Motility of the left colon in children and adolescents with functional constpation; a retrospective comparison between solid‐state and water‐perfused colonic manometry

Background

Using water‐perfused (WP) high‐resolution manometry, we recently demonstrated that children with functional constipation (FC) lacked the postprandial increase in distal colonic cyclic motor patterns that was observed in healthy adults. Our aim was to determine if similar results could be detected using a solid‐state (SS) manometry catheter.

Methods

We performed a retrospective analysis of 19 children with FC (median age 11.1 years, 58% male) who underwent colonic manometry with a SS catheter (36 sensors, 3 cm apart). Data were compared with previously published data using a WP catheter (36 sensors, 1.5 cm apart) recorded from 18 children with FC (median age 15 years; 28% male).

Key Results

The cyclic motor patterns recorded by the SS catheter did not differ from those previously recorded by the WP catheter. There was no detected increase in this activity in response to the meal in either group. Long‐single motor patterns were recorded in most patients (n = 16, 84%) with the SS catheter. The number of these events did not differ from the WP recordings. In the SS data, HAPCs were observed in 4 children prior to the meal, in 5 after the meal. This did not differ significantly from the WP data.

Conclusions & Inferences

These data recorded by SS manometry did not differ from WP manometry data. Regardless of the catheter used, both studies revealed an abnormal colonic response to a meal, indicating a pathology which is not related to the catheter used to record these data.     

β‐Catenin regulates membrane potential in muscle cells by regulating the α2 subunit of Na,K‐ATPase

Muscle β‐catenin has been shown to play a role in the formation of the neuromuscular junction (NMJ). Our previous studies showed that muscle‐specific conditional knockout of β‐catenin (HSA‐β‐cat^?/?) results in early postnatal death in mice. To understand the underlying mechanisms, we investigated the electrophysiological properties of muscle cells from HSA‐β‐cat^?/? and control mice, and found that, in the absence of muscle β‐catenin, the resting membrane potential (RMP) depolarised in muscle cells from the diaphragm, gastrocnemius and extensor digitorum longus muscles. Furthermore, in a primary line of mouse myoblasts (C2C12 cells) transfected with small‐interfering RNAs targeting β‐catenin, the RMP was depolarised as well. Finally, the expression levels of the α2 subunit of sodium/potassium adenosine triphosphatase were reduced by β‐catenin knockdown in vitro or deletion in vivo. These results suggest a possible mechanism underlying the depolarised RMP in the absence of muscle β‐catenin, and provide additional evidence supporting a role for β‐catenin in the development of NMJs.     

Factors associated with successful decrease and discontinuation of antegrade continence enemas (ACE) in children with defecation disorders: a study evaluating the effect of ACE on colon motility

Background Antegrade continence enemas (ACE) have been used in the treatment of defecation disorders in children; little is known on their effect on colon motility and the utility of the colon manometry (CM) predicting long‐term ACE outcomes. Methods Retrospective review of children with constipation undergoing CM before and after ACE to evaluate CM changes and their utility on predicting ACE outcome. Key Results A total of 40 patients (mean age 8.8 SD 3 years and 53% female patients) were included; 39 of 40 responded to the ACE. Of these 39, 14 (36%) were dependent and 25 (64%) had decreased it (11 of those or 28% discontinued it). On repeat CM we found a significant increase in the fasting (P < 0.01) and postprandial (P = 0.03) motility index, number of bisacodyl‐induced high amplitude propagating contractions (HAPCs) (P = 0.03), and total HAPCs (P = 0.02). Gastrocolonic response to a meal, propagation and normalization of HAPCs improved in 28%, 58%, and 33%, respectively, with CM normalizing in 33% of patients. The baseline CM did not predict ACE outcome. The presence of normal HAPCs on the repeat CM was associated with ACE decrease. Progression and normalization of HAPCs (P = 0.01 and 0.02, respectively) and CM normalization (P = 0.01) on repeat CM were individually associated with ACE decrease. No CM change was associated with ACE discontinuation. Multivariate analysis showed that older age and HAPC normalization on CM predict ACE decrease and older age is the only predictor for ACE discontinuation. Conclusions & Inferences Colon motility improves after ACE and the changes on the repeat CM may assist in predicting ACE outcome.     

Deficit in acoustic signal‐in‐noise detection in glycine receptor α3 subunit knockout mice

Hearing is an essential sense for communication in animals and humans. Normal function of the cochlea of higher vertebrates relies on a fine‐tuned interplay of afferent and efferent innervation of both inner and outer hair cells. Efferent inhibition is controlled via olivocochlear feedback loops, mediated mainly by acetylcholine, γ‐aminobutyric acid (GABA) and glycine, and is one of the first sites affected by synapto‐ and neuropathy in the development of hearing loss. While the functions of acetylcholine, GABA and other inhibitory transmitters within these feedback loops are at least partially understood, especially the function of glycine still remains elusive. To address this question, we investigated hearing in glycine receptor (GlyR) α3 knockout (KO) and wildtype (WT) mice. We found no differences in pure tone hearing thresholds at 11.3 and 16 kHz between the two groups as assessed by auditory brainstem response (ABR) measurements. Detailed analysis of the ABR waves at 11.3 kHz, however, revealed a latency decrease of wave III and an amplitude increase of wave IV in KO compared to WT animals. GlyRα3 KO animals showed significantly impaired prepulse inhibition of the auditory startle response in a noisy environment, indicating that GlyRα3‐mediated glycinergic inhibition is important for signal‐in‐noise detection.     

Nitrergic–purinergic interactions in rat distal colon motility

Responses of rat distal colon circular muscle strips to exogenous nitric oxide (NO) and adenosine 5'-triphosphate (ATP) and to electrical field stimulation (EFS) were assessed in the absence/presence of various agents that interfere with nitrergic-purinergic pathways. Exogenous NO (10-6 to 10-4 mol L-1) elicited concentration-dependent, tetrodotoxin (TTX)-insensitive relaxations. The soluble guanylyl-cyclase (sGC) inhibitor 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced duration and amplitude; the small conductance Ca2+-sensitive K+ (SK)-channel blocker apamin (APA) only shortened the relaxations. ODQ + APA showed a marked inhibitory effect on duration and amplitude. TTX, APA, the NO-synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) and the purinergic receptor P2Y antagonist Reactive Blue 2 (RB2) shortened the relaxations by exogenous ATP (10-3 mol L-1) but did not influence the amplitude. ODQ had no effect. TTX + l-NAME did not yield a more pronounced inhibitory effect than TTX alone. The effect of ATP-gamma-S was similar to that of ATP. Electrical field stimulation (EFS) (40 V, 0.05 ms, 0.5-4 Hz for 30 s) yielded TTX-sensitive relaxations that were not altered by l-NAME, ODQ or RB2. APA shortened the relaxations. l-NAME + APA nearly abolished these relaxations. ODQ + APA and RB2 +l-NAME reduced the duration. These results suggest that distinct sets of small conductance SK-channels are involved in the amplitude and the duration of the relaxations and that NO increases their sensitivity to NO and ATP via guanosine 3',5'-cyclic monophosphate (cGMP). ATP elicits relaxations via P2Y receptors with subsequent activation of SK-channels and induces neuronal release of NO. Both nitrergic and purinergic pathways must be blocked to inhibit EFS-induced relaxations.     

Na+/Ca2+ exchanger 2‐heterozygote knockout mice display decreased acetylcholine release and altered colonic motility in vivo

Background The Na⁺/Ca²⁺ exchanger (NCX) is a plasma membrane transporter involved in regulating intracellular Ca²⁺ concentrations. NCX is critical for Ca²⁺ regulation in cardiac muscle, vascular smooth muscle, and nerve fibers. However, little is known about the physiological role of NCX in the myenteric neurons and smooth muscles of the gastrointestinal tract. Methods To determine the role of NCX1 and NCX2 in gastrointestinal tissues, we examined electric field stimulation (EFS)‐induced responses in the longitudinal smooth muscle of the distal colon in NCX1‐ and NCX2‐heterozygote knockout mice. Key Results We found that the amplitudes of EFS‐induced relaxation that persisted during EFS were greater in NCX2 heterozygous mice (HET) than in wild‐type mice (WT). Under the nonadrenergic, noncholinergic (NANC) condition, EFS‐induced relaxation in NCX2 HET was similar in amplitude to that of WT. In addition, an NCX inhibitor, YM‐244769 enhanced EFS‐induced relaxation but did not affect EFS‐induced relaxation under the NANC condition, as in NCX2 HET. Unlike NCX2 HET, NCX1 HET displayed no marked changes in colonic motility. These results indicate that cholinergic function in the colon is altered in NCX2 HET. The magnitude of acetylcholine (ACh)‐induced contraction in NCX2 HET was similar to that in WT. In contrast, EFS‐induced ACh release was reduced in NCX2 HET compared with that in WT. Conclusions & Inferences In this study, we demonstrate that NCX2 regulates colonic motility by altering ACh release onto the myenteric neurons of the distal colon.     

Methamphetamine‐induced up‐regulation of α2/δ subunit of voltage‐gated calcium channels is regulated by DA receptors

This study was carried out to determine the roles of dopamine D1 and D2 receptors on the up‐regulation of α₂/δ subunit of voltage‐gated Ca²⁺ channels (VGCCs) induced by methamphetamine (METH). In the conditioned place preference paradigm, METH‐induced place preference suppressed with gabapentin, an antagonist for α₂/δ subunit. Under these conditions, the increase in α₂/δ subunit expression was found in the frontal cortex and limbic forebrain. In addition, the METH‐induced place preference was significantly attenuated by dopamine D1 and D2 receptor antagonists, SCH23390 and sulpiride, respectively. The expression of α₂/δ subunit protein and its mRNA was significantly enhanced in the METH‐treated cortical neurons. These increases in protein and mRNA of α₂/δ subunit were completely abolished by SCH23390 and sulpiride with simultaneous exposure to METH. These findings indicate that up‐regulation of α₂/δ subunit is regulated through the activation of dopamine D1 and D2 receptors during METH treatment. Synapse 64:822–828, 2010. © 2010 Wiley‐Liss, Inc.     

Distention of the colon is associated with initiation of propagated contractions in children

Abstract  The presence of high-amplitude propagating contractions (HAPCs) has been identified as a marker of colonic neuromuscular integrity. The physiologic mechanisms of HAPCs initiation have yet to be determined. Distention secondary to colonic filling has been hypothesized as physiologic initiator. The aim of this study was to study the effect of intraluminal balloon distention in the colon of children with defecatory disorders. Colonic manometry was performed with a polyethylene balloon situated at the proximal end of the catheter, which was placed in the most proximal colonic segment reached during colonoscopy. A stepwise pressure controlled distention of the balloon was performed using barostat computer (10–50 mmHg). Propagated contractions were defined as those that migrated over at least three recording sites. They were divided into HAPCs, amplitude >60 mmHg and low-amplitude propagating contractions (LAPCs), amplitude <60 mmHg. Children with spontaneous HAPCs or HAPCs after bisacodyl provocation were considered to have normal motility. Twenty children completed the study. Among the 14 children with normal colonic motility, balloon distention elicited HAPCs in four and LAPCs in 10 children. No HAPC were elicited in six children with abnormal motility and LAPCs were seen in four of them. The balloon-induced propagated contractions had similar characteristics as those occurring spontaneously and after bisacodyl provocation but the pressure needed to elicit them and their amplitude was inconsistent. These findings suggest that intraluminal distention can trigger propagated contractions in children. This mechanism of action for induction of propagated contractions is not as consistent as the motor response found in response to bisacodyl administration.     

Amyotrophic lateral sclerosis patient antibodies label Ca2+ channel α1 subunit

Sporadic amyotrophic lateral sclerosis is an idiopathic human degenerative disease of spinal cord and brain motor neurons. Prior studies demonstrated that most patients with amyotrophic lateral sclerosis posses immunoglobulins that bind to purified L-type voltage-gated calcium channels, that titers of anti–voltage-gated calcium channel antibodies correlate with disease progression rates, and that amyotrophic lateral sclerosis patient-derived antibodies (ALS IgG) produce electrophysiological changes in the function of voltage-gated calcium channels. Using Western transfer immunoblots and enzyme-linked immunosorbent assays, the calcium ionophore–forming α₁ subunig of the voltage-gated calcium channel is now identified as the major voltage-gated calcium channel antigen to which ALS IgG binds. Additionally, the binding of an L-type voltage-gated calcium channel α₁ subunit–directed monoclonal antibody, which itself mimics the effects of ALS IgG on skeletal muscle voltage-gated calcium channel currents, is selectively prevented by preaddition of ALS IgG. Voltage-gated calcium channel–binding IgG from patients with Lambert-Eaton myasthenic syndrome appears to be differentiated from ALS IgG by the reactivity of the former to both α₁ and β subunits of the calcium channel. These assays provide further evidence linking amyotrophic lateral sclerosis to an autoimmune process, and suggest one means to differentiate immunoglobulins from patients with amyotrophic lateral sclerosis from those of patients with another autoimmune disease expressing calcium channel antibodies.     

α‐Synuclein mediates alterations in membrane conductance: a potential role for α‐synuclein oligomers in cell vulnerability

α‐Synuclein has been linked to the pathogenesis of Parkinson’s disease and other synucleinopathies through its propensity to form toxic oligomers. The exact mechanism for oligomeric synuclein‐directed cell vulnerability has not been fully elucidated, but one hypothesis portends the formation of synuclein‐containing pores within cell membranes leading to leak channel‐mediated calcium influx and subsequent cell death. Here we demonstrate synuclein‐induced formation of sodium dodecyl sulfate‐stable oligomers, intracellular synuclein‐positive aggregates, alterations in membrane conductance reminiscent of leak channels and subsequent cytotoxicity in a dopaminergic‐like cell line. Furthermore we demonstrate that the synuclein‐induced membrane conductance changes are blocked by direct extracellular application of an anti‐synuclein antibody. The work presented here confirms that synuclein overexpression leads to membrane conductance changes and demonstrates for the first time through antibody‐blocking studies that synuclein plays a direct role in the formation of leak channels.     

Impaired glutamate recycling and GluN2B‐mediated neuronal calcium overload in mice lacking TGF‐β1 in the CNS

Transforming growth factor β1 (TGF‐β1) is a pleiotropic cytokine expressed throughout the CNS. Previous studies demonstrated that TGF‐β1 contributes to maintain neuronal survival, but mechanistically this effect is not well understood. We generated a CNS‐specific TGF‐β1‐deficient mouse model to investigate the functional consequences of TGF‐β1‐deficiency in the adult mouse brain. We found that depletion of TGF‐β1 in the CNS resulted in a loss of the astrocyte glutamate transporter (GluT) proteins GLT‐1 (EAAT2) and GLAST (EAAT1) and decreased glutamate uptake in the mouse hippocampus. Treatment with TGF‐β1 induced the expression of GLAST and GLT‐1 in cultured astrocytes and enhanced astroglial glutamate uptake. Similar to GLT‐1‐deficient mice, CNS‐TGF‐β1‐deficient mice had reduced brain weight and neuronal loss in the CA1 hippocampal region. CNS‐TGF‐β1‐deficient mice showed GluN2B‐dependent aberrant synaptic plasticity in the CA1 area of the hippocampus similar to the glutamate transport inhibitor DL‐TBOA and these mice were highly sensitive to excitotoxic injury. In addition, hippocampal neurons from TGF‐β1‐deficient mice had elevated GluN2B‐mediated calcium signals in response to extrasynaptic glutamate receptor stimulation, whereas cells treated with TGF‐β1 exhibited reduced GluN2B‐mediated calcium signals. In summary, our study demonstrates a previously unrecognized function of TGF‐β1 in the CNS to control extracellular glutamate homeostasis and GluN2B‐mediated calcium responses in the mouse hippocampus.