Quantitative mRNA analysis of adrenergic receptor subtypes in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation

Barbara Kobel Clinic for Ruminants, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Berne, Bremgartenstrasse 109a, 3001 Berne, Switzerland.

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Ladina Engel Clinic for Ruminants, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Berne, Bremgartenstrasse 109a, 3001 Berne, Switzerland.

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Edgar C. Ontsouka Clinic for Ruminants, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Berne, Bremgartenstrasse 109a, 3001 Berne, Switzerland.
Division of Nutrition and Physiology, Institute of Animal Genetics, Nutrition and Housing,Vetsuisse Faculty, University of Berne, Bremgartenstrasse 109a, 3001 Berne, Switzerland.

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Hans U. Graber Clinic for Ruminants, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Berne, Bremgartenstrasse 109a, 3001 Berne, Switzerland.

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Jürg W. Blum Division of Nutrition and Physiology, Institute of Animal Genetics, Nutrition and Housing,Vetsuisse Faculty, University of Berne, Bremgartenstrasse 109a, 3001 Berne, Switzerland.

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Adrian Steiner Clinic for Ruminants, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Berne, Bremgartenstrasse 109a, 3001 Berne, Switzerland.

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Mireille Meylan Clinic for Ruminants, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Berne, Bremgartenstrasse 109a, 3001 Berne, Switzerland.

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Abstract

Objective—To investigate the distribution of mRNA coding for 9 adrenoceptor subtypes in the intestines of healthy dairy cows and cows with cecal dilatationdislocation (CDD).

Sample Population—Full-thickness specimens of the intestinal wall were obtained from the ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy (control) cows (group 2, specimens collected during laparotomy; group 3, specimens collected after slaughter).

Procedures—Concentrations of mRNA for 9 adrenoceptor subtypes (α1A, α1B, α1D, α2AD, α2B, α2C, β1, β2, and β3) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed relative to mRNA expression of a housekeeping gene.

Results—Expression of mRNA for α1B-, α2AD-, α2B-, β1-, and β2-adrenoceptors was significantly lower in cows with CDD than in control cows. In the ileum, these receptors all had lower mRNA expression in cows with CDD than in control cows. The same effect was detected in the ELSC for mRNA for α2AD-, α2B-, β1-, and β2-adrenoceptors, and in the cecum and PLAC for α2B- and β2-adrenoceptors. Groups did not differ significantly for α1A-adrenoceptors. The mRNA expression for α1D-, α2C-, and β3-adrenoceptors was extremely low in all groups.

Conclusions and Clinical Relevance—Differences in expression of mRNA coding for adrenoceptors, most pronounced in the ileum and spiral colon, between cows with CDD and control cows support the hypothesis of an implication of adrenergic mechanisms in the pathogenesis of CDD in dairy cows.

Abstract

Objective—To investigate the distribution of mRNA coding for 9 adrenoceptor subtypes in the intestines of healthy dairy cows and cows with cecal dilatationdislocation (CDD).

Sample Population—Full-thickness specimens of the intestinal wall were obtained from the ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy (control) cows (group 2, specimens collected during laparotomy; group 3, specimens collected after slaughter).

Procedures—Concentrations of mRNA for 9 adrenoceptor subtypes (α1A, α1B, α1D, α2AD, α2B, α2C, β1, β2, and β3) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed relative to mRNA expression of a housekeeping gene.

Results—Expression of mRNA for α1B-, α2AD-, α2B-, β1-, and β2-adrenoceptors was significantly lower in cows with CDD than in control cows. In the ileum, these receptors all had lower mRNA expression in cows with CDD than in control cows. The same effect was detected in the ELSC for mRNA for α2AD-, α2B-, β1-, and β2-adrenoceptors, and in the cecum and PLAC for α2B- and β2-adrenoceptors. Groups did not differ significantly for α1A-adrenoceptors. The mRNA expression for α1D-, α2C-, and β3-adrenoceptors was extremely low in all groups.

Conclusions and Clinical Relevance—Differences in expression of mRNA coding for adrenoceptors, most pronounced in the ileum and spiral colon, between cows with CDD and control cows support the hypothesis of an implication of adrenergic mechanisms in the pathogenesis of CDD in dairy cows.

Cecal dilatation-dislocation is a disorder that primarily affects dairy cows. It has been described numerous times in cows in Europe and North America.

It is a common and economically important disease, and the prevalence of CDD is similar to that of abomasal displacement in cows in Switzerland.1 Several studies2–7 have evaluated the clinical signs, prevention, and treatment of CDD, but the pathogenesis of the disease still remains poorly understood. Originally, CDD was believed to be caused primarily by motility dysfunction in the cecum, leading to accumulation of digesta and gas, and by subsequent dilatation and secondary displacement of the cecum.2,5,8,9 However, cows with delayed recovery or recurrence after surgical treatment of CDD had a motility pattern in the cecum and PLAC that was strikingly similar to that recorded in a cow with an obstruction of the distal portion of the PLAC as a result of a phytobezoar occluding a cannula implanted in that region.10,11 This led to the hypothesis that a decrease in motility in a more distal part of the intestine (ie, the spiral colon), rather than in the cecum, may be the cause of delayed recovery or recurrence after CDD and may also be implicated in the pathogenesis of the disease.10

In general, stimulation of the sympathetic nervous system causes inhibition of gastrointestinal motility in vivo and in vitro.12 These effects are believed to be mediated by inhibition of acetylcholine release from intrinsic cholinergic neurons and by a direct action on smooth-muscle cells.12,13 Adrenergic pathways are involved in the control of several key functions of the GIT, such as modulation of motility, secretion, absorption, and vascular tone,12,14–17 which leads to the assumption that the adrenergic pathways may also play a role in the pathogenesis of CDD. In fact, the role of adrenergic receptors in intestinal motility in cattle has barely been investigated. In most studies in ruminants, experiments have focused on the ruminoreticular area and were conducted mainly in small ruminants. The best known effect of α-adrenoceptors on GIT motility in ruminants is the inhibitory effect of α2-adrenoceptor agonists, such as xylazine and detomidine, on ruminoreticular motility.18–21 Even in human medicine in which the use of adrenoceptor agonists and antagonists is widespread for treatment of patients with circulatory and respiratory disorders, the role of the various adrenoceptors on GIT motility is poorly understood.12

Adrenoceptors are G-protein–coupled receptors that mediate the effects of the adrenergic system in response to activation by the endogenous catecholamines epinephrine and norepinephrine.22 They are classified in 9 subtypes (α1A, α1B, α1D, α2AD, α2B, α2C, β1, β2, and β3).23 The distribution of mRNA coding for these 9 subtypes in the GIT of healthy dairy cows has been described elsewhere.24

The objective of the study reported here was to investigate the expression of mRNA coding for the 9 adrenoceptor subtypes in the ileum, cecum, PLAC, and ELSC of dairy cows with CDD and to compare results with those obtained for healthy dairy cows. For this purpose, we used a reverse transcriptase-PCR technique for quantitative analysis of mRNA coding for bovine adrenoceptor subtypes.23 We hypothesized that the distribution of mRNA coding for adrenoceptor subtypes in intestinal tissues would differ between cows with CDD and healthy dairy cows. A confirmation of this hypothesis would indicate a possible involvement of adrenoceptor subtypes in the pathogenesis of CDD, which would open possibilities for investigations focused on treatment of CDD because selective agonists or antagonists of adrenoceptor subtypes may be of use to support or even replace invasive surgical interventions.

Materials and Methods

Sample population—Full-thickness specimens of the intestinal wall were obtained from the ileum, cecum, PLAC, and ELSC of 3 groups of cows. Group 1 comprised 15 cows with CDD. Fifteen healthy (control) cows were allocated into 2 groups (group 2, biopsy specimens of the intestinal wall collected during laparotomy; group 3, specimens of the intestinal wall collected after slaughter). Informed consent was obtained for all client-owned cows. The project was approved by the Swiss Board for Animal Welfare and Protection.

Group 1 consisted of 15 dairy cows (lactating and nonlactating) referred to the Clinic for Ruminants, Vetsuisse Faculty of Berne, Switzerland, for treatment of naturally developing CDD. Medication by veterinarians prior to referral of the cows was not homogeneous and included spasmolytics (primarily metamizol), the prokinetic drug neostigmine, purgatives such as mineral oil or salt solutions (eg, sodium sulfate), IV administration of fluids, a combination of these treatments, or no treatment and immediate referral. At our facility, the diagnosis of CDD was confirmed and the cows immediately underwent surgery. The only preoperative treatment consisted of IV administration of fluids, as needed. No pathologic findings (other than those caused by CDD) were observed during physical examination of the 15 cows of group 1. Cows with concomitant diseases in addition to CDD were not included in the study.

The 15 control cows comprised 2 groups. Group 2 consisted of 7 healthy lactating dairy cows that were part of another research project at our facility. No preoperative treatments were administered to the cows of this group. Group 3 consisted of 8 healthy dairy cows that were culled and sent to slaughter. Cows of group 3 were healthy, as assessed at time of arrival at the slaughterhouse, and had been culled for reasons not related to GIT disease.

This was not a case-controlled study; therefore, no matching was performed between cows with and without CDD. Cows in all groups were of similar age and stage of lactation, and breed distribution was similar in all groups. The 30 cows were from 30 herds, thus minimizing the risk of systematic herd effects. All cows were not allowed access to feed for several hours before sample collection (cows of group 1 because of CDD, cows of group 2 because food was removed for 24 hours before surgery, and cows of group 3 because of transport and lairage time at the slaughterhouse, where water but no food was provided).

Collection of samples—For cows of groups 1 and 2, samples of intestinal specimens were collected during routine laparotomy via the right flank with the cows in a standing position. In cows of group 1, full-thickness samples of the intestinal wall were collected during routine surgery for correction of CDD. After routine enterotomy of the cecum and drainage of cecal contents, the ileum (40 cm orad to the ileocecal valve), cecal body (midway between the ileocecal valve and cecal apex), PLAC (40 cm aborad to the ileocecal valve), and ELSC were each exteriorized through the incision. At each site, a full-thickness biopsy specimen of approximately 0.5 cm2 was dissected, and the intestinal wall was closed by use of 2 inverting Cushing sutures of polyglyconate 3-0.a The specimens were immediately rinsed with ice-cold PBS solution (pH, 7.4), stored in 2 mL of RNA stabilization solutionb at 4°C for 24 hours, and then frozen at −20°C until assayed. The abdomen was closed in a routine manner. Postoperative care consisted of administration of antimicrobials for 5 days and IV administration of fluids or spasmolytics, as needed. Cows were discharged from our facility after recovery from surgery.

For group 2, samples were obtained in a manner similar to that for group 1. Samples were collected from the same locations as described for group 1. Samples were collected during laparotomy performed as part of another research project on GIT motility in cattle. Postoperative care of cows of group 2 consisted of administration of antimicrobials for 5 days after surgery.

Because of practical constraints, we were able to perform the surgery and obtain samples from only 7 healthy cows for group 2. Therefore, another control group (group 3) was needed. Group 3 consisted of 8 healthy dairy cows, which enabled us to achieve the same number of cows with and without CDD. For group 3, tissue samples were collected from the same intestinal locations as for groups 1 and 2. Samples were obtained within minutes after the cows were stunned at a local slaughterhouse . For each location, a fullthickness specimen of approximately 0.5 cm2 was immediately dissected, rinsed with PBS solution, and stored in RNA stabilization solution,b as described for group 1.

Experimental procedure—Specimens for all groups were processed in the same manner. Total cellular RNA from the specimens was isolated by homogenizing the tissues for 5 minutes by use of a glass-bead cell disrupterc in 1 mL of extraction solutiond/80 to 100 mg of tissue, with a total of 180 to 200 mg of tissue. Specimens were then incubated for 10 minutes at ambient temperature. We then added 200 μL of chloroform/mL of extraction solution.d Specimens were vortexed for 15 seconds, and the tissue homogenates were allowed to sit for 10 minutes at ambient temperature. Homogenates were centrifuged (12,000 X g for 15 minutes at 4°C). The RNA in the upper aqueous phase (approx 400 μL) was transferred to new 1.5-mL tubes, precipitated by the addition of 500 μL of iso-2-propanol, incubated for 15 minutes at ambient temperature, and centrifuged at 12,000 X g for 10 minutes at 4°C. Supernatant was decanted, and RNA pellets were washed twice with 75% ethanol; each wash was followed by centrifugation at 9,200 X g for 8 minutes at 4°C. The pellets were completely dried during 5 minutes at ambient temperature and then diluted in 25 μL of RNase-free water. Total extracted RNA was quantified by use of UV spectroscopye (optical density, 260 nm), and the stock solution was diluted by the addition of RNase-free water to create a working solution of 100 ng/μL. Quality of recovered RNA was considered acceptable25 when the value for the optical density at 260 nm divided by the optical density at 280 nm was > 1.9.

Synthesis of first-strand cDNA was performed as described elsewhere23 by use of 200 units of reverse transcriptasef and 100 pmol of random hexamer primers.g The final concentration of cDNA was 25 ng/μL. Primer (forward and reverse) sequences of the adrenoceptor subtypes (α1A, α1B, α1D, α2AD, α2B, α2C, β1, β2, and β3) were obtained from another publication.23 A master mix was prepared with 6.4 μL of H2O, 1.2 μL of 4mM MgCl2, 0.2 μL of 4-pmol forward primer, 0.2 μL of 4-pmol reverse primer, and 1.0 μL of DNA binding dye.h Nine microliters of the master mix was placed in glass capillaries, and 1 μL of reverse-transcribed RNA was added as a PCR template. Product-specific PCR cycle conditions for all adrenoceptor subtypes and the housekeeping gene (ie, GAPDH) were as described elsewhere.23 After the last amplification cycle, PCR products were controlled by use of a melting curve analysis to ensure the specific amplification products were generated.

The quantification of mRNA coding for the housekeeping gene GAPDH was used as a reference to standardize gene expression for the investigated cDNA samples. Amounts of GADPH were determined for each cow and each tissue sample. Expression of mRNA coding for the adrenoceptors was reported as the percentage of mRNA expression for GADPH, which was calculated as follows:

article image

where CPAR is the crossing point for adrenoceptor amplification and CPGAPDH is the crossing point for GAPDH amplification.23 Reverse-transcribed RNA of pooled brain tissues (cortex, thalamus, and hypothalamus) from healthy dairy cows was added to each PCR assay to monitor variation among assays (interassay control samples), as described elsewhere.25,26

Statistical analysis—For all parametric statistical analyses, mRNA expression of each adrenoceptor subtype was logarithmically transformed to obtain a normal distribution. Correctness of the transformations was verified by use of quantile-versus-quantile plots and Lilliefors tests. Preliminary statistical evaluation of the results revealed significant differences between groups 2 and 3 (healthy cows for which samples were collected during laparotomy or after slaughter); thus, data for the 2 control groups could not be pooled for comparison with data for group 1 (cows with CDD), and further statistical analysis was performed for each group separately. To compare the logarithm of mRNA expression among various tissues and groups, a 2-factor ANOVA was performed for each adrenoceptor subtype with repeated measurements on tissue and fixed effects on group.

The general linear model approach was used in combination with weighted least squares. Weights were introduced to correct for the unequal variances that were observed for the 3 groups. Fit of the models was evaluated by plotting the residuals versus the fitted values and by use of quantile-versus-quantile plots of the residuals. Factors were compared by calculating the difference of the corresponding factor effects together with the corresponding SD. These values were then used to perform t tests. For a single tissue, the logarithmic expression of group was evaluated by use of a single-factor ANOVA. Degrees of freedom were adjusted in accordance with the method of Welch because variances of the 3 groups were unequal. Pairwise comparisons were then performed by use of t tests with nonpooled SD. Significance was defined as values of P ≤ 0.05. For all statistical analyses in which multiple testing was necessary, P values were adjusted in accordance with the method of Holm. Statistical software packagesi,j were used for computations.

Results

Relative expression of mRNA coding for adrenoceptors in the intestines of cows was low in all 3 groups and all tissues (Table 1). Relative abundance of mRNA coding for α1D-, α2C-, and β3-adrenoceptor subtypes was extremely low (CPAR values ranging from 33 to 35 cycles). Thus, statistical analysis was not performed for these 3 adrenoceptor subtypes because sufficient precision of the results was not warranted at these extremely low amounts of expression. Distribution of mRNA expression for the remaining adrenoceptor subtypes was plotted (Figure 1).

Table 1—

Relative amounts of mRNA coding for adrenoceptor subtypes in the intestinal tract of 15 cows with CDD (group 1) and 15 healthy dairy cows (groups 2 and 3).*

Intestinal locationAdrenoreceptor subtypeGroup 1Group 2Group 3
Ileum
α1A0.0012–0.03470.0010–0.02590.0020–0.0072
α1B0.0361–0.13090.1896–0.38040.2468–0.4009
α1D0.0900–0.17190.0837–0.37750.2929–0.4439
α2AD0.3448–1.71661.4500–2.56481.9648–4.0471
α2B0.0058–0.01950.0769–0.15160.1408–0.3624
α2C0.0049–0.01190.0057–0.02790.0370–0.0518
β10.0356–0.11980.1670–0.27800.1719–0.2553
β20.2201–0.69641.5383–2.38621.3721–2.5237
β30.0100–0.05350.0359–0.10380.0281–0.0666
Cecum
α1A0.0034–0.01690.0037–0.01650.0009–0.0030
α1B0.0972–0.20190.1100–0.26210.1397–0.2172
α1D0.0487–0.14910.0741–0.17810.0815–0.1980
α2AD0.3988–0.60030.4496–0.59410.4851–0.6744
α2B0.0059–0.01090.0234–0.04130.1203–0.1683
α2C0.0046–0.02230.0146–0.02190.0155–0.0543
β10.0731–0.31820.1492–0.30040.1810–0.2971
β20.3645–0.73401.7664–2.18450.8942–1.7679
β30.0068–0.03370.0232–0.06310.0154–0.0521
PLAC
α1A0.0046–0.03430.0018–0.00640.0020–0.0098
α1B0.0841–0.23100.1386–0.52140.1504–0.2005
α1D0.0477–0.16070.0711–0.22280.0712–0.1465
α2AD0.3476–0.81170.4069–0.64120.7189–0.9021
α2B0.0096–0.02780.0271–0.04630.0794–0.1179
α2C0.0095–0.01470.0222–0.06640.0205–0.0572
β10.0814–0.21970.2160–0.36100.1701–0.3469
β20.3325–0.52441.1723–2.35470.8947–2.0824
β30.0107–0.03100.0278–0.08030.0223–0.0505
ELSC
α1A0.0010–0.04190.0018–0.01870.0009–0.0044
α1B0.0749–0.27550.1676–0.41670.1782–0.2518
α1D0.0623–0.20190.1203–0.27290.1211–0.4376
α2AD0.2696–0.54120.4460–0.71150.6757–1.1146
α2B0.0052–0.02700.0342–0.06650.1001–0.1831
α2C0.0066–0.01460.0195–0.10000.0354–0.0523
β10.0324–0.25340.2430–0.36870.1567–0.2434
β20.2563–0.76002.3327–2.96671.1151–2.5427
β30.0279–0.09050.0361–0.10340.0328–0.0659

Values are expressed as interquartile ranges (25th to 75th percentiles) of the percentage for the housekeeping gene GADPH.

Group 2 comprised 7 healthy dairy cows from which samples were collected during routine laparotomy. Group 3 comprised 8 healthy dairy cows that were culled for reasons not related to GIT disease and from which samples were collected immediately after stunning at a slaughterhouse.

Figure 1—
Figure 1—

Box-and-whisker plots of the expression of mRNA coding for 6 adrenergic receptor (AR) subtypes (α1A, α1B, α2AD, α2B, β1, and β2) in samples of the intestinal wall (pooled results for the ileum, cecum, PLAC, and ELSC) obtained from dairy cows with CDD (group 1 [white boxes]), samples from healthy dairy cows obtained during laparotomy (group 2 [light-gray boxes]), and samples from healthy cull dairy cows obtained immediately after stunning during slaughter (group 3 [dark-gray boxes]). Results represent the logarithm of the results for the adrenoceptors relative to results for the housekeeping gene GAPDH. The boxes represent the range of the central 50% of the values (ie, 25th to 75th percentiles), the horizontal line within each box represents the median value, and the whiskers indicate the range of values that are within the inner fences (inner fence = box hinge ± [1.5 X {box hinge – median}]). a–cWithin each adrenoceptor, values with different letters differ significantly (P ≤ 0.05).

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1367

Statistical analysis was conducted for the remaining adrenoceptor subtypes. Analysis revealed no significant differences among groups and no group-by-tissue interaction in expression of mRNA coding for the α1Aadrenoceptor subtype.

For the α1B-adrenoceptor subtype, there was a significant (P = 0.008) effect of group (Figure 2). Relative mRNA expression was significantly lower in the intestines of cows with CDD (group 1) than in intestines of both control groups (P = 0.019 and 0.017 for groups 2 and 3, respectively), but there was no significant difference between samples collected from healthy dairy cows during laparotomy (group 2) and those collected after slaughter (group 3). Because there was a significant (P = 0.003) group-by-tissue interaction, statistical analysis was conducted for each tissue separately. Samples of the ileum for group 1 differed significantly from samples of the ileum of groups 2 (P = 0.004) and 3 (P < 0.001), whereas no significant effect was detected for samples of ileum between groups 2 and 3. No effect of group was found for the remaining tissues.

Figure 2—
Figure 2—

Box-and-whisker plots of the expression of mRNA coding for the α1B-adrenoceptor subtype in 4 locations of the intestine (ileum, cecum, PLAC, ELSC) of cows with CDD and 2 groups of healthy control cows. a,bWithin each location, values with different letters differ significantly (P ≤ 0.05). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1367

Expression of mRNA coding for the α2AD-adrenoceptor subtype revealed significant effects for group (P = 0.002) and tissue (P < 0.001), but there was no group-by-tissue interaction. Overall analysis for all tissues revealed significantly (P = 0.002) lower values for group 1, compared with values for group 3; however, there was no significant difference in values between groups 2 and 3 (Figure 1). A pattern for lower values in group 1, compared with values for group 2, was evident; however, the values did not differ significantly (P = 0.074) between groups 1 and 2. Additional separate statistical analysis for each location revealed differences among groups in the ileum and ELSC but not in the cecum and PLAC. In the ileum, values for group 1 differed significantly from values for groups 2 (P = 0.031) and 3 (P = 0.031), whereas values did not differ significantly between groups 2 and 3. In the ELSC, mRNA expression for α2AD-adrenoceptors for group 1 differed significantly (P = 0.007), compared with expression for group 3, but there was not a significant difference in expression between groups 1 and 2, nor between groups 2 and 3 (Figure 3).

Figure 3—
Figure 3—

Box-and-whisker plots of the expression of mRNA coding for the α2AD-adrenoceptor subtype in 4 locations of the intestine of cows with CDD and 2 groups of healthy control cows. a,bWithin each location, values with different letters differ significantly (P ≤ 0.05). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1367

Statistical analysis for expression of mRNA coding for α2B-adrenoceptors revealed a significant (P < 0.001) effect of group as well as a significant (P = 0.03) tissue-by-group interaction. In the overall analysis, values of group 1 were significantly (P < 0.001) lower than those of groups 2 and 3. In addition, expression for group 2 differed significantly (P < 0.001), compared with expression for group 3, with lower mRNA expression for group 2 (Figure 1). Additional separate statistical analysis for each location revealed significant (P < 0.001) differences among all groups in all tissues (Figure 4). The only exception was in the PLAC, where values for groups 1 and 2 did not differ significantly.

Figure 4—
Figure 4—

Box-and-whisker plots of the expression of mRNA coding for the α2B-adrenoceptor subtype in 4 locations of the intestine of cows with CDD and 2 groups of healthy control cows. a–cWithin each location, values with different letters differ significantly (P ≤ 0.05). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1367

A significant (P = 0.002) effect of group was detected for expression of mRNA coding for β1-adrenoceptors, but there was no significant effect of tissue or group-by-tissue interaction. Overall analysis with all locations revealed significantly lower mRNA expression for group 1, compared with expression for groups 2 (P = 0.002) and 3 (P = 0.005), whereas expression did not differ significantly between groups 2 and 3 (Figure 1). There were no significant differences among groups for expression in the cecum and PLAC, but a significant difference was detected for expression between groups 1 and 2 in the ileum (P = 0.008) and ELSC (P = 0.016) and between groups 1 and 3 in the ileum (P = 0.004). Expression did not differ significantly between groups 2 and 3 in the ileum and ELSC or between groups 1 and 3 in the ELSC (Figure 5).

Figure 5—
Figure 5—

Box-and-whisker plots of the expression of mRNA coding for the β1-adrenoceptor subtype in 4 locations of the intestine of cows with CDD and 2 groups of healthy control cows. a,bWithin each location, values with different letters differ significantly (P ≤ 0.05). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1367

A significant (P < 0.001) effect of group was detected for expression of mRNA coding for β2-adrenoceptors, but there was not a significant effect of tissue or a group-by-tissue interaction. Overall statistical analysis of all locations revealed significant differences among all 3 groups (P < 0.001 between groups 1 and 2, P < 0.001 between groups 1 and 3, and P = 0.04 between groups 2 and 3), with the most pronounced difference being the distinctly lower values for group 1, compared with values for groups 2 and 3 (Figure 1). Similarly, additional statistical analysis revealed differences among all groups in the cecum (P < 0.001 between groups 1 and 2, P = 0.003 between groups 1 and 3, and P = 0.03 between groups 2 and 3) and ELSC (P < 0.001 between groups 1 and 2, P < 0.001 between groups 1 and 3, and P = 0.02 between groups 2 and 3).

In the ileum and PLAC, expression for group 1 differed significantly (P < 0.001), compared with expression for groups 2 and 3, but no significant difference was detected for expression between groups 2 and 3 (Figure 6).

Figure 6—
Figure 6—

Box-and-whisker plots of the expression of mRNA coding for the β2-adrenoceptor subtype in 4 locations of the intestine of cows with CDD and 2 groups of healthy control cows. a–cWithin each location, values with different letters differ significantly (P ≤ 0.05). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1367

Discussion

Analysis of results of the study reported here revealed generally low steady-state expression of mRNA coding for all adrenoceptor subtypes and a significantly lower expression of mRNA coding for the adrenoceptor subtypes α1B, α2AD, α2B, β1, and β2 in cows with CDD, compared with expression in healthy control cows. In the GIT, adrenoceptors play a role in the regulation of several functions, including motility, secretion, absorption, and vascular tone.12,14–17 The amount of expression, functions of adrenoceptors, or both are regulated through various mechanisms and can vary in physiologic and pathologic situations.27,28 In hypothyroid rats, expression of α1-, β1-, and β2-adrenoceptors in several tissues differs from the expression of the same receptors in healthy rats.29 Reduced expression of mRNA coding for the adrenoceptor subtypes α1B, α2AD, α2B, β1, and β2 in cows with CDD observed in the study reported here may reflect involvement of these adrenoceptor subtypes in the pathogenesis of the disease. However, whether these differences indicate intrinsic variability among cows (possibly leading to an increase or decrease in the risk of developing CDD) or downregulation of mRNA expression for adrenoceptor subtypes before, during, or after dilatation of the large intestine remains unknown.

In the GIT, α1-adrenoceptor subtypes are located on postjunctional smooth-muscle cells and, to a lesser extent, intrinsic neurons.12 In general, these receptors have been only poorly investigated. Most studies have not focused on adrenoceptor subtypes, probably because of a lack of specific subtype agonists and antagonists. Distinct differences in expression and function of the α1-adrenoceptor among species and locations have been described.30–32 For example, α1-adrenoceptor agonists exert a peripheral excitatory action on motility in the ileum, proximal portion of the cecum, and colon in sheep but are inhibitory in the cecal corpus and apex,30 whereas α1-adrenoceptor ago nists inhibit motility in the jejunum, ileum, and parts of the colon in horses.31 In the colon of pigs, norepinephrine appears to stimulate the secretion of sodium and chloride ions via α1-adrenoceptor subtypes.32 Several studies have evaluated α1-adrenoceptors in the GIT of sheep, goats, swine, horses, humans, and rodents, but we are aware of only 1 study24 in which expression of mRNA coding for the various subtypes at several locations of the GIT of cattle has been reported.

In the study reported here, relative expression of mRNA coding for the α1A-adrenoceptor subtype did not differ among the 3 groups. This adrenoceptor subtype appears to be implicated in the maintenance of basal vascular tone and arterial blood pressure in several species.33–35 To our knowledge, no studies have been conducted to determine the role of α1A-adrenoceptors in the GIT.

In the overall statistical analysis, relative expression of mRNA coding for the α1B-adrenoceptor subtype was significantly lower in cows with CDD, compared with expression for both control groups. Separate statistical analysis for each location confirmed results of the overall analysis for the ileum (ie, a significant difference between group 1 and both control groups), whereas no difference among groups was found for the remaining locations. In contrast to the α1A-adrenoceptor, the α1B-adrenoceptor subtype plays a less important role in the regulation of blood pressure and vascular tone but appears to participate in responses to exogenous impulses, such as prolonged tissue ischemia.33,36 The same receptor subtype also leads to translocation of the α1D-adrenoceptor subtype (which is typically located within the cell) to the cell membrane. This indicates that there are interactions among adrenoceptor subtypes.37

The α2-adrenoceptors may be found at presynaptic or postsynaptic locations. At the presynaptic location, they act as autoreceptors to inhibit norepinephrine release from adrenergic nerves or as heteroceptors to modulate the release of other neurotransmitters (eg, acetylcholine) from nerve terminals.12 An important postjunctional location of these adrenoceptors is on enterocytes, where they control absorption of water and electrolytes.12 The effects of α2-adrenoceptor agonists, primarily xylazine, on GIT motility have been established. In horses, xylazine can act as an inhibitory or excitatory mediator, depending on the location of the activated adrenoceptors, but its effect on GIT motility is mostly inhibitory.38–41 In cows, xylazine reduces myoelectric activity of the ileum, cecum, and PLAC42 and causes atony of the reticulum.19 Furthermore, α2-adrenoceptor agonists inhibit motility of the ileum, cecum, and proximal portion of the colon in sheep30 and inhibit motility of the ruminoreticular area in goats,43 sheep,18,21,44 and cattle.19,21 The α2-adrenoceptor agonist clonidine decreases bicarbonate secretion from the duodenal mucosa in humans45 and increases absorption of water and electrolytes in the ileum of rabbits,14 which confirms a role of these adrenoceptors in the regulation of secretion and absorption in the GIT.

In the study reported here, relative expression of mRNA coding for the α2AD-adrenoceptor was significantly lower in cows of group 1, compared with expression for cows of group 3. Expression of mRNA for the α2AD-adrenoceptor for group 2 was between values for groups 1 and 3, and values for group 2 did not differ significantly, compared with values for the other 2 groups, in the overall statistical analysis. Separate statistical analysis for each location confirmed a significant difference for the ELSC between groups 1 and 3 and no difference between group 2 and either of the other groups. However, relative expression of mRNA coding for the α2AD-adrenoceptor was significantly lower in the ileum of cows with CDD, compared with expression for either of the control groups. Because tissue samples were not collected in exactly the same manner from cows of group 1 (biopsy during laparotomy) and group 3 (tissue samples collected after stunning during slaughter), the difference observed in the ELSC is difficult to interpret because it may potentially reflect variation in methods. In contrast, the significant difference between groups 1 and 2 for samples obtained from the ileum, which were collected in a similar manner during surgery in both groups, indicates an actual difference between cows with CDD and healthy control cows. Significant differences were not evident for the cecum and PLAC.

In groups 2 and 3, expression of mRNA coding for the α2AD-adrenoceptor was higher in the ileum than in any other location. This confirms the results of another study24 in which our laboratory group detected significantly higher expression of mRNA coding for this adrenoceptor in the ileum of healthy cull dairy cows, compared with expression in the cecum, PLAC, and ELSC. In the study reported here, this effect appeared to be reduced in cows with CDD because of the diminished expression of mRNA in the ileum of sick cows. In a study46 in knockout mice, α2AD-adrenoceptors mediated the inhibition of intestinal motility. Other authors47 have described mediation of muscle contraction in the circular smooth muscle of the colon of dogs through these receptors.47 A role of this adrenoceptor subtype in the regulation of electrolyte transport in the ileum of pigs has also been suggested.32 The α2AD-adrenoceptor subtype plays an important role in the regulation of blood pressure in several species and has been detected in the vascular endothelium of several species33,48,49 and on platelets of humans.48 Thus, the α2AD-adrenoceptor affects the regulation of several intestinal functions in various species. In fact, most of the classical functions mediated by α2-adrenoceptors, such as hypotension, sedation, analgesia, hypothermia, and anesthetic-sparing effects, are mediated primarily by the α2AD-adrenoceptor subtype.50

The tissue samples analyzed in the study reported here included cells directly affecting the function of the GIT (smooth-muscle cells, nerve cells, and enterocytes) as well as cells from blood vessels, leukocytes, and platelets. Interpretation of the results was hampered by the fact that the cell type or types with reduced expression of mRNA coding for the α2AD-adrenoceptor subtype in cows with CDD remain unknown.

Relative expression of mRNA coding for the α2B-adrenoceptor subtype differed significantly among all locations and all groups, except for the PLAC in groups 1 and 2. In group 1, the expression of mRNA coding for this subtype was lowest in all locations. The next lowest expression was detected for group 2, whereas group 3 had the highest expression. Data from a study50 in knockout mice indicated a possible role of this receptor subtype in developmental and reproductive processes. Furthermore, it may play a role in vasoconstrictor responses to α2-adrenoceptor agonists (ie, such responses were not observed in α2B-adrenoceptor knockout mice).51 To our knowledge, no investigations have been conducted on the role of this adrenoceptor subtype in the GIT.

Differences between the 2 control groups were unexpected but may have been caused by differences in blood content or coagulation in the samples, at least for the adrenoceptor subtypes expressed on blood cells and platelets. Furthermore, there were differences in the manner in which cows of the 2 control groups were stressed before and during collection of samples. Cows of group 3 were stressed by transport to the slaughterhouse and the stunning process, whereas cows of group 2 were stressed by clinical examination and surgery. An interaction between stress and adrenoceptor-mediated modulation of body functions has been described.52 Thus, differences in the types of stress may lead to differences in expression of mRNA coding for adrenoceptors. In contrast, differences between groups 1 and 2 were not affected by differences in the method of sample collection and stress situations because they were identical for these 2 groups; therefore, we believe that results for groups 1 and 2 truly reflect differences between cows with CDD and healthy control cows.

The β1-and β2-adrenoceptors are found primarily on smooth-muscle cells, but the former may also be found on enteric neurons.12 These adrenoceptors predominantly inhibit motility of the GIT12,15,18,53,k and also influence secretion12,15 and absorption.15–17 The involvement of β1- and β2-adrenoceptors in hormone secretion in the colon of rats54 as well as the role of these adrenoceptors in the regulation of glucose absorption in the intestines of rats and absorption of water, sodium, chloride, and fructose in the GIT of humans have been described.15–17 The β-adrenoceptors have not been investigated in depth in ruminants; however, they appear to play a role in modulation of ruminoreticular and duodenojejunal motility in sheep18 and motility of the PLAC in cattle.55

In the study reported here, relative expression of mRNA coding for the β1-adrenoceptor subtype was significantly lower in cows with CDD, compared with expression for both control groups, because of differences in the ileum, where mRNA expression was significantly lower in cows with CDD than in healthy control cows, and in the ELSC, where mRNA expression of group 1 was significantly lower than for group 2. In the ELSC, expression for group 1 did not differ from expression for group 3, probably because of the wide variation observed in values from cows with CDD. Several studies12,15,55–57 have been conducted on the effects of the β1-adrenoceptor subtype in the GIT. They reveal major inhibitory effects in several species (humans, cats, rabbits, rats, and mice). In sheep, activation of the β1-adrenoceptor by its agonist dobutamin leads to inhibition of ruminoreticular and abomasal motility as well as to an increase in duodenojejunal motility.18 Furthermore, it has been suggested60 that epinephrine and norepinephrine stimulate β1-adrenoceptors, which causes secretion of potassium ions in the colon of rabbits.

Statistical analysis revealed significant differences in expression of mRNA coding for the β2-adrenoceptor subtype between cows with CDD and healthy control cows in all investigated locations. Similar to actions of the β1-subtype, the β2-subtype predominantly has inhibitory effects on motility of the GIT. In rabbits and horses, activation of β2-adrenoceptors causes inhibition of intestinal motility12; the same effect was detected in the colon of cats and rats.57,60 The β2-adrenoceptor subtype is also expressed on lymphocytes and macrophages.61,62 Similar to results for the α2B-adrenoceptor subtype, in which there were significant differences between the 2 control groups for all locations, the differences in mRNA expression for the β2-adrenoceptor observed between the 2 control groups for the cecum and ELSC were not expected. The mRNA coding for the α2B-adrenoceptor had significantly lower expression for group 2, compared with expression for group 3, in all tissues, whereas the opposite was observed for the expression of mRNA for the β2-adrenoceptor in the cecum and ELSC. As mentioned previously, differences in the sample collection methods and stress situations of the cows could have been responsible for these effects. Furthermore, a systemic effect of disease on the investigated tissues that led to reduced expression of mRNA for these receptor subtypes cannot be excluded. Measurements of mRNA expression in tissues not directly affected by CDD may be of use to address this issue in future studies.

Ideally, the study reported here should have been performed with 2 equal groups of cows and with all cows subjected to the same procedures (ie, groups 1 and 2). Furthermore, matching on the basis of herd, stage of lactation, breed, and nutrition factors for each cow with CDD would have allowed us to reliably exclude potential effects of these factors on the study results. However, such a study design could not be applied because of practical constraints (related primarily to animal welfare issues) that limited the number of cows in group 2. Therefore, the control group was completed by the inclusion of healthy cull cows from which samples were collected immediately after stunning during slaughter (group 3). Although this approach precluded matching and exclusion of most effects other than for CDD itself, the fact that significant differences were detected between the 2 control groups also provided valuable information for subsequent studies because analysis of these results indicated that expression of mRNA coding for adrenoceptor subtypes is not the same in intestinal tissues of otherwise healthy cows when the samples are collected from live cows or immediately after cows are stunned during slaughter. This observation indicates that caution should be used when interpreting results of mRNA measurements in samples collected after death because amounts of receptors in tissues collected after death may not necessarily reflect mRNA expression in vivo.

With regard to results of the study reported here, stringent methods were used for statistical analysis. However, significant differences between cows with CDD and healthy cows were found despite the small sample size and inability to pool results for the 2 control groups. Thus, we are confident that the differences observed, especially between groups 1 and 2, are actual differences reflecting the effects of CDD on mRNA expression of adrenoceptors in the intestines of cows.

Relative expression of mRNA coding for α1D, α2C, and β3 was extremely low and therefore not analyzed statistically. Although the method used here (real-time reverse transcriptase-PCR assay) is the technique of choice for measurements of mRNA in extremely low abundance, the detection limit of this kinetic PCR assay is approximately at crossing-point values < 33 (ie, 33 amplification cycles are needed for PCR products to become detectable).63 Gene copy detection is inconsistent above this value. In the study reported here, CPAR values of mRNA coding for α1D-, α2C-, and β3-adrenoceptor subtypes ranged from 33 to 35. Thus, the method used in this study did not allow us to gain additional insights into mRNA expression of these 3 adrenoceptor subtypes in the intestinal tract of healthy cows and cows with CDD. In another study24 conducted by our laboratory group in which we focused on expression of mRNA coding for adrenoceptor subtypes in 10 locations of the GIT of healthy dairy cows by use of real-time reverse transcriptase-PCR assay, the lowest relative concentrations of mRNA were detected for these same 3 adrenoceptor subtypes.

The study reported here revealed significantly lower steady-state amounts of mRNA coding for several adrenoceptor subtypes in cows with CDD, compared with amounts for healthy control cows. Significant differences between healthy and affected cows were dependent on adrenoceptor subtypes and locations within the GIT, with lower values for the adrenoceptor subtypes α1B, α2AD, α2B, β1, and β2 in cows with CDD, compared with values for those adrenoceptor subtypes in the control groups. The fact that significant differences between group 1 and the healthy control groups were predominantly localized in the ileum and ELSC is in accordance with results for an in vivo study10 in cows with CDD in which one third of the cows did not have normal myoelectric motor complex activity in the ileum 1 day after surgery for correction of CDD. The myoelectric motor complex of the small intestine influences motility patterns of the more distal segments of the GIT64; thus, a disorder of motility in the ileum may lead to motility disturbances in more distal segments of the GIT (ie, cecum, PLAC, and ELSC). Furthermore, results observed for the ELSC support the hypothesis that delayed recovery and recurrence of CDD after surgery may be a consequence of a motility disorder in the spiral colon, rather than in the cecum.10

Abundance of mRNA does not necessarily reflect protein expression or receptor function. However, to our knowledge, investigations of the expression of adrenoceptor proteins and the function of specific subtypes have been limited because of a lack of specific agonists and antagonists for several adrenoceptor sub types used in binding studies, immunhistochemical analysis, or organ bath studies. Therefore, analysis of mRNA expression by use of a real-time reverse transcriptase-PCR assay was warranted because it allowed us to investigate each subtype separately despite extremely low mRNA expression.

Because adrenoceptors have predominantly inhibitory actions on GIT motility,12,13 an increase (rather than a decrease) in expression of mRNA for adrenoceptors may have been expected in cows with motility disorders such as CDD, compared with expression for healthy cows. However, the results reported here reflect dynamic control of genes coding for adrenoceptors. Because intestinal biopsy specimens were collected at a relatively late time point during the course of the disease (ie, during surgical correction of the condition), it is possible that mRNA expression for adrenoceptor subtypes reported here differed from expression at earlier time points during development of the disease, if these receptors are implicated in the pathogenesis of CDD. For example, there could have been an initial increase in adrenoceptor subtypes during development of CDD that were later downregulated once the intestine was distended.

Measurable amounts of mRNA in cells are influenced by the rate of gene transcription and by mRNA stability (ie, rate of transcript degradation). The differences between groups observed in this study could have resulted from a change in rates of gene transcription or transcript degradation. However, the methods used did not allow us to determine whether the observed changes resulted from altered rates of synthesis or degradation of the transcripts studied. The fact that almost no information about the regulation of mRNA expression for adrenoceptor subtypes in the GIT during health and disease is available, even in humans, makes it even more difficult to interpret the results of our study.

Finally, the full-thickness specimens of intestinal wall analyzed included cells that directly affect the function of the GIT, such as smooth muscle cells, enterocytes and nerve cells, as well as cells from blood vessels, leukocytes, and platelets. To reduce the number of cell types included in the samples, the mucosa could have been removed so that we would have analyzed only the seromuscular layers. However, adrenoceptors located in the mucosa play an important role in the regulation of secretion and absorption, and we did not want to exclude these receptors from the analyses. Furthermore, removing the mucosa would have removed receptors on enterocytes but not on other cells such as leukocytes; thus, it would not necessarily have become easier to interpret the results in terms of potential effects on motility.

Despite some inherent limitations as a result of the methods used, analysis of results for the study reported here indicates that CDD influences mRNA expression for the adrenoceptor subtypes α1B, α2AD, α2B, β1, and β2 in the intestines of cows, which, in turn, suggests that these adrenoceptors are involved in the regulation of GIT functions in cattle and, possibly, in the pathogenesis of CDD. Thus, on the basis of the significant differences observed between cows with CDD and healthy control cows, additional investigations of these receptors to examine protein expression and function are warranted. Also, studies with adequate techniques (eg, immunhistochemical analysis) are necessary to determine the cell types that have reduced expression of mRNA coding for adrenoceptors in cows with CDD. Such studies may lead to a better comprehension of the pathogenesis of CDD and, eventually, to new therapeutic approaches, which could include administration of agonists or antagonists of specific adrenoceptor subtypes for the treatment of cows with CDD.

ABBREVIATIONS

CDD

Cecal dilatation-dislocation

PLAC

Proximal loop of the ascending colon

GIT

Gastrointestinal tract

ELSC

External loop of the spiral colon

GAPDH

Glyceraldehyde phosphate dehydrogenase

a.

Maxon 3-0, Tüscher AG, Berne, Switzerland.

b.

RNAlater, Ambion Inc, Austin, Tex.

c.

Mini-beadbeater, Biospec Products, Bartlesville, Okla.

d.

TriFast, PeqLab Biotechnologie GmBh, Erlangen, Germany.

e.

Biophotometer, Eppendorf, Netheler-Hinz, Hamburg, Germany.

f.

Moloney murine leukemia virus reverse transcriptase (MMLV-RT), Promega Corp, Madison, Wis.

g.

Random hexamer primers, MBI Fermentas, St Leon-Rot, Germany.

h.

Light cycler fast start DNA master SYBR green I, Roche Diagnostics, F Hoffmann la Roche Ltd, Basel, Switzerland.

i.

R Development Core Team. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing, 2003.

j.

Systat, version 10.0, Systat Software Inc, Richmond, Calif.

k.

Brikas P, Fioramonti J, Bueno L. Central and peripheral beta-adrenergic control of gastrointestinal motility in sheep (abstr). Reprod Nutr Dev 1990;(suppl 2):216s.

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