Role of ubiquitin–proteasome proteolysis in muscle fiber destruction in experimental chloroquine‐induced myopathy

Previous studies have documented the presence of rimmed vacuoles, atrophic fibers, and increased lysosomal cathepsin activity in skeletal muscle from animal models of chloroquine‐induced myopathy, suggesting that muscle fibers in this type of myopathy may be degraded via the lysosomal‐proteolysis pathway. Given recent evidence of abnormal ubiquitin accumulation in rimmed vacuoles, in this study we chose to examine the significance of the ubiquitin–proteasome proteolytic system in the process of muscle fiber destruction in experimental chloroquine myopathy. Expression of ubiquitin, 26S proteasome proteins, and ubiquitin ligases, such as muscle‐specific RING finger‐1 (MuRF‐1) and atrogin‐1/muscle atrophy F‐box protein (MAFbx), was analyzed in innervated and denervated rat soleus muscles after treatment with either saline or chloroquine. Abnormal accumulation of rimmed vacuoles was observed only in chloroquine‐treated denervated muscles. Ubiquitin and proteasome immunostaining, and ubiquitin, MuRF‐1, and atrogin‐1/MAFbx mRNAs were significantly increased in denervated soleus muscles from saline‐ and chloroquine‐treated rats when compared with contralateral innervated muscles. Further, ubiquitin and ubiquitin ligase mRNA levels were higher in denervated muscles from chloroquine‐treated rats when compared with saline‐treated rats. These data demonstrate increased proteasomes and ubiquitin in denervated muscles from chloroquine‐treated rats and suggest that the ubiquitin–proteasome proteolysis pathway as well as the lysosomal‐proteolysis pathway mediate muscle fiber destruction in experimental chloroquine myopathy. Muscle Nerve 39: 521–528, 2009     

Effects of tail suspension on serum testosterone and molecular targets regulating muscle mass

Introduction: The contribution of reduced testosterone levels to tail suspension (TS)‐induced muscle atrophy remains equivocal. The molecular mechanism by which testosterone regulates muscle mass during TS has not been investigated. Methods: Effects of TS on serum testosterone levels, muscle mass, and expression of muscle atrophy‐ and hypertrophy‐inducing targets were measured in soleus (SOL) and extensor digitorum longus (EDL) muscles after testosterone administration during 1, 5, and 14 days of TS in male mice. Results: TS produced an increase followed by a transient drop in testosterone levels. Muscle atrophy was associated with downregulation of Igf1 and upregulation of Mstn, Redd1, Atrogin‐1, and MuRF1 mRNA with clear differences in Igf1, Mstn, and MAFbx/Atrogin‐1 gene expression between SOL and EDL. Testosterone supplementation did not affect muscle mass or protein expression levels during TS. Conclusions The known anabolic effects of testosterone are not sufficient to ameliorate loss of muscle mass during TS. Muscle Nerve 52 : 278–288, 2015     

New mouse model of skeletal muscle atrophy using spiral wire immobilization

Introduction: Disuse‐induced skeletal muscle atrophy is a serious concern; however, there is not an effective mouse model to elucidate the molecular mechanisms. We developed a noninvasive atrophy model in mice. Methods: After the ankle joints of mice were bandaged into a bilateral plantar flexed position, either bilateral or unilateral hindlimbs were immobilized by wrapping in bonsai steel wire. Results: After 3, 5, or 10 days of immobilization of the hip, knee, and ankle, the weight of the soleus and plantaris muscles decreased significantly in both bilateral and unilateral immobilization. MAFbx/atrogin‐1 and MuRF1 mRNA was found to have significantly increased in both muscles, consistent with disuse‐induced atrophy. Notably, the procedure did not result in either edema or necrosis in the fixed hindlimbs. Conclusions: This method allows repeated, direct access to the immobilized muscle, making it a useful procedure for concurrent application and assessment of various therapeutic interventions. Muscle Nerve 54 : 788–791, 2016     

Effects of nandrolone on denervation atrophy depend upon time after nerve transection

Anabolic steroids prevent disuse atrophy and reverse atrophy caused by glucocorticoids. To determine whether these beneficial effects extend to denervation atrophy, we tested whether nandrolone blocked denervation atrophy acutely or reversed subacute denervation atrophy. We also tested the association of such anabolic effects with expression of MAFbx, MuRF1 (both of which accelerate denervation atrophy), and IGF-1 (which prevents such atrophy). When begun at the time of denervation, nandrolone did not alter atrophy or expression of MAFbx, MuRF1, or IGF-1 measured 3, 7, or 14 days thereafter. When nandrolone administration was begun 28 days after denervation, atrophy was significantly reduced 7 and 28 days later (16% and 30%, respectively), and this was associated with significant reductions in expression of MAFbx and MuRF1, without alterations in the expression of IGF-1. The findings indicate that the actions of nandrolone depend on time after nerve transection and that the timing of anabolic steroid administration is an important determinant of responses of atrophying muscle to these agents.     

Radiopacity of intracerebral hemorrhage correlates with perihemorrhagic edema

Background: Experimental evidence indicates that iron plays a key role in edema formation after intracerebral hemorrhage (ICH). We investigated the relationship between ICH radiopacity on CT as a marker of hematoma iron content and perihemorrhagic edema (PHE) after ICH. Methods: We retrospectively investigated patients with spontaneous lobar and ganglionic supratentorial ICH who received follow‐up CT scans during the first 7 days after symptom onset (d1, d2–4, d5–7). Measurements of ICH and edema volumes were taken using a semiautomatic threshold‐based volumetric algorithm. Radiopacity of the blood clot was determined using the mean Hounsfield unit (HU) count of the ICH. Results: A total of 117 patients aged 71.92 ± 11.55 years with spontaneous ICH (34.63 ± 32.44 ml) were included in the analysis. Mean ICH radiopacity was 59.7 ± 3.4 HU. We found significantly larger relative PHE at d2–4 (1.7 ± 0.9 vs. 1.3 ± 0.8; P = 0.032) and d5–7 (2.0 ± 1.3 vs. 1.3 ± 0.9; P = 0.007) and larger peak relative PHE (2.3 ± 1.6 vs. 1.6 ± 1.1; P = 0.006) in patients with ICH radiopacity >60 HU (n = 59), as compared to patients with ICH radiopacity <60 HU (n = 58). Conclusions: Higher ICH radiopacity, reflecting higher in vivo hematoma iron content, is associated with more PHE after ICH.     

Lack of caspase‐3 attenuates immobilization‐induced muscle atrophy and loss of tension generation along with mitigation of apoptosis and inflammation

Introduction: Immobilization by casting induces disuse muscle atrophy (DMA). Methods: Using wild type (WT) and caspase‐3 knockout (KO) mice, we evaluated the effect of caspase‐3 on muscle mass, apoptosis, and inflammation during DMA. Results: Caspase‐3 deficiency significantly attenuated muscle mass decrease [gastrocnemius: 28 ± 1% in KO vs. 41 ± 3% in WT; soleus: 47 ± 2% in KO vs. 56 ± 2% in WT; (P < 0.05)] and gastrocnemius twitch tension decrease (23 ± 4% in KO vs. 36 ± 3% in WT, P < 0.05) at day 14 in immobilized vs. contralateral hindlimb. Lack of caspase‐3 decreased immobilization‐induced increased apoptotic myonuclei (3.2‐fold) and macrophage infiltration (2.2‐fold) in soleus muscle and attenuated increased monocyte chemoattractant protein‐1 mRNA expression (2‐fold in KO vs. 18‐fold in WT) in gastrocnemius. Conclusions: Caspase‐3 plays a key role in DMA and associated decreased tension, presumably by acting on the apoptosis and inflammation pathways. Muscle Nerve 47: 711–721, 2013     

Elevation of heme oxygenase‐1 by proteasome inhibition affords dopaminergic neuroprotection

Postmortem studies have shown that heme oxygenase‐1 (HO‐1) immunoreactivity is increased in patients with Parkinson disease. HO‐1 expression is highly upregulated by a variety of stress. Since the proteasome activity is decreased in patients with Parkinson disease, we investigated whether proteasome activity regulates HO‐1 content. MG‐132, a proteasome inhibitor, increased the amount of HO‐1 protein mainly in astrocytes of primary mesencephalic cultures. Quantitative RT‐PCR analysis revealed that lactacystin upregulated HO‐1 mRNA expression. Proteasome inhibition with MG132 also increased the cytomegalovirus promoter‐driven expression of Flag‐HO‐1 protein and resulted in an accumulation of ubiquitinated Flag‐HO‐1 in Flag‐HO‐1‐overexpressing PC12 cells. In addition, a cycloheximide chase assay demonstrated that the degradation of Flag‐HO‐1 protein was slowed by MG‐132. Next, the function of HO‐1 which was upregulated by proteasome inhibitors was examined. Proteasome inhibitors protected dopaminergic neurons from 6‐hydroxydopamine (6‐OHDA)‐induced toxicity and this neuroprotection was abrogated by co‐treatment with zinc protoporphyrin IX, a HO‐1 inhibitor. Furthermore, 6‐OHDA‐induced toxicity was blocked by bilirubin and carbon monoxide, products of the HO‐1‐catalyzed degradation of heme. These results suggest that mesencephalic HO‐1 protein level is regulated by proteasome activity and the elevation by proteasome inhibition affords neuroprotection. © 2010 Wiley‐Liss, Inc.     

Orodispersible sublingual piribedil to abort OFF episodes: A single dose placebo‐controlled,randomized, double‐blind,cross‐over study

S90049, a novel sublingual formulation of the non‐ergoline D₂‐D₃ agonist piribedil, has a pharmacokinetic profile promising to provide rapid relief on motor signs in Parkinson's disease (PD). We assessed the efficacy and safety of S90049 in aborting OFF episodes responding to subcutaneous apomorphine in PD patients with motor fluctuations. This was a single‐dose double‐blind double‐placebo 3 × 3 cross‐over study. Optimal tested doses were determined during a previous open‐label titration phase (S90049 median dose: 60 mg, apomorphine: 5 mg). Primary endpoint was the maximal change versus baseline in UPDRS motor score (ΔUPDRS III) assessed after drug administration following an overnight withdrawal of antiparkinsonian medications. Thirty patients (age: 60 ± 8 years, PD duration: 12 ± 6 years, UPDRS III OFF: 37 ± 15) participated. S90049 wassuperior to placebo on ΔUPDRS III (?13 ± 12 versus ?7 ± 9 respectively; estimated difference ?5.2, 95% Confidence Interval (CI)[?10.4;0.05], P = 0.05). This was also true for secondary outcomes: number of patients switching from OFF to ON (17 on S90049 vs. 8 on placebo, P = 0.03), time to turn ON (P = 0.013) and duration of the ON phase (P = 0.03). In the 17 patients who switched ON on S90049, ΔUPDRS III was similar on S90049 (?21.2 ± 10.1) and apomorphine (?23.6 ± 14.1) (estimated difference: 4.0 95% CI [?2.9;10.9]). S90049 was well tolerated: no serious or unexpected adverse event occurred. A single dose of up to 60 mg of S90049 given sublingually was superior to placebo in improving UPDRS III and aborting a practical OFF in patients with advanced PD. Testing greater doses might improve response rate. © 2009 Movement Disorder Society     

Metabolic effects of adjunctive aripiprazole in clozapine‐treated patients with schizophrenia

Fan X, Borba CPC, Copeland P, Hayden D, Freudenreich O, Goff DC, Henderson DC. Metabolic effects of adjunctive aripiprazole in clozapine‐treated patients with schizophrenia. Objective: This study examined the effects of adjunctive aripiprazole therapy on metabolism in clozapine‐treated patients with schizophrenia. Method: In an 8‐week randomized, double‐blind, placebo‐controlled study, subjects received either aripiprazole (15 mg/day) or placebo. At baseline and week 8, metabolic parameters were assessed by the frequently sampled intravenous glucose tolerance test, nuclear magnetic resonance spectroscopy and whole‐body dual‐energy X‐ray absorptiometry (DXA). Results: Thirty subjects completed the study (16 in the aripiprazole group and 14 in the placebo group). Glucose effectiveness measured by the frequently sampled intravenous glucose tolerance test improved significantly in the aripiprazole group (0.003 ± 0.006 vs. ?0.005 ± 0.007/min, P = 0.010). The aripiprazole group showed significant reductions in both plasma low‐density lipoprotein (LDL) levels (?15.1 ± 19.8 vs. 4.4 ± 22.5 mg/dl, P = 0.019) and LDL particle numbers (?376 ± 632 vs. ?36 ± 301 nm , P = 0.035). Further, there was a significant reduction in the lean mass (?1125 ± 1620 vs. 607 ± 1578 g, P = 0.011) measured by whole‐body DXA scan in the aripiprazole group. All values were expressed as mean ± standard deviation, aripiprazole vs. placebo. Conclusion: Adjunctive therapy with aripiprazole may have some metabolic benefits in clozapine‐treated patients with schizophrenia.     

Rituximab in refractory myasthenia gravis: Extended prospective study results

Introduction: Rituximab appears to be beneficial in treatment‐refractory myasthenia gravis (MG); however, prospective, long‐term durability data are lacking. Methods: In this prospective, open‐label study of rituximab in refractory MG, 22 patients (10 nicotinic acetylcholine receptor, 9 muscle‐specific tyrosine kinase, 3 seronegative) received rituximab at baseline, with repeat cycles driven by clinical worsening. Manual muscle testing (MMT) scores and CD19/CD20⁺ B‐cell counts were serially monitored. Results: At mean follow‐up of 28.8 ± 19.0 months (range, 6–66), mean MMT scores declined from 10.6 ± 5.4 to 3.3 ± 3.1 (P < 0.0001). Mean prednisone dosage declined from 25.2 ± 15.1 to 7.3 ± 7.1 mg/d (P = 0.002). Ten relapses occurred, with average time to first relapse of 17.1 ± 5.5 months (range, 9–23). CD19/CD20⁺ count recovery did not predict relapse. Three patients experienced prolonged B‐cell depletion (range, 24–45 months) after 1 cycle. Discussion: Sustained clinical improvement was associated with rituximab after 1 cycle, with prolonged time to relapse and reduction in steroid dosage. Muscle Nerve 58 : 453–456, 2018     

Comparison of voxel‐based specific region analysis of Alzheimer's disease and simple computed tomography evaluation of medial temporal atrophy in psychiatric patients

Background: The present study was designed to find a good alternative to the voxel‐based specific region analysis of Alzheimer's disease (VSRAD) on magnetic resonance images, which has high accuracy for discriminating between very early Alzheimer's disease (AD) patients and healthy controls. Because magnetic resonance imaging is not necessarily available in ordinary psychiatric hospitals in Japan, this type of study is of clinical significance. Methods: In 30 psychiatric inpatients with a variety of diagnoses, the mean area of the inferior horn (Area), the mean width of the medial temporal lobe (Width), and their ratio (Area/Width) were measured on computed tomography (CT) section that was parallel to the orbitomeatus line. These three indices of medial temporal atrophy were compared separately with Z‐scores from VSRAD, which indicate the degree to which the mean values deviate from control. Specifically, higher Z‐scores indicate a smaller volume of the medical temporal lobe. Results: The mean (± SD) of the CT indices Area, Width, and Area/Width were 39.6 ± 22.1 mm², 10.3 ± 3.01 mm, and 4.65 ± 4.03, respectively. The mean (± SD) of the Z‐scores from VSRAD was 1.47 ± 0.76. The CT indices were all significantly correlated with the Z‐scores while controlling for age (Area, r = 0.38, d.f. = 27, P = 0.04; Width, r = ?0.45, d.f. = 27, P = 0.01; Area/Width, r = 0.57, d.f. = 27, P = 0.001). Of the three CT indices, Area/Width had the largest area under the curve (AUC) in receiver operating characteristic (ROC) curve analysis regarding Z‐scores ≥1.0 as the gold standard for the detection of subtle medial temporal atrophy. Conclusion: Although we cannot conclude that Area/Width from CT scans is a perfect alternative to Z‐scores from VSRAD ≥1.0 because of its lower sensitivity, it was found that we can expect Z‐scores ≥1.0 at an Area/Width ratio ≥4.0. Further research with a larger sample size and prospective design is required to confirm the usefulness of our CT method in the evaluation of medial temporal atrophy in psychiatric patients.     

Vastus lateralis NA+‐K+‐ATpase activity,protein, and isoform distribution in chronic obstructive pulmonary disease

In this study we investigate the hypothesis that protein abundance, isoform distribution, and maximal catalytic activity of sodium–potassium–adenosine triphosphatase (Na⁺‐K⁺‐ATPase) would be altered in muscle of patients with moderate to severe chronic obstructive pulmonary disease (COPD). Tissue samples were obtained from the vastus lateralis of 10 patients with COPD (mean ± SE: age = 67 ± 2.9 years; FEV₁ = 39 ± 5.5%) and 10 healthy, matched controls (CON: age = 68 ± 2 years; FEV₁ = 114 ± 4.2%). The samples were assessed for maximal catalytic activity (V_max) of the enzyme using the K⁺‐stimulated 3‐O‐methylfluorescein‐phosphatase (3‐O‐MFPase) assay, enzyme abundance using the [³H]‐ouabain assay, and isoform content of both α (α₁, α₂, α₃) and β (β₁, β₂, β₃) using Western blot techniques. A 19.4% lower (P < 0.05) V_max was observed in COPD compared with CON (90.7 ± 6.7 vs. 73.1 ± 4.7 nmol · mg protein^?1 h^?1). No differences between groups were observed for pump concentration (259 ± 15 vs. 243 ± 17 pmol · g wet weight). For the isoforms, α₁ was decreased by 28% (P < 0.05), and α₂ was increased by 12% (P < 0.05) in COPD compared with CON. No differences between groups were observed for α₃ or for the β isoforms. We conclude that moderate COPD compromises V_max, which occurs in the absence of changes in pump abundance. The reduction in V_max could be due to a shift in isoform expression (α₁, α₂), alterations in intrinsic regulation, or to structural changes in the enzyme. The changes observed in the catalytic activity of the pump could have major effects on membrane excitability and fatigability, which are typically compromised in COPD. Muscle Nerve, 2009     

Expression of stromal‐cell‐derived factor‐1 (SDF‐1): a predictor of ischaemic stroke?

Background and purpose: Platelet stromal‐cell‐derived factor‐1 (SDF‐1) plays a pivotal role in angiogenesis and the regeneration of ischaemic tissue through the regulation of haematopoietic progenitor cells and is upregulated at the sites of vascular injury and platelet activation. Thus, SDF‐1 has recently been discussed as a predictor in ischaemic diseases such as acute myocardial infarction. However, no clinical data pertinent to the investigation of the platelet SDF‐1 expression in patients with stroke are available. Methods: We consecutively evaluated 196 patients who were admitted to the stroke unit with symptoms suspected for stroke. Surface expression of the platelet activation markers (P‐selectin and GPIb) and the expression of platelet‐bound SDF‐1 were determined by two‐colour whole blood flow cytometry. Results: Patients with transient ischaemic attack (TIA) as well as with ischaemic stroke showed similar levels of SDF‐1 expression on hospital admission compared with patients with non‐ischaemic (NI) events and with 30 healthy controls (TIA (mean fluorescence intensity ± SD): 31.5 ± 18.2 vs. NI: 26.4 ± 15.7; P = 0.361; stroke: 28.7 ± 19.8 vs. NI; P = 0.943; control: 26.1 ± 11.3; P > 0.05 compared with all). Platelet SDF‐1 expression showed a trend with the severity of stroke according to National Institute of Health Stroke Scale score (r = 0.125; P = 0.085), but significantly correlated with the peak levels of C‐reactive protein (r = 0.218; P = 0.002) and with the levels of platelet activation (P‐selectin: r = 0.389; P = 0.001). Multifactorial analysis of covariance revealed a significant influence on platelet SDF‐1 expression by smoking (P = 0.019). Conclusions: Platelet SDF‐1 surface expression did not show any significant difference in patients with TIA and ischaemic stroke compared with patients with NI events. Thus, single biomarker evaluation of platelet SDF‐1 surface expression is not helpful to predict ischaemic stroke.     

Low levels of mutant ubiquitin are degraded by the proteasome in vivo

The ubiquitin‐proteasome system fulfills a pivotal role in regulating intracellular protein turnover. Impairment of this system is implicated in the pathogenesis of neurodegenerative diseases characterized by ubiquitin‐ containing proteinaceous deposits. UBB⁺¹, a mutant ubiquitin, is one of the proteins accumulating in the neuropathological hallmarks of tauopathies, including Alzheimer's disease, and polyglutamine diseases. In vitro, UBB⁺¹ properties shift from a proteasomal ubiquitin‐fusion degradation substrate at low expression levels to a proteasome inhibitor at high expression levels. Here we report on a novel transgenic mouse line (line 6663) expressing low levels of neuronal UBB⁺¹. In these mice, UBB⁺¹ protein is scarcely detectable in the neuronal cell population. Accumulation of UBB⁺¹ commences only after intracranial infusion of the proteasome inhibitors lactacystin or MG262, showing that, at these low expression levels, the UBB⁺¹ protein is a substrate for proteasomal degradation in vivo. In addition, accumulation of the protein serves as a reporter for proteasome inhibition. These findings strengthen our proposition that, in healthy brain, UBB⁺¹ is continuously degraded and disease‐related UBB⁺¹ accumulation serves as an endogenous marker for proteasomal dysfunction. This novel transgenic line can give more insight into the intrinsic properties of UBB⁺¹ and its role in neurodegenerative disease. © 2010 Wiley‐Liss, Inc.     

Butyrate prevents muscle atrophy after sciatic nerve crush

Introduction: Histone deacetylases (HDACs) have been implicated in neurogenic muscle atrophy, but the mechanisms by which HDAC inhibitors might have beneficial effects are not defined. Methods: We used sciatic nerve crush to determine the effect of butyrate on denervation‐induced gene expression and oxidative stress. Results: Butyrate treatment initiated 3 weeks before injury and continued 1 week after injury increases histone acetylation and reduces muscle atrophy after nerve crush. Butyrate delivered only after nerve crush similarly prevented muscle atrophy. Butyrate had no effect on the increase in histone deacetylase 4 (HDAC4) protein levels following nerve crush but prevented the increase in expression of myogenin, MuRF1, and atrogin‐1. Butyrate did not affect mitochondrial reactive oxygen species production, but it increased antioxidant enzyme activity, reduced proteasome activity, and reduced oxidative damage following nerve injury. Conclusions: These data suggest that HDAC inhibitors are promising pharmacological agents for treating neurogenic muscle atrophy. Muscle Nerve 52: 859–868, 2015     

Manganese‐induced downregulation of astroglial glutamine transporter SNAT3 involves ubiquitin‐mediated proteolytic system

SNAT3 is a major facilitator of glutamine (Gln) efflux from astrocytes, supplying Gln to neurons for neurotransmitter synthesis. Our previous investigations have shown that, in primary cortical astrocyte cultures, SNAT3 protein is degraded after exposure to manganese (Mn²⁺). The present studies were performed to identify the processes responsible for this effect. One of the well‐established mechanisms for protein‐level regulation is posttranslational modification via ubiquitination, which leads to the rapid degradation of proteins by the 26S proteasome pathway. Here, we show that astrocytic SNAT3 directly interacts with the ubiquitin ligase, Nedd4‐2 (neural precursor cells expressed developmentally downregulated 4‐2), and that Mn²⁺ increases both Nedd4‐2 mRNA and protein levels. Additionally, we have found that Mn²⁺ exposure elevates astrocytic ubiquitin B mRNA expression, free ubiquitin protein levels, and total protein ubiquitination. Furthermore, Mn²⁺ effectively decreases astrocytic mRNA expression and the phosphorylation of serum and glucocorticoid‐inducible kinase, a regulatory protein, which, in the active phosphorylated form, is responsible for the phosphorylation and subsequent inactivation of Nedd4‐2. Additional findings establish that Mn²⁺ increases astrocytic caspase‐like proteolytic proteasome activity and that the Mn²⁺‐dependent degradation of SNAT3 protein is blocked by the proteasome inhibitors, N‐acetyl‐leu‐leu‐norleucinal and lactacystin. Combined, these results demonstrate that Mn²⁺‐induced SNAT3 protein degradation and the dysregulation of Gln homeostasis in primary astrocyte cultures proceeds through the ubiquitin‐mediated proteolytic system. © 2010 Wiley‐Liss, Inc.     

Origin and modulation of circular smooth muscle layer contractions in the porcine esophagus

Background The origin and modulation mechanisms controlling timing and amplitude of esophageal body peristalsis are not fully understood. We aimed to characterize the neurotransmitters involved in the origin and modulation of circular smooth muscle esophageal body (EB) contractions. Methods Responses of porcine EB strips to electrical stimulation of motor neurons (MNs) were assessed in organ baths and with microelectrodes. The effect of antagonists of inhibitory (L‐NAME 1 mmol L^?1, MRS2179 10 μmol L^?1) and excitatory neurotransmitters (atropine 1 μmol L^?1; SR140333 1 μmol L^?1‐NK₁ra‐, GR94800 1 μmol L^?1‐NK₂ra‐) and of ganglionic neurotransmitters (hexamethonium 100 μmol L^?1, ondansetron 1 μmol L^?1, NF279 10 μmol L^?1) were characterized. Key Results Electrical field stimulation (EFS) induced a frequency‐dependent off‐contraction (16.8 ± 0.8 g) following a latency period. Latency was significantly reduced by L‐NAME (?66.1 ± 4.1%) and MRS2179 (?25.9 ± 5.6%), and strongly increased by atropine (+36.8 ± 5.8%). Amplitude was reduced by L‐NAME (?69.9 ± 10.4%), MRS2179 (?34.1 ± 6.0%), atropine (?42.3 ± 4.7%), hexamethonium (?18.9 ± 3.3%), NF279 (?20.7 ± 3.5%), ondansetron (?16.3 ± 3.2%), GR94800 (?28.0 ± 4.8%) SR140333 (?20.9 ± 7.1%), and α‐chymotrypsin (?31.3 ± 7.0%). The EFS induced a monophasic nitrergic inhibitory junction potential. Conclusions & Inferences Our results suggest that timing (latency) and amplitude of esophageal contractions are determined by a balance of complex interactions between excitatory and inhibitory MNs. Latency depends on the activation of inhibitory MNs releasing NO and a minor purinergic contribution through P2Y₁ receptors, and excitatory MNs releasing ACh. Amplitude depends on a major contribution of excitatory MNs releasing ACh and tachykinins, and also on inhibitory MNs releasing NO, ATP or related purines, and peptidergic neurotransmitters acting as strong modulators of the excitatory neuroeffector transmission.