Atropine can induce autophagy independent of the M3 muscarinic acetylcholine receptor

Background: No other effects of atropine other than as an antagonist of muscarinic acetylcholine receptor (mAChR) have been found. Methods: In this study, human kidneyepithelial cells were treated with different physiological regulators. Results: Subsequently, it was found that atropine could significantly induce autophagy as demonstrated by the appearance of autophagosome-like double- or single-membrane vesicles in the cytoplasm ofhost cells and the number of GFP-LC3 dots. In addition, increased conversion of the autophagy marker protein LC3-I and LC3-II and increased p62/SQSTM1 indicatedincomplete autophagy. In addition, atropine induced autophagosome levels in a dose-dependent manner within a certain concentration range in human kidney epithelial cells. In atropine-treated mouse skeletal muscle cells containing nicotinic acetylcholinereceptors and rat cardiac muscle cells containing mAchR, atropine induced autophagy in mouse skeletal muscle cells but not in rat cardiac muscle cells. Furthermore, atropine did not induce autophagy in tissue cells containing mAchR in vivo but did in tissue cells not containing mAchR. Conclusion: This study expands the application and understanding of atropine’s action mechanism in the field of medicine.

Nicotinic acetylcholine signaling is required for motor learning but not for rehabilitation from spinal cord injury

Therapeutic intervention for spinal cord injury is limited,with many approaches relying on strengthening the remaining substrate and driving recovery through rehabilitative training.As compared with learning novel compensatory strategies,rehabilitation focuses on resto ring movements lost to injury.Whether rehabilitation of previously learned movements after spinal cord injury requires the molecular mechanisms of motor learning,or if it engages previously trained motor circuits without requiring novel learning remains an open question.In this study,mice we re randomly assigned to receive intrape ritoneal injection with the pan-nicotinic,non-competitive antagonist mecamylamine and the nicotinicα7 subunit selective antagonist methyllycaconitine citrate salt or vehicle(normal saline)prior to motor learning assays,then randomly reassigned after motor learning for rehabilitation study post-injury.Ce rvical spinal co rd dorsal column lesion was used as a model of in complete injury.Results of this study showed that nicotinic acetylcholine signaling was required for motor learning of the single pellet-reaching task but it was dispensable for the rehabilitation of the same task after injury.Our findings indicate that critical diffe rences exist between the molecular mechanisms supporting compensatory motor learning strategies and the restoration of behavior lost to spinal cord injury.

Anisodine hydrobromide alleviates oxidative stress caused by hypoxia/reoxygenation in human cerebral microvascular endothelial cells predominantly via inhibition of muscarinic acetylcholine receptor 4

Background:Anisodine hydrobromide(AT3),an anti-cholinergic agent,could be delivered to the brain across the blood-brain barrier and has been used clinically for the treatment of cerebral ischemia/reperfusion injury.Endothelial dysfunction can be caused by hypoxia/reoxygenation(H/R)via oxidative stress and metabolic alterations.The present study investigated whether AT3 regulates the production of nitric oxide(NO)and reactive oxygen species(ROS),and the HIF-1αpathway via regulation of muscarinic acetylcholine receptors(mAChRs)in brain microvascular endothelial cells after H/R exposure.Methods:Under H/R conditions,hCMEC/D3 cerebral microvascular endothelial cells were treated with AT3.Specific inhibitors of M2-and M4-mAChRs were used to explore the mechanism by which AT3 influences oxidative stress in endothelial cells.Then,mAChRs expression was detected by western blotting and NO production was detected by Greiss reaction.The intracellular ROS level was measured using DCFH-DA probes.The expression of hypoxia-inducible transcription factor 1α(HIF-1α)was also detected.Results:While H/R induced the expression of M2-and M4-mAChRs,AT3 suppressed the H/R-upregulated M2-and M4-mAChRs.H/R also induced the production of NO,ROS,and apoptosis.AT3 and M4-mAChR inhibitors inhibited the H/R-induced production of NO and ROS and apoptosis.HIF-1αwas induced by H/R,but was suppressed by AT3.Conclusion:Thus,the in vitro evidence shows that AT3 protects against H/R injury in cerebral microvascular endothelial cells via inhibition of HIF-1α,NO and ROS,predominantly through the downregulation of M4-mAChR.The findings offer novel understandings regarding AT3-mediated attenuation of endothelial cell apoptosis and cerebral ischemia/reperfusion injury.

Effect of differential rearing environments on nicotine-stimulated locomotor activity and nicotinic acetylcholine receptor subtypes

OBJECTIVE Individuals vary in sensitivity to the behavioral effects of nicotine,resulting in differences in their vulnerability to addiction.The role of rearing environment in determining individual sensitivity to nicotine is unclear.The neuropharmacological mechanisms mediating the effect of rearing environment on the actions of nicotine are also understood.Thus,the contribution of rearing environment in determining the sensitivity to the locomotor effects of nicotine and regulating α4β2*-and α7-nicotinic acetylcholine(n ACh) receptor expressionwas determined in rats reared in isolated(IC) or enriched(EC) conditions.METHODS To measure locomotor activity,adolescent rats(postnatal day 21-51)were injected with saline(1 mL·kg^(-1)) or nicotine(0.3 mg·kg^(-1)) subcutaneously,then placed in chamberswhere ambulatory activity was monitored for 30-min by computer for 14 daily sessions.α4β2*-andα7-n ACh receptor expression in the mesolimbic dopamine pathway was determined by quantitative autoradiography of [125 I]-epibatidine and [125 I]-bungarotoxinbinding,respectively,in 16 μmol·L^(-1) coronal sections.Values for receptor expression in fmol are ±s of 8 brains and compared by two-tailed,unpaired t-test with P<0.05 considered significant.RESULTS EC-rats are similarly sensitive as IC-rats to the locomotor effects of nicotine.[125 I]-epibatidine binding in the ventral tegmental area of EC-rats was reduced(2.8±0.3 fmo L) compared to IC-rats(4.0±0.4 fmo L);there was no difference in the nucleus accumbens.There was no difference between EC-and IC-rats in α7-n ACh receptor expression in the mesolimbic dopamine pathway.CONCLUSION Rearing environment differentially regulates n ACh receptor subtypes in EC and IC rats.These data suggest regulation of n ACh receptors by environmental factors may be a mechanism for the protective effect of enrichment against altered sensitivity to nicotine in genetically vulnerable individuals.The characterization of these mechanisms will aid in development of novel pharmacological tools mimicking the protection afforded by environmental enrichment in nicotine-sensitive individuals.

Microcalorimetric Study of Acetylcholine and Acetylthiocholine Hydrolysis by Acetylcholinesterase

Acetylcholinesterase (AChE) is an important enzyme responsible for the cleavage of acetylcholine. Studies of the activity of this enzyme use an artificial substrate, acetylthiocholine, because a product of its catalysis, thiocholine, readily generates a light absorbing product upon reaction with Elman’s reagent 5,5’-dithiobis-(2-nitrobenzoic acid (DTNB). The hydrolysis of acetylcholine cannot be assayed with this method. The isothermal titration calorimetry can assay the hydrolysis of both substrates, without requiring additional reagents other than the enzyme and the substrate. To compare kinetic values obtained in the hydrolysis of acetylcholine (ACh) and acetylthiocholine (ATCh), with carbaryl acting as inhibitor, a calorimetric technique was used to evaluate kinetic properties of the two reactions. This method can show the hydrolysis of both substrates by the heat exchange that occurs during catalysis. In addition, it allowed the assessment of the AChE inhibition by carbaryl, a common insecticide. The results show a similarity between values obtained with both substrates, which are slightly higher for acetylcholine, the enzyme natural substrate. Enzymatic parameters values from ATCh and ACh were similar to each other and inhibitory constants using carbaryl were also similar, displaying that any approach to ACh is feasible using ATCh. The results obtained from ITC show the precision achieved by the calorimetric method.

Septic encephalopathy: When cytokines interact with acetylcholine in the brain

Sepsis-associated encephalopathy(SAE) is a brain dysfunction that occurs secondary to infection in the bo characterized by alteration of consciousness, ranging from delirium to coma, seizure or focal neurological signs. S involves a number of mechanisms, including neuroinflammation, in which the interaction between cytokines a acetylcholine results in neuronal loss and alterations in cholinergic signaling. Moreover, the interaction also occurs the periphery, accelerating a type of immunosuppressive state. Although its diagnosis is not specific in biochemis and imaging tests, it could potentiate severe outcomes, including increased mortality, cognitive decline, progress immunosuppression, cholinergic anti-inflammatory deficiency, and even metabolic and hydroelectrolyte imbalan Therefore, the bilateral communication between SAE and the multiple peripheral organs and especially the immu system should be emphasized in sepsis management.

AduoLa Fuzhenglin Down-regulates Microwave-induced Expression of β_1-adrenergic Receptor and Muscarinic Type 2 Acetylcholine Receptor in Myocardial Cells of Rats

This paper is aimed to study the effect of ADL on expression of ~z-AR and Mz-AchR in myocardial cells of rats exposed to microwave radiation. Immunohistochemistry, Western blot and image analysis were used to detect the expression of ~I-AR and Mz-AchR in myocardial cells at 7 and 14 d after microwave exposure. The results show that the expression level was higher in microwave exposure group and 0.75 g/（kg.d） ADL group than in sham operation group and significantly lower in 1.5 and 3.0 g/（kg.d） ADL groups than in microwave group. So we have a conclusion that the expression of I^z-AR and Mz-AchR is down-regulated in myocardial cells of rats exposed to microwave radiation. ADL can protect rats against microwave-induced heart tissue injury.

Possible implications of dysregulated nicotinic acetylcholine receptor diffusion and nanocluster formation in myasthenia gravis

Myasthenia gravis is a rare and invalidating disease affecting the neuromuscular junction of voluntary muscles.The classical form of this autoimmune disease is characterized by the presence of antibodies against the most abundant protein in the neuromuscular junction,the nicotinic acetylcholine receptor.Other variants of the disease involve autoimmune attack of non-receptor scaffolding proteins or enzymes essential for building or maintaining the integrity of this peripheral synapse.This review summarizes the participation of the above proteins in building the neuromuscular junction and the destruction of this cholinergic synapse by autoimmune aggression in myasthenia gravis.The review also covers the application of a powerful biophysical technique,superresolution optical microscopy,to image the nicotinic receptor in live cells and follow its motional dynamics.The hypothesis is entertained that anomalous nanocluster formation by antibody crosslinking may lead to accelerated endocytic internalization and elevated turnover of the receptor,as observed in myasthenia gravis.

Insensitive Acetylcholine Receptor Conferring Resistance of Plutella xylostella to Nereistoxin Insecticides

The combinative rate measurement of （3-[I125] iodotyrosyl） α-bungarotoxin was applied in the analysis of the relation between nerve acetylcholine receptor and three types of insecticide resistance in diamondback moth, Plutella xylostella （L.）. In the dimehypo-resistant strain and in the cartap-resistant strain, the nerve acetylcholine receptor showed the remarkable insensitivity to dimehypo and cartap, of which the binding rate to ligand was approximately 66 and 60%, respectively, of the susceptible strain. The sensitivity to deltamethrin in the deltamethrin-resistant strain did not show visible change. These results indicated that the decline in the sensitivity of nerve acetylcholine receptor to insecticide might be a potential mechanism to nereistoxin insecticides resistance in the diamondback moth.

Acetylcholine secretion by motor neuron-like cells from umbilical cord mesenchymal stem cells

Umbilical cord mesenchymal stem cells were isolated by a double enzyme digestion method. The third passage of umbilical cord mesenchymal stem cells was induced with heparin and/or basic fi- broblast growth factor. Results confirmed that cell morphology did not change after induction with basic fibroblast growth factor alone. However, neuronal morphology was visible, and micro- tubule-associated protein-2 expression and acetylcholine levels increased following induction with heparin alone or heparin combined with basic fibroblast growth factor. Hb9 and choline acetyl- transferase expression was high following inductive with heparin combined with basic fibroblast growth factor. Results indicate that the inductive effect of basic fibroblast growth factor alone was not obvious. Heparin combined with basic fibroblast growth factor noticeably promoted the differen- tiation of umbilical cord mesenchymal stem cells into motor neuron-like cells. Simultaneously, um- bilical cord mesenchymal stem cells could secrete acetylcholine.

Targeting α7 nicotinic acetylcholine receptors: a future potential for neuroprotection from traumatic brain injury

Traumatic brain injury （TBI） poses a significant socioeconomic burden in the world. The long lasting consequences in cognitive impairments are often underreported and its mechanisms are unclear. In this perspective, cholinergic dysfunction and thera-peutic strategy targeting this will be reviewed. Novel agents that can target specific subtype of acetylcholine receptors have been developed over the recent years and are at various stages of development, which include AR-R 17779, GTS-21, SSR- 180711A, AR-R17779, and PNU-282987. A detailed review on this topic has been previously published （Shin and Dixon, 2015）.

Effect of Schisandra chinensis polysaccharide on intracerebral acetylcholinesterase and monoamine neurotransmitters in a D-galactose-induced aging brain mouse model

BACKGROUND： The most prominent characteristic of brain aging is decreased learning and memory ability. The functions of learning and memory are closely related to intracerebral acetylcholinesterase （ACHE） and monoamine neurotransmitter activity. Previous studies have shown that Schisandra chinensis polysaccharide has an anti-aging effect. OBJECTIVE： To explore the effects of Schisandra chinensis polysaccharide on AChE activity and monoamine neurotransmitter content, as well as learning and memory ability in a D-galactose-induced aging mouse brain model compared with the positive control drug Kangnaoling. DESIGN, TIME AND SETTING： Completely randomized, controlled experiment based on neurobiochemistry was performed at the Pharmacological Laboratory, Henan University of Traditional Chinese Medicine from September to December 2003. MATERIALS： Schisandra chinensis was purchased from Henan Provincial Medicinal Company. Schisandra chinensis polysaccharide was obtained by water extraction and alcohol precipitation. Kangnaoling pellets were provided by Liaoning Tianlong Pharmaceutical （batch No. 20030804; state drug permit No. H21023095）. A total of 50 six-week-old Kunming mice were randomly divided into five groups： blank control, model, Kangnaoling, high and low dosage Schisandra chinensis polysaccharide groups, with 10 mice per group. METHODS： Mice in the blank control group were subcutaneously injected with 0.5 mL/20 g normal saline into the nape of the neck each day, while the remaining mice were subcutaneously injected with 5% D-galactose saline solution （0.5 mL/20 g） in the nape for 40 days to induce a brain aging model. On day 11, mice in the high and low dosage Schisandra chinensis polysaccharide groups were intragastrically infused with 20 mg/mL and 10 mg/mL Schisandra chinensis polysaccharide solution （0.2 mL/10 g）, respectively. Mice from the Kangnaoling group were intragastrically infused with 35 mg/mL Kangnaoling suspension （0.2 mL/10 g）, and the mice in the model group were intragastrically infused with the same volume of normal saline （0.2 mL/10 g） once per day for 30 consecutive days. MAIN OUTCOME MEASURES： Two hours after the final administration, pathohistological changes in the cerebral cortex and hippocampus were observed using hematoxylin ＆ eosin staining. AChE activity was detected using chromatometry. Monoamine neurotransmitter content was measured using fluorimetry. Learning and memory was measured using the step down test and darkness avoidance test. RESULTS： Both Schisandra chinensis polysaccharide and Kangnaoling improved pathological injury to the cerebral cortex and hippocampus in a mouse model of brain aging. Compared with the blank control group, AChE activity and content of norepinephrine （NA）, dopamine （DA）, and 5-hydroxytryptamine （5-HT） were significantly decreased in the model group （P 〈 0.01 ）. In contrast, AChE activity and NA, DA, and 5-HT levels significantly increased in the Kangnaoling and high dosage Schisandra chinensis polysaccharide groups （P 〈 0.01）, while NA levels significantly increased in the low dosage Schisandra chinensis polysaccharide group （P 〈 0.01）. Drug treatment improved learning and memory abilities （P 〈 0.01 or P 〈 0.05）. CONCLUSION： Schisandra chinensis polysaccharide significantly increased levels of central neurotransmitters and improved learning and memory in a mouse model of brain aging. The effects of Schisandra chinensis polysaccharide were equal to that of Kangnaoling pellets.

Effect of green flickering light on myopia development and expression of M1 muscarinic acetylcholine receptor in guinea pigs

AIM： To investigate the effects of green flickering light on refractive development and expression of muscarinic acetylcholine receptor（mAChR） M1 in the eyes of guinea pigs.METHODS： Thirty guinea pigs（15-20 days old） were randomly divided into three groups（n=10/group）. Animals in group I were raised in a completely closed carton with green flickering light illumination. Those in group II were kept in the open top closed carton under normal natural light. Guinea pigs were raised in a sight-widen cage under normal natural light in group III. The refractive status and axial length were measured before and after 8 weeks＇ illumination. Moreover, total RNA extracted from retinal, choroidal, and scleral tissues were determined by real-time reverse transcription polymerase chain reaction（RT-PCR）. The expressions of the receptor M1 were also explored in the retina, choroid, and sclera using immunohistochemistry.RESULTS： There was a remarkable reduction in refractive error and increase in axial length after 8-weeks＇ green flickering light stimulation（P〈0.001）. The expression of M1 receptor mRNA in sclera and retina in myopia group were remarkably lower than that in group II and III（P〈0.01）. Significant reduced expression of M1 receptor stimulated by green flickering light in retina and sclera tissues were also observed（P〈0.05）. However, there was no M1 receptor expression in choroid in 3 groups.CONCLUSION： Myopia can be induced by 8 weeks＇ green flickering light exposure in the animal model. M1 receptor may be involved causally or protectively in myopia development.

Effects of acetylcholine injection on electric activities of pain-related neurons in the locus ceruleus of healthy rats

BACKGROUND： A large number of investigations have shown that acetylcholine （ACh） and the nucleus locus coeruleus （LC） play an important role in the modulation of pain in rats; however, there is no concrete evidence addressing the relationship between ACh injection into the LC and the electrical activities of pain-related neurons in the LC of healthy rats. OBJECTIVE： To study changes in the discharge of pain-related neurons in the LC following injection of ACh, or its M receptor antagonist, atropine, and to investigate the role of ACh and the LC in the pain signaling pathway. DESIGN, TIME AND SETTING： A randomized, controlled, neuroelectrophysiological animal experiment was performed from November 2007 to December 2008, in the Physiological Laboratory of Harbin Medical University, China. MATERIALS： Acetylcholine chloride was obtained from Shanghai San＇aisi Reagent Co., Ltd., China atropine was purchased from Tianjin Jinyao Amino Acid Co., Ltd., China. METHODS： This study was divided into two sections as follows： （1） 46 adult Wistar rats were randomly assigned into an ACh group and a control group, with 23 rats in each. （2） 34 adult Wistar rats were randomly assigned to an atropine group and a control group, with 17 rats in each. The sciatic nerve was stimulated by a series of electrical impulses, serving as peripheral noxious stimuli. Electrical changes in pain-related neurons in the LC were measured by glass microelectrodes. The LC of rats in the ACh and atropine groups were injected with 2 μg/μL ACh or 0.5 μg/μL atropine, respectively, in 1 μL volume. Rats in the control groups received injection of 1 pL physiological saline within 4 minutes. MAIN OUTCOME MEASURES： To measure the net increase in the discharge value, latency and complete inhibitory duration of pain-related neurons before and after administration of ACh or atropine. RESULTS： The injection of ACh into the LC increased the pain-evoked discharge frequency and shortened the latency of the pain-excitation neurons. It decreased the pain-evoked discharged frequency and prolonged the inhibitory duration of pain-inhibition neurons. Injection of atropine into LC blocked the effects of ACh. CONCLUSION： ACh strengthened the response of pain-related neurons in LC of rats to noxious stimulation, exhibiting the effects of facilitated pain. This indicates that ACh and LC play an important role in the modulation of algesia.

Activities of nicotinic acetylcholine receptors modulate neurotransmission and synaptic architecture

The cholinergic system is involved in a broad spectrum of brain function, and its failure has been implicated in Alzheimer＇s disease. Acetylcholine transduces signals through muscarinic and nicotinic acetylcholine receptors, both of which influence synaptic plasticity and cognition. However, the mechanisms that relate the rapid gating of nicotinic acetylcholine receptors to persistent changes in brain function have remained elusive. Recent evidence indicates that nicotinic acetylcholine receptors activities affect synaptic morphology and density, which result in persistent rearrangements of neural connectivity. Further investigations of the relationships between nicotinic acetylcholine receptors and rearrangements of neural circuitry in the central nervous system may help understand the pathogenesis of Alzheimer＇s disease.

Altered acetylcholinesterase levels in the spinal cord anterior horn and dorsal root ganglion following sciatic nerve ischemia

BACKGROUND： Peripheral nerve ischemia has been shown to result in ischemic fiber degenera-tion and axoplasmic transport disturbance. However, the effect on acetylcholinesterase （AChE） ex- pression in relevant cells following sciatic nerve ischemia remains unclear. OBJECTIVE： To observe AChE concentration changes following peripheral nerve ischemia. DESIGN, TIME AND SETTING： The present comparative observation, neuroanatomical experiment was performed at the Central Laboratory Animal of Chengde Medical College between 2006 and 2007. MATERIALS： A total of 20 healthy, adult, Wistar rats were randomized into two groups （n = 10）： 8-day ischemia and 14-day ischemia. METHODS： Ischemia injury was induced in the unilateral sciatic nerve （experimental side） through ligation of the common iliac artery. The contralateral side received no intervention, and served as the control side. Rats in the 8-day ischemia and 14-day ischemia groups were allowed to survive for 8 and 14 days, respectively. MAIN OUTCOME MEASURES： The L5 lumbar spinal cord and the L5 dorsal root ganglion were removed from both sides and sectioned utilizing a Leica vibrating slicer. AChE cellular expression was detected using Karnovsky-Root, and the number of AChE-positive cells and average gray value were analyzed using a MiVnt image analysis system. RESULTS： In the 8-day ischemia group, AChE-positive cell numbers were significantly less in the dorsal root ganglion and spinal cord anterior horn of the experimental side, but the average gray value was significantly greater, compared with the control side （P 〈 0.05）. These changes were more significant in the 14-day ischemia group than in the 8-day ischemia group （P 〈 0.01）. CONCLUSION： Peripheral nerve ischemia leads to decreased AChE expression in the associated cells in a time-dependent manner.

DESENSITIZATION OF ACETYLCHOLINE ON THE INHIBITION EFFECTS OF BLOOD PRE SSURE IN ANESTHETIZED CANINE

Objective To investigate the desensitization of acetylcholine (ACh) on the inhibition effects of blood pressure (BP) in anesthe tized canine and build a model for studying desensitization in vivo. Methods Through changing the intervals (120, 100, 80, 60, 40, 20 seconds) of twice ACh administration (each was 15μg·kg -1,i.v.), the desensitization on the effect of systemic blood pressure of the first ACh in jection towards the subsequent ACh administration was observed. Results When ACh administration intervals were 40, 60, 80 , 100 seconds, the percentages of desensitization of ACh on systemic blood press ure were significantly increased (P<0.05). However, as the intervals were 20 and 120 seconds, the effects of twice ACh administration had no significant dif ference (P>0.05). Conclusion The results indicated that ACh contents in blo od might influence the action of next ACh administration. To some extent, the hi gher the concentration of ACh in blood, the bigger the ratio of desensitization of exogenous ACh is. In addition, this method of twice drug administration could be used as a model of studying desensitization in vivo.

Acetylcholine receptor pathway in lung cancer: New twists to an old story

Genome-wide association studies revealed that allelic variation in the α5-α3-β4 nicotine acetylcholine receptor(n ACh R) cluster on chromosome 15q24-15q25.1 was associated with lung cancer risk. n ACh Rs are membrane ligand-gated cation channels whose activation is triggered by the binding of the endogenous neurotransmitter acetylcholine(ACh) or other biologic compounds including nicotine. n ACh Rs have been found on lung cancer cells, underscoring the idea that the non-neuronal n ACh R pathway has important implications for lung cancer. Several studies focusing on the treatment with n ACh R antagonists with improved selectivity might trigger novel strategies for the intervention and prevention of lung cancer. Here we review the genetic risk factors for lung cancer in the n ACh R gene cluster, the roles of nicotine receptors, and the molecular mechanisms of acetylcholine receptor pathways to lead to more opportunities for intervention and prevention of lung cancer.

Acetylcholine-induced calcium oscillation in isolated outer hair cells in guinea pig

Objective This study is to explore the relationship between acetylcholine(ACh)-induced calcium release from intracellular Ca2+ stores and function of outer hair cell(OHC) motors, in an attempt to elucidate the mechanism of OHC electromotility at resting state. Methods OHCs were isolated from adult guinea pig (200-300 g) cochlea and loaded with Fluo-3/AM. The cells were treated with ACh/dHBSS, ACh/HBSS, dHBSS only or HBSS only. Intracellular [Ca2+]i variations in cells under the four treatments were observed using an Ar-Kr laser scan confocal microscope. Results [Ca2+]i oscillations were recorded in five OHCs treated with ACh/dHBSS but not in other cells. This is the first time that Ach-excited [Ca2+]i oscillations are reported in guinea pig OHCs independent of extracellular calcium. Conclusions ACh-excited [Ca2+]i oscillations in OHCs originates from intracellular calcium release and may play a crucial role in maintaining active mechanical motility of the OHC at resting and modulating OHC electromotility.

“Warming yang and invigorating qi” acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis

Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction.＂Warming yang and invigorating qi＂ acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis.However,few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction.Here,we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following ＂warming yang and invigorating qi＂ acupuncture therapy.Needles were inserted at acupressure points Shousanli（LI10）,Zusanli（ST36）,Pishu（BL20）,and Shenshu（BL23） once daily for 7 consecutive days.The treatment was repeated after 1 day of rest.We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment.This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide.These findings suggest that ＂warming yang and invigorating qi＂ acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis.