ICC‐MY coordinate smooth muscle electrical and mechanical activity in the murine small intestine

Background Animals carrying genetic mutations have provided powerful insights into the role of interstitial cells of Cajal (ICC) in motility. One classic model is the W/W^V mouse which carries loss‐of‐function mutations in c‐kit alleles, but retains minimal function of the tyrosine kinase. Previous studies have documented loss of slow waves and aberrant motility in the small intestine of W/W^V mice where myenteric ICC (ICC‐MY) are significantly depleted. Methods Here, we used morphological and electrophysiological techniques to further assess the loss of ICC around the circumference of the small intestine and determine consequences of losing ICC‐MY on electrical activity, Ca²⁺ transients and contractions of the longitudinal muscle (LM). Key Results In wild‐type mice, there was coherent propagation of Ca²⁺ transients through the ICC‐MY network and spread of this activity to the LM. In short segments of small intestine in vitro and in exteriorized segments, slow waves coordiated smoothly propagating Ca²⁺ waves and contractions in the LM of wild‐type mice. In W/W^V mice, Ca²⁺ waves were initiated at variable sites along and around intestinal segments and propagated without constraint unless they collided with other Ca²⁺ waves. This activity resulted in abrupt, uncoordinated contractions. Conclusions & Inferences These results show how dominance of pacemaking by ICC‐MY coordinates propagating con‐tractions and regulates the spontaneous activity of smooth muscle.     

Ca2+/calmodulin‐dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ‐aminobutyric acid type a receptor inhibition

The fast inhibitory synaptic transmission mediated by the γ‐aminobutyric acid type A receptor (GABA_AR) within spinal dorsal horn exerts a gating control over the synaptic conveyance of nociceptive information from the periphery to higher brain regions. Although a large body of evidence has demonstrated that the impairment of GABAergic inhibition alone is sufficient to elicit pain hypersensitivity in intact animals, the underlying mechanisms remain to be characterized. The present study shows that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is an important signaling protein downstream of reduced GABAergic inhibition. We found that pharmacological removal of inhibition by intrathecal application of the GABA_AR antagonist bicuculline significantly enhanced the autophosphorylation of CaMKII at Thr286 in spinal dorsal horn of mice. In addition to increased CaMKII activity, bicuculline also promoted CaMKII interaction with N‐methyl‐D‐aspartate (NMDA)‐subtype glutamate receptors and induced the translocation of CaMKII from cytosolic compartments to the synaptosomal membrane fraction. Immunoblotting analysis revealed that the phosphorylation levels of NMDA receptor NR2B subunit at Ser1303 and of AMPA‐subtype glutamate receptor GluR1 subunit at Ser831, two important CaMKII phosphorylation sites, were substantially enhanced after bicuculline application. Behavioral tests illustrated that intrathecal administration of the CaMKII inhibitor KN‐93, NMDA receptor antagonist D‐APV, or AMPA receptor antagonist GYKI 52466 effectively ameliorated the mechanical allodynia evoked by bicuculline. These data thus indicate that CaMKII signaling is critical for the reduced inhibition to evoke spinal sensitization. © 2013 Wiley Periodicals, Inc.     

Increased colonic motility in a rat model of irritable bowel syndrome is associated with up‐regulation of L‐type calcium channels in colonic smooth muscle cells

Objective This paper aimed to investigate the relationship between up‐regulation of L‐type calcium channels and altered motility disorder in a rat model of irritable bowel syndrome (IBS). Methods Male Sprague–Dawley rats were subjected to neonatal maternal separation (NMS) from postnatal day 2–14 or normal handling (NH), and used when weighted 250–300 g. Colonic smooth muscle contractions was studied in an organ bath system. L‐type Ca²⁺ channel α_1c subunit expression in smooth muscles from rat colon were studied by immunofluorescence and Western blotting analysis. The intracellular calcium concentration ([Ca²⁺]_i) of enzymatically isolated single colonic smooth muscle cell was studied with laser confocal fluorescent microscopy. Results The fecal pellets during 1 h water avoidance stress (WAS) were significantly increased; the amplitude of spontaneous contractions and contractions induced by Bay K 8644 (10 nm –1 μm ), KCl (10–60 mm ) and ACh (100 nm –10 μm ) were significantly increased in NMS rats, when comparing with that of NH rats. [Ca²⁺]i induced by Bay K 8644 (1 μm ), KCl (40 mm ), and ACh (10 μm ) significantly increased in muscle cells of NMS rats than NH rats. Further, α_1c protein expression was significantly up‐regulated in colonic smooth muscle of NMS rats than NH rats. Conclusion These results suggest that NMS lead to up‐regulation of L‐type Ca²⁺ channels expression in the colon, which contributes to the colonic motility disorder. Our findings provide direct evidence to help understanding the underlying mechanism of chronic stress‐induced colonic motility disorder in IBS.     

Synchronous phosphorylation of CPI-17 and MYPT1 is essential for inducing Ca(2+) sensitization in intestinal smooth muscle

Background Myosin phosphatase activity is regulated by mechanisms involving the phosphorylation of CPI‐17 and MYPT1, primarily based on studies with tonic‐type vascular smooth muscles. This study examined how these mechanisms contribute to the regulation of contraction of a phasic‐type intestinal smooth muscle. Methods Phosphorylation levels, tension, and Ca²⁺ sensitization was detected in rat ileal smooth muscle. Key Results In rat ileal smooth muscle, phosphorylation level of CPI‐17 at Thr³⁸ and MYPT1 at Thr⁸⁵³, but not MYPT1 at Thr⁶⁹⁶, were increased with carbachol (1 μmol L^?1) accompanied with muscle contraction. The PKC inhibitor Go6976 (1 μmol L^?1) inhibited the carbachol‐induced phosphorylation of CPI‐17, whereas the Rho‐associated kinase (ROCK) inhibitor, Y‐27632 (10 μmol L^?1) inhibited the carbachol‐induced phosphorylation of both CPI‐17 and MYPT1. Application of Go6976 or Y‐27632 alone inhibited the carbachol‐induced contraction; however, the combined application of these inhibitors did not inhibit the contraction in an additive manner. In β‐escin‐permeabilized ileal strip, treatment with antiphosphorylated antibodies for CPI‐17 at Thr³⁸ and MYPT1 at Thr⁸⁵³ and Thr⁶⁹⁶ alone almost completely abolished the Ca²⁺ sensitization due to carbachol with GTP. Conclusions & Inferences In conclusion, receptor stimulation increases the Ca²⁺ sensitivity of contractile elements through CPI‐17 phosphorylation via the PKC/ROCK pathways and MYPT1 phosphorylation via the ROCK pathway, when these mechanisms operate cooperatively and/or synchronously in intestinal smooth muscle.     

Ca2+/calmodulin-dependent protein kinase II in the spinal cord contributes to neuropathic pain in a rat model of mononeuropathy

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to subserve activity-dependent neuronal plasticity in the central nervous system. To examine in vivo the implication of spinal CaMKII activity in the generation and development of neuropathic pain after peripheral nerve injury, we used an animal model of mononeuropathy, the chronic constriction injury (CCI) model, in the rat. We found that, 3 days after CCI, the total CaMKII (tCaMKII) immunoreactivity increased in the superficial laminae of the spinal cord and this increase continued for up to 14 days. The immunoreactivity of phosphorylated CaMKII showed an increase from 1 day after CCI, which preceded the up-regulation of tCaMKII. A non-selective N-methyl-d-aspartate receptor antagonist, MK801, significantly attenuated the increase of tCaMKII and phosphorylated CaMKII. Moreover, intrathecal administration of an inhibitor of CaMKII, KN93, before the CCI surgery attenuated the development of thermal hyperalgesia and mechanical allodynia. In addition, KN93 significantly reduced the nociceptive behavior in phase II of the formalin test. These findings demonstrate that the activity of CaMKII in spinal neurons is elevated after peripheral nerve injury and may be involved in central sensitization. The alteration of CaMKII is considered to be a neuroplastic change that occurs in spinal neurons that contributes to neuropathic pain, suggesting the potential for the development of novel therapeutics for neuropathic pain that target CaMKII.     

Specific distribution of Ca / calmodulin-dependent protein kinase II α and β isoforms in some structures of the rat forebrain

The immunohistochemical distribution of Ca²⁺ / calmodulin-dependent protein kinase II (CaM kinase II) α and β isoforms in the rat forebrain was examined by using monoclonal antibodies specific to each isoform. The present study confirmed that α and β immunoreactives are localized only in neuronal elements. At the light microscopic level, specific distribution patterns of these isoforms and staining characteristics were recognized in some regions of the forebrain as follows. Firstly, α-immunoreactive neurons were more homogeneously distributed throughout the cellular layers of the cerebral cortex (i.e., layers II-IV) than β-immunoreactive ones. Secondly, neurons in the globus pallidus were immunostained by the anti-β antibody, but not by the anti-α antibody. Thirdly, neurons in the medial habenular nucleus, the subthalamic nucleus and the reticular thalamic nucleus were more densely stained with the anti-β antibody than with the anti-α antibody. However, marked differences were not observed in the hippocampal formation at the light microscopic level. The electron microscopic analysi of the cerebral cortex demostrated that subcellular localizations of α- and β-immunoreactive products within the cortical neurons were quite dissimilar: (i) the nucleus was stained only with the anti-α antibody, but not with the antiβ antibody, and (ii) β-immunoreactive products were more sporadically localized in the cytoplasms of the perikarya and dendrited than the α-immunoreactive ones. These rigional and subcellular differences between the distribution patterns of α and β immunoreactivities suggest the functional diversity of CaM kinase II α and β isoforms in the central system.     

Normal oxidative phosphorylation in intestinal smooth muscle of childhood chronic intestinal pseudo‐obstruction

Background Chronic intestinal pseudo‐obstruction (CIPO) is a severe disease of the digestive tract motility. In pediatric population, CIPO remains of unknown origin for most patients. Chronic intestinal pseudo‐obstruction is also a common feature in the course of mitochondrial oxidative phosphorylation disorders related for some patients to mutations in TYMP, POLG1, mtDNA tRNA^leu(UUR) or tRNA^lys genes. We hypothesized that CIPOs could be the presenting symptom of respiratory chain enzyme deficiency and thus we investigated oxidative phosphorylation in small bowel and/or colon smooth muscle of primary CIPO children. Methods We studied eight children with CIPO and 12 pediatric controls. We collected clinical, radiological and pathological data and measured respiratory chain enzymatic activity in isolated smooth muscle of the small bowel and/or the colon. We also sequenced TYMP, POLG, mtDNA tRNA^leu(UUR) and tRNA^lys genes. Key Results Neither pathological nor radiological data were in favor of a mitochondrial dysfunction. No respiratory chain enzyme deficiency was detected in CIPO children. In myogenic CIPO, respiratory enzymes and citrate synthase activities were increased in small bowel and/or colon whereas no abnormality was noted in neurogenic and unclassified CIPO. Levels of enzyme activities were higher in control small bowel than in control colon muscle. Sequencing of TYMP, POLG, mtDNA tRNA^leu(UUR) and tRNA^lys genes and POLG gene did not reveal mutation for any of the patients. Conclusions & Inferences The normal enzymatic activities as the lack of radiological and genetic abnormalities indicate that, at variance with adult patients, oxidative phosphorylation deficiency is not a common cause of childhood CIPO.     

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.     

Glial Na+‐dependent ion transporters in pathophysiological conditions

Sodium dynamics are essential for regulating functional processes in glial cells. Indeed, glial Na⁺ signaling influences and regulates important glial activities, and plays a role in neuron‐glia interaction under physiological conditions or in response to injury of the central nervous system (CNS). Emerging studies indicate that Na⁺ pumps and Na⁺‐dependent ion transporters in astrocytes, microglia, and oligodendrocytes regulate Na⁺ homeostasis and play a fundamental role in modulating glial activities in neurological diseases. In this review, we first briefly introduced the emerging roles of each glial cell type in the pathophysiology of cerebral ischemia, Alzheimer's disease, epilepsy, Parkinson's disease, Amyotrophic Lateral Sclerosis, and myelin diseases. Then, we discussed the current knowledge on the main roles played by the different glial Na⁺‐dependent ion transporters, including Na⁺/K⁺ ATPase, Na⁺/Ca²⁺ exchangers, Na⁺/H⁺ exchangers, Na⁺‐K⁺‐Cl^? cotransporters, and Na⁺‐ cotransporter in the pathophysiology of the diverse CNS diseases. We highlighted their contributions in cell survival, synaptic pathology, gliotransmission, pH homeostasis, and their role in glial activation, migration, gliosis, inflammation, and tissue repair processes. Therefore, this review summarizes the foundation work for targeting Na⁺‐dependent ion transporters in glia as a novel strategy to control important glial activities associated with Na⁺ dynamics in different neurological disorders. GLIA 2016;64:1677–1697     

Enzyme replacement therapy in adult‐onset glycogenosis II: Is quantitative muscle MRI helpful?

Although it has been shown that muscle magnetic resonance imaging (MRI) improves the phenotypic characterization of patients with neuromuscular disorders and allows accurate quantification of muscle and adipose tissue distribution, to date quantitative MRI has not been used to assess the therapeutic response in clinical trials of neuromuscular diseases. We discuss quantitative MRI findings after a 6‐month course of enzyme replacement therapy administered to nine patients with adult‐onset glycogenosis II. Muscle Nerve 40: 122–125, 2009     

Depression of Ca2+/calmodulin‐dependent protein kinase II in dorsal root ganglion neurons after spinal nerve ligation

The enzyme calcium/calmodulin‐dependent protein kinase II (CaMKII) is associated with memory and its α isoform is critical for development of activity‐induced synaptic changes. Therefore, we hypothesized that CaMKII is involved in altered function of dorsal root ganglion (DRG) neurons after neuronal injury. To test this hypothesis, Sprague–Dawley rats were made hyperalgesic by L5 and L6 spinal nerve ligation (SNL), and changes in total phosphorylated and unphosphorylated CaMKII (tCaMKII) and phosphorylated form of its α isoform (pCaMKIIα) were analyzed using immunochemistry in different subpopulations of DRG. SNL did not induce any changes in tCaMKII between experimental groups, while the overall percentage of pCaMKIIα‐positive neurons in injured L5 DRG SNL (24.8%) decreased significantly when compared to control (41.7%). SNL did not change the percentage of pCaMKIIα/N52 colabeled neurons but decreased the percentage of N52‐negative nonmyelinated neurons that expressed pCaMKIIα from 27% in control animals to 11% after axotomy. We also observed a significant decrease in the percentage of small nonpeptidergic neurons labeled with IB4 (37.6% in control vs. 4.0% in L5 SNL DRG), as well as a decrease in the percentage of pCaMKIIα/IB4 colabeled neurons in injured L5 DRGs (27% in control vs. 1% in L5 DRG of SNL group). Our results show that reduction in pCaMKIIα levels following peripheral injury is due to the loss of IB4‐positive neurons. These results indicate that diminished afferent activity after axotomy may lead to decreased phosphorylation of CaMKIIα. J. Comp. Neurol. 518:64–74, 2010. © 2009 Wiley‐Liss, Inc.