was the most potent agonist in the present study.
Neither 5-HT2A nor 5-HT2C receptors are involved in increasing the cyclic AMP levels in the cultured astrocytes, despite evidence for these cells expressing mRNA for both receptor subtypes and functional 5-HT2A receptors (Deecher et al., 1993; Hirst et al., 1994). This was confirmed with the selective 5-HT2 receptor agonist (DOI) which was inactive and the selective 5-HT2 receptor antagonist (ketanserin) which did not inhibit 5-CT stimulated cyclic AMP accumulation.
The effects observed are unlikely to be due to stimulation of 5-HT4 receptors as cisapride (10 μM), a 5-HT4 receptor agonist, was inactive. Also the affinity of 5-CT for 5-HT4 receptors is approximately 200 fold lower than the values obtained in the present study (Boess & Martin, 1994).
This implies that the receptors most likely to cause the cyclic AMP accumulation are 5-HT6 and/or 5-HT7. With the current lack of selective antagonists it is not possible to clearly discriminate between the 5-HT6 and 5-HT7 receptors based on the order of antagonist affinity. The antagonist profile observed in this study is characteristic of the 5-HT6 and 5-HT7 receptor subtypes, both of which exhibit high affinities towards me-thiothepin, clozapine, mianserin and ritanserin (Monsma et al., 1993; Shen et al., 1993; Plassat et al., 1993; Lovenberg et al., 1993). However, the potency of mesulergine (p Ki 7.58) indicates 5-HT7 receptors. Cloned rat 5-HT6 receptors (Monsma et al., 1993) and humans 5-HT6 receptors (Kohen et al., 1996) exhibit a lower affinity for mesulergine (p Ki values of 5.76 and 5.42, respectively), as opposed to all data on 5-HT7 receptors (p Ki 7.6–8.2) (Bard et al., 1993; Lovenberg et al., 1993; Plassat et al., 1993; Shen et al., 1993). In addition, these two receptors can be discriminated by the relative order of potency of 5-CT and 5-HT. The higher potency of 5-CT compared to 5-HT (pEC50 values of 7.81 and 6.68, respectively) indicates that this effect is likely to be mediated by 5-HT7 receptors. These values are comparable to those obtained for cyclic AMP accumulation in HeLa cells transfected with the rat 5-HT7 receptor cDNA (7.89 and 6.81 for 5-CT and 5-HT, respectively) (Lovenberg et al., 1993). Thus the rank order of agonist potency, 5-CT > 5-MeOT> 5-HT, obtained in the present study indicates expression of a functional 5-HT7 receptor.
It is of interest to note that despite the consistent cyclic AMP responses observed, there was no detectable [3H]-5-CT binding to the thalamic/hypothalamic astrocyte cultures (data not shown). This implies that the level of 5-HT7 receptor expression by the cultured astrocytes is below that detectable by this type of assay i.e. below approximately 20 fmol mg−1 protein. This observation is anomalous but not un-precendented; Giles et al. (1994) have shown functional 5-HT1B receptor expression in transfected CHO cells which mediated inhibition of forskolin induced cyclic AMP levels but they did not observe any specific binding of [3H]-5-HT or [125I]-cyano-pindolol.
The RT-PCR studies (Figure 3) demonstrate the presence of 5-HT7 receptor messenger RNA confirming that the thalamic/ hypothalamic astrocytes have the capacity to express this receptor subtype. 5-HT7 receptor messenger RNA was detected in RNA extracted from astrocytes cultured from all the regions investigated, but since the RT-PCR data are not quantitative no comments can be made on regional differences in mRNA expression levels. 5-HT6 receptor specific primers also amplified a cDNA frgament corresponding to this receptor subtype. However, as discussed above, the pharmacological profile of this receptor was not clearly observed in the thalamic/hypothalamic astrocytes. With the lack of subtype selective compounds, the expression of functional 5-HT6 receptors cannot be ruled out. It is possible that this receptor is expressed at a higher density on astrocytes cultured from other brain regions. However, a full pharmacological profile of the astro-cytic 5-HT receptor was only determined for astrocytes derived from the thalamic/hypothalamic area. For example, in the astrocytes derived from the cerebellum the pEC50 value for 5-CT was 6.1 compared to 7.7 in the thalamic/hypothalamic astrocytes. This lower figure could be indicative of a different receptor profile in astrocytes cultured from other brain regions.
Astrocytes cultured from the brain area incorporating the thalamus and hypothalamus showed the greatest magnitude of response to 5-HT and 5-CT, this was significantly greater than the response observed in astrocytes derived from the brain stem and colliculus (Table 2). This result is consistent with the 5-HT7 receptor mRNA and protein distribution in the adult rat brain, where the highest levels are detected in the thalamus (Gustafson et al., 1996). Thus, a regional correlation could be proposed between the in vivo expression of the receptor and the responses of astrocytes cultured from different regions of the neonatal rat brain.
Such regional variation has been previously documented in astrocyte responses to neurotransmitters and neuropeptides (Wilkin et al., 1990). Astrocytes could be analogous to neurones in terms of regionally defined phenotype heterogeneity. Moreover, the regional heterogeneity of astrocytes may reflect the different functional requirements exacted by the different neuronal populations with which they are associated. Thus, local populations of astrocytes may be biochemically specialised to interact with particular neurones and respond selectively to extracellular stimulation. Our results add to the list of neurotransmitter receptors expressed by cultured astrocytes (Kimelberg, 1995).
The data presented here raise the question of whether 5-HT7 receptor expression by the astrocytes in vitro reflects the ability of the cells to express this receptor in vivo. Interestingly, the earliest accounts of 5-HT stimulating an accumulation of cyclic AMP were from Fillion and colleagues (1980) with a glial membrane fraction derived from horse striatum. The structural complexity of the mammalian brain has often precluded definitive studies of the expression of neurotransmitter receptors by astrocytes in vivo. In spite of this, receptors for several neurotransmitters have been shown including α1-, α2-, β1-and β2-adrenoceptors, GABAA receptors, purinoceptors, histamine receptors and tachykinin receptors (Kettenman & Ransom, 1995). The issue of astroglial 5-HT receptor expression in vivo requires further study and is currently under investigation in this laboratory.
At the present time physiological roles for astrocytic 5-HT7 receptors remain speculative although these cells have been suggested to play a role in the development of the 5-hydroxytryptaminergic system within the central nervous system (Whitaker-Azmitia, 1991). At the cellular level, there is accumulating evidence that glia are critical for the establishment, organization and maintenance of neuronal systems (Kettenman & Ransom, 1995). These glial functions may be exerted on the 5-hydroxytryptaminergic system in part through the action of S-100β, a calcium binding protein, which is synthesized by astrocytes in situ and has been shown to have neurotrophic activity on 5-hydroxytryptaminergic neurones (Donato, 1991). Transcription of the S-100 gene is regulated through a conserved cyclic AMP response element (Montminy et al., 1990), thus 5-HT receptor-mediated increases in intracellular cyclic AMP levels could affect the expression of this protein.
In summary, the present study has shown that cultured astrocytes derived from the thalamic/hypothalamic area express functional 5-HT receptors positively coupled to adenylyl cyclase. The pharmacological profile of this receptor suggested it to be of the 5-HT7 subtype. Messenger RNA corresponding to this receptor was detected by RT-PCR. However, 5-HT6 receptor mRNA was also detected and expression of this receptor by the astrocytes cannot be ruled out in the absence of selective compounds. Regional heterogeneity in the magnitude of the cyclic AMP accumulation was observed with the greatest response in thalamic/hypothalamic astrocytes. To our knowledge, these data provide the first evidence for the presence of 5-HT receptors positively coupled to adenylyl cyclase on cultured astrocytes.