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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
,2,3,3
* Clinic for Ruminants, and
and
Division of Nutrition and Physiology, Institute of Animal Genetics, Nutrition and Housing, Vetsuisse Faculty, University of Berne, CH-3012 Berne, Switzerland
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: dairy cow • gastrointestinal tract • 5-hydroxytryptamine (serotonin) receptor
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Gershon, 1999; Taniyama et al., 2000). In humans and rats, the 5-HTR4 subtype, a G protein-coupled receptor, includes several splice variants that may have different locations and pharmacological profiles (Gerald et al., 1995; Blondel et al., 1998; Bender et al., 2000). The 5-HTR4 has been identified in various organs and tissues (Hedge and Eglen, 1996; Lefebvre et al., 1998; Ontsouka et al., 2004b).
In monogastrics, 5-HTR4 mediate smooth muscle contraction (Tomita et al., 1997; Prins et al., 2000a,b; Ono et al., 2005) or relaxation (Baxter et al., 1991; Tam et al., 1994; Kuemmerle et al., 1995) of esophagus, stomach, and intestine, depending on receptor location and species. In ruminants, 5-HTR4 mediate ruminal relaxation (van Miert and van Duin, 1998) or contraction of the abomasal wall (Spring et al., 2003). The functional role of 5-HTR4 in motility of other bovine GIT locations, mainly in the intestine, remains unclear.
The mRNA levels of 5-HTR4 were less in the spiral colon of cows with cecal dilation-dislocation (CDD) than in healthy controls (Engel et al., 2006). Given the prevalence of motility disorders such as CDD in lactating dairy cows and the role that 5-HTR4 may play in their pathogenesis, the investigation of its expression in muscle layers of various GIT sites from the abomasum to the spiral colon of healthy dairy cows was warranted and will serve as a basis to assess the role of 5-HTR4 in diseased cows.
Using binding and mRNA studies, we tested the hypotheses that the expression of 5-HTR4 differs among smooth muscle layers from various GIT locations in healthy cows and that mRNA and protein expression are correlated.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The project was approved by the Swiss Board for Animal Welfare and Protection. Healthy dairy cows that had been sold for slaughter (n = 8) were included in the current study. Full-thickness samples (segments of approximately 15 cm in length) from the fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of spiral colon (ELSC) were collected from each cow within minutes after stunning for slaughter, and placed onto a solid support submerged in chilled PBS (consisting of 8 g of NaCl, 200 mg of KCl, 2.6 g of Na2HPO4·7H2O, and 240 mg of KH2PO4 per liter, pH 7.4). The mucosal and submucosal layers were immediately scraped, and the complete removal of these layers was controlled histologically for each sample individually. The muscle tissues were evaluated histologically according to a previously described procedure by use of a grid method (Ontsouka et al., 2004a). The prepared muscle tissues consisted of more than 97% smooth muscle cells (data not shown) and were subsequently used for analysis of 5-HTR4 mRNA and protein levels.
After collection and preparation, 200 mg of muscle samples from the fundus abomasi, pylorus, ileum, cecum, PLAC, and ELSC were immediately transferred into RNAlater (Ambion Inc., Austin, TX) RNA stabilization buffer and stored at 4°C for 24 h, followed by freezing at –20°C until RNA processing. In addition, 15 g of tissues from the aforementioned GIT locations were collected for the radioligand binding studies and stored in ice-cold 50 mM Tris HCl buffer (pH 7.4) containing 6 mM MgCl2 and 1 mM ethylene glycol-bis-(ß-Amino-ethyl ether) N, N’-tetra-acetic acid (EGTA), and supplemented with a protease inhibitory cocktail containing chymotrypsin (1.5 µg/mL), thermolysin (0.8 µg/mL), papain (1 mg/mL), pronase (1.5 µg/mL), pancreatic extract (15 µg/ mL), and trypsin (0.2 µg/mL; Roche Biochemicals, Basel, Switzerland).
Real Time Reverse Transcription-PCR
Materials and procedures for RNA extraction and quantification and for reverse-transcription PCR were as described previously (Reist et al., 2003). Primers used for the amplification of mRNA for 5-HTR4 and for the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) were taken from earlier publications (Inderwies et al., 2003; Reist et al., 2003). For PCR procedures, pooled cDNA of bovine brain samples collected from 3 dairy cows other than those used in the current study was included as a positive control, allowing for verification of the specificity of the amplified product and for correction of interrun variations (Reist et al., 2003). An additional negative control (nonreverse-transcribed RNA) was added to ensure that no genomic DNA was am-plified.
The mRNA quantification was performed by using the recorded crossing point (CPt) values during PCR amplification of 5-HTR4 and of GAPDH products. The CPt is the amplification cycle number at which the measured signal becomes significantly greater than background, and thus reliably detectable. As previously described (Reist et al., 2003; Meylan et al., 2004; Engel et al., 2006), quantitative mRNA expression levels (E) of 5- HTR4 were obtained by relating the CPt values of 5-HTR4 to those of GAPDH:
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Radioligand Binding for 5-HTR4
Membrane suspensions of muscle tissues from the fundus abomasi, pylorus, ileum, cecum, PLAC, and ELSC were prepared as previously described (Carron et al., 2005; Ontsouka et al., 2006). The assay buffer consisted of 50 mM Tris HCl buffer at pH 7.4, with 6 mM MgCl2, 1 mM EGTA, and 10 µM of pargyline (a monoamine inhibitor; Sigma Aldrich, Steinheim, Germany). The binding studies were performed using the prototypic 5-HTR4 antagonist [3H]GR113808 (84 Ci/mmol; Amersham Biosciences, Buckinghamshire, UK) described in several other studies (Grossman et al., 1993; Blondel et al., 1998; Bender et al., 2000).
In preliminary studies, the appropriate conditions for binding assays with the 5-HTR4 antagonist [3H]GR113808 were determined using suspended membranes from the colonic muscle layer and brain preparations (pooled cortex, hypothalamus, and thalamus) of dairy cows and full-thickness jejunum of young calves. The competition of [3H]GR113808-specific binding on intestinal membranes was tested by use of increasing concentrations of 1) unlabelled GR113808 (Tocris Bioscience, Bristol, UK), 2) the 5-HTR agonist serotonin creatine sulphate (Sigma Aldrich, Steinheim, Germany), and 3) the 5-HTR4 antagonist RS23597-190 (Tocris Bioscience, Bristol, UK). The saturation binding assays were performed using up to 10 nM [3H]GR113808; [3H]GR113808 binding assays were tested at 4, 24, and 37°C.
According to the results of these preliminary investigations, all subsequent binding assays in the current study were performed with membrane protein in a concentration of 200 µg/mL and with an incubation step at 24°C for 30 min. Saturation binding of [3H]GR113808 in suspended membranes from 3 selected GIT locations (fundus abomasi, ileum, and ELSC) were performed using concentrations up to 1 nM, which is in the range used for [3H]GR113808 binding assays in other species (Ansanay et al., 1996; Arranz et al., 1998; Bender et al., 2000).
Competitive Binding Assays.
Suspended membrane preparations from the fundus abomasi, pylorus, ileum, cecum, PLAC, and ELSC were incubated with 0.3 nM [3H]GR113808 at 24°C for 30 min in the presence of increasing concentrations of the unlabelled GR113808 (10–14 to 10–5 M) under constant shaking. All experiments were performed in triplicate using the assay buffer described above. The termination of binding, filtration, and determination of bound [3H]-activity was as described previously (Carron et al., 2005; Ontsouka et al., 2006). The inhibition constants (Ki) of unlabelled GR113808 were calculated using the equation of Cheng and Prusoff (1973) and were evaluated by weighted, least squares, curve fitting using a software program (GraphPad Software Inc., San Diego, CA). The F-test was used to compare 1- and 2-site receptor binding models. Based on that, a 2-site receptor model was judged appropriate to fit our data only if the P value was less than 0.05. Furthermore, the GIT locations for which the competitive binding of 0.3 nM [3H]GR113808 had shown the best fit curves (with r2 > 0.85) were selected for further testing of the saturation binding assays.
Saturation Binding Assays. Based on competitive binding assays, membranes of smooth muscle tissues (200 µg/mL) from the fundus abomasi, ileum, and ELSC were incubated with 0.02 to 1 nM [3H]GR113808 in the absence (total binding; TB) and presence (nonspecific binding, NSB) of 10 µM unlabelled GR113808 (as a competitor) at 24°C for 30 min under constant shaking. The specific binding (SB) was calculated as the difference between TB and NSB. All experiments were performed in triplicate using the assay buffer described above. The percentage SB of [3H]GR113808 in the assays using suspended membranes from the ileum and ELSC was >60%, but it was between 55 and 60% in suspended membranes from the fundus abomasi. The SB was expressed as fmol of [3H]GR113808 bound per milligram of membrane protein. Equilibrium binding data were plotted as a function of the [3H]GR113808 concentration (saturation curve). Maximal binding capacity (Bmax) and the dissociation constant (KD) were calculated by weighted, least squares, curve fitting using the GraphPad software, as described in the preceding section. In addition, Hill coefficients were calculated with GraphPad software.
Statistical Analyses
Data are presented as means ± SEM. Statistical analysis was carried out using SAS (SAS Inst. Inc., Cary, NC). Before statistical evaluation, the data were log10 transformed to achieve a normal distribution. Effects of location were tested for significance by ANOVA using the GLM procedure of SAS, and the Bonferroni’s correction was used to adjust for multiple comparisons. Pearson’s correlation coefficients in the CORR procedure of SAS were used to calculate correlations between mRNA and receptor densities. The level of significant difference was set at a P-value 
0.05. Values of 0.05 < P < 0.1 were considered as trends.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The mRNA levels of 5-HTR4 were detected in muscle layers from the fundus abomasi, pylorus, ileum, cecum, PLAC, and ELSC (Figure 1). The mRNA levels of 5-HTR4 were affected by GIT location (P < 0.05), with greater levels in muscle layers from the ELSC and ileum than from the PLAC (P = 0.05 and P = 0.07, respectively).
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The competition of [3H]GR113808 specific binding by unlabelled GR113808 on membranes from smooth muscle tissues of the ileum and PLAC were best fit by a 1-site receptor model, whereas the competition of [3H]GR113808 specific binding on membranes from smooth muscle tissues of the fundus abomasi, pylorus, cecum, and ELSC were best fit by a 2-site receptor model, indicating the presence of high- and low-affinity binding sites (Figure 2 and Table 1). The Ki for the ileum was close to the Ki of high-affinity binding sites for fundus abomasi, pylorus, cecum, ELSC, whereas the Ki for the PLAC was similar to the Ki of low-affinity binding sites for aforementioned locations (Table 1). The curves of [3H]GR113808 competitive bindings were best fit for the fundus abomasi, ileum, and PLAC, with R2 values > 0.85 (Table 1).
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The specific binding of [3H]GR113808 to bovine intestinal tissues (ELSC) could not be fully saturated even using up to 10 nM [3H]GR113808 (Figure 3A). The 5-HTR4 numbers using 0.02 to 1 nM [3H]GR113808 did not differ (P = 0.17) among fundus abomasi, ileum, and ELSC (Figure 3B to 3D; Table 2). The KD did not differ significantly (P = 0.23) among the 3 locations. The Hill coefficients were ileum > fundus abomasi > ELSC (Table 2).
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There was no significant association between mRNA levels and receptor densities of 5-HTR4 in the fundus abomasi, ileum, and ELSC (r = 0.14; P = 0.71).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), and it is one of the most abundantly expressed 5-HT receptors in the bovine GIT at the mRNA level (Reist et al., 2003; Meylan et al., 2004). Further-more, various effects on intestinal motility mediated by 5-HTR4 have been documented in other species (Plaza et al., 1996; Tomita et al., 1997; Ono et al., 2005). In the current study, 5-HTR4 mRNA levels and densities have been measured in smooth muscle cells from longitudinal and circular layers, but also from nerve terminals innervating these cells. Precise localization of the receptors within the intestinal wall was not the aim of this study and would require different methods, such as immunohistochemistry, insitu hybridization, or fractionation of intestinal samples. Based on histological evaluation of the prepared samples (Ontsouka et al., 2004a), they consisted almost exclusively of smooth muscle cells; thus the interpretation of the results reported here may hold true primarily for the muscular layers of the GIT. Given the high identity between partial sequences of bovine and human 5-HTR4 (Reist et al., 2003), the binding properties of bovine 5-HTR4 were studied using the labeled 5-HTR4 antagonist [3H]GR113808, as previously done in human studies (Grossman et al., 1993; Blondel et al., 1998; Bender et al., 2000).
As expected from our previous studies (Reist et al., 2003; Meylan et al., 2004; Engel et al., 2006), mRNA for 5-HTR4 were measurable in smooth muscle tissues from the fundus abomasi, pylorus, ileum, cecum, PLAC, and ELSC in relative low abundancies because the recorded CPt values during PCR amplification were not detected below 30 cycles (details not shown). The mRNA levels of 5-HTR4 in smooth muscle tissues from the PLAC were even less than in those from ileum and ELSC. This is in contradiction with a previous study (Engel et al., 2006) in which reduced mRNA levels of 5-HTR4 had been reported in the ELSC compared with the ileum, cecum and PLAC. The discrepancy between the 2 studies may be due, at least in part, to the nature of the investigated samples. The mRNA levels of 5-HTR4 had been measured in full-thickness intestinal wall tissues in the previous study, whereas the mucosa and submucosa had been removed from investigated samples in the current study. According to studies performed previously in our laboratory (Reist et al., 2003; Meylan et al., 2004; Engel et al., 2006), it appears that mRNA synthesis, turnover rates of 5-HTR4 within intestinal layers (that is, epithelial and muscle layers), or both, differ in a site-specific manner along the bovine GIT. In the current study, the accuracy and reliability of mRNA measurements were verified by determination of the mRNA levels of 5-HTR4 in pooled brain samples, which were similar as in previous studies (Reist et al., 2003; Engel et al., 2006).
The presence of binding sites for 5-HTR4 in smooth muscle tissues from the fundus abomasi, pylorus, ileum, cecum, PLAC, and ELSC, as predicted by the mRNA analyses, were demonstrated by the inhibition of [3H]GR113808 specific binding induced by the unlabelled 5-HTR4 antagonist GR113808. The competition of [3H]GR113808 specific binding by unlabelled GR113808 on suspended membranes of smooth muscle tissues from the fundus abomasi, pylorus, cecum, and ELSC revealed the existence of high- and low-affinity binding sites. The distribution of receptor fractions corresponding to high-and low-affinity binding sites varied along the GIT. Similar curves based on [3H]GR113808 competitive binding were also seen using suspended membranes from bovine brain samples (unpublished observations). The inhibition of [3H]GR113808 specific binding on ileal membranes showed the existence of high-affinity binding sites, whereas low-affinity binding sites were not detected. In contrast, the inhibition of [3H]GR113808 specific binding in the PLAC demonstrated the existence of low-affinity binding sites only. These results indicate the presence of a heterogeneous population of 5-HTR4 binding sites in bovine gastrointestinal tissues, and tissue-specific distribution of low- and high-affinity binding sites. Although the precise nature of low-affinity binding sites is not determined, this situation may imply a variable importance (in a location-specific manner) of 5- HTR4 with regard to the function of smooth muscle cells for digestive functions along the GIT. Organ bath studies performed in species other than cattle (Kaumann, 1993; Prins et al., 1999, 2000a) showed that 5-HTR4 is present at nanomolar concentrations. Based on radioligand binding methods in the current study, the obtained Ki values of high-affinity binding sites were also in the nanomolar range. Interestingly, competitive binding of [3H]GR113808 by another 5-HTR4 antagonist, RS23597-190, on colonic membranes during the preliminary studies was inhibited according to a 2-site receptor model and the Ki values remained in a range as reported in the current study (unpublished observations), supporting data using unlabelled GR113808. However, the binding of [3H]GR113808 was also competitively inhibited in a 1-site receptor model by 5-HT with Ki values in the millimolar range (unpublished observations). Together, these observations imply the presence of high- and low-affinity binding sites.
The presence of binding sites corresponding to 5-HTR4 in muscle tissues along the bovine GIT is per se of importance because the mRNA levels of 5-HTR4 have been found to be reduced in ELSC of cows suffering from CDD in comparison with healthy animals (Engel et al., 2006). However, it is not clear whether the reduced 5-HTR4 mRNA levels in diseased cows primarily resulted from downregulation of receptor mRNA expression in cells situated in muscle layers (smooth muscle cells directly, cells of nerve terminals at neuromuscular junctions, or both) or in the epithelial layers because full-thickness specimens were used. In contrast, in the current study, the mucosa was scraped off because we thought that the receptors situated in the smooth muscle layers, rather than in the mucosa, are likely directly involved in the regulation of GIT motility. Nevertheless, the downregulation of 5-HTR4 mRNA expression observed in the ELSC of cows with CDD, compared with healthy cows, is of great interest because myoelectric studies of intestinal activity in dairy cows after surgical correction of CDD have shown that delayed recovery or relapse after CDD is likely due to a motility disturbance not in the cecum itself, but in more distal segments of the intestine, i.e., in the spiral colon (Stocker et al., 1997). Therefore, differences in mRNA expression levels in the ELSC between healthy animals and those with CDD indicated that 5-HTR4 may be implicated in the pathogenesis of the disease. Taken together, the findings of previous reports and of the current study indicate the involvement of 5-HTR4 in the regulation of intestinal motility in dairy cows, in health and disease. However, potential differences between tissues from healthy and diseased animals now need to be investigated at the protein level, even more because no significant correlation between mRNA and protein expression levels was demonstrated for 5-HTR4 in the current study. Indeed, the confirmation of the presence of 5-HTR4 binding sites in the bovine GIT indicates that prokinetic drugs acting at 5-HTR4 may be of use to stimulate the motility of the bovine GIT and thus prevent the occurrence, relapse, or both of motility disorders such as CDD.
In preliminary studies, Bmax of 5-HTR4 could not be accurately measured because the specific binding of [3H]GR113808 to 5-HTR4 was not fully saturated even using up to 10 nM [3H]GR113808. The situation in the bovine is thus greatly different from humans, where saturation binding of 5-HTR4 was fully completed with lesser amounts of [3H]GR113808 (Bender et al., 2000; Bach et al., 2001). The reasons for such discrepancies are unknown. Although we have previously reported a high similarity between human and bovine sequences of 5-HTR4 (Reist et al., 2003), this comparison was based on the alignment of partial coding sequences in a well-conserved region. Indeed, modifications in 5-HTR4 due to splicing variations are located toward the C-terminal tails, as reported in other species (Blondel et al., 1998; Claeysen et al., 1999). In human tissues, variants of 5- HTR4 (a to h) were identified with varying mRNA levels and functions not only between each other but between tissues as well (Bender et al., 2000). The competitive binding of [3H]GR113808 in cloned human 5-HTR4a and 5-HTR4b revealed nearly a similar pharmacological profile of these variants or variations in binding affinity for [3H]GR113808, with 5-HTR4b > 5-HTR4a (Bender et al., 2000; Bach et al., 2001). Moreover, variations may additionally occur within the coding sequence of 5-HTR4 as demonstrated in the porcine species (Ullmer et al., 1995). Based on this information, it cannot be excluded that differential splicing may occur in the 7 transmembrane spanning regions, which presumably contain the ligand binding domain. This may influence binding to the G-protein, as it has been demonstrated for other G-protein coupled receptors (Spengler et al., 1993). In this respect, neither our previous nor the present studies have allowed to gain more insight into this point because appropriate additional investigations targeted at 5-HTR4 variants were not performed in the context of the current study.
Given that competitive binding of [3H]GR113808 demonstrated the presence of high- and low-affinity binding sites, we assessed receptor densities using lesser amounts of [3H]GR113808 in order to mainly measure and compare high-affinity binding sites in the fundus abomasi, ileum, and ELSC. Our data clearly showed that densities of 5-HTR4 and the affinity (KD) of [3H]GR113808 did not differ among muscle layers from the fundus abomasi, ileum, and ELSC, but KD values in our study were 14- to 20-fold less than in a study with human heart tissue (Bach et al., 2001) and 2- to 4-fold less than in a study with human cortex (Lai et al., 2003). Moreover, Hill coefficients for samples from the ileum were very close to unity, demonstrating the interaction of [3H]GR113808 with a single population of binding sites. In contrast, the Hill coefficients of the saturation binding in the fundus abomasi and ELSC (fundus abomasi > ELSC) were only moderately close to 1.
Our study also showed that 5-HTR4 mRNA levels and the corresponding receptor protein densities in the ileum, fundus abomasi, and ELSC were not associated. This may indicate the occurrence of posttranslational modifications of 5-HTR4. Nevertheless, our results might be biased by the fact that the saturation binding assays using 0.02 to 1 nM [3H]GR113808 likely allowed mainly for binding of high-affinity sites; in contrast, the mRNA analyses indistinctly recorded mRNA corresponding to both low- and high-affinity binding sites.
In conclusion, 5-HTR4 receptor mRNA and binding ites are present in the smooth muscle layers of the bovine GIT and may play a role with respect to gastrointestinal motility. The effects of this receptor subtype may be different among GIT locations due to differences in the amount of high- relative to low-affinity binding sites. The 5-HTR4 mRNA levels did not mirror the receptor densities at the protein level.
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Footnotes |
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3 Present address: Veterinary Physiology, Vetsuisse Faculty, University of Berne, Switzerland.
2 Corresponding author: edgar.ontsouka{at}physio.unibe.ch
Received for publication April 10, 2006. Accepted for publication July 28, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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- and ß-adrenergic receptor subtypes in the mammary gland of dairy cows. Domest. Anim. Endocrinol. 24:123–135.[CrossRef][Medline]-adrenoceptors in young cattle. Domest. Anim. Endocrinol. 30:170–184.[CrossRef][Medline]
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