Agonist-induced desensitization of adenylyl cyclase activity mediated by 5-hydroxytryptamine7 receptors in rat frontocortical astrocytes

doi:10.1016/S0006-8993(97)01185-2

Brain Research

Volume 784, Issues 1–2, 16 February 1998, Pages 57-62

https://doi.org/10.1016/S0006-8993(97)01185-2 Get rights and content

Abstract

Our previous study has demonstrated that astrocytes derived from the rat frontal cortex contain 5-hydroxytryptamine (5-HT)₇ receptors positively coupled to adenylyl cyclase. In this study, we observed a desensitization of 5-HT₇ receptors induced by a treatment with agonists (0.1, 1, and 10 μM, 0.5 to 3.5 h). Maximum responses, but not the EC₅₀ values, in the concentration–response curve of 5-HT-induced cyclic AMP formation were decreased after pretreatment with 5-HT. Pretreatment with 5-carboxamidotryptamine (5-CT) elicited a potent desensitization of 5-HT-induced cyclic AMP formation. Neither 2-methyl-5-HT nor α-methyl-5-HT caused the desensitization. When the astrocytes were treated with isoproterenol, N-ethylcarboxamidoadenosine, and dibutyryl cyclic AMP (all of which increase intracellular cyclic AMP levels), 5-HT-induced cyclic AMP responses were not affected. Conversely, adenylyl cyclase activity mediated by either isoproterenol or N-ethylcarboxamidoadenosine was attenuated by pretreatment with each of these agonists, but not 5-HT. In addition, our study showed that the administration of 5-HT, 5-CT, and 8-hydroxy-2-(di-n-propylamino)tetralin to the astrocytes stimulated cyclic AMP formation both in the presence and absence of forskolin, whereas in neuron-rich cultures of the frontal cortex, these agonists did not change basal cyclic AMP levels and decreased forskolin-stimulated cyclic AMP formation. Neurons may predominantly contain 5-HT_1A receptors that are negatively coupled to adenylyl cyclase. These results suggest that 5-HT₇ receptors are highly expressed in astrocytes but not in neuronal cells, and that pretreatment with their agonists results in a homologous desensitization of the receptors.

Introduction

5-Hydroxytryptamine (5-HT) receptor subtypes have been classified by operational, transductional, and structural criteria into seven major groups 6, 10. The most recent addition of the classification is the 5-HT₇ receptor, which has been cloned in the rat 9, 13, 14, mouse [12], guinea-pig [18], and in humans [2]. These receptors contain seven predicted transmembrane domains and are positively coupled to adenylyl cyclase activation 9, 13, 14. Messenger RNA for 5-HT₇ receptors has been detected in high abundance in the hypothalamus, thalamus, cortex, hippocampus, and other limbic areas 5, 9, 13, 14, 18. The functional significance of 5-HT₇ receptors in the central nervous system, however, is not fully understood.

In a previous study we used reverse transcription and polymerase chain reaction (RT–PCR) to demonstrate that cultured astrocytes derived from the rat frontal cortex express 5-HT₇ receptors and that the addition of serotonergic agonists to the astrocytes elicits the activation of adenylyl cyclase [17]. The rank order of efficacy of the agonists on cyclic AMP formation was: 5-carboxamidotryptamine (5-CT)>5-HT>5-methoxytryptamine (5-MeOT). This is in agreement with other mammalian cell culture experiments 13, 14, 18. In addition, cyclic AMP formation induced by 5-HT was inhibited by methiothepin, a potent inhibitor of 5-HT₇ receptors, but not the antagonists of 5-HT₁, 5-HT₂, 5-HT₃, and 5-HT₄ receptors [17]. Conversely, we have found that the administration of 5-HT to neuron-rich cultures of the rat frontal cortex did not undergo any changes in cyclic AMP levels (unpublished data). It is possible that 5-HT₇ receptors are selectively and/or abundantly expressed in astrocytes, rather than in other brain cells, and may be involved in astroglial function. We are the first to observe and compare the effects of serotonergic agonists on adenylyl cyclase activation in astrocytes and neuron-rich cultures derived from the rat frontal cortex. The administration of 5-HT increased cyclic AMP formation in astrocytes and decreased cyclic AMP formation in neuron-rich cultures.

Several investigators have characterized the homologous desensitization of G protein-coupled receptors, such as β₂-adrenergic receptors, muscarinic acetylcholine receptors, and 5-HT₂ receptors 3, 7, 8, 11, 19. It has been shown that the mechanisms of the desensitization process induced by agonists are involved in several different phenomena such as the phosphorylation of receptors by either protein kinase A (PKA), protein kinase C (PKC), or a certain receptor kinase, the sequestering of receptors to an internal compartment, and the modulation of receptor expression. Recently, it has been reported that the 5-HT₄ and 5-HT₆ receptor subtypes, which are positively coupled to adenylyl cyclase, are desensitized by pretreatment with serotonergic agonists in mouse colliculi neurons [1]and in neuroblastoma NCB.20 [4]. The present study examined whether 5-HT₇ receptors are desensitized by agonists by investigating the effects of pretreatment with serotonergic agonists on 5-HT₇ receptor-mediated adenylyl cyclase activation in astrocytes. Our results suggest that 5-HT₇ receptors undergo homologous desensitization by their agonists in a time- and concentration-dependent manner.

Section snippets

Materials

5-HT hydrochloride and isoproterenol hydrochloride were obtained from Sigma Chemical (St. Louis, MO, USA). 5-CT, 5-MeOT, 2-methyl-5-HT, α-methyl-5-HT, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), N-ethylcarboxamidoadenosine (NECA), and phorbol 12-myristate 13-acetate (PMA) were obtained from Research Biochemicals (Natick, MA, USA). Forskolin and staurosporine were obtained from Wako Pure Chemical (Osaka, Japan). Dibutyryl cyclic AMP was obtained from Yamasa (Chiba, Japan). N-[2-(p

Differences in serotonergic agonist-mediated cyclic AMP responses in astrocytes and in neuron-rich cultures

The addition of 5-HT and 5-CT (1 and 10 μM) increased cyclic AMP levels in astrocytes by 5- to 7-fold. 5-HT synergistically activated cyclic AMP formation in the presence of 1 μM forskolin. Similarly, 8-OH-DPAT led to a slight elevation of basal cyclic AMP levels and significantly increased forskolin-stimulated cyclic AMP formation (Fig. 1). In contrast, neither 5-HT, 5-CT, nor 8-OH-DPAT significantly affected basal cyclic AMP levels in neuron-rich cultures, even at concentrations of 10 μM;

Discussion

In the present study, the administration of 5-CT and 5-HT to rat frontocortical astrocytes potently increased cyclic AMP formation. 8-OH-DPAT slightly increased basal cyclic AMP levels and significantly enhanced forskolin-stimulated cyclic AMP formation. In contrast, the administrations of these agonists to neuron-rich cultures failed to change basal cyclic AMP levels and decreased forskolin-stimulated cyclic AMP formation. 8-OH-DPAT is a well-known agonist of 5-HT_1A receptors. 5-HT_1A receptors

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We found that astrocyte calcium signaling can be elicited by 5-HT. Also, the SSRIs citalopram and fluoxetine induce calcium signals in about 1/3 of all astrocytes, even when neuronal signal propagation is inhibited. Astrocytic responses to 5-HT have a unique pattern and they could mostly not be evoked twice. We determined that glutamate is a substance that can interfere with 5-HT-induced calcium signals in astrocytes since after stimulation by glutamate, astrocytes did not show a response to 5-HT.
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This study compared the use of adapter G-proteins to link G_s coupled G-protein receptors to a Ca²⁺ signal, enabling high throughput functional studies using a fluorescent imaging plate reader (FLIPR, Molecular Devices).The pharmacological profile of the human 5-hydroxytryptamine (5-HT₇) receptor was studied using the adapter G-proteins G_α16 and G_qs5 and compared to previously published adenylyl cyclase and receptor binding data. Human embryonic kidney (HEK) 293 cells stably expressing the human 5-HT_7(a) receptor were transiently transfected with the adapter G-proteins. Changes in intracellular Ca²⁺ were monitored using the fluorescent Ca²⁺-indicator Fluo-4. 5-Carboxamidotryptamine (5-CT) induced an increase in fluorescence in transfected cells only, which was attenuated by N-ethylmalaeimide and abolished by thapsigargin, consistent with a G-protein mediated mobilisation of intracellular Ca²⁺. The pharmacological profile of agonists at the 5-HT₇ receptor was similar using either adapter G-protein. Agonist potency estimates were similar to that reported in binding studies but were greater than that seen in adenylyl cyclase studies. 8-Hydroxy-N,N-dipropylaminotetralin (8-OH-DPAT) and tryptamine acted as partial agonists using the adapter G-proteins, but were full agonists in recombinant systems using adenylyl cyclase. meta-Chlorophenylpiperazine (mCPP) and trifluoro-methylphenyl piperazine (TFMPP) were antagonists on intracellular Ca²⁺. Antagonist pharmacological profiles were similar between adapter G-proteins, receptor binding, and adenylyl cyclase studies. These results show that adapter G-proteins can be used to study G_s-linked receptors using the high throughput FLIPR system measuring changes in intracellular Ca²⁺ and provide novel information on mCPP and 8-OH-DPAT.
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¹: Present address: Department of Pharmacology, University of Washington, Seattle, WA 98195, USA.

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Research reportAgonist-induced desensitization of adenylyl cyclase activity mediated by 5-hydroxytryptamine7 receptors in rat frontocortical astrocytes

Abstract

Introduction

Section snippets

Materials

Differences in serotonergic agonist-mediated cyclic AMP responses in astrocytes and in neuron-rich cultures

Discussion

J. Biol. Chem.

J. Biol. Chem.

Neuron

Neuropharamocol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Characterization of homologous 5-hydroxytryptamine4 receptor desensitization in colliculi neurons

Mol. Pharmacol.

Serotonin receptor-mediated activation of adenylate cyclase in the neuroblastoma NCB.20: a novel 5-hydroxytryptamine receptor

Mol. Pharmacol.

Research report
Agonist-induced desensitization of adenylyl cyclase activity mediated by 5-hydroxytryptamine₇ receptors in rat frontocortical astrocytes

Characterization of homologous 5-hydroxytryptamine₄ receptor desensitization in colliculi neurons