• View in gallery
    Figure 1—

    Specific binding of 1nM of 3H-RX821002 to increasing concentrations of pooled, suspended smooth muscle specimens from the bovine spiral colon incubated at 23°C for 15 minutes.

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    Figure 2—

    Competition of specific binding of 3H-RX821002 to membrane preparations from the ileum (A) and spiral colon (B). Suspended ileal and colonic smooth muscle specimens were incubated with 0.25nM of 3H-RX821002 and various concentrations of blocking agents of α1,2- (phentolamine, closed squares), α1A-(5-methylurapidil, open squares), α2AD-(BRL44408, closed inverted triangles), α2B-(imiloxan; closed circles; prazosin, open circles), and α2C-(MK-912, closed diamonds; rauwolscine, open triangles) AR subtypes. Data are the mean values of 6 independent experiments performed with individual ileal and colonic membranes.

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    Figure 3—

    Saturation of specific binding of 3H-RX821002 to α2AD-(open circles), α2B- (inverted open triangles), and α2C-(open squares) AR subtypes on membrane preparations from the bovine ileum (A; dashed line) and spiral colon (B; solid line) from a representative cow. Specific binding was calculated by subtracting the nonspecific binding (as determined by incubation of 3H-RX821002 with membrane protein in the presence of excessive amounts of BRL44408, imiloxan, and MK-912 that have high selectivity and blocking capacity for α2AD-, α2B-, and α2C-AR subtypes, respectively) from total binding (as determined by total incubation of 3H-RX821002 with tissue protein in the absence of receptor subtype-selective blocking agents).

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mRNA expression and binding sites for α2-adrenergic receptor subtypes in muscle layers of the ileum and spiral colon of dairy cows

Edgar C. OntsoukaClinic for Ruminants, Vetsuisse Faculty of Berne, and the 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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Jürg W. BlumDivision of Nutrition and Physiology, Institute of Animal Genetics, Nutrition, and Housing, Vetsuisse Faculty, University of Berne, CH-3012 Berne, Switzerland.

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Adrian SteinerClinic for Ruminants, Vetsuisse Faculty of Berne, University of Berne, CH-3012 Berne, Switzerland.

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Mireille MeylanClinic for Ruminants, Vetsuisse Faculty of Berne, University of Berne, CH-3012 Berne, Switzerland.

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Abstract

Objective—To measure maximum binding capacity (Bmax) and levels of mRNA expression for α2-adrenergic receptor (AR) subtypes in ileal and colonic muscle layers of healthy dairy cows.

Sample Population—Ileal and colonic muscle specimens from 6 freshly slaughtered cows.

Procedures—Ileal and colonic muscle layers were obtained by scraping the mucosa and submucosa from full-thickness tissue specimens. Level of mRNA expression for α2-AR subtypes was measured by real-time reverse transcriptase-PCR analysis and expressed relative to the mean mRNA expression of glyceraldehyde phosphate dehydrogenase, ubiquitin, and 18S ribosomal RNA. Binding studies were performed with tritiated RX821002 (3H-RX821002) and subtype-selective ligands as competitors.

Results—mRNA expression for α2AD-, α2B-, and α2C-AR subtypes was similar in ileal and colonic muscle layers. The mRNA expression for α2AD-AR was significantly greater than that for α2B- and α2C-AR subtypes, representing 92%, 6%, and 2%, respectively, of the total mRNA. Binding competition of 3H-RX821002 with BRL44408, imiloxan, and MK-912 was best fitted by a 1-site model. The Bmax of α2AD- and α2C-AR sub-types was greater than that of α2B-AR. The Bmax and level of mRNA expression were only correlated (r = 0.8) for α2AD-AR. Ratio of Bmax to mRNA expression for α2C-AR was similar to that for α2B-AR, but significantly greater than for α2AD-AR.

Conclusions and Clinical Relevance—Subtypes of α2-AR in bovine intestinal muscle layers are represented by a mixture of α2AD- and α2C-ARs and of α2B-AR at a lower density. Information provided here may help in clarification of the role of AR subtypes in α2-adrenergic mechanisms regulating bovine intestinal motility.

Abstract

Objective—To measure maximum binding capacity (Bmax) and levels of mRNA expression for α2-adrenergic receptor (AR) subtypes in ileal and colonic muscle layers of healthy dairy cows.

Sample Population—Ileal and colonic muscle specimens from 6 freshly slaughtered cows.

Procedures—Ileal and colonic muscle layers were obtained by scraping the mucosa and submucosa from full-thickness tissue specimens. Level of mRNA expression for α2-AR subtypes was measured by real-time reverse transcriptase-PCR analysis and expressed relative to the mean mRNA expression of glyceraldehyde phosphate dehydrogenase, ubiquitin, and 18S ribosomal RNA. Binding studies were performed with tritiated RX821002 (3H-RX821002) and subtype-selective ligands as competitors.

Results—mRNA expression for α2AD-, α2B-, and α2C-AR subtypes was similar in ileal and colonic muscle layers. The mRNA expression for α2AD-AR was significantly greater than that for α2B- and α2C-AR subtypes, representing 92%, 6%, and 2%, respectively, of the total mRNA. Binding competition of 3H-RX821002 with BRL44408, imiloxan, and MK-912 was best fitted by a 1-site model. The Bmax of α2AD- and α2C-AR sub-types was greater than that of α2B-AR. The Bmax and level of mRNA expression were only correlated (r = 0.8) for α2AD-AR. Ratio of Bmax to mRNA expression for α2C-AR was similar to that for α2B-AR, but significantly greater than for α2AD-AR.

Conclusions and Clinical Relevance—Subtypes of α2-AR in bovine intestinal muscle layers are represented by a mixture of α2AD- and α2C-ARs and of α2B-AR at a lower density. Information provided here may help in clarification of the role of AR subtypes in α2-adrenergic mechanisms regulating bovine intestinal motility.

Epinephrine released from the adrenal medulla, norepinephrine released by the sympathetic nervous system, and synthetic α- and β-adrenergic receptor agonists influence various functions upon binding to the specific receptors that are widely distributed in the GIT. The α2-AR subtypes are G-protein–coupled receptors mediating primarily the inhibition of adenylate cyclase.1–3 The α2-AR located on intestinal smooth-muscle and epithelial cells mediate intestinal motility and regulate growth and secretory function of epithelial cells.4–8 Furthermore, the GIT is supplied by sympathetic noradrenergic terminals that express α2-AR. As shown in various species, these receptors are also present in cholinergic neurons, mediating the inhibition of GIT motility by inhibiting the release of acetylcholine.5,9–11

Mammals possess 3 genetic α2-AR subtypes (α2A, α2B, and α2C). The subtype α2A (present in humans, rabbits, and pigs) is identical with the α2D subtype (present in rats, mice, guinea pigs, and cattle); these are interspecies variants of the same receptor.12–14 The designation α2AD-AR is generally used in cattle.13,15 Several ligands with variable α2-AR subtype selectivity, as characterized predominantly in humans, mice, and rats, are available and allow the study of these subtypes at the protein level and investigation of their biological effects.

Studies16–22 performed in ruminants, including cattle, have demonstrated that α2-adrenergic agonists such as xylazine inhibit motility of the reticulorumen. In contrast, the role of these receptors in the regulation of bovine intestinal motility is poorly understood. Nevertheless, motility disturbances such as CDD are highly prevalent in dairy herds. A myoelectric study23 of the intestinal activity in lactating dairy cows after surgical correction of CDD has shown that delayed recovery or relapse after CDD might likely be the result of motility disturbances that are not in the cecum itself but in more distal segments of the intestine (ie, in the spiral colon). However, myoelectric activity observed in the bovine large intestine is initiated or modulated by motility patterns in the distal portion of the small intestine (ie, in the ileum).24 Therefore, because α2-ARs may be involved in the pathogenesis of CDD, investigation of these receptors in the intestine, mainly in the ileum and spiral colon, of healthy cows is of importance to further assess their role, especially in diseased cows.

Recently, by use of real-time RT-PCR analysis, we have reported site-specific distribution and variable abundance of mRNA coding for AR subtypes of the α1, α2-, and β-AR families in full-thickness specimens of the bovine GIT.15 However, caution is warranted for the interpretation of these results with regard to the effects of uneven mRNA distribution in the various GIT segments on motility, because the measured mRNA had been extracted from full-thickness samples from the abomasal and intestinal walls. Thus, mRNA from different cell types was measured (eg, also from epithelial cells, which are likely not directly involved in the regulation of GIT motility).25,26 Furthermore, mRNA expression does not necessarily reflect receptor protein expression.27 Therefore, combined investigations of α2-AR at the mRNA and protein levels in intestinal muscle layers were warranted to better assess their role in the regulation of GIT motility.

To our knowledge, radioligand binding studies evaluating the densities of individual α2-AR subtypes in bovine GIT tissues are not available to date. Therefore, the purpose of the study reported here was to measure mRNA expression for α2AD-, α2B-, and α2C-ARs and the corresponding abundance of binding sites in muscle layers of the ileum and spiral colon of healthy dairy cows. The hypotheses tested were that the expression of α2-AR subtypes (1) differs between tissues and (2) differs within the α2-AR family and (3) that associations exist between the expression of receptor subtypes at the mRNA and protein levels.

Materials and Methods

Sample preparation— Six healthy dairy cows that had been sold for slaughter were included in the present study. The protocol was approved by the Research Board of the Vetsuisse Faculty. Segments (approx 15 cm in length) of full-thickness samples from the ileum and external loop of the spiral colon were collected within minutes after stunning for slaughter and placed onto a solid support that was sub-merged with chilled 1X PBS solution (pH, 7.4). The mucosal and submucosal layers were immediately scraped to remove the mucosa and submucosa from the muscle layers. Each individual specimen of muscle tissue was evaluated histologically according to the previously described procedure by use of a grid method.28 Prepared muscle tissues consisted of > 97% of smooth muscle cells (data not shown) and were subsequently used for receptor analysis at the mRNA and protein levels.

After collection and preparation, 200 mg of ileal and colonic muscle specimens was immediately transferred into RNA stabilization buffera and stored at 4°C for 24 hours, followed by freezing at −20°C until RNA processing.

Furthermore, an additional 15 g of ileal and colonic muscles was collected for the radioligand binding studies and stored in ice-cold 50mM Tris HCl buffer (pH, 7.4) containing 6mM MgCl2 and 1mM EGTA, and supplemented with a protease inhibitory cocktail.b

Real-time RT-PCR analysis of α2-AR subtypes and house-keeping genes—Materials and procedures for RNA extraction and quantification and for RT-PCR analysis were as described previously.29,30 Primers used for the amplification of bovine α2AD-, α2B-, and α2C-ARs of GAPDH, 18S ribosomal RNA, and ubiquitin (housekeeping genes) were determined from earlier publications.13,29 The mRNA expression of α2-ARs and house-keeping genes was measured during 3 independent runs. However, PCR analysis of ileal and colonic muscle specimens was always performed simultaneously within a run. During PCR analysis, a pooled complementary DNA sample of bovine brain specimens from another study was included as a positive control, which allowed for verification of the specificity of amplified products and for correction of interrun variation. An additional negative control (non–reverse-transcribed RNA) was added to make sure that no genomic DNA was amplified.

Quantity of mRNA coding for α2AD-, α2B-, and α2C-ARs was expressed relative to the mean amount of mRNA expression of multiple housekeeping genes, as described previously.31 In brief, the recorded CPs during PCR amplification of α2-AR subtypes and housekeeping genes were transformed into relative quantities as described by other investigators.32 The CP represents the amplification cycle number at which the measured signal becomes significantly higher than the background and is detectable. Then, the geNorm algorithm, which relies on the principle that the expression ratio of 2 ideal housekeeping genes must be identical among samples, was used to calculate the normalization factor for each sample as a mean pairwise variation between a particular housekeeping gene and all others. The amount of mRNA expression of α2-AR subtypes in each individual sample was obtained by dividing their respective quantities by the normalization factor.

Binding studies for α2-AR subtypes—The preparation of receptor membrane suspensions from ileal and colonic muscle specimens was performed as recently described.30,33 The α2-AR binding was studied by use of 3H-RX821002,c a selective α2-adrenergic antagonist.26,34–37 The linearity of 3H-RX821002 binding in relation to the concentration of intestinal membrane suspension was tested to define the optimal membrane concentration in the linear range for later application in the α2-AR binding assays.

Competitive binding assays—Assays were performed in triplicate to characterize the α2-AR subtypes. Membrane suspensions (100 μL) from each cow were incubated with a fixed concentration of 3H-RX821002 (50 μL; 0.25nM) and various concentrations (ranging from 10−10.5 to 10−6 M; 50 μL) of several α2-AR ligands,d namely BRL44408 (α2AD), imiloxan (α2B), MK-912 (α2C), prazosin (α2B), rauwolscine (α2C), and phentolamine (α1 and α2), which were used as competitors at 23°C for 15 minutes under constant shaking. In addition, the inhibitory potency of 5-methylurapidil,d an α1A-AR blocking agent, was also tested under the described conditions. All substances were diluted in Tris-HCl buffer, as described. The termination of binding and filtration, and the determination of bound tritium activity, was as described previously.30,33

The Ki of individual competitors was calculated by use of the following equation38:

article image

The IC50 values were evaluated by use of a weighted least squares curve fitting with a commercial software program.e The KD was the Ki of 3H-RX821002 derived from saturation binding assays, and C was the concentration of 3H-RX821002 used in these assays.

Saturation binding assays—The saturation binding assays of 3H-RX821002 were performed to determine the binding capacities at α2AD-, α2B-, and α2C-AR sites. Membrane suspensions (100 mL) were incubated with increasing concentrations of 3H-RX821002 (ranging from 0.03 to 2nM) with or without excessive amounts of chosen α2AD-, α2B-, and α2C-AR–selective blocking agents (competitors), BRL44408 (50 μL; 60nM), imiloxan (50 μL; 2μM), and MK-912 (50 μL; 20nM), respectively. The aforementioned concentrations were 20 times higher than the calculated Ki of competitors on the basis of competition binding assays and served to calculate nonspecific binding to respective α2-AR subtypes. The specific binding was calculated by subtracting nonspecific binding (after incubation with excessive concentrations of BRL44408, imiloxan, or MK-912) from total binding (after incubation without these competitors).

Procedures used to determine radioligand binding were similar to those described for competitive binding assays. Specific binding to α2AD-, α2B-, and α2C-AR represented 73%, 61%, and 67% of total binding, respectively, and was expressed as femtomoles of bound 3H-RX821002 per milligram of membrane protein. Equilibrium binding data were plotted as a function of 3H-RX821002 concentration (saturation curve). The Bmax and KD were calculated with a weighted least squares curve fitting by use of a commercial software program.e

Statistical analysis—For evaluation of results, a commercial software program was used.f Data were log10 transformed to reach normal distribution prior to analysis. Differences in mRNA expression, Bmax, and KD between the ileum and colon were identified by use of the paired t test. Differences in mRNA expression, Bmax, and KD among α2-AR subtypes were localized by use of a 2-way ANOVA and the mixed-model procedure.f The model was as follows:

article image

where Yijk represents the measured value, μ represents the general mean, subtype represents the receptor subtype, tissue represents the location in the intestine (ie, ileum or spiral colon), subtype × tissue represents the interaction between receptor and intestinal tissue, and eijk represents the residual error. The receptor subtype and tissue were used as fixed effects, and the individual cows were used as random effects. Correction according to Bonferroni was used to adjust for repeated testing. Pearson correlation coefficientsf were used to calculate correlations between mRNA expression and Bmax values within α2-AR subtypes. Data are presented as means ± SEM; the level of significance was set at P < 0.05.

Results

mRNA Expression for α2-AR subtypes and for housekeeping genes—Overall analysis revealed a significant (P = 0.002) effect of receptor subtypes, without a significant effect of tissue or tissue × receptor interaction. The mRNA expression of α2AD-, α2B-, and α2C-ARs did not differ between muscle preparations from the ileum or spiral colon (Table 1). However, mRNA expression for α2AD-AR was significantly higher than that for α2B- and α2C-ARs (P = 0.005 for α2B and P = 0.002 for α2C). The α2AD-AR subtype accounted for 92%, the α2B-AR subtype for 6%, and the α2C-AR sub-type for 2% of the total α2-AR mRNA. Mean amounts of GAPDH, ubiquitin, and 18S ribosomal RNA in ileal versus colonic muscle specimens were closely correlated (data not shown; r = 0.87; P = 0.001).

Table 1—

Mean ± SEM (n = 6) mRNA expression for α2-AR subtypes in bovine intestinal muscle tissues.*

α2-AR subtypesMuscle tissue
IleumSpiral colon
α2AD0.892 ± 0.311a0.680 ± 0.064a
α2B0.087 ± 0.039b0.036 ± 0.014b
α2C0.015 ± 0.006b0.022 ± 0.004b

mRNA expression is expressed relative to the mean mRNA expression of GAPDH, ubiquitin, and 18S ribosomal RNA.29

Different superscript letters within a row or column indicate significant (P < 0.05) differences between the ileum and spiral colon for a particular α2-AR subtype (row) or amongα2-AR subtypes within a particular tissue (column), respectively.

Binding studies—A strong positive correlation (R2 = 0.996) was found between the specific binding of 3H-RX821002 with various concentrations of colonic muscle tissue membranes (Figure 1). On the basis of these findings, the membrane preparation at 1 mg/mL, lying within the linear range, was applied for all α2-AR binding assays. Similar results were obtained with ileal membrane suspensions (data not shown).

Figure 1—
Figure 1—

Specific binding of 1nM of 3H-RX821002 to increasing concentrations of pooled, suspended smooth muscle specimens from the bovine spiral colon incubated at 23°C for 15 minutes.

Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1883

Competition binding assays—The specific binding of 3H-RX821002 to α2-AR was inhibited by various α2-AR blocking agents, but 5-methylurapidil, a blocking agent for α1A-AR, failed to inhibit 3H-RX821002 binding at concentrations up to 10−6 M (Figure 2). Binding curves derived from ileal and colonic membrane data were best fitted by a 1-site receptor model. The tested blocking agents differed slightly in their potency to inhibit the specific binding of 3H-RX821002 to α2-AR (Table 2). However, Ki values did not differ between the ileum and colon.

Figure 2—
Figure 2—

Competition of specific binding of 3H-RX821002 to membrane preparations from the ileum (A) and spiral colon (B). Suspended ileal and colonic smooth muscle specimens were incubated with 0.25nM of 3H-RX821002 and various concentrations of blocking agents of α1,2- (phentolamine, closed squares), α1A-(5-methylurapidil, open squares), α2AD-(BRL44408, closed inverted triangles), α2B-(imiloxan; closed circles; prazosin, open circles), and α2C-(MK-912, closed diamonds; rauwolscine, open triangles) AR subtypes. Data are the mean values of 6 independent experiments performed with individual ileal and colonic membranes.

Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1883

Table 2—

Mean ± SEM (n = 6) Ki values of several ligands forα2-AR subtypes in bovine intestinal muscle tissues.*

Ligands (nmol/L)Ki value
IleumSpiral colon
Phentolamine2.7 ± 0.42.7 ± 0.6
BRL444082.3 ± 0.72.1 ± 0.7
Imiloxan13 ± 1013 ± 10
Prazosin120 ± 100110 ± 100
MK-9120.55 ± 0.090.78 ± 0.06
Rauwolscine12 ± 089 ± 1

IC50 values were converted to Ki values by use of KD values of 0.25nM as derived from saturation binding assays.

Saturation binding assays—The specific binding 3H-RX821002 to α2-AR subtypes on membrane preparations from the ileum and spiral colon was saturable (Figure 3). The Bmax for α2AD-, α2B-, and α2C-ARs did not differ between the ileum and spiral colon (Table 3 and 4). However, Bmax for α2AD- and α2C-ARs was higher than that for α2B-AR (P = 0.044 for α2AD and P = 0.053 for α2C). The KD value for α2B-AR was significantly (P = 0.028 for α2AD and P = 0.042 for α2C) lower than that for α2AD- and α2C-ARs.

Figure 3—
Figure 3—

Saturation of specific binding of 3H-RX821002 to α2AD-(open circles), α2B- (inverted open triangles), and α2C-(open squares) AR subtypes on membrane preparations from the bovine ileum (A; dashed line) and spiral colon (B; solid line) from a representative cow. Specific binding was calculated by subtracting the nonspecific binding (as determined by incubation of 3H-RX821002 with membrane protein in the presence of excessive amounts of BRL44408, imiloxan, and MK-912 that have high selectivity and blocking capacity for α2AD-, α2B-, and α2C-AR subtypes, respectively) from total binding (as determined by total incubation of 3H-RX821002 with tissue protein in the absence of receptor subtype-selective blocking agents).

Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1883

Table 3—

Mean ± SEM (n = 6) binding characteristic values of α2-AR subtypes in bovine intestinal muscle tissues.

Binding characteristicsMuscle tissue
IleumSpiral colon
Bmax
 α2AD-AR (fmol/mg protein)83 ± 24a76 ± 11a
 α2B-AR (fmol/mg protein)48 ± 12b46 ± 8b
 α2C-AR (fmol/mg protein)86 ± 22a69 ± 11a
KD
 α2AD-AR (nmol/L)0.32 ± 0.03a0.28 ± 0.03a
 α2B-AR (nmol/L)0.18 ± 0.02b0.16 ± 0.02b
 α2C-AR (nmol/L)0.30 ± 0.02a0.28 ± 0.02a

fmol = Femtomoles.

See Table 1 for remainder key.

Table 4—

Mean ± SEM (n = 6) CP values of PCR amplification of α2-AR subtypes and housekeeping genes in bovine intestinal muscle tissues.

VariableCP value
IleumSpiral colon
α2-AR subtypes
 α2AD32.11 ± 0.55a30.27 ± 0.26a
 α2B33.92 ± 0.21b34.94 ± 0.34b
 α2C35.07 ± 0.71b35.42 ± 0.43b
Housekeeping genes*
 Ubiquitin23.48 ± 0.4721.70 ± 0.22
 18S ribosomal RNA23.89 ± 0.5623.98 ± 0.61
 GAPDH25.31 ± 0.8123.45 ± 0.12

Expression levels of housekeeping genes were not analyzed for differences among tissues, as these genes are involved in various cell functions.

See Table 1 for remainder of key.

Associations between mRNA and protein expression among α2-AR subtypes—The Bmax of α2AD-AR was positively correlated (r = 0.8, P = 0.003) with mRNA expression of α2AD-AR, but the Bmax and mRNA expression of α2B-AR (r = 0.2; P = 0.5) and α2C-AR (r = −0.6; P = 0.1) were not correlated. Mean values (± SEM) of the Bmax-to-mRNA ratio were 101 ± 10, 1,507 ± 576, and 9,250 ± 5,256 for α2AD-, α2B-, and α2C-ARs, respectively. The Bmax-to-mRNA ratio for α2B- and α2C-ARs was significantly (P = 0.042 for α2B and P = 0.023 for α2C) greater than that for α2AD-AR.

Discussion

To our knowledge, this is the first report on α2AD, α2B-, and α2C-AR subtype mRNA expression in bovine intestinal muscle layers combined with radioligand binding assays. In our study, muscle layers were prepared by use of a conventional scraping method. On the basis of our experience, it is an easy and rapid way to isolate smooth-muscle specimens, which allows rapid handling of the samples to reduce RNA and protein degradation. In contrast to a previous study,15 we did not find significant differences in mRNA expression for any of the α2-AR subtypes between muscle tissue specimens from the ileum and spiral colon of cows. However, the distribution of receptor subtypes in muscle layers of our study remained similar to that seen in full-thickness tissue specimens, implying either a low abundance of receptors in the removed mucosa or a similar distribution of receptors through all intestinal layers. Indeed, in our study, the mucosal and submucosal layers had been removed from the investigated samples, whereas in the previous study,15 mRNA expression had been measured from full-thickness intestinal wall specimens. In the study reported here, the main result for mRNA expression is that the α2AD-AR is the predominant receptor, comprising > 90% of the entire α2-AR population. At the mRNA level, the α2B- and α2C-AR subtypes accounted for < 10% of the total α2-AR population (with α2B-AR > α2C-AR but no significant difference between these 2 receptors). This distribution of mRNA coding for α2-AR subtypes is in agreement with that reported for full-thickness bovine intestinal preparations or in the bovine mammary gland,13,15 but it differs markedly from the situation in the liver.30 A comparison of mRNA expression for these receptors between longitudinal and circular muscle layers or in relation to the neurones supplying the musculature was beyond the scope of this study.

The presence of receptor proteins corresponding to α2AD-, α2B-, and α2C-ARs, as predicted by the measured mRNA transcripts, was clearly confirmed by inhibition of 3H-RX821002 specific binding with the selective subtype antagonists BRL44408, imiloxan, and MK-912 for the evaluation of α2AD-, α2B-, and α2C-ARs, respectively. The resulting competition binding curves were best fitted with a 1-site receptor model, indicating the existence of single binding sites. The lig- and 3H-RX821002 had high selectivity for α2-AR, whereas the α1-adrenergic antagonist 5-methylurapidil failed to inhibit 3H-RX821002 specific binding. Moreover, the calculated Ki values were close among BRL44408, imiloxan, and MK-912, demonstrating that 3H-RX821002 had a similar affinity for α2AD-, α2B-, and α2C-ARs, respectively. These results confirm the presence of α2AD-, α2B-, and α2C-AR proteins. Given that the specific binding of 3H-RX821002 in concentrations ranging up to 2nM was saturable, this range was applied to measure the densities (Bmax values) of α2AD, α2B-, and α2C-ARs. Binding sites for α2AD-, α2B-, and α2C-ARs did not differ between muscle membranes from the bovine ileum and spiral colon. However, in contrast to results of the mRNA analyses showing a clear predominance of α2AD-AR (> 90% of the total mRNA population), Bmax of α2AD-AR was similar to that of α2C-AR, while Bmax of α2B-AR was lowest. To estimate the receptor densities, saturation-binding assays were additionally performed with other ligands (as competitors) also having high selectivity for α2B- and α2C-ARs, such as prazosin and rauwolscine, respectively. Data obtained (not shown) were similar to those reported in our study, thus supporting the results obtained with 3H-RX821002.

Although we have clearly demonstrated the presence of all 3 α2-AR subtypes in bovine intestinal smooth muscle layers, the variable densities of AR subtypes might imply a variable importance with respect to motility regulation in the bovine intestine. Interestingly, the affinity of 3H-RX821002 to bovine α2-AR was similar to that reported for rats, as illustrated by the obtained KD values.36 Several studies10,11,39,40performed in laboratory animals have reported the predominance of the α2AD-AR subtype in α2-AR–mediated inhibitory functions, inclusively in the intestine. Nevertheless, in mice lacking the α2AD-AR gene, the existence of non-α2AD–AR binding sites was documented. These binding sites have been described as being either specific for α2C-AR41 or α2B-AR.42 In our study, results of the competition binding assays proved the existence of sites corresponding to all 3 genetic α2-AR subtypes. However, receptor proteins present in muscle layers of the bovine intestinal wall appeared to be predominantly a mixture of α2AD- and α2C-ARs. Interestingly, as demonstrated by KD values, the α2B-AR, which is the least expressed receptor protein, had a higher affinity to the tritiated antagonists than the 2 more abundant subtypes. The biological importance of this finding, beyond the binding characteristics of different receptor subtypes, is difficult to interpret without functional studies. As we found a positive correlation between mRNA expression and density of binding sites for α2AD-AR, but not for α2B- and α2C-ARs, this may indicate different receptor turnover rates in the α2-AR family.

The presence of these receptor proteins in ileal and colonic smooth muscle layers is in itself of great importance, as the mRNA expression of α2AD- and α2B-ARs has been found to be reduced in the intestine of cows with CDD, compared with healthy cows.43 Taken together, these findings predict the involvement of the α2-AR in the regulation of intestinal motility in health and disease.

In conclusion, no significant difference was found between the ileum and spiral colon of healthy dairy cows with respect to α2-AR mRNA and protein expression in smooth muscle tissues. The mRNA transcripts for α2AD-, α2B-, and α2C-ARs were variably expressed (ie, α2AD >> α2B > α2C) in both localizations. The mRNA expression for individual receptor subtypes did not a priori mirror the corresponding density of binding sites (ie, Bmax α2AD-AR=Bmax α2C-AR > Bmax α2B-AR). These results represent a first step in the study of the role of AR subtypes in α2-adrenergic mechanisms regulating intestinal motility in health and disease of cattle.

ABBREVIATIONS

GIT

Gastrointestinal tract

AR

Adrenergic receptor

CDD

Cecal dilation-dislocation

RT-PCR

Reverse transcriptase-PCR

GAPDH

Glyceraldehyde phosphate dehydrogenase

CP

Crossing point

3H-RX821002

Tritiated RX821002 (selective α2-adrenergic receptor antagonist)

Ki

Dissociation constant

IC50

Molar concentration of a drug that inhibits specific binding by 50%

KD

Equilibrium Ki

Bmax

Maximum binding capacity

a.

RNA later, Ambion Inc, Austin, Tex.

b.

Protease inhibitor cocktails, Roche Biochemicals, Basel, Switzerland.

c.

[3H]-RX821002 (56Ci/mmol), Amersham Bioscience, Chalfont St Giles, Buckinghamshire, UK.

d.

Grogg Chemie, Stettlen-Deisswil, Switzerland.

e.

GraphPad Software Inc, San Diego, Calif.

f.

SAS System for Windows, version 8.2, SAS Institute Inc, Cary, NC.

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Contributor Notes

Drs. Ontsouka's and Blum's present address is Veterinary Physiology, Vetsuisse Faculty, University of Berne, Switzerland.

Supported by a grant from the Department for Clinical Veterinary Medicine, Vetsuisse Faculty of Berne, University of Berne, Switzerland.

The authors thank Prof U. Honegger for technical assistance.

Address correspondence to Dr. Meylan.