Aerobic exercise training upregulates skeletal muscle calpain and ubiquitin-proteasome systems in healthy mice

Cunha TF, Moreira JB, Paixao NA, Campos JC, Monteiro AW, Bacurau AV, Bueno CR Jr., Ferreira JC, Brum PC. Aerobic exercise training upregulates skeletal muscle calpain and ubiquitin-proteasome systems in healthy mice. J Appl Physiol 112: 1839-1846, 2012. First published March 29, 2012; doi:10.1152/japplphysiol.00346.2011.-Aerobic exercise training (AET) is an important mechanical stimulus that modulates skeletal muscle protein turnover, leading to structural rearrangement. Since the ubiquitin-proteasome system (UPS) and calpain system are major proteolytic pathways involved in protein turnover, we aimed to investigate the effects of intensity-controlled AET on the skeletal muscle UPS and calpain system and their association to training-induced structural adaptations. Long-lasting effects of AET were studied in C57BL/6J mice after 2 or 8 wk of AET. Plantaris cross-sectional area (CSA) and capillarization were assessed by myosin ATPase staining. mRNA and protein expression levels of main components of the UPS and calpain system were evaluated in plantaris by real-time PCR and Western immunoblotting, respectively. No proteolytic system activation was observed after 2 wk of AET. Eight weeks of AET resulted in improved running capacity, plantaris capillarization, and CSA. Muscle RING finger-1 mRNA expression was increased in 8-wk-trained mice. Accordingly, elevated 26S proteasome activity was observed in the 8-wk-trained group, without accumulation of ubiquitinated or carbonylated proteins. In addition, calpain abundance was increased by 8 wk of AET, whereas no difference was observed in its endogenous inhibitor calpastatin. Taken together, our findings indicate that skeletal muscle enhancements, as evidenced by increased running capacity, plantaris capillarization, and CSA, occurred in spite of the upregulated UPS and calpain system, suggesting that overactivation of skeletal muscle proteolytic systems is not restricted to atrophying states. Our data provide evidence for the contribution of the UPS and calpain system to metabolic turnover of myofibrillar proteins and skeletal muscle adaptations to AET.

Transiting exoplanets from the CoRoT space mission XIX. CoRoT-23b: a dense hot Jupiter on an eccentric orbit

We report the detection of CoRoT-23b, a hot Jupiter transiting in front of its host star with a period of 3.6314 +/- 0.0001 days. This planet was discovered thanks to photometric data secured with the CoRoT satellite, combined with spectroscopic radial velocity (RV) measurements. A photometric search for possible background eclipsing binaries conducted at CFHT and OGS concluded with a very low risk of false positives. The usual techniques of combining RV and transit data simultaneously were used to derive stellar and planetary parameters. The planet has a mass of M-p = 2.8 +/- 0.3 M-Jup, a radius of R-pl = 1.05 +/- 0.13 R-Jup, a density of approximate to 3 gcm(-3). RV data also clearly reveal a nonzero eccentricity of e = 0.16 +/- 0.02. The planet orbits a mature G0 main sequence star of V = 15.5 mag, with a mass M-star = 1.14 +/- 0.08 M-circle dot, a radius R-star = 1. 61 +/- 0.18 R-circle dot and quasi-solar abundances. The age of the system is evaluated to be 7 Gyr, not far from the transition to subgiant, in agreement with the rather large stellar radius. The two features of a significant eccentricity of the orbit and of a fairly high density are fairly uncommon for a hot Jupiter. The high density is, however, consistent with a model of contraction of a planet at this mass, given the age of the system. On the other hand, at such an age, circularization is expected to be completed. In fact, we show that for this planetary mass and orbital distance, any initial eccentricity should not totally vanish after 7 Gyr, as long as the tidal quality factor Q(p) is more than a few 10(5), a value that is the lower bound of the usually expected range. Even if CoRoT-23b features a density and an eccentricity that are atypical of a hot Jupiter, it is thus not an enigmatic object.

Trunk, Pelvis, Hip, and Knee Kinematics, Hip Strength, and Gluteal Muscle Activation During a Single-Leg Squat in Males and Females With and Without Patellofemoral Pain Syndrome

STUDY DESIGN: Controlled laboratory study using a cross-sectional design. OBJECTIVES: To determine whether there are any differences between the sexes in trunk, pelvis, hip, and knee kinematics, hip strength, and gluteal muscle activation during the performance of a single-leg squat in individuals with patellofemoral pain syndrome (PFPS) and control participants. BACKGROUND: Though there is a greater incidence of PFPS in females, PFPS is also quite common in males. Trunk kinematics may affect hip and knee function; however, there is a lack of studies of the influence of the trunk in individuals with PFPS. METHODS: Eighty subjects were distributed into 4 groups: females with PFPS, female controls, males with PFPS, and male controls. Trunk, pelvis, hip, and knee kinematics and gluteal muscle activation were evaluated during a single-leg squat. Hip abduction and external rotation eccentric strength was measured on an isokinetic dynamometer. Group differences were assessed using a 2-way multivariate analysis of variance (sex by PFPS status). RESULTS: Compared to controls, subjects with PFPS had greater ipsilateral trunk lean (mean +/- SD, 9.3 degrees +/- 5.30 degrees versus 6.7 degrees +/- 3.0 degrees; P = .012), contralateral pelvic drop (10.3 degrees +/- 4.7 degrees versus 7.4 degrees 3.8 degrees; P = .003), hip adduction (14.8 degrees +/- 7.8 degrees versus 10.8 degrees +/- 5.6 degrees; P<.0001), and knee abduction (9.2 degrees +/- 5.0 degrees versus 5.8 degrees +/- 3.4 degrees; P<.0001) when performing a single-leg squat. Subjects with PFPS also had 18% less hip abduction and 17% less hip external rotation strength. Compared to female controls, females with PFPS had more hip internal rotation (P<.05) and less muscle activation of the gluteus medius (P = .017) during the single-leg squat. CONCLUSION: Despite many similarities in findings for males and females with PFPS, there may be specific sex differences that warrant consideration in future studies and when clinically evaluating and treating females with PFPS. J Orthop Sports Phys Ther 2012;42(6):491-501, Epub 8 March 2012. doi:10.2519/jospt.2012.3987

Models of passive and active dendrite motoneuron pools and their differences in muscle force control

Motoneuron (MN) dendrites may be changed from a passive to an active state by increasing the levels of spinal cord neuromodulators, which activate persistent inward currents (PICs). These exert a powerful influence on MN behavior and modify the motor control both in normal and pathological conditions. Motoneuronal PICs are believed to induce nonlinear phenomena such as the genesis of extra torque and torque hysteresis in response to percutaneous electrical stimulation or tendon vibration in humans. An existing large-scale neuromuscular simulator was expanded to include MN models that have a capability to change their dynamic behaviors depending on the neuromodulation level. The simulation results indicated that the variability (standard deviation) of a maintained force depended on the level of neuromodulatory activity. A force with lower variability was obtained when the motoneuronal network was under a strong influence of PICs, suggesting a functional role in postural and precision tasks. In an additional set of simulations when PICs were active in the dendrites of the MN models, the results successfully reproduced experimental results reported from humans. Extra torque was evoked by the self-sustained discharge of spinal MNs, whereas differences in recruitment and de-recruitment levels of the MNs were the main reason behind torque and electromyogram (EMG) hysteresis. Finally, simulations were also used to study the influence of inhibitory inputs on a MN pool that was under the effect of PICs. The results showed that inhibition was of great importance in the production of a phasic force, requiring a reduced co-contraction of agonist and antagonist muscles. These results show the richness of functionally relevant behaviors that can arise from a MN pool under the action of PICs.

The role of reactive oxygen species in the modulation of the contraction induced by angiotensin II in carotid artery from diabetic rat

The modulation played by reactive oxygen species on the angiotensin II-induced contraction in type I-diabetic rat carotid was investigated. Concentration-response curves for angiotensin II were obtained in endothelium-intact or endothelium-denuded carotid from control or streptozotocin-induced diabetic rats, pre-treated with tiron (superoxide scavenger), PEG-catalase (hydrogen peroxide scavenger), dimethylthiourea (hydroxyl scavenger), apocynin [NAD(P) H oxidase inhibitor], SC560 (cyclooxygenase-1 inhibitor), SC236 (cyclooxygenase-2 inhibitor) or Y-27632 (Rho-kinase inhibitor). Reactive oxygen species were measured by flow cytometry in dihydroethidium (DHE)-loaded endothelial cells. Cyclooxygenase and AT1-receptor expression was assessed by immunohistochemistry. Diabetes increased the angiotensin II-induced contraction but reduced the agonist potency in rat carotid. Endothelium removal, tiron or apocynin restored the angiotensin II-induced contraction in diabetic rat carotid to control levels. PEG-catalase, DMTU or SC560 reduced the angiotensin II-induced contraction in diabetic rat carotid at the same extent. SC236 restored the angiotensin II potency in diabetic rat carotid. Y-27632 reduced the angiotensin II-induced contraction in endothelium-intact or -denuded diabetic rat carotid. Diabetes increased the DHE-fluorescence of carotid endothelial cells. Apocynin reduced the DHE-fluorescence of endothelial cells from diabetic rat carotid to control levels. Diabetes increased the muscular cyclooxygenase-2 expression but reduced the muscular AT1-receptor expression in rat carotid. In summary, hydroxyl radical, hydrogen peroxide and superoxide anion-derived from endothelial NAD(P) H oxidase mediate the hyperreactivity to angiotensin II in type I-diabetic rat carotid, involving the participation of cyclooxygenase-1 and Rho-kinase. Moreover, increased muscular cyclooxygenase-2 expression in type I-diabetic rat carotid seems to be related to the local reduced AT1-receptor expression and the reduced angiotensin II potency. (C) 2011 Elsevier B. V. All rights reserved.

The immediate effects of local and adjacent acupuncture on the tibialis anterior muscle: a human study

Abstract Background This study compares the immediate effects of local and adjacent acupuncture on the tibialis anterior muscle and the amount of force generated or strength in Kilogram Force (KGF) evaluated by a surface electromyography. Methods The study consisted of a single blinded trial of 30 subjects assigned to two groups: local acupoint (ST36) and adjacent acupoint (SP9). Bipolar surface electrodes were placed on the tibialis anterior muscle, while a force transducer was attached to the foot of the subject and to the floor. An electromyograph (EMG) connected to a computer registered the KGF and root mean square (RMS) before and after acupuncture at maximum isometric contraction. The RMS values and surface electrodes were analyzed with Student's t-test. Results Thirty subjects were selected from a total of 56 volunteers according to specific inclusion and exclusion criteria and were assigned to one of the two groups for acupuncture. A significant decrease in the RMS values was observed in both ST36 (t = -3.80, P = 0,001) and SP9 (t = 6.24, P = 0.001) groups after acupuncture. There was a decrease in force in the ST36 group after acupuncture (t = -2.98, P = 0.006). The RMS values did not have a significant difference (t = 0.36, P = 0.71); however, there was a significant decrease in strength after acupuncture in the ST36 group compared to the SP9 group (t = 2.51, P = 0.01). No adverse events were found. Conclusion Acupuncture at the local acupoint ST36 or adjacent acupoints SP9 reduced the tibialis anterior electromyography muscle activity. However, acupuncture at SP9 did not decrease muscle strength while acupuncture at ST36 did.

Mechanical Alterations in airway smooth muscle after interaction with indoor pollutant – A mathematical model.

The viscoelasticity of mammalian lung is determined by the mechanical properties and structural regulation of the airway smooth muscle (ASM). The exposure to polluted air may deteriorate these properties with harmful consequences to individual health. Formaldehyde (FA) is an important indoor pollutant found among volatile organic compounds. This pollutant permeates through the smooth muscle tissue forming covalent bonds between proteins in the extracellular matrix and intracellular protein structure changing mechanical properties of ASM and inducing asthma symptoms, such as airway hyperresponsiveness, even at low concentrations. In the experimental scenario, the mechanical effect of FA is the stiffening of the tissue, but the mechanism behind this effect is not fully understood. Thus, the aim of this study is to reproduce the mechanical behavior of the ASM, such as contraction and stretching, under FA action or not. For this, it was created a two-dimensional viscoelastic network model based on Voronoi tessellation solved using Runge-Kutta method of fourth order. The equilibrium configuration was reached when the forces in different parts of the network were equal. This model simulates the mechanical behavior of ASM through of a network of dashpots and springs. This dashpot-spring mechanical coupling mimics the composition of the actomyosin machinery of ASM through the contraction of springs to a minimum length. We hypothesized that formation of covalent bonds, due to the FA action, can be represented in the model by a simple change in the elastic constant of the springs, while the action of methacholine (MCh) reduce the equilibrium length of the spring. A sigmoid curve of tension as a function of MCh doses was obtained, showing increased tension when the muscle strip was exposed to FA. Our simulations suggest that FA, at a concentration of 0.1 ppm, can affect the elastic properties of the smooth muscle ¯bers by a factor of 120%. We also analyze the dynamic mechanical properties, observing the viscous and elastic behavior of the network. Finally, the proposed model, although simple, incorporates the phenomenology of both MCh and FA and reproduces experimental results observed with in vitro exposure of smooth muscle to FA. Thus, this new mechanical approach incorporates several well know features of the contractile system of the cells in a tissue level model. The model can also be used in different biological scales.

Mechanical alterations in airway smooth muscle after interaction with indoor pollutant - A mathematical model

The viscoelasticity of mammalian lung is determined by the mechanical properties and structural regulation of the airway smooth muscle (ASM). The exposure to polluted air may deteriorate these properties with harmful consequences to individual health. Formaldehyde (FA) is an important indoor pollutant found among volatile organic compounds. This pollutant permeates through the smooth muscle tissue forming covalent bonds between proteins in the extracellular matrix and intracellular protein structure changing mechanical properties of ASM and inducing asthma symptoms, such as airway hyperresponsiveness, even at low concentrations. In the experimental scenario, the mechanical effect of FA is the stiffening of the tissue, but the mechanism behind this effect is not fully w1derstood. Thus, the aim of this study is to reproduce the mechanical behavior of the ASM, such as contraction and stretching, under FA action or not. For this, it was created a two-dimensional viscoelastic network model based on Voronoi tessellation solved using Runge-Kutta method of fourth order. The equilibrium configuration was reached when the forces in different parts of the network were equal. This model simulates the mechanical behavior of ASM through of a network of dashpots and springs. This dashpot-spring mechanical coupling mimics the composition of the actomyosin machinery of ASM through the contraction of springs to a minimum length. We hypothesized that formation of covalent bonds, due to the FA action, can be represented in the model by a simple change in the elastic constant of the springs, while the action of methacholinc (MCh) reduce the equilibrium length of the spring. A sigmoid curve of tension as a function of MCh doses was obtained, showing increased tension when the muscle strip was exposed to FA. Our simulations suggest that FA, at a concentration of 0.1 ppm, can affect the elastic properties of the smooth muscle fibers by a factor of 120%. We also analyze the dynamic mechanical properties, observing the viscous and elastic behavior of the network. Finally, the proposed model, although simple, ir1corporates the phenomenology of both MCh and FA and reproduces experirnental results observed with ir1 vitro exposure of smooth muscle to .FA. Thus, this new mechanical approach incorporates several well know features of the contractile system of the cells ir1 a tissue level model. The model can also be used in different biological scales.

Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.

Effects of eccentric cycle ergometry in alpine skiers

Eccentric cycling, where the goal is to resist the pedals, which are driven by a motor, increases muscle strength and size in untrained subjects. We hypothesized that it could also be beneficial for athletes, particularly in alpine skiing, which involves predominantly eccentric contractions at longer muscle lengths. We investigated the effects of replacing part of regular weight training with eccentric cycling in junior male alpine skiers using a matched-pair design. Control subjects ( N=7) executed 1-h weight sessions 3 times per week, which included 4-5 sets of 4 leg exercises. The eccentric group ( N=8) performed only 3 sets, followed by continuous sessions on the eccentric ergometer for the remaining 20 min. After 6 weeks, lean thigh mass increased significantly only in the eccentric group. There was a groupxtime effect on squat-jump height favouring the eccentric group, which also experienced a 6.5% improvement in countermovement-jump height. The ability to finely modulate muscle force during variable eccentric cycling improved 50% (p=0.004) only in the eccentric group. Although eccentric cycling did not significantly enhance isometric leg strength, we believe it is beneficial for alpine skiers because it provides an efficient means for hypertrophy while closely mimicking the type of muscle actions encountered while skiing.

Remodeling of calcium handling in skeletal muscle through PGC-1?: impact on force, fatigability, and fiber type

Regular endurance exercise remodels skeletal muscle, largely through the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). PGC-1α promotes fiber type switching and resistance to fatigue. Intracellular calcium levels might play a role in both adaptive phenomena, yet a role for PGC-1α in the adaptation of calcium handling in skeletal muscle remains unknown. Using mice with transgenic overexpression of PGC-1α, we now investigated the effect of PGC-1α on calcium handling in skeletal muscle. We demonstrate that PGC-1α induces a quantitative reduction in calcium release from the sarcoplasmic reticulum by diminishing the expression of calcium-releasing molecules. Concomitantly, maximal muscle force is reduced in vivo and ex vivo. In addition, PGC-1α overexpression delays calcium clearance from the myoplasm by interfering with multiple mechanisms involved in calcium removal, leading to higher myoplasmic calcium levels following contraction. During prolonged muscle activity, the delayed calcium clearance might facilitate force production in mice overexpressing PGC-1α. Our results reveal a novel role of PGC-1α in altering the contractile properties of skeletal muscle by modulating calcium handling. Importantly, our findings indicate PGC-1α to be both down- as well as upstream of calcium signaling in this tissue. Overall, our findings suggest that in the adaptation to chronic exercise, PGC-1α reduces maximal force, increases resistance to fatigue, and drives fiber type switching partly through remodeling of calcium transients, in addition to promoting slow-type myofibrillar protein expression and adequate energy supply.

Different molecular and structural adaptations with eccentric and conventional strength training in elderly men and women

Reprogramming of gene expression contributes to structural and functional adaptation of muscle tissue in response to altered use. The aim of this study was to investigate mechanisms for observed improvements in leg extension strength, gain in relative thigh muscle mass and loss of body and thigh fat content in response to eccentric and conventional strength training in elderly men (n = 14) and women (n = 14; average age of the men and women: 80.1 ± 3.7 years) by means of structural and molecular analyses. Biopsies were collected from m. vastus lateralis in the resting state before and after 12 weeks of training with two weekly resistance exercise sessions (RET) or eccentric ergometer sessions (EET). Gene expression was analyzed using custom-designed low-density PCR arrays. Muscle ultrastructure was evaluated using EM morphometry. Gain in thigh muscle mass was paralleled by an increase in muscle fiber cross-sectional area (hypertrophy) with RET but not with EET, where muscle growth is likely occurring by the addition of sarcomeres in series or by hyperplasia. The expression of transcripts encoding factors involved in muscle growth, repair and remodeling (e.g., IGF-1, HGF, MYOG, MYH3) was increased to a larger extent after EET than RET. MicroRNA 1 expression was decreased independent of the training modality, and was paralleled by an increased expression of IGF-1 representing a potential target. IGF-1 is a potent promoter of muscle growth, and its regulation by microRNA 1 may have contributed to the gain of muscle mass observed in our subjects. EET depressed genes encoding mitochondrial and metabolic transcripts. The changes of several metabolic and mitochondrial transcripts correlated significantly with changes in mitochondrial volume density. Intramyocellular lipid content was decreased after EET concomitantly with total body fat. Changes in intramyocellular lipid content correlated with changes in body fat content with both RET and EET. In the elderly, RET and EET lead to distinct molecular and structural adaptations which might contribute to the observed small quantitative differences in functional tests and body composition parameters. EET seems to be particularly convenient for the elderly with regard to improvements in body composition and strength but at the expense of reducing muscular oxidative capacity.

Immediate and delayed transplantation of mesenchymal stem cells improve muscle force after skeletal muscle injury in rats

Mesenchymal stem cell (MSC) therapy is a promising approach for regaining muscle function after trauma. Prior to clinical application, the ideal time of transplantation has to be determined. We investigated the effects of immediate and delayed transplantation. Sprague-Dawley rats received a crush trauma to the left soleus muscle. Treatment groups were transplanted locally with 2 × 10(6) autologous MSCs, either immediately or 7 days after trauma. Saline was used as sham therapy. Contraction force tests and histological analyses were performed 4 weeks after injury. GFP-labelled MSCs were followed after transplantation. The traumatized soleus muscles of the sham group displayed a reduction of twitch forces to 36 ± 17% and of tetanic forces to 29 ± 11% of the non-injured right control side, respectively. Delayed MSC transplantation resulted in a significant improvement of contraction maxima in both stimulation modes (twitch, p = 0.011; tetany, p = 0.014). Immediate transplantation showed a significant increase in twitch forces to 59 ± 17% (p = 0.043). There was no significant difference in contraction forces between muscles treated by immediate and delayed cell transplantation. We were able to identify MSCs in the interstitium of the injured muscles up to 4 weeks after transplantation. Despite the fundamental differences of the local environment, which MSCs encounter after transplantation, similar results could be obtained with respect to functional muscle regeneration. We believe that transplanted MSCs residing in the interstitial compartment evolve their regenerative capabilities through paracrine pathways. Our data suggest a large time window of the therapeutical measures.

A novel steroid-like compound F90927 exerting positive-inotropic effects in cardiac muscle

Here we report a novel steroid-like compound F90363, exhibiting positive inotropy in vivo and in vitro in various cardiac muscle preparations. F90363 is a racemic mixture composed of the stereoisomers (-)-F90926 and (+)-F90927. Only F90927 exerted positive inotropy, while F90926 induced a weak negative inotropy, but only at concentrations 10(3) times higher than F90927 and most likely resulting from an unspecific interaction. The rapid time course of the action of F90927 suggested a direct interaction with a cellular target rather than a genomic alteration. We could identify the L-type Ca2+ current I(Ca(L)) as a main target of F90927, while excluding other components of cardiac Ca2+ signalling as potential contributors. In addition, several other signaling pathways known to lead to positive inotropy (e.g. alpha- and beta-adrenergic stimulation, cAMP pathways) could be excluded as targets of F90927. However, vessel contraction and stiffening of the cardiac muscle at high doses (>30 microM, 0.36 mg kg(-1), respectively) prevent the use of F90927 as a candidate for drug development. Since the compound may still find valuable applications in research, the aim of the present study was to identify the cellular target and the mechanism of inotropy of F90927.

In vitro effects of bethanechol on specimens of intestinal smooth muscle obtained from the duodenum and jejunum of healthy dairy cows

OBJECTIVE: To describe the in vitro effects of bethanechol on contractility of smooth muscle preparations from the small intestines of healthy cows and define the muscarinic receptor subtypes involved in mediating contraction. SAMPLE POPULATION: Tissue samples from the duodenum and jejunum collected immediately after slaughter of 40 healthy cows. PROCEDURES: Cumulative concentration-response curves were determined for the muscarinic receptor agonist bethanechol with or without prior incubation with subtype-specific receptor antagonists in an organ bath. Effects of bethanechol and antagonists and the influence of intestinal location on basal tone, maximal amplitude (A(max)), and area under the curve (AUC) were evaluated. RESULTS: Bethanechol induced a significant, concentration-dependent increase in all preparations and variables. The effect of bethanechol was more pronounced in jejunal than in duodenal samples and in circular than in longitudinal preparations. Significant inhibition of the effects of bethanechol was observed after prior incubation with muscarinic receptor subtype M(3) antagonists (more commonly for basal tone than for A(max) and AUC). The M(2) receptor antagonists partly inhibited the response to bethanechol, especially for basal tone. The M(3) receptor antagonists were generally more potent than the M(2) receptor antagonists. In a protection experiment, an M(3) receptor antagonist was less potent than when used in combination with an M(2) receptor antagonist. Receptor antagonists for M(1) and M(4) did not affect contractility variables. CONCLUSIONS AND CLINICAL RELEVANCE: Bethanechol acting on muscarinic receptor sub-types M(2) and M(3) may be of clinical use as a prokinetic drug for motility disorders of the duodenum and jejunum in dairy cows.