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Novel aspects of cholinergic regulation of colonic ion transport.

Bader S, Diener M - Pharmacol Res Perspect (2015)

Bottom Line: As a strong acetylcholinesterase activity was found in colonic epithelium, the effect of choline on I sc was examined.Although choline proved to be only a partial agonist, it concentration-dependently desensitized the response to acetylcholine, suggesting that it might act as a modulator of cholinergically induced anion secretion.Thus the cholinergic regulation of colonic ion transport - up to now solely explained by cholinergic submucosal neurons stimulating epithelial muscarinic receptors - is more complex than previously assumed.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Physiology and Biochemistry, Justus-Liebig-University Giessen Giessen, Germany.

ABSTRACT
Nicotinic receptors are not only expressed by excitable tissues, but have been identified in various epithelia. One aim of this study was to investigate the expression of nicotinic receptors and their involvement in the regulation of ion transport across colonic epithelium. Ussing chamber experiments with putative nicotinic agonists and antagonists were performed at rat colon combined with reverse transcription polymerase chain reaction (RT-PCR) detection of nicotinic receptor subunits within the epithelium. Dimethylphenylpiperazinium (DMPP) and nicotine induced a tetrodotoxin-resistant anion secretion leading to an increase in short-circuit current (I sc) across colonic mucosa. The response was suppressed by the nicotinic receptor antagonist hexamethonium. RT-PCR experiments revealed the expression of α2, α4, α5, α6, α7, α10, and β4 nicotinic receptor subunits in colonic epithelium. Choline, the product of acetylcholine hydrolysis, is known for its affinity to several nicotinic receptor subtypes. As a strong acetylcholinesterase activity was found in colonic epithelium, the effect of choline on I sc was examined. Choline induced a concentration-dependent, tetrodotoxin-resistant chloride secretion which was, however, resistant against hexamethonium, but was inhibited by atropine. Experiments with inhibitors of muscarinic M1 and M3 receptors revealed that choline-evoked secretion was mainly due to a stimulation of epithelial M3 receptors. Although choline proved to be only a partial agonist, it concentration-dependently desensitized the response to acetylcholine, suggesting that it might act as a modulator of cholinergically induced anion secretion. Thus the cholinergic regulation of colonic ion transport - up to now solely explained by cholinergic submucosal neurons stimulating epithelial muscarinic receptors - is more complex than previously assumed.

No MeSH data available.


Related in: MedlinePlus

RT-PCR detection of mRNA expression of nicotinic acetylcholine receptor subunits in isolated rat colonic crypts. The agarose gel shows bands of cDNA fragments amplified using primers for α2 (154 bp), α4 (197 bp), α5 (146 bp), α6 (142 bp), α7 (446 bp), α10 (168 bp), β2 (116 bp), and β4 (300 bp). cDNA from spinal cord was used as positive, water instead of cDNA as negative control. The efficiency of RNA isolation and cDNA synthesis was verified by GAPDH-specific primers (303 bp). Each RT-PCR reaction was performed in at least three independent experiments. The mRNA for β2 was only inconsistently detected in two of five experiments, whereas the signals for the other nicotinic receptor subunits depicted here were found consistently. Cry, colonic crypts; SC, spinal cord; RT-PCR, reverse transcription polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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fig01: RT-PCR detection of mRNA expression of nicotinic acetylcholine receptor subunits in isolated rat colonic crypts. The agarose gel shows bands of cDNA fragments amplified using primers for α2 (154 bp), α4 (197 bp), α5 (146 bp), α6 (142 bp), α7 (446 bp), α10 (168 bp), β2 (116 bp), and β4 (300 bp). cDNA from spinal cord was used as positive, water instead of cDNA as negative control. The efficiency of RNA isolation and cDNA synthesis was verified by GAPDH-specific primers (303 bp). Each RT-PCR reaction was performed in at least three independent experiments. The mRNA for β2 was only inconsistently detected in two of five experiments, whereas the signals for the other nicotinic receptor subunits depicted here were found consistently. Cry, colonic crypts; SC, spinal cord; RT-PCR, reverse transcription polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Mentions: To determine which of the nicotinic receptor subunits are expressed by rat colonic epithelium, RT-PCR was performed using mRNA from isolated crypts, that is, epithelial cells devoid of neuronal contamination, as starting material. Skeletal muscle was used as positive control for α1- and β1-subunit, for all the other subunits spinal cord served as positive control. The following subunits were consistently (in each of n ≥ 3 independent assays for each mRNA) detected in colonic epithelial cells: α2, α4, α5, α6, α7, α10, and β4. The mRNA for β2 was only inconsistently detected in two of five experiments (Fig.1). Not detectable in colonic epithelium, but in control tissues were the α1-, α3-, β1-, and β3-subunits. The α9 subunit was neither detectable in the positive control nor in the colonic epithelium, although different primers from the literature (Glowatzki et al. 1995; Hecker et al. 2009; Mikulski et al. 2010; Schirmer et al. 2011) were tested (see Discussion for possible reasons). Water controls without template did not show any amplificates (Fig.1).


Novel aspects of cholinergic regulation of colonic ion transport.

Bader S, Diener M - Pharmacol Res Perspect (2015)

RT-PCR detection of mRNA expression of nicotinic acetylcholine receptor subunits in isolated rat colonic crypts. The agarose gel shows bands of cDNA fragments amplified using primers for α2 (154 bp), α4 (197 bp), α5 (146 bp), α6 (142 bp), α7 (446 bp), α10 (168 bp), β2 (116 bp), and β4 (300 bp). cDNA from spinal cord was used as positive, water instead of cDNA as negative control. The efficiency of RNA isolation and cDNA synthesis was verified by GAPDH-specific primers (303 bp). Each RT-PCR reaction was performed in at least three independent experiments. The mRNA for β2 was only inconsistently detected in two of five experiments, whereas the signals for the other nicotinic receptor subunits depicted here were found consistently. Cry, colonic crypts; SC, spinal cord; RT-PCR, reverse transcription polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492755&req=5

fig01: RT-PCR detection of mRNA expression of nicotinic acetylcholine receptor subunits in isolated rat colonic crypts. The agarose gel shows bands of cDNA fragments amplified using primers for α2 (154 bp), α4 (197 bp), α5 (146 bp), α6 (142 bp), α7 (446 bp), α10 (168 bp), β2 (116 bp), and β4 (300 bp). cDNA from spinal cord was used as positive, water instead of cDNA as negative control. The efficiency of RNA isolation and cDNA synthesis was verified by GAPDH-specific primers (303 bp). Each RT-PCR reaction was performed in at least three independent experiments. The mRNA for β2 was only inconsistently detected in two of five experiments, whereas the signals for the other nicotinic receptor subunits depicted here were found consistently. Cry, colonic crypts; SC, spinal cord; RT-PCR, reverse transcription polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Mentions: To determine which of the nicotinic receptor subunits are expressed by rat colonic epithelium, RT-PCR was performed using mRNA from isolated crypts, that is, epithelial cells devoid of neuronal contamination, as starting material. Skeletal muscle was used as positive control for α1- and β1-subunit, for all the other subunits spinal cord served as positive control. The following subunits were consistently (in each of n ≥ 3 independent assays for each mRNA) detected in colonic epithelial cells: α2, α4, α5, α6, α7, α10, and β4. The mRNA for β2 was only inconsistently detected in two of five experiments (Fig.1). Not detectable in colonic epithelium, but in control tissues were the α1-, α3-, β1-, and β3-subunits. The α9 subunit was neither detectable in the positive control nor in the colonic epithelium, although different primers from the literature (Glowatzki et al. 1995; Hecker et al. 2009; Mikulski et al. 2010; Schirmer et al. 2011) were tested (see Discussion for possible reasons). Water controls without template did not show any amplificates (Fig.1).

Bottom Line: As a strong acetylcholinesterase activity was found in colonic epithelium, the effect of choline on I sc was examined.Although choline proved to be only a partial agonist, it concentration-dependently desensitized the response to acetylcholine, suggesting that it might act as a modulator of cholinergically induced anion secretion.Thus the cholinergic regulation of colonic ion transport - up to now solely explained by cholinergic submucosal neurons stimulating epithelial muscarinic receptors - is more complex than previously assumed.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Physiology and Biochemistry, Justus-Liebig-University Giessen Giessen, Germany.

ABSTRACT
Nicotinic receptors are not only expressed by excitable tissues, but have been identified in various epithelia. One aim of this study was to investigate the expression of nicotinic receptors and their involvement in the regulation of ion transport across colonic epithelium. Ussing chamber experiments with putative nicotinic agonists and antagonists were performed at rat colon combined with reverse transcription polymerase chain reaction (RT-PCR) detection of nicotinic receptor subunits within the epithelium. Dimethylphenylpiperazinium (DMPP) and nicotine induced a tetrodotoxin-resistant anion secretion leading to an increase in short-circuit current (I sc) across colonic mucosa. The response was suppressed by the nicotinic receptor antagonist hexamethonium. RT-PCR experiments revealed the expression of α2, α4, α5, α6, α7, α10, and β4 nicotinic receptor subunits in colonic epithelium. Choline, the product of acetylcholine hydrolysis, is known for its affinity to several nicotinic receptor subtypes. As a strong acetylcholinesterase activity was found in colonic epithelium, the effect of choline on I sc was examined. Choline induced a concentration-dependent, tetrodotoxin-resistant chloride secretion which was, however, resistant against hexamethonium, but was inhibited by atropine. Experiments with inhibitors of muscarinic M1 and M3 receptors revealed that choline-evoked secretion was mainly due to a stimulation of epithelial M3 receptors. Although choline proved to be only a partial agonist, it concentration-dependently desensitized the response to acetylcholine, suggesting that it might act as a modulator of cholinergically induced anion secretion. Thus the cholinergic regulation of colonic ion transport - up to now solely explained by cholinergic submucosal neurons stimulating epithelial muscarinic receptors - is more complex than previously assumed.

No MeSH data available.


Related in: MedlinePlus