Pharmacological characterization of serotonin 5-HT3 receptor-mediated electrical response in cultured mouse neuroblastoma cells

doi:10.1016/0028-3908(88)90048-2

Neuropharmacology

Volume 27, Issue 3, March 1988, Pages 301-307

https://doi.org/10.1016/0028-3908(88)90048-2 Get rights and content

Abstract

The aim of this study was to investigate the pharmacological characteristics of the 5-hydroxytryptamine-(5-HT)-induced electrical response in cultured neuroblastoma N1E-115 cells of the mouse. In these cells 5-HT induces a transient membrane depolarization, which is associated with a transient inward current, that has been recorded in voltage clamp experiments on whole cells. The peak amplitude of the inward current depends on the concentration of 5-HT applied. Maximum peak inward current was evoked by 10 μM 5-HT and half maximum effect by 2 μM. Responses to 5-HT were blocked by nanomolar concentrations of selective 5-HT₃-receptor antagonists, whereas the selective agonist 2-methyl-5-HT mimicked the membrane depolarization induced by 5-HT. A number of agonists and antagonists, which are known to act on 5-HT₁-like, 5-HT₂, dopaminergic and adrenergic receptors failed to affect the response to 5-HT in neuroblastoma cells. Observed antagonistic effects of SCH 23390 [(R) − (+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hemimaleate] and haloperidol are discussed. The inhibitory effect of the 5-HT₃ receptor antagonist, ICS 205–930 [(3α-tropanyl)-1H-indole-3-carboxylic acid ester] has been demonstrated. When cells were exposed to 0.1 nM ICS 205–930 the maximum evoked response was reduced by about 50%, but a surmountable shift of the concentration-response curve of 5-HT was not observed. The kinetics of the 5-HT-induced inward current remained unchanged in the presence of ICS 205–930. Recovery from the block by ICS 205–930 was very slow. The apparent non-competitive nature of this antagonism is discussed.

The amplitude of the membrane depolarization evoked by ionophoretic application of 5-HT decreased in the presence of a small concentration of 5-HT. Dopamine (DA) also induced a depolarizing response in N1E-115 cells. Responses to ionophoretically applied 5-HT and dopamine were both potently desensitized by 5-HT. Dopamine induced densensitization only at much greater concentrations.

It is concluded that in cultured neuroblastoma N1E-115 cells of the mouse, the 5-HT-induced membrane depolarization and the associated inward current are mediated by a single population of 5-HT₃ receptor-mediated ion channels. These cells are an appropriate in vitro model to study the molecular mechanisms underlying the functioning of 5-HT₃ receptors.

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The figure showing simulated current was generated in Prism Software using exported simulated traces. To study the effects of 5-HI, we examined whole-cell currents activated by 5-HT in N1E-115 neuroblastoma cells expressing the native mouse 5-HT3 receptor (Neijt et al., 1988; Hope et al., 1993), and subsequently in HEK 293 cells transiently expressing the recombinant mouse 5-HT3A receptor. Inward current with a rapid onset was observed upon application of a maximally efficacious concentration of 5-HT (30 μM) for 10 s to either N1E-115 cells or transfected HEK 293 cells (Fig. 1A, left).
Allosteric modulation of ligand-gated ion channels can play important roles in shaping synaptic transmission. The function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT₃) receptor, a member of the Cys-loop ligand-gated ion channel superfamily, is modulated by a variety of compounds such as alcohols, anesthetics and 5-hydroxyindole (5-HI). In this study, the molecular determinants of allosteric modulation by 5-HI were explored in N1E-115 neuroblastoma cells expressing the native 5-HT₃ receptor and HEK 293 cells transfected with the recombinant 5-HT_3A receptor using molecular biology and whole-cell patch-clamp techniques. 5-HI potentiated 5-HT-activated currents in both N1E-115 cells and HEK 293 cells, and significantly decreased current desensitization and deactivation. Substitution of Leu293 (L293, L15′) in the second transmembrane domain (TM2) with cysteine (L293C) or serine (L293S) abolished 5-HI modulation. Other mutations in the TM2 domain, such as D298A and T284F, failed to alter 5-HI modulation. The L293S mutation enhanced dopamine efficacy and converted 5-HI into a partial agonist at the mutant receptor. These data suggest that 5-HI stabilizes the 5-HT_3A receptor in the open state by decreasing both desensitization and 5-HT unbinding/channel closing; and L293 is a common site for both channel gating and allosteric modulation by 5-HI. Our observations also indicate existence of a second 5-HI recognition site on the 5-HT_3A receptor, which may overlap with the 5-HT binding site and is not involved in the positive modulation by 5-HI. These findings support the idea that there are two discrete sites for 5-HI allosteric modulation and direct activation in the 5-HT_3A receptor.
Tachyphylaxis to 5-HT<inf>3</inf>-receptor-mediated activation of vagal afferents is prevented by co-activation of 5-HT<inf>2</inf> receptors
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Functional studies have provided evidence that 5-HT₃ ion-channel receptors (5-HT₃Rs) on vagal cardiopulmonary afferents mediating the Bezold–Jarisch reflex (BJR) rapidly desensitize upon repeated exposure to selective 5-HT₃R agonists. G-protein-coupled 5-HT₂ receptors (5-HT₂Rs) also exist on vagal afferents, although activation of these receptors does not elicit the BJR. However, there is in vivo evidence that 5-HT₂Rs may regulate the activity of 5-HT₃Rs. The aim of this study was to determine whether co-activation of 5-HT₂Rs prevents desensitization of 5-HT₃Rs mediating the BJR in conscious rats. The principal findings were that (1) tachyphylaxis rapidly developed to the BJR-mediated hemodynamic responses elicited by successive injections of 5-HT₃R agonists and (2) co-injection of the selective 5-HT₂R agonist, α-methyl-5-HT, prevented tachyphylaxis to the BJR-mediated hemodynamic responses elicited by the 5-HT₃R agonists. Additional studies provided evidence that (1) tachyphylaxis to the 5-HT₃R agonists was not due to impairment of the central or efferent processing of the BJR, and (2) the pressor responses elicited by α-methyl-5-HT were not responsible for preventing tachyphylaxis to the BJR reflex responses elicited by 5-HT₃R agonists. These results suggest that the loss of response to 5-HT₃R agonists is due to desensitization of 5-HT₃Rs on vagal afferents mediating the BJR and that co-activation of 5-HT₂Rs prevents the desensitization of these 5-HT₃Rs.
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5-Hydroxytryptamine 3 (5-HT₃) and α7 nicotinic receptors share high sequence homology and pharmacological cross-reactivity. An assessment of the potential role of α7 receptors in many neurophysiological processes, and hence their therapeutic value, requires the development of selective α7 receptor agonists. We used a recently reported selective α7 receptor agonist, (R)-(−)-5′Phenylspiro[1-azabicyclo[2.2.2] octane-3,2′-(3′H)furo[2,3-b]pyridine (PSAB-OFP) and confirmed its activity on human recombinant α7 receptors. However, PSAB-OFP also displayed high affinity binding to 5-HT₃ receptors. To assess the functional activity of PSAB-OFP on 5-HT₃ receptors we studied recombinant human 5-HT₃ receptors expressed in Xenopus oocytes, as well as native mouse 5-HT₃ receptors expressed in N1E-115 neuroblastoma cells, using whole-cell patch clamp and Ca²⁺ imaging. Our results show that PSAB-OFP is an equipotent, partial agonist of both α7 and 5-HT₃ receptors. We conclude that it will be necessary to identify the determinant of this overlapping pharmacology in order to develop more selective α7 receptor ligands.
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Using selective antibodies to visualize the short isoform of the 5-HT_3A receptor, we report here that both native and cloned 5-HT_3A(S) receptors formed clusters associated with F-actin in all cell types studied. NG 108-15 cells expressing native 5-HT_3A(S) receptors, COS-7 cells transiently expressing 5-HT_3A(S) subunits, and CHO cells stably transfected with a plasmid encoding the 5-HT_3A(S) sequence all exhibited similar surface receptor topology with 5-HT_3A(S) receptor cluster accumulation in F-actin-rich lamellipodia and microspikes. Colocalization and coclustering of 5-HT_3A(S) subunits and F-actin were also observed in transfected hippocampal neurons. Treatment of the neurons with latrunculin-A, a compound altering F-actin polymerization, demonstrated that 5-HT_3A(S) receptor cluster size and topology were dependent on F-actin integrity. These results suggest that the anchoring of 5-HT_3A(S) receptor clusters to the cytoskeletal network probably plays a key role in the physiological regulation of the receptor topology and dynamics, as is the case for other members of the 4-TMD ion channel receptor family.
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Pharmacological characterization of serotonin 5-HT3 receptor-mediated electrical response in cultured mouse neuroblastoma cells

Abstract

Brain Res.

Eur. J. Pharmac.

Eur. J. Pharmac.

Eur. J. Pharmac.

Neurotransmitter synthesis by neuroblastoma clones

Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine

Neuropharmacology

The antipsychotic potential of GR 38032F, a selective antagonist of 5-HT3 receptors in the central nervous system

Br. J. Pharmac.

ICS 205–930: a highly selective and potent antagonist at peripheral neuronal 5-hydroxytryptamine (5-HT) receptors

Br. J. Pharmac.

Serotonin (5-HT) enhances hippocampal noradrenaline (NA) release: Evidence for facilitatory 5-HT receptors within the CNS

Naunyn-Schmiedebergs Arch. Pharmac.

MDL 72222: a potent and highly selective antagonist at neuronal 5-hydroxytryptamine receptors

Naunyn-Schmiedebergs Arch. Pharmac.

Characterization of the effect of 5-hydroxytryptamine on N1E-115 neuroblastoma cells

Br. J. Pharmac.

5-Hydroxytryptamine can antagonize the ability of metoclopramide and SCH 23390 to enhance electrical field stimulation-evoked contractions of guinea-pig isolated stomach muscle

J. Pharm. Pharmac.

Pharmacological characterization of serotonin 5-HT₃ receptor-mediated electrical response in cultured mouse neuroblastoma cells

The antipsychotic potential of GR 38032F, a selective antagonist of 5-HT₃ receptors in the central nervous system