GABAA receptor subunit messenger RNA expression in the enteric nervous system of the rat: implications for functional diversity of enteric GABAA receptors

doi:10.1016/S0306-4522(99)00174-8

Neuroscience

Volume 93, Issue 3, August 1999, Pages 1159-1165

https://doi.org/10.1016/S0306-4522(99)00174-8 Get rights and content

Abstract

GABAergic neurons occur in the myenteric plexus and submucosa and their innervations of the gut, where GABA stimulates motor neurons, and non-neural cells via “central type” GABA_A receptors. These receptors occur on half of the neurons in the rat intestine. The GABA_A receptor is a ligand-gated chloride channel constructed from different subunit families (α, β, γ, δ, ϵ). In rat these exist as subtypes, α_1–6, β_1–3, γ_1–3 and δ, defining the clinically relevant pharmacological features of GABA_A receptors. However, the identity, distribution, and abundance of enteric GABA_A receptor subunits are unknown. To identify and map the regional expression of GABA_A receptor subunit messenger RNAs in the enteric nervous system, we assayed enteric RNA from the ileum of Sprague–Dawley rats by reverse transcription–polymerase chain reaction for α_1–6, β_1–3, γ_1–3, and δ subunit messenger RNAs. Subunit messenger RNA localization, was probed by in situ hybridization. Reverse transcription–polymerase chain reaction analysis of RNA from myenteric and submucosal nerve layers revealed the expression α₁, α₃, α₅, β₂, β₃, γ₁ and γ₃ subunit messenger RNAs. Little α₄ and α₆ and no α₂, β₁, γ₂ or δ subunit messenger RNA were detected. In situ hybridization revealed that transcripts for α₁, α₃, α₅ and β₂ subunits occur in both myenteric and submucous ganglia. However, β₃ messenger RNA was found only in myenteric plexus. The γ₁ subunit messenger RNA was also restricted to the cells in the myenteric plexus while γ₃ was found in cells of both nerve layers.

In this study of the subunit messenger RNA expression profile of GABA_A receptors within the enteric nerve layers we show an abundant, diverse and widespread distribution that is unique in comparison to the CNS. The distinctive and heterogeneous distribution of enteric GABA_A subunits may be important in the integration of neural control of gut function.

Section snippets

Experimental procedures

All tissues used in this study were obtained from male Sprague–Dawley rats (125–175 g; Charles River, Canada) killed by decapitation according to accepted standards of the University of Ottawa and National Research Council animal care commitees. Each animal was subjected to a laparotomy and the small intestine exposed for resection of the ileum. Segments of the proximal ileum (6 cm in length) were removed into an oxygenated Krebs' buffer, maintained at 37°C and pH 7.2. The individual segments

Reverse transcription–polymerase chain reaction

GABA subunit mRNAs were detected using subunit-specific primers in RNA isolated from homogenates of gut wall free of mucosa. After reverse transcription (RT) and PCR amplification of the cDNA, bands of the predicted size were observed in ethidium bromide-stained agarose gels. This assay consistently detected α₁, α₃, α₅, β₂, β₃, γ₁ and γ₃ subunit mRNAs (Fig. 1). However, α₄ and α₆ message were detected at threshold levels in two out of six samples. This was not thought to be the result of

Discussion

In order to determine how the enteric GABAergic system is integrated in the neural control of gut function we have undertaken the first comprehensive study of the subunit expression profile of GABA_A receptors within the nerve networks of the intestine. This information provides a framework for further functional and pharmacological studies since we have found a distinctive and heterogeneous distribution of GABA_A receptor subunit mRNAs. In contrast to the CNS, α₃, γ₁ and γ₃ subunit mRNAs were

Acknowledgements

This work was supported by the Medical Research Council of Canada; National Research Council of Canada; Canadian Association of Gastroenterology Studentship to R.S.

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GABAA receptor subunit messenger RNA expression in the enteric nervous system of the rat: implications for functional diversity of enteric GABAA receptors

Abstract

Section snippets

Experimental procedures

Reverse transcription–polymerase chain reaction

Discussion

Acknowledgements

Eur. J. Pharmac.

Brain Res.

Gastroenterology

Eur. J. Pharmac.

Gastroenterology

Regul. Pept.

Gastroenterology

Trends Neurosci.

Eur. J. Pharmac.

J. auton. nerv. Syst.

Neurosci. Lett.

Neurosci. Lett.

J. auton. nerv. Syst.

J. auton. nerv. Syst.

J. auton. nerv. Syst.

Gastroenterology

J. auton. nerv. Syst.

Neuroscience

Eur. J. Pharmac.

Neurosci. Lett.

Fedn Eur. biochem. Socs Lett.

Brain Res.

Molec. Brain Res.

Assembly of GABAA receptor subunits: α1β1 and α1β1γ2S subunits produce unique ion channels with dissimilar single-channel properties

J. Neurosci.

Alfaxalone, pentobarbital and diazepam potentiate gamma- aminobutyric acid-induced depolarizations in single myenteric neurons of guinea pig intestine

J. Pharmac. exp. Ther.

GABAA receptor subtypes: from pharmacology to molecular biology

Fedn Am. Socs exp. Biol. J.

Actions of γ-aminobutyric acid on neurones of guinea-pig myenteric plexus

Br. J. Pharmac.

Modulation by general anaesthetics of rat GABAA receptors comprised of alpha 1 beta 3 and beta 3 subunits expressed in human embryonic kidney 293 cells

Br. J. Pharmac.

Gastric endocrine cells: gene expression, processing, and targeting of active products

Physiol. Rev.

GABA_A receptor subunit messenger RNA expression in the enteric nervous system of the rat: implications for functional diversity of enteric GABA_A receptors

Assembly of GABA_A receptor subunits: α1β1 and α1β1γ2S subunits produce unique ion channels with dissimilar single-channel properties

Modulation by general anaesthetics of rat GABA_A receptors comprised of alpha 1 beta 3 and beta 3 subunits expressed in human embryonic kidney 293 cells