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Distribution of 5-HT3, 5-HT4, and 5-HT7 Receptors Along the Human Colon.

Yaakob NS, Chinkwo KA, Chetty N, Coupar IM, Irving HR - J Neurogastroenterol Motil (2015)

Bottom Line: The 5-HT3 receptor subunits were found in all tissues throughout the colon and ileum.The 5-HT3 receptor E subunit was mainly found in the mucosa layers.The major differences in 5-HT receptor distribution within the human colon are in relation to the mucosa and muscular tissue layers where the 5-HT3 receptor E subunit is predominantly found in the mucosal layer which may be of therapeutic relevance.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville campus), Parkville Victoria, Australia.

ABSTRACT

Background/aims: Several disorders of the gastrointestinal tract are associated with abnormal serotonin (5-HT) signaling or metabolism where the 5-HT3 and 5-HT4 receptors are clinically relevant. The aim was to examine the distribution of 5-HT3, 5-HT4, and 5-HT7 receptors in the normal human colon and how this is associated with receptor interacting chaperone 3, G protein coupled receptor kin-ases, and protein LIN-7 homologs to extend previous observations limited to the sigmoid colon or the upper intestine.

Methods: Samples from ascending, transverse, descending, and sigmoid human colon were dissected into 3 separate layers (mucosa, lon-gitudinal, and circular muscles) and ileum samples were dissected into mucosa and muscle layers (n = 20). Complementary DNA was synthesized by reverse transcription from extracted RNA and expression was determined by quantitative or end point polymerase chain reaction.

Results: The 5-HT3 receptor subunits were found in all tissues throughout the colon and ileum. The A subunit was detected in all sam-ples and the C subunit was expressed at similar levels while the B subunit was expressed at lower levels and less frequently. The 5-HT3 receptor E subunit was mainly found in the mucosa layers. All splice variants of the 5-HT4 and 5-HT7 receptors were expressed throughout the colon although the 5-HT4 receptor d, g, and i variants were expressed less often.

Conclusions: The major differences in 5-HT receptor distribution within the human colon are in relation to the mucosa and muscular tissue layers where the 5-HT3 receptor E subunit is predominantly found in the mucosal layer which may be of therapeutic relevance.

No MeSH data available.


Distribution of serotonin type 3 (5-HT3) receptor subunits and receptor interacting chaperone 3 (RIC3) transcripts in human intestinal tissue layers. (A) Comparison of the relative expression levels of transcripts of RIC3 and 5-HT3 receptor subunits in the mucosal and muscle tissue layers in the human ileum (n = 4). (B) Comparison of the relative expression levels of transcripts of RIC3 and 5-HT3 receptor subunits in the mucosal and muscle layers (circular and longitudinal) in tissue samples obtained from throughout the human colon (n = 16). Data are expressed as a ratio relative to β-actin as described in the Material and Methods section. Bars indicate the mean. 5-HT3 receptor D subunit (HTR3D) transcripts were not detected in any tissue tested. RIC3 and 5-HT3 receptor subunit transcripts are expressed at significantly lower levels than glyceraldehyde dehydrogenase (GAPDH) transcripts (P < 0.001 one-way ANOVA followed by Tukey’s multiple comparisons test) in all tissues. The letters above the x-axis (v, w, x, y, and z representing the highest to lowest level respectively) indicate that the transcripts are found at significantly different levels (P < 0.05 one-way ANOVA followed by Tukey’s multiple comparisons test) in the ileum or colon tissue layers (ie, transcripts in the different layers with an x underneath are expressed at the same level; x is the highest and z the lowest level in [A] while v is the highest and z the lowest level in [B]).
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f1-jnm-21-361: Distribution of serotonin type 3 (5-HT3) receptor subunits and receptor interacting chaperone 3 (RIC3) transcripts in human intestinal tissue layers. (A) Comparison of the relative expression levels of transcripts of RIC3 and 5-HT3 receptor subunits in the mucosal and muscle tissue layers in the human ileum (n = 4). (B) Comparison of the relative expression levels of transcripts of RIC3 and 5-HT3 receptor subunits in the mucosal and muscle layers (circular and longitudinal) in tissue samples obtained from throughout the human colon (n = 16). Data are expressed as a ratio relative to β-actin as described in the Material and Methods section. Bars indicate the mean. 5-HT3 receptor D subunit (HTR3D) transcripts were not detected in any tissue tested. RIC3 and 5-HT3 receptor subunit transcripts are expressed at significantly lower levels than glyceraldehyde dehydrogenase (GAPDH) transcripts (P < 0.001 one-way ANOVA followed by Tukey’s multiple comparisons test) in all tissues. The letters above the x-axis (v, w, x, y, and z representing the highest to lowest level respectively) indicate that the transcripts are found at significantly different levels (P < 0.05 one-way ANOVA followed by Tukey’s multiple comparisons test) in the ileum or colon tissue layers (ie, transcripts in the different layers with an x underneath are expressed at the same level; x is the highest and z the lowest level in [A] while v is the highest and z the lowest level in [B]).

Mentions: The distribution of 5-HT3 receptor subunits was examined using quantitative reverse transcript PCR on samples obtained from throughout the length of the colon and the ileum area of the small intestine. In all regions, expression was examined in both the mucosal and muscular tissue layers and reported relative to expression of β-actin. GAPDH was used as a second housekeeping gene for comparison with a previous study where the relative expression of the 5-HT3 receptors in the sigmoid colon was reported.22 Control RNA samples incubated without reverse transcriptase and then amplified with GAPDH primers were also undertaken to demonstrate that there was no DNA contamination of the RNA extractions (data not shown). GAPDH levels of expression were consistently and significantly higher than those of the 5-HT3 receptor subunits or RIC3 in both ileum and colon (P < 0.05 one-way ANOVA; Fig. 1) which is consistent with the previous study.22 The 5-HT3 receptor D subunit was consistently not detected in either ileum or colon samples which is in agreement with prior studies where transcripts of HTR3D were only evident at very low levels.23,25 In the ileum, RIC3 and 5-HT3 receptor A subunit transcripts were found in all samples with the other subunits being less prevalent (Fig. 1A). Transcript levels of RIC3 were significantly higher than 5-HT3 receptor B subunits in both mucosa and muscle layers and also the mucosa levels of the A subunit and the C subunit in the muscle layer. No significant differences were observed in the expression levels of the 5-HT3 receptor subunits with the major exception that the E subunit was only found in the mucosa layer (Fig. 1A).


Distribution of 5-HT3, 5-HT4, and 5-HT7 Receptors Along the Human Colon.

Yaakob NS, Chinkwo KA, Chetty N, Coupar IM, Irving HR - J Neurogastroenterol Motil (2015)

Distribution of serotonin type 3 (5-HT3) receptor subunits and receptor interacting chaperone 3 (RIC3) transcripts in human intestinal tissue layers. (A) Comparison of the relative expression levels of transcripts of RIC3 and 5-HT3 receptor subunits in the mucosal and muscle tissue layers in the human ileum (n = 4). (B) Comparison of the relative expression levels of transcripts of RIC3 and 5-HT3 receptor subunits in the mucosal and muscle layers (circular and longitudinal) in tissue samples obtained from throughout the human colon (n = 16). Data are expressed as a ratio relative to β-actin as described in the Material and Methods section. Bars indicate the mean. 5-HT3 receptor D subunit (HTR3D) transcripts were not detected in any tissue tested. RIC3 and 5-HT3 receptor subunit transcripts are expressed at significantly lower levels than glyceraldehyde dehydrogenase (GAPDH) transcripts (P < 0.001 one-way ANOVA followed by Tukey’s multiple comparisons test) in all tissues. The letters above the x-axis (v, w, x, y, and z representing the highest to lowest level respectively) indicate that the transcripts are found at significantly different levels (P < 0.05 one-way ANOVA followed by Tukey’s multiple comparisons test) in the ileum or colon tissue layers (ie, transcripts in the different layers with an x underneath are expressed at the same level; x is the highest and z the lowest level in [A] while v is the highest and z the lowest level in [B]).
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f1-jnm-21-361: Distribution of serotonin type 3 (5-HT3) receptor subunits and receptor interacting chaperone 3 (RIC3) transcripts in human intestinal tissue layers. (A) Comparison of the relative expression levels of transcripts of RIC3 and 5-HT3 receptor subunits in the mucosal and muscle tissue layers in the human ileum (n = 4). (B) Comparison of the relative expression levels of transcripts of RIC3 and 5-HT3 receptor subunits in the mucosal and muscle layers (circular and longitudinal) in tissue samples obtained from throughout the human colon (n = 16). Data are expressed as a ratio relative to β-actin as described in the Material and Methods section. Bars indicate the mean. 5-HT3 receptor D subunit (HTR3D) transcripts were not detected in any tissue tested. RIC3 and 5-HT3 receptor subunit transcripts are expressed at significantly lower levels than glyceraldehyde dehydrogenase (GAPDH) transcripts (P < 0.001 one-way ANOVA followed by Tukey’s multiple comparisons test) in all tissues. The letters above the x-axis (v, w, x, y, and z representing the highest to lowest level respectively) indicate that the transcripts are found at significantly different levels (P < 0.05 one-way ANOVA followed by Tukey’s multiple comparisons test) in the ileum or colon tissue layers (ie, transcripts in the different layers with an x underneath are expressed at the same level; x is the highest and z the lowest level in [A] while v is the highest and z the lowest level in [B]).
Mentions: The distribution of 5-HT3 receptor subunits was examined using quantitative reverse transcript PCR on samples obtained from throughout the length of the colon and the ileum area of the small intestine. In all regions, expression was examined in both the mucosal and muscular tissue layers and reported relative to expression of β-actin. GAPDH was used as a second housekeeping gene for comparison with a previous study where the relative expression of the 5-HT3 receptors in the sigmoid colon was reported.22 Control RNA samples incubated without reverse transcriptase and then amplified with GAPDH primers were also undertaken to demonstrate that there was no DNA contamination of the RNA extractions (data not shown). GAPDH levels of expression were consistently and significantly higher than those of the 5-HT3 receptor subunits or RIC3 in both ileum and colon (P < 0.05 one-way ANOVA; Fig. 1) which is consistent with the previous study.22 The 5-HT3 receptor D subunit was consistently not detected in either ileum or colon samples which is in agreement with prior studies where transcripts of HTR3D were only evident at very low levels.23,25 In the ileum, RIC3 and 5-HT3 receptor A subunit transcripts were found in all samples with the other subunits being less prevalent (Fig. 1A). Transcript levels of RIC3 were significantly higher than 5-HT3 receptor B subunits in both mucosa and muscle layers and also the mucosa levels of the A subunit and the C subunit in the muscle layer. No significant differences were observed in the expression levels of the 5-HT3 receptor subunits with the major exception that the E subunit was only found in the mucosa layer (Fig. 1A).

Bottom Line: The 5-HT3 receptor subunits were found in all tissues throughout the colon and ileum.The 5-HT3 receptor E subunit was mainly found in the mucosa layers.The major differences in 5-HT receptor distribution within the human colon are in relation to the mucosa and muscular tissue layers where the 5-HT3 receptor E subunit is predominantly found in the mucosal layer which may be of therapeutic relevance.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville campus), Parkville Victoria, Australia.

ABSTRACT

Background/aims: Several disorders of the gastrointestinal tract are associated with abnormal serotonin (5-HT) signaling or metabolism where the 5-HT3 and 5-HT4 receptors are clinically relevant. The aim was to examine the distribution of 5-HT3, 5-HT4, and 5-HT7 receptors in the normal human colon and how this is associated with receptor interacting chaperone 3, G protein coupled receptor kin-ases, and protein LIN-7 homologs to extend previous observations limited to the sigmoid colon or the upper intestine.

Methods: Samples from ascending, transverse, descending, and sigmoid human colon were dissected into 3 separate layers (mucosa, lon-gitudinal, and circular muscles) and ileum samples were dissected into mucosa and muscle layers (n = 20). Complementary DNA was synthesized by reverse transcription from extracted RNA and expression was determined by quantitative or end point polymerase chain reaction.

Results: The 5-HT3 receptor subunits were found in all tissues throughout the colon and ileum. The A subunit was detected in all sam-ples and the C subunit was expressed at similar levels while the B subunit was expressed at lower levels and less frequently. The 5-HT3 receptor E subunit was mainly found in the mucosa layers. All splice variants of the 5-HT4 and 5-HT7 receptors were expressed throughout the colon although the 5-HT4 receptor d, g, and i variants were expressed less often.

Conclusions: The major differences in 5-HT receptor distribution within the human colon are in relation to the mucosa and muscular tissue layers where the 5-HT3 receptor E subunit is predominantly found in the mucosal layer which may be of therapeutic relevance.

No MeSH data available.