Ca2+ signalling pathways activated by acetylcholine in mouse C2C12 myotubes

In mouse C2C12 myotubes acetylcholine (ACh) elevates the concentration of myoplasmic Ca²⁺ ([Ca²⁺]_i) by inducing Ca²⁺ influx through transmittergated and voltage-gated channels, and by mobilizing Ca²⁺ from internal stores. The relative contribution of each of these ACh-activated sources to the global [Ca²⁺]_i elevation was estimated. We found that Ca²⁺ entry through voltage- and ACh-gated channels accounts for roughly 80% of the total [Ca²⁺]_i increment, while mobilization from internal caffeine-sensitive and inositol-trisphosphate (InsP ₃ ^–) sensitive stores contributes the remaining 20% to the maximal [Ca²⁺]_i increment. Furthermore, we found that ACh-induced mobilization from InsP ₃-sensitive stores also develops in embryonic chick myotubes. The differential importance of the Ca²⁺ signalling pathways activated by ACh during myogenesis is discussed.     

Ca2+ spike initiation from sensitized inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in megakaryocytes

Ca²⁺-mediated Ca²⁺ spikes were analysed in fura-2-loaded megakaryocytes. Direct Ca²⁺ loading using whole-cell dialysis induced an all-or-none Ca²⁺ spike on top of a tonic increase in cellular Ca²⁺ concentration ([Ca²⁺]_i) with a latency of 3–7 s. The latency decreased with increasingly higher concentrations of Ca²⁺ in the dialysing solution. Spike size and its initiation did not correlate with the tonic level of [Ca²⁺]_i. Thapsigargin completely abolished the Ca²⁺-induced spike initiation, suggesting that Ca²⁺ spikes originate from thapsigargin-sensitive Ca²⁺ pools. An inhibitor of phosphatidylinositide-specific phospholipase C (PLC), 2-nitro-4-carboxyphenyl-N,N-diphenyl-carbamate prolonged the latency without changes of spike size in most cases (6/9 cells), but abolished the spike initiation in the other cells (3/9). The results suggest that an increase in [Ca²⁺]_i charges up the inositol-1,4,5-trisphosphate(InsP ₃)- and thapsigargin-sensitive Ca²⁺ pools which progressively sensitize to low or slightly elevated levels of InsP₃ by the action of Ca²⁺-dependent PLC until a critical Ca²⁺ content is reached, and then the Ca²⁺ spike is triggered. Thus, the limiting step of Ca²⁺ spike triggering is the initial filling process and the level of InsP₃ in megakaryocytes.     

Calcium permeability of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells

The fractional contribution of Ca to current flow through neuronal-type nicotinic acetylcholine receptor channels was determined by quantitative fluorescence microfluorimetry using fura-2. The method, which has been applied already to several types of cells and channels is described in detail here. At –70 mV and 2 mM external Ca concentration it was found that Ca contributes 2.5% to the net current. The fractional contribution was found to be voltage dependent, increasing at negative potentials e-fold for a 110 mV potential difference. Total non-specific cation current was found to have a bell-shaped dependence on external Ca concentration peaking at 2 mM.     

The desensitization of the embryonic mouse muscle acetylcholine receptor depends on the cellular environment

The rate of desensitization of nicotinic acetylcholine (ACh) receptor (nAChR), an important characteristic of nAChR function, was studied in myotubes of the mouse C2C12 cell line at different times after fusion, by measuring the decay of ACh-evoked currents (I _ACh) under various patch-clamp configurations. We observed a progressive slowing of I _ACh decay rate (half-decay time rose from about 0.5 s to over 5 s) in myotubes of increasing size (i.e. age) under all experimental conditions, except in outside-out patches, when I _ACh decayed as fast as in the smallest myotubes. Single-channel conductance (about 35 pS) and open time (about 3.5 ms), measured in outside-out and cell-attached patches, were independent of myotube size. In Xenopus oocytes injected with poly(A⁺)RNA extracted from C2C12 myoblasts or mature myotubes, I _ACh decay was about 50 times slower than in myotubes. Neither cAMP-dependent nor diacylglycerol-dependent protein kinases, actin nor microtubule polymerization state influenced I _ACh decay. Our data indicate that the cellular environment, but not readily dialysable cytosolic factors, markedly influences the functional behaviour of nAChR.     

Monoclonal antibody to β2 subunit of neuronal nicotinic receptor depresses the postjunctional non-contractile Ca mobilization in the mouse diaphragm muscle

The involvement of subtypes of nicotinic acetylcholine receptor (nAChR) in the postjunctional non-contractile Ca²⁺ mobilization was investigated in mouse diaphragm muscles treated with an anticholinesterase, using monoclonal antibodies (mAbs) to nAChR subunits. mAb 210 (specific for 1 subunit of muscle nAChR) depressed contractile Ca²⁺ transients without affecting non-contractile Ca²⁺ transients. mAb 270 (specific for β2 subunit of neuronal nAChR) depressed only non-contractile Ca²⁺ transients. mAb 210 did not completely block the ACh-activated channel currents in flexor digitorum brevis muscle cells. The present findings indicate that the anti-β2 mAb 270-related subtype of nAChR may postsynaptically operate the non-contractile Ca²⁺ mobilization at the neuromuscular junction, suggesting the involvement of a subtype different from the usual muscle-type nAChR.     

Effect of TPEN on the calcium release of cultured C2C12 mouse myotubes

N,N,N',N'-tetrakis(2-pyridylmethyl)-ethilenediamine (TPEN) is a membrane permeable heavy metal chelator that has been used to study intracellular calcium homeostasis but its exact mode of action is still unresolved. Here we examine the effects of TPEN on the Ca(2+) release from and the Ca(2+) uptake into the sarcoplasmic reticulum (SR) of cultured C2C12 skeletal muscle cells. Low concentrations (50 microM) of the drug evoked Ca(2+) transients in approximately 60% of C2C12 myotubes, while at high concentrations (500 microM) it significantly reduced the size of both depolarization-and caffeine-induced Ca(2+) transients, decreased the rate constant of decay and the calculated pump activity but failed to induce Ca(2+) transients. Experiments at low extracellular [Ca(2+)] revealed that it is the total rather than free TPEN concentration that is responsible for the observed effects. TPEN does not modify Ca(2+) release by Zn(2+) chelation, as evidenced by the unaltered effect seen after the removal of Zn(2+) from the extracellular space of the cells by chelating with EDPA. These findings provide experimental evidence that TPEN directly modifies both the release of Ca(2+) from the SR and its removal from the myoplasm.     

Desensitization of acetylcholine induced inositol 1,4,5-trisphosphate formation in neuroblastoma SH-SY5Y cells following repetitive acetylcholine stimulations

In this study, the desensitization of acetylcholine-induced inositol 1,4,5-trisphosphate [I(1,4,5)P₃] formation, upon short-time prestimulations, was investigated in cultures of human neuroblastoma SH-SY5Y cells. Four repeated stimulations for 10 seconds with 10 μM acetylcholine were necessary to induce a desensitization of the I(1,4,5)P₃ formation. The desensitization was observed 4 hours after the initiation of repetitive stimulations. The same effect was obtained by a single prestimulation with 1 mM acetylcholine. Preincubation of the cells with phorbol 12-myristate 13-acetate (PMA) markedly down-regulated the acetylcholine-induced I(1,4,5)P₃ formation. However, the protein kinase C (PKC) inhibitors H7 and staurosporine did not influence the desensitization induced by four repeated stimulations with 20 μM acetylcholine. These results indicate that the signal transduction can be desensitized following repeated stimulations with sub-maximal concentrations of receptor agonist and although activation of PKC can induce the same down-regulation, PKC is most likely not involved in the desensitization induced by repetitive acetylcholine-stimulations.     

Inositol trisphosphate may access calcium from stores not coupled to muscarinic receptors in Xenopus oocytes

Oocytes of a large fraction of Xenopus females exhibit a complex response to acetylcholine (ACh) consisting of rapid, transient and prolonged, slow chloride currents. Frequent consecutive challenges or a single prolonged challenge with ACh result in a marked decrease in response amplitudes, i. e. refractoriness. In ACh-refractory oocytes, the response to injected inositol 1,4,5,-trisphosphate (InsP ₃,), the intracellular mediator of the ACh response, is not affected. Similarly, InsP ₃-evoked responses were obtained in oocytes that lacked muscarinic response or that lost their responsiveness as a result of progesterone-induced maturation. To investigate the mechanism of this phenomenon, we have depleted intracellular calcium stores by repeated challenges with ACh in calcium-free medium. Disappearance of the ACh response through depletion of the ACh-coupled calcium store did not prevent a subsequent response to InsP ₃. These results imply that InsP ₃ can mobilize calcium from other stores, not depleted by previous exposure to ACh. This finding is further reinforced by our results that demonstrate that ACh causes ⁴⁵Ca efflux in responsive oocytes, while InsP ₃ in supramaximal concentrations does not induce ⁴⁵Ca efflux. Indeed, InsP ₃ can induce ⁴⁵Ca efflux only when more than 2 pmol/oocyte is injected. This is also the concentration of InsP ₃ that desensitizes the InsP ₃ response. These data suggest that InsP ₃ also releases cellular calcium from stores different from those mobilized by ACh.     

Adaptive control of intracellular Ca2+ release in C2C12 mouse myotubes

Laser scanning confocal imaging was used to monitor release of Ca²⁺ from localized regions in a skeletal muscle cell line with sparsely distributed Ca²⁺ release sites. The goal was to distinguish between two schemes proposed to explain the phenomenon of “quantal” Ca²⁺ release from caffeine-sensitive Ca²⁺ stores in muscle and other tissues: (1) all-or-none (true quantal) Ca²⁺ release from functionally discrete stores that have different sensitivities to caffeine; or (2) adaptive behavior of individual release sites, each responding transiently and repeatedly to incremental caffeine doses. Our results showed that Ca²⁺ release induced by K⁺ or caffeine occurs in discrete loci within the cell. The image areas and fluorescence intensities of some of these evoked local signals were similar to those of Ca²⁺ sparks that were observed under resting conditions and which are believed to be due to spontaneous activation of single release units. In contrast to the expectations imposed by quantal models, incremental doses of caffeine activated the same sets of release sites throughout the cell. Ca²⁺ release, at a given site, triggered by a submaximal dose of caffeine was transient and could be reactivated by addition of a higher caffeine dose, showing the same type of adaptive behavior as measured globally from larger areas of the cell. These results suggest that incremental Ca²⁺ release is accounted for by adaptive behavior of individual Ca²⁺ release sites. Received: 9 August 1995/Received in revised form and accepted: 13 October 1995     

IP3R在LH-EGFR诱导小鼠卵母细胞第一次减数分裂恢复中的作用

目的:探究1,4,5-三磷酸肌醇受体(IP3R)在促黄体素-表皮生长因子受体(LH-EGFR)信号诱导卵母细胞第一次减数分裂恢复中的作用及机制。方法:构建小鼠卵丘-卵母细胞复合物(COCs)体外培养模型以及卵泡培养模型,研究IP3R特异性抑制剂2-氨乙基二苯基硼酸脂(2-APB)及heparin对LH/EGF诱导减数分裂恢复以及EGF诱导卵丘扩展的影响,利用real-time PCR技术检测卵丘扩展因子的表达,钙离子荧光探针Fluo 3-AM检测胞内钙离子水平变化,酶联免疫法检测胞内cGMP水平变化。结果:IP3R抑制剂2-APB和heparin能够有效抑制EGF诱导的减数分裂恢复(P<0.05),同时显著降低了COCs内cGMP的含量(P<0.05);2-APB和Heparin还能够抑制EGF诱导的卵丘扩展,同时显著降低卵丘扩展因子的mRNA表达水平(P<0.05);激活IP3R可升高细胞内钙离子水平,而2-APB及heparin能够有效降低EGF升高的卵丘细胞内钙离子水平(P<0.05);利用卵泡培养模型进一步的研究表明,2-APB及heparin同样显著抑制了LH诱导的卵母细胞减数分裂恢复(P<0.05)。结论:LH-EGFR信号通路通过IP3R升高卵丘细胞内钙离子水平,诱导卵母细胞第一次减数分裂的恢复。     

钙通道阻滞剂对大鼠肾上腺嗜铬细胞烟碱受体通道电流的影响

目的:研究钙通道阻滞剂(CCB)对大鼠肾上腺嗜铬细胞(RAMC) 烟碱受体通道电流(I_NIC)的作用。方法: 利用全细胞膜片钳技术,用烟碱(NIC)诱发RAMC的NIC受体通道电流,分别观察硝苯吡啶(NIF)、ω-conotoxin GVIA和ω-agatoxin IVA急性灌洗细胞前后I_NIC的变化。结果: 不同类型和浓度的CCB分别灌洗RAMC ,均能明显抑制NIC所诱发的细胞I_NIC,灌洗5 min时 10 μmol/L NIF、400 nmol/L ω-conotoxin GVIA和100 nmol/L ω-agatoxin IVA对I_NIC的峰值抑制率分别为(61.7±5.1)%、(29.3±7.4)%和(17.6±7.5)%。结论: 不同类型的CCB急性作用于RAMC能明显阻滞NIC所诱发的I_NIC,提示CCB可能通过直接阻滞NIC受体通道来抑制RAMC儿茶酚胺的分泌过程。     

2-Aminoethoxydiphenyl borate inhibits agonist-induced Ca2+ signals by blocking inositol trisphosphate formation in acutely dissociated mouse pancreatic acinar cells

Evidence suggests that 2-aminoethoxydiphenyl borate (2-APB) modulates intracellular Ca²⁺ signals in a complex manner. 2-APB inhibits or potentiates intracellular Ca²⁺ signals in different cell types, perhaps through different mechanisms. Here, we report a novel mechanism underlying 2-APB-induced inhibition of agonist-activated Ca²⁺ oscillations in mouse pancreatic acinar cells, using patch-clamp and biochemical techniques. Pre-treatment of the cells with 100 µM 2-APB completely abolished ACh- but not inositol trisphosphate (InsP₃)-induced Ca²⁺ oscillations, suggesting that the mechanism of inhibition occurs between cytoplasmic receptors and InsP₃ receptor activation. In addition, 100 µM 2-APB significantly inhibited ACh-induced phospholipase C (PLC) activation. These findings indicate that, in mouse pancreatic acinar cells, in addition to modulating InsP₃ receptors and blocking the store-operated Ca²⁺ pathway, high concentrations of 2-APB also inhibit agonist-induced Ca²⁺ signals by reducing InsP₃ formation.     

Human nicotinic acetylcholine receptor: the influence of second messengers on activation and desensitization

The amplitude and time course of acetylcholine(ACh)-induced membrane current were determined in cells of the human medulloblastoma cell line TE 671. ACh was applied and washed out very rapidly (about 50 ms) by a shift of a cell between two streams of solution, one of which contained the transmitter. ACh-induced current was recorded in the whole-cell mode of patch clamping. The time course of activation of the ACh-induced current could not be resolved because the method of ACh application was still too slow. Desensitization started immediately with ACh application; it could be described by two time constants. With an ACh concentration of 3 μM, the fast time constant was about 0.5 s and the slow time constant was about 3.9 s. When the ACh concentration was raised in steps to 100 mM, the peak amplitude of the current increased, reached a maximum at 1 mM and decreased again. The rate of desensitization was directly correlated with increasing ACh concentration. Current amplitude and desensitization time constants were not affected by intracellular application of cAMP or of a catalytic subunit of a cAMP-dependent protein kinase. When the intracellular calcium concentration was raised at a constant magnesium concentration, desensitization time constants remained unaffected, but the current amplitude decreased. This decrease is not caused by a decrease in single-channel conductance, therefore it may represent a decrease in the number of activatable channels.     

A major role of the nicotinic acetylcholine receptor gene CHRNA2 in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is unlikely

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is known to be caused by mutations in the transmembrane regions of the neuronal nicotinic acetylcholine receptor (nAChR) genes CHRNA4 and CHRNB2. A third nAChR gene, CHRNA2, has been recently reported as mutated in an Italian family with nocturnal frontal lobe epilepsy, nocturnal wandering and ictal fear. We have now evaluated the role of CHRNA2 in families with "classical" ADNFLE. Mutation screening was performed in 47 families by amplification and subsequent sequencing of part of CHRNA2 exon 6 containing transmembrane regions 1-3. Detected variants were tested in a case-control association study. No mutations were identified in the parts of CHRNA2 that contribute to the ion pore. Sequencing identified a novel synonymous nucleotide exchange (c.771C/T) that was also present in two of 78 controls and is therefore likely to be non-pathogenic. The absence of mutations in our sample of 47 families renders a major role of CHRNA2 in ADNFLE unlikely.     

Parvalbumin-containing interneurons of the human cerebral cortex express nicotinic acetylcholine receptor proteins

Cholinergic fibers from the basal forebrain are known to contact cholinoceptive cortical pyramidal neurons. Recent electrophysiological studies have revealed that nicotinic acetylcholine receptors are also present in human cerebrocortical interneurons. A direct visualization of nicotinic receptor subunits in cortical interneurons has, however, not yet been performed. We have applied double-immunofluorescence using antibodies against parvalbumin --a marker for the Chandelier and basket cell subpopulation of interneurons--and to the alpha4 and alpha7 subunit proteins of the nicotinic acetylcholine receptor. The vast majority of the parvalbuminergic interneurons was immunoreactive for the alpha4 and the alpha7 nicotinic acetylcholine receptor. Provided these receptors would be functional--as suggested by recent electrophysiological findings--the connectivity pattern of cholinergic afferents appears much more complex than thought before. Not only direct cholinergic impact on cortical projection neurons but also the indirect modulation of these by cholinergic corticopetal fibers contacting intrinsic cortical cells would be possible.     

Cholinergic responses in cloned human TE671/RD tumour cells

The cholinergic responses of the human tumour cell line TE671/RD were examined using digital Ca²⁺ imaging fluorescence microscopy and patch-clamp measurements. In response to stimulation of the muscarinic acetylcholine (ACh) receptor (mAChR), the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) rose about two-fold, in parallel with inositol 1,4,5-trisphosphate accumulation, measured by chromatographic techniques. By contrast, there was no increment of [Ca²⁺]_i upon stimulation of the nicotinic ACh receptor (nAChR), nor after caffeine application. Electrophysiological experiments showed that TE671/RD cells lack functional voltage-activated Ca²⁺ channels. The stimulation of the nAChR induced transient whole-cell currents (I _ACh). Little or no current was detected in isotonic extracellular Ca²⁺, with Cs⁺ in the patch pipette. Cell pretreatment with muscarine reduced I _ACh by about 20%, without consistent modifications of current kinetics. Muscarine applied to the extra-patch membrane under the cell-attached configuration had no obvious effect on ACh-evoked unitary events. In conclusion, in human TE671/ RD cells, muscarinic stimulation increases [Ca²⁺]_i, while nicotinic stimulation does not. In addition, the nAChR exhibits peculiar ion permeability properties and is not functionally regulated by the breakdown of phosphoinositides.     

Decreased vesicular acetylcholine transporter and alpha(4)beta(2) nicotinic receptor density in the rat brain following 192 IgG-saporin immunolesioning

Degeneration of cholinergic neurons is a well known characteristic of Alzheimer's disease (AD). Two radioligands were studied in a rat model of cholinergic degeneration to evaluate their potential efficacy for molecular imaging of AD. Following specific cholinergic-cell immunolesioning with 192 IgG-saporin (SAP), ex vivo autoradiography was performed with (123)IBVM, a radioligand which targets the vesicular acetylcholine transporter (VAChT). Following the decay of (123)I, the same animals had in vitro autoradiography performed with (125)I-A-85380, a marker for nicotinic acetylcholine receptors (nAChRs). As expected significant, widespread decreases in (123)IBVM uptake were observed in SAP treated animals. Moderate but significant reductions in (125)I-A-85380 binding in the hippocampus (Hip) and cerebellum (Cbm) were also observed following SAP immunolesioning. The results with (123)IBVM confirm and extend previous work investigating the uptake of radioiodinated IBVM in this animal model. The results with (125)I-A-85380 are unique and are in contrast with work performed in this animal model with other nAChR radioligands, indicating the favourable properties of this radioligand for molecular imaging.     

Potentiation of the nicotinic acetylcholine receptor by aluminum in mammalian neurons

Aluminum (Al³⁺), a known neurotoxic substance, has long been implicated in the pathogenesis of Alzheimer’s disease and other neurodegenerative diseases. Al³⁺ targets many ligand-gated and voltage-gated ion channels and modulates their functions. In the present study, the actions of Al³⁺ on the nicotinic acetylcholine receptor (nAChR) were investigated by whole-cell patch clamp technique in acutely isolated rat trigeminal ganglion neurons. We observed that Al³⁺ potentiated nicotine-evoked inward currents in a concentration-dependent manner (10–1000 μM). The effects of Al³⁺ on nicotine-evoked currents were voltage independent. Al³⁺ appeared to increase the affinity of nicotine to nAChR but not the efficacy. Al³⁺ reduced the agonist concentration producing a half-maximal response (EC₅₀) for nicotine from 74.4±1.9 μM to 32.9±2.6 μM, but did not alter the threshold nor maximal response. On the contrary, another trivalent cation, Ga³⁺, had little effect on nicotine-evoked currents. The present results indicated that Al³⁺ enhanced the function of nAChR and this potentiation might underlie the neurological alteration induced by Al³⁺.