Neuromuscular synaptic function in mice lacking major subsets of gangliosides

Gangliosides are a family of sialylated glycosphingolipids enriched in the outer leaflet of neuronal membranes, in particular at synapses. Therefore, they have been hypothesized to play a functional role in synaptic transmission. We have measured in detail the electrophysiological parameters of synaptic transmission at the neuromuscular junction (NMJ) ex vivo of a GD3-synthase knockout mouse, expressing only the O- and a-series gangliosides, as well as of a GM2/GD2-synthase*GD3-synthase double-knockout (dKO) mouse, lacking all gangliosides except GM3. No major synaptic deficits were found in either null-mutant. However, some extra degree of rundown of acetylcholine release at high intensity use was present at the dKO NMJ and a temperature-specific increase in acetylcholine release at 35 degrees C was observed in GD3-synthase knockout NMJs, compared with wild-type. These results indicate that synaptic transmission at the NMJ is not crucially dependent on the particular presence of most ganglioside family members and remains largely intact in the sole presence of GM3 ganglioside. Rather, presynaptic gangliosides appear to play a modulating role in temperature- and use-dependent fine-tuning of transmitter output.     

The effect of acetylcholine on the function and structure of the developing mammalian neuromuscular junction

Isolated soleus muscles from rats aged 9–12 days were exposed to acetylcholine for 2 h in normal Krebs solution. This treatment caused changes in the ultrastructural appearance of the neuro-muscular junction and a significant reduction of axon profiles per endplate. Nevertheless, most neuro-muscular junctions remained functional, since the ratio of the indirectly to directly elicited contraction was not reduced.If muscles were exposed to acetylcholine in Krebs solutions containing 12m M Ca²⁺ instead of the normal 1.9 m M, the ultrastructural changes produced by acetylcholine were more severe, and the number of axon terminals per endplate was further reduced so that many endplates became completely denervated. This was also reflected in the impaired function of the nerve-muscle preparation; the ratio of the indirectly to directly elicited contraction decreased and about 40% of the muscles fibres became functionally denervated. Addition of curare to the incubating medium prevented the functional deterioration of the preparation.Addition of the protease inhibitors leupeptin and pepstatin protected the nerve terminals from the damaging effects of acetylcholine in Krebs solution containing 12m M Ca²⁺ and the number of axon profiles per endplate remained normal. The functional deterioration was also much reduced when protease inhibitors were included in the incubation medium.These results suggest that acetylcholine causes the activation and release of proteolytic enzymes in developing muscles. The response is mediated by calcium and may have a role in the removal of superfluous nerve-muscle contacts during development.     

Adenosine A2B and A3 receptor location at the mouse neuromuscular junction

To date, four subtypes of adenosine receptors have been cloned (A₁R, A_2AR, A_2BR, and A₃R). In a previous study we used confocal immunocytochemistry to identify A₁R and A_2AR receptors at mouse neuromuscular junctions (NMJs). The data shows that these receptors are localized differently in the three cells (muscle, nerve and glia) that configure the NMJs. A₁R localizes in the terminal teloglial Schwann cell and nerve terminal, whereas A_2AR localizes in the postsynaptic muscle and in the axon and nerve terminal. Here, we use Western blotting to investigate the presence of A_2BR and A₃R receptors in striated muscle and immunohistochemistry to localize them in the three cells of the adult neuromuscular synapse. The data show that A_2BR and A₃R receptors are present in the nerve terminal and muscle cells at the NMJs. Neither A_2BR nor A₃R receptors are localized in the Schwann cells. Thus, the four subtypes of adenosine receptors are present in the motor endings. The presence of these receptors in the neuromuscular synapse allows the receptors to be involved in the modulation of transmitter release.     

Sleep and its homeostatic regulation in mice lacking the adenosine A1 receptor

Sleep deprivation (SD) increases extracellular adenosine levels in the basal forebrain, and pharmacological manipulations that increase extracellular adenosine in the same area promote sleep. As pharmacological evidence indicates that the effect is mediated through adenosine A1 receptors (A1R), we expected A1R knockout (KO) mice to have reduced rebound sleep after SD. Male homozygous A1R KO mice, wild-type (WT) mice, and heterozygotes (HET) from a mixed 129/C57BL background were implanted during anesthesia with electrodes for electroencephalography (EEG) and electromyography (EMG). After 1 week of recovery, they were allowed to adapt to recording leads for 2 weeks. EEG and EMG were recorded continuously. All genotypes had a pronounced diurnal sleep/wake rhythm after 2 weeks of adaptation. We then analyzed 24 h of baseline recording, 6 h of SD starting at light onset, and 42 h of recovery recording. Neither rapid eye movement sleep (REM sleep) nor non-REM sleep (NREMS) amounts differed significantly between the groups. SD for 6 h induced a strong NREMS rebound in all three groups. NREMS time and accumulated EEG delta power were equal in WT, HET and KO. Systemic administration of the selective A1R antagonist 8-cyclopentyltheophylline (8-CPT) inhibited sleep for 30 min in WT, whereas saline and 8-CPT both inhibited sleep in KO. We conclude that constitutional lack of adenosine A1R does not prevent the homeostatic regulation of sleep.     

Increased and decreased activity elicits specific morphological adaptations of the neuromuscular junction

The objective of this investigation was to compare the effects of decreased vs. increased activity on the neuromuscular system. Twenty-four young adult (7 weeks old) Sprague-Dawley rats were randomly assigned to three treatment groups (N=8/group). Increased activity was achieved by treadmill running for up to 1 h/day. Decreased activity was induced by muscle unloading via the hindlimb suspension model. Control animals engaged in normal weight bearing and ambulatory activity. At the end of the 10 week intervention period, animals were killed and soleus muscles were removed, quickly frozen, and examined using cytofluorescent (neuromuscular junctions) and histochemical (myofibers) procedures. Pre-synaptic morphology was quantified by measuring nerve terminal branching, and post-synaptic assessment was conducted by staining acetylcholine receptors at the endplate. Myofiber profiles of solei were compiled by determining fiber size (cross-sectional area) and fiber type composition. Results show that exercise training significantly (P相似文献   

Nerve terminal contributes to acetylcholine receptor organization at the dystrophic neuromuscular junction of mdx mice

Changes in the distribution of acetylcholine receptors have been reported to occur at the neuromuscular junction of mdx mice and may be a consequence of muscle fiber regeneration rather than the absence of dystrophin. In the present study, we examined whether the nerve terminal determines the fate of acetylcholine receptor distribution in the dystrophic muscle fibers of mdx mice. The left sternomastoid muscle of young (1‐month‐old) and adult (6‐month‐old) mdx mice was injected with 60 μl lidocaine hydrochloride to induce muscle degeneration‐regeneration. Some mice had their sternomastoid muscle denervated at the time of lidocaine injection. After 10 days of muscle denervation, nerve terminals and acetylcholine receptors were labeled with 4‐Di‐2‐ASP and rhodamine‐α‐bungarotoxin, respectively, for confocal microscopy. In young mdx mice, 75% (n = 137 endplates) of the receptors were distributed in islands. The same was observed in 100% (n = 114 endplates) of the adult junctions. In denervated‐regenerated fibers of young mice, the receptors were distributed as branches in 89% of the endplates (n = 90). In denervated‐regenerated fibers of adult mice, the receptors were distributed in islands in 100% of the endplates (n = 100). These findings show that nerve‐dependent mechanisms are also involved in the changes in receptor distribution in young dystrophic muscles. In older dystrophic muscles, other factors may play a role in receptor distribution. Anat Rec 290:181–187, 2007. © 2007 Wiley‐Liss, Inc.     

Intraganglionic laminar endings in the rat esophagus contain purinergic P2X2 and P2X3 receptor immunoreactivity

Intraganglionic laminar endings (IGLEs) represent the most prominent vagal afferent terminal structures throughout the gastrointestinal tract. They are most prominent in the esophagus and stomach, but can be found down to the distal colon. Their role as mechanosensors as proposed on anatomical grounds was recently substantiated in elegant functional experiments. There is evidence that vagal mechanosensors in the esophagus and stomach respond to ATP. Thus, the present study aimed at detecting purinergic receptors on IGLEs. IGLEs in the rat esophagus were identified by immunohistochemistry for calretinin and sections were co-incubated with antibodies directed against P2X₂ or P2X₃ receptors. Also, double label immunocytochemistry for purinergic receptors and calcitonin gene-related peptide as a marker for spinal afferents was performed. Terminal nerve fibers immunoreactive for P2X₂ and P2X₃, respectively, were observed between outer and inner layers of the tunica muscularis, covering myenteric ganglia totally or partly. Both P2X₂ and P2X₃ receptor immunoreactivities were highly co-localized with calretinin positive IGLEs as shown by confocal laser scanning microscopy. Numerous calcitonin gene-related peptide immunostained fibers were found to closely approach and intermingle with P2X immunopositive IGLEs. However, there was never co-staining for either of the purinergic receptors and calcitonin gene-related peptide within the same fibers. P2X₃ but not P2X₂ immunoreactivity was also observed within nerve fiber arborizations in the mucosa of the pharynx. In the nodose ganglion, 8.9±1.1% of P2X₂ and 7.2±1.3% of P2X₃ immunopositive neurons, respectively, co-stained for calretinin. On the other hand, 63.4±4.6% and 60.1±5.3% of calretinin positive cell bodies contained P2X₂ and P2X₃ receptor immunoreactivity, respectively. These results indicate that IGLEs are equipped with both P2X₂ and P2X₃ receptors. Thus, they may act as chemosensors or their mechanosensory properties may be modulated by ATP. It is also suggested that spinal afferents innervating the esophagus are equipped with neither P2X₂ nor P2X₃ purinergic receptors.     

Synaptic terminals from mice midbrain exhibit functional P2X7 receptor

P2X(7) receptor has been recently localized in mice cerebellar granule neuron fibers. Here, the expression of this subunit has been detected in wild type mice midbrain, by quantitative real time-polymerase chain reaction, immunocytochemistry and Western blot assays. The functionality of this P2X(7) subunit has been confirmed using microfluorimetric experiments in isolated synaptic terminals from mice midbrain. 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP) was 30-fold more potent than ATP and EC(50) values were 20 microM and 630 microM respectively. Brilliant Blue G (BBG) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) produced an inhibition in the responses induced by BzATP, with IC(50) values of 0.027 nM and 2.23 nM, respectively. In addition, P2X(7) inhibitors as ZnSO(4), BBG and suramin abolished partially or totally the responses induced by the physiological agonist ATP. According to immunochemical and PCR assays the presence of a "P2X(7)-like" protein in synaptosomes from validated P2X(7) knockout (KO) model have been detected. In KO animals, BzATP was sixfold more potent than ATP and the EC(50) values were 87 microM and 590 microM respectively. BBG and KN-62 also produced an inhibition in the responses induced by BzATP, with IC(50) value of 0.61 nM and 118 nM respectively, both of them higher than in wild type mice. Moreover, the calcium mobilization ability of native P2X(7) receptors was higher in control compared with KO mice. These biochemical and pharmacological experiments are consistent with the presence of a functional P2X(7) receptor in wild type mice midbrain, and the existence of a less efficient "P2X(7)-like" receptor in the KO model.     

Distribution of P2X1, P2X2, and P2X3 receptor subunits in rat primary afferents: relation to population markers and specific cell types

We determined the co-expression of immunoreactivity (IR) for ATP-receptor subunits (P2X1, P2X2, and P2X3), neuropeptides, neurofilament (NF), and binding of the isolectin B(4) from Griffonia simplicifolia type one (GS-I-B(4)) in adult dorsal root ganglion neurons. P2X1-IR was expressed primarily in small DRG neurons. Most P2X1-IR neurons expressed neuropeptides and/or GS-I-B(4)-binding, but lacked NF-IR. P2X1-IR overlapped with P2X3-IR, though each was also found alone. P2X2-IR was expressed in many P2X3-IR small neurons, as well as a group of medium to large neurons that lacked either P2X3-IR or GS-I-B(4)-binding. A novel visible four-channel fluorescence technique revealed a unique population of P2X2/3-IR neurons that lacked GS-I-B(4)-binding but expressed NF-IR. Co-expression of P2X1, and P2X3 in individual neurons was also demonstrated. We examined P2X subunit-IR on individual recorded neurons that had been classified by current signature in vitro. Types 1, 2, 4 5, and 7 expressed distinct patterns of P2X-IR that corresponded to patterns identified in DRG sections, and had distinct responses to ATP. Types with rapid ATP currents (types 2, 5, and 7) displayed P2X3-IR and/or P2X1-IR. Types with slow ATP currents (types 1 and 4) displayed P2X2/3-IR. Type 1 neurons also displayed P2X1-IR. This study demonstrates that the correlation between physiological responses to ATP and the expression of particular P2X receptor subunits derived from expression systems is also present in native neurons, and also suggests that novel functional subunit combinations likely exist.     

Modulation of P2X7 receptor expression in macrophages from mineral oil-injected mice

P2X7 receptor activation is involved in a number of pro-inflammatory responses in macrophages and other immune cells. Their expression can be positively modulated with lipopolysaccharide (LPS) and TNFalpha, reinforcing their role during inflammation. We investigated the effect of substances capable of recruiting macrophages into the peritoneal cavity of mice (mineral oil and thioglycolate) on P2X7 receptor expression and function, addressing whether these stimuli can interfere with multinucleated giant cell (MGC) formation, ATP-induced apoptosis, plasma membrane permeabilization and nitric oxide production. It was demonstrated that mineral oil treatment reduces P2X7-dependent MGC formation, whereas thioglycolate treatment does not. Mineral oil treatment reduced P2X7 receptor expression, down-modulating ATP-induced apoptosis, permeabilization and nitric oxide production. In conclusion, mineral oil down modulated P2X7 expression and consequently P2X7-associated phenomena, but thioglycolate did not. These effects might be associated with the unpleasant side effects already described during long-term administration of mineral oil for cosmetic purposes or as a laxative and could be useful in understanding the mechanism of recycling and modulation of P2 receptors present in other situations of immunopathological interest.     

Expression and function of HLA-DR3 and DQ8 in transgenic mice lacking functional H2-M

H2-M or HLA-DM are non-classical class II molecules encoded by the MHC and play an important role during antigen presentation. They catalyze exchange of CLIP (Class II-associated invariant chain peptide) or other low-affinity peptides bound to class II molecules for peptides capable of more efficient binding. The phenotype of mice lacking H2-M is determined by the allotype of the MHC class II molecules expressed. In general, H2-M deficiency does not affect the surface expression of mature class II molecules. The class II molecules in such cases predominantly contain CLIP in their peptide-binding groove. In some mice strains, H2-M deficiency results in defective CD4+ T-cell development accompanied by defective responses to conventional antigens and superantigens. Even though the HLA class II molecules show similar dependency for HLA-DM for presenting antigens in vitro, their interaction in vivo is not known. By using transgenic approach we show here that DQ8 and DR3 are expressed at normal levels in H2-M-deficient mice and the CD4+ T-cell development is unaltered. However, the ability of DQ8 molecules to present peptide antigens is compromised in a H2-M-deficient state. Presentation of exogenous bacterial superantigens by both DQ8 and DR3 is unaffected in H2-M-deficient mice. Unexpectedly, Staphylococcal Enterotoxin B-induced systemic IFN-gamma production was significantly higher in H2-M-deficient DQ8/DR3 transgenic mice and these mice were susceptible to SEB-induced toxic shock at doses that are non-lethal to H2-M-sufficient counterparts.     

Adaptive changes in the expression of central opioid receptors in mice lacking the dopamine D2 receptor gene

On the basis of numerous studies that have described interactions between the dopaminergic and opioidergic systems, we have investigated whether genetic deletion of dopamine D2 receptors (D2R) might influence the expression of central opioid receptors. The levels of mu, delta, kappa and nociceptin opioid peptide receptors were determined in the brains and spinal cords of D2R knockout mice using quantitative autoradiography. The significant changes in opioid receptor binding found in the brains of heterozygous and homozygous mice were mainly restricted to the basal ganglia. In homozygous mice, a down-regulation of mu and delta receptors was observed in the striatal and pallidal areas. This alteration may be an adaptive response to the increase in enkephalin levels previously described in the striatum of these mutant mice. On the contrary, an up-regulation of kappa receptors was found in the striatal and nigral regions and might be related to a change in dynorphin levels. Significant increases in nociceptin receptor binding were also observed in homozygous mice in brain areas involved in motor behavior. At the spinal level, only kappa and nociceptin receptor binding showed significant overall differences between genotypes. The functional consequences of these adaptive changes are discussed in relation to the findings of behavioral and neurochemical studies reported to date in D2R knockout mice.     

Ventilatory pattern and chemosensitivity in M1 and M3 muscarinic receptor knockout mice

Acetylcholine (ACh) acting through muscarinic receptors is thought to be involved in the control of breathing, notably in central and peripheral chemosensory afferents and in regulations related to sleep-wake states. By using whole-body plethysmography, we compared baseline breathing at rest and ventilatory responses to acute exposure (5 min) to moderate hypoxia (10% O(2)) and hypercapnia (3 and 5% CO(2)) in mice lacking either the M(1) or the M(3) muscarinic receptor, and in wild-type matched controls. M(1) knockout mice showed normal minute ventilation (V(E)) but elevated tidal volume (V(T)) at rest, and normal chemosensory ventilatory responses to hypoxia and hypercapnia. M(3) knockout mice had elevated V(E) and V(T) at rest, a reduced V(T) response slope to hypercapnia, and blunted V(E) and frequency responses to hypoxia. The results suggest that M(1) and M(3) muscarinic receptors play significant roles in the regulation of tidal volume at rest and that the afferent pathway originating from peripheral chemoreceptors involves M(3) receptors.     

被动训练促进失神经肌萎缩模型大鼠骨骼肌结构和功能的恢复

文题释义：肌肉萎缩：是指横纹肌营养障碍,肌肉纤维变细甚至消失等导致的肌肉体积缩小。多由肌肉本身疾患或神经系统功能障碍所致,病因主要有：神经源性肌萎缩、肌源性肌萎缩、失用性肌萎缩和其他原因性肌萎缩。肌肉营养状况除肌肉组织本身的病理变化外,更与神经系统有密切关系。脊髓疾病常导致肌肉营养不良而发生肌肉萎缩。 肌卫星细胞：是一类存在于肌细胞基底膜与肌膜之间的成体干细胞,作为肌源性干细胞在肌肉组织损伤后,能够在激活后发挥良好的增殖、分化能力,在骨骼肌损伤的修复和再生过程中发挥重要作用。 背景：炎症细胞或炎性因子参与失神经损伤后骨骼肌肌卫星细胞的增殖和分化,在失神经骨骼肌肌组织病理过程中起着重要的作用。 目的：研究被动康复训练对失神经萎缩大鼠骨骼肌结构、功能以及肌动蛋白和炎症因子表达的影响。 方法：将30只SD大鼠平均分为假手术组、模型组和训练组,模型组及训练组大鼠暴露坐骨神经并剪断,假手术组只暴露而不剪断坐骨神经。造模后2个月始用自制滚筒对训练组大鼠进行被动康复训练2个月,用肌肉湿质量比和BBB评分评估肌肉萎缩的程度及运动功能,苏木精-伊红染色观察肌纤维微细结构及横截面积,免疫组化染色检测各组腓肠肌肌动蛋白及肿瘤坏死因子α、白细胞介素6及白细胞介素1β表达。实验经沈阳医学院实验动物福利伦理委员会的审批,批准文号为SYYXY2015010601。结果与结论：①训练组BBB评分高于模型组;②训练组腓肠肌湿质量高于模型组但肌纤维的横截面积却低于模型组(P < 0.001,P < 0.05),训练组腓肠肌肌动蛋白表达高于模型组(P < 0.001);③训练组炎症因子肿瘤坏死因子α、白细胞介素6及白细胞介素1β的表达水平低于模型组(P < 0.001或P < 0.05);④结果说明,被动训练有助于失神经萎缩肌肉结构和功能的恢复,降低炎症因子的水平防止肌肉的进一步萎缩,提高骨骼肌的肌力。 ORCID: 0000-0002-9303-8651(王世杨) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Alteration of dorsal root ganglion P2X3 receptor expression and function following spinal nerve ligation in the rat

One subtype of ATP-gated ion channel, the P2X₃ receptor, is expressed primarily on peripheral sensory neurons. While it is known that P2X₃ receptors can participate in certain forms of nociceptive signaling, their involvement in neuropathic pain transmission is not known. We have examined the expression and function of P2X₃ receptors in a rat spinal nerve ligation model of neuropathic pain. Fourteen days following L5/L6 spinal nerve ligation, the corresponding dorsal root ganglia (DRG) were removed from animals exhibiting mechanical allodynia, and these were studied using immunohistochemical and electrophysiological techniques. Using a polyclonal antibody to label the P2X₃ receptor, a significant reduction in neuronal P2X₃ immunoreactivity was observed in the ipsilateral (injured) L5 and L6 DRG following nerve ligation. In vitro electrophysiological analysis of acutely isolated DRG neurons revealed a similar decrease in functional P2X₃-containing receptors. In small diameter (22–25 μm) neurons, a significant reduction in the number of cells exhibiting a response to α,β-meATP was observed. However, a subset of small diameter neurons retained P2X₃ responses of equal amplitude to those recorded from naive and sham control DRG neurons. Interestingly, P2X₃ immunoreactivity and P2X₃-like responses were also detected in a subset of larger diameter (50 μm) neurons and the number and amplitude of these responses were unchanged after spinal nerve ligation. These results suggest that, while there appears to be a decrease in fast desensitizing P2X₃ receptors following L5/L6 nerve ligation injury, certain subsets of small and large DRG neurons maintain normal P2X₃ receptor expression and function. These remaining receptors may provide a P2X₃ receptor-mediated component to neuropathic pain. Electronic Publication     

Presynaptic inhibition of spontaneous acetylcholine release mediated by P2Y receptors at the mouse neuromuscular junction

At the neuromuscular junction, ATP is co-released with the neurotransmitter acetylcholine (ACh) and once in the synaptic space, it is degraded to the presynaptically active metabolite adenosine. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of extracellular ATP (100 muM) and the slowly hydrolysable ATP analog 5'-adenylylimidodiphosphate lithium (betagamma-imido ATP) (30 muM) on miniature end-plate potential (MEPP) frequency. We found that application of ATP and betagamma-imido ATP decreased spontaneous secretion by 45.3% and 55.9% respectively. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A(1) adenosine receptor antagonist and alpha,beta-methylene ADP sodium salt (alphabeta-MeADP), which is an inhibitor of ecto-5'-nucleotidase, did not prevent the inhibitory effect of ATP, demonstrating that the nucleotide is able to modulate spontaneous ACh release through a mechanism independent of the action of adenosine. Blockade of Ca(2+) channels by both, Cd(2+) or the combined application of nitrendipine and omega-conotoxin GVIA (omega-CgTx) (L-type and N-type Ca(2+) channel antagonists, respectively) prevented the effect of betagamma-imido ATP, indicating that the nucleotide modulates Ca(2+) influx through the voltage-dependent Ca(2+) channels related to spontaneous secretion. betagamma-Imido ATP-induced modulation was antagonized by the non-specific P2 receptor antagonist suramin and the P2Y receptor antagonist 1-amino-4-[[4-[[4-chloro-6-[[3(or4)-sulfophenyl] amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl] amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid (reactive blue-2), but not by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS), which has a preferential antagonist effect on P2X receptors. Pertussis toxin and N-ethylmaleimide (NEM), which are blockers of G(i/o) proteins, prevented the action of the nucleotide, suggesting that the effect is mediated by P2Y receptors coupled to G(i/o) proteins. The protein kinase C (PKC) antagonist chelerythrine and the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) occluded the effect of betagamma-imido ATP, while the protein kinase A (PKA) antagonist KT-5720 and the inhibitor of the calcium/calmodulin-dependent protein kinase II (CAMKII) KN-62 failed to do so. betagamma-Imido ATP did not affect 10, 15 and 20 mM K(+)-evoked release and application of reactive blue-2 before incubation in high K(+) induced a higher asynchronous secretion. Thus, our results show that at mammalian neuromuscular junctions, ATP induces presynaptic inhibition of spontaneous ACh release due to the modulation of Ca(2+) channels related to tonic secretion through the activation of P2Y receptors coupled to G(i/o) proteins. We also demonstrated that at increasing degrees of membrane depolarization evoked by K(+), endogenously released ATP induces presynaptic inhibition as a means of preventing excessive neurotransmitter secretion.     

Dual excitatory actions of acetylcholine at the neuromuscular junction in the guinea-pig vas deferens

The action of acetylcholine (ACh) on the smooth muscle of guinea-pig vas deferens was studied using the sucrose-gap method. ACh, when applied at a concentration of 10^–6 M, evoked a depolarization of the smooth muscle membrane which was slow in time course (slow depolarization). When ACh was applied at higher concentrations, another depolarization which was fast in time course (fast depolarization) occurred, overlapping the early part of the slow depolarization. The magnitudes of both depolarizations were concentration-dependent on ACh. TTX and adrenergic receptor antagonists had little effect on either depolarizations, while guanethidine and nicotinic receptor antagonists mainly suppressed the fast depolarization. In contrast, atropine suppressed the slow depolarization. The membrane conductance observed by current application, was reduced during the slow depolarization, and the reversal potential of the depolarization was 18.3 mV negative to the resting membrane potential. Whereas, the reversal potential of the fast depolarization was 27.6 mV positive to the resting membrane potential. This reversal potential was quite similar to that of the adenosine triphosphate (ATP)-induced depolarization, previously observed in the same tissue. From these observations, it is suggested that in the guinea-pig vas deferens, ACh acts on nicotinic receptors at the sympathetic postganglionic nerve terminal, causing the release mostly of a non-adrenergic transmitter, probably ATP. In addition, ACh also acts on muscarinic receptors on the smooth muscle membrane, inducing membrane depolarization resulting from a reduction of the membrane conductance to potassium ions.     

ATP elicits inward currents in isolated vasopressinergic neurohypophysial terminals via P2X2 and P2X3 receptors

Effects of extracellular adenosine tri-phosphate (ATP) on ionic currents were investigated using the perforated-patch whole-cell recording technique on isolated terminals of the Hypothalamic Neurohypophysial System (HNS). ATP induced a current response in 70% of these isolated terminals. This inwardly-rectifying, inactivating current had an apparent reversal near 0 mV and was dose-dependent on ATP with an EC₅₀=9.6±1.0 M. In addition, current amplitudes measured at maximal ATP concentrations and optimum holding potentials had a current density of 70.8 pA pF^–1 and were greatly inhibited by suramin and PPADS. Different purinergic receptor agonists were tested, with the following efficacy: ATP 2-methylthioATP > ATP--S > Bz-Bz-ATP > ,-methylene-ATP > ,-methylene-ATP. However, UTP and ADP were ineffective. These data suggest the involvement of a P2X purinergic receptor in the ATP-induced responses. Immunocytochemical labeling in vasopressinergic terminals indicates the existence of P2X_{2,3,4, and 7}, but not P2X₆ receptors. Additionally, P2X_{2 and 3} were not found in terminals which labeled for oxytocin. In summary, the EC₅₀, decay, inactivation, and pharmacology indicate that a functional mixture of P2X_{2 and 3} homomeric receptors mediate the majority of the ATP responses in vasopressinergic HNS terminals. We speculate that the characteristics of these types of receptors reflect the function of co-released ATP in the terminal compartment of these and other CNS neurons.