A. M. Petrov

Reactive oxygen species contribute to the presynaptic action of extracellular ATP at the frog neuromuscular junction

During normal cell metabolism the production of intracellular ATP is associated with the generation of reactive oxygen species (ROS), which appear to be important signalling molecules. Both ATP and ROS can be released extracellularly by skeletal muscle during intense activity. Using voltage clamp recording combined with imaging and biochemical assay of ROS, we tested the hypothesis that at the neuromuscular junction extracellular ATP generates ROS to inhibit transmitter release from motor nerve endings. We found that ATP produced the presynaptic inhibitory action on multiquantal end‐plate currents. The inhibitory action of ATP (but not that of adenosine) was significantly reduced by several antioxidants or extracellular catalase, which breaks down H2O2. Consistent with these data, the depressant effect of ATP was dramatically potentiated by the pro‐oxidant Fe2+. Exogenous H2O2 reproduced the depressant effects of ATP and showed similar sensitivity to anti‐ and pro‐oxidants. While NO also inhibited synaptic transmission, inhibitors of the NO‐producing cascade did not prevent the depressant action of ATP. The ferrous oxidation in xylenol orange assay showed the increase of ROS production by ATP and 2‐MeSADP but not by adenosine. Suramin, a non‐selective antagonist of P2 receptors, and pertussis toxin prevented the action of ATP on ROS production. Likewise, imaging with the ROS‐sensitive dye carboxy‐2′,7′‐dichlorodihydrofluorescein revealed increased production of ROS in the muscle treated with ATP or ADP while UTP or adenosine had no effect. Thus, generation of ROS contributed to the ATP‐mediated negative feedback mechanism controlling quantal secretion of ACh from the motor nerve endings.

Distinct α2 Na,K-ATPase membrane pools are differently involved in early skeletal muscle remodeling during disuse.

Location, location, location. The Na-K pump of skeletal muscle is regulated differently at neuromuscular junctions.

Role of membrane cholesterol in spontaneous exocytosis at frog neuromuscular synapses: reactive oxygen species–calcium interplay

Cholesterol depletion increases reactive oxygen species (ROS) levels in extra‐ and intracellular space through NAPDH oxidase activation, which is accompanied by synaptic lipid oxidation. ROS production due to extraction of cholesterol involves both an enhancement of synaptic vesicle exocytosis and an increase in cytosolic [Ca2+]i. An ROS‐dependent rise in [Ca2+]i is suppressed by inhibitors of the transient receptor potential vanilloid channel and leads to an increase in synaptic vesicle exocytosis. The ROS–calcium pathway might influence synaptic vesicle exocytosis via activation of phosphatase protein 2B (calcineurin). The results help us better understand why a decrease of membrane cholesterol increases spontaneous synaptic vesicle exocytosis.

The Role of Cholesterol in the Exo- and Endocytosis of Synaptic Vesicles in Frog Motor Nerve Endings

Experiments on frog neuromuscular preparations using electrophysiological (two-electrode voltage clamping) and optical (with the fluorescent endocytic stain FM1-43) methods were performed to study the importance of membrane cholesterol in the exo- and endocytic cycle of synaptic vesicles (SV) in motor nerve endings in conditions of prolonged rhythmic stimulation of the motor nerve (20 impulses/sec, 3 min). Extraction of cholesterol from the superficial plasma membranes using methyl-β-cyclodextrin (1 mM) led to marked changes in SV recycling. There was weakening of SV exocytosis and suppression of processes leading to the recovery of SV populations with rapid readiness to release neurotransmitter. When cholesterol was leached from the outer membranes and the membranes of SV undergoing recycling, these effects were supplemented by impairments to SV endocytosis and recycling. Thus, plasma membrane cholesterol plays a key role in the processes of exocytosis, while the efficiency of endocytosis depends on cholesterol in SV membranes.

β2-adrenoceptor agonist-evoked reactive oxygen species generation in mouse atria: implication in delayed inotropic effect

Fenoterol, a β2-adrenoceptor agonist, has anti-apoptotic action in cardiomyocytes and induces a specific pattern of downstream signaling. We have previously reported that exposure to fenoterol (5 μM) results in a delayed positive inotropic effect which is related to changes in both Ca2+ transient and NO. Here, the changes in reactive oxygen species (ROS) production in response to the fenoterol administration and the involvement of ROS in effect of this agonist on contractility were investigated in mouse isolated atria. Stimulation of β2-adrenoceptor increases a level of extracellular ROS, while intracellular ROS level rises only after removal of fenoterol from the bath. NADPH-oxidase inhibitor (apocynin) prevents the increase in ROS production and the Nox2 isoform is immunofluorescently colocalized with β2-adrenoceptor at the atrial myocytes. Treatments with antioxidants (N-acetyl-L-cysteine, NADPH inhibitors, exogenous catalases) significantly inhibit the fenoterol induced increase in the contraction amplitude, probably by attenuating Ca2+ transient and up-regulating NO production. ROS generated in a β2-adrenoceptor-dependent manner can potentiate the activity of some Ca2+ channels. Indeed, inhibition of ryanodine receptors, TRPV-or L-type Ca2+- channels shows a similar efficacy in reduction of positive inotropic effect of both fenoterol and H2O2. In addition, detection of mitochondrial ROS indicates that fenoterol triggers a slow increase in ROS which is prevented by rotenone, but rotenone has no impact on the inotropic effect of fenoterol. We suggest that stimulation of β2-adrenoceptor with fenoterol causes the activation of NADPH-oxidase and after the agonist removal extracellularly generated ROS penetrates into the cell, increasing the atrial contractions probably via Ca2+ channels.

Effects of 5α-cholestan-3-one on the synaptic vesicle cycle at the mouse neuromuscular junction.

We have investigated the effects of 5α-cholesten-3-one (5Ch3, 200 nM) on synaptic transmission in mouse diaphragm. 5Ch3 had no impact on the amplitude or frequency of miniature endplate currents (MEPCs, spontaneous secretion), but decreased the amplitude of EPCs (evoked secretion) triggered by single action potentials. Treatment with 5Ch3 increased the depression of EPC amplitude and slowed the unloading of the dye FM1-43 from synaptic vesicles (exocytosis rate) during high-frequency stimulation. The estimated recycling time of vesicles did not change, suggesting that the decline of synaptic efficiency was due to the reduction in the size of the population of vesicles involved in release. The effects of 5Ch3 on synaptic transmission may be related to changes in the phase properties of the membrane. We have found that 5Ch3 reduces the staining of synaptic regions with the B-subunit of cholera toxin (a marker of lipid rafts) and increases the fluorescence of 22-NBD-cholesterol, indicating a phase change within the membrane. Manipulations of membrane cholesterol (saturation or depletion) strongly reduced the influence of 5Ch3 on both FM1-43 dye unloading and staining with the B-subunit of cholera toxin. Thus, 5Ch3 reduces the number of vesicles which are actively recruited during synaptic transmission and alters membrane properties. These effects of 5Ch3 depend on membrane cholesterol.

Cholesterol regulates contractility and inotropic response to β2-adrenoceptor agonist in the mouse atria: Involvement of Gi-protein–Akt–NO-pathway

Majority of cardiac β2-adrenoceptors is located in cholesterol-rich microdomains. Here, we have investigated the underlying mechanisms by which a slight to moderate cholesterol depletion with methyl-β-cyclodextrin (MβCD, 1 and 5mM) interferes with contractility and inotropic effect of β2-adrenergic agonist (fenoterol, 50μM) in the mouse atria. Treatment with MβCD itself increased amplitude of Ca2+ transient but did not change the contraction amplitude due to a clamping action of elevated NO. Cholesterol depletion significantly attenuated the positive inotropic response to fenoterol which is accompanied by increase in NO generation and decrease in Ca2+ transient. Influence of 1mM MβCD on the fenoterol-driven changes in both contractility and NO level was strongly attenuated by inhibition of Gi-protein (pertussis toxin), Akt (Akt 1/2 kinase inhibitor) or NO-synthase (L-NAME). After exposure to 5mM MβCD, pertussis toxin or Akt inhibitor could recover the β2-agonist effects on contractility, NO production and Ca2+ transient, while L-NAME only reduced NO level. An adenylyl cyclase activator (forskolin, 50nM) had no influence on the MβCD-induced changes in the β2-agonist effects. Obtained results suggest that slight cholesterol depletion upregulates Gi-protein/Akt/NO-synthase signaling that attenuates the positive inotropic response to β2-adrenergic stimulation without altering the Ca2+ transient. Whilst moderate cholesterol depletion additionally could suppress the enhancement of the Ca2+ transient amplitude caused by the β2-adrenergic agonist administration in Gi-protein/Akt-dependent but NO-independent manner.

Membrane lipid rafts are disturbed in the response of rat skeletal muscle to short-term disuse

Marked loss of skeletal muscle mass occurs under various conditions of disuse, but the molecular and cellular mechanisms leading to atrophy are not completely understood. We investigate early molecular events that might play a role in skeletal muscle remodeling during mechanical unloading (disuse). The effects of acute (6-12 h) hindlimb suspension on the soleus muscles from adult rats were examined. The integrity of plasma membrane lipid rafts was tested utilizing cholera toxin B subunit or fluorescent sterols. In addition, resting intracellular Ca2+ level was analyzed. Acute disuse disturbed the plasma membrane lipid-ordered phase throughout the sarcolemma and was more pronounced in junctional membrane regions. Ouabain (1 µM), which specifically inhibits the Na-K-ATPase α2 isozyme in rodent skeletal muscles, produced similar lipid raft changes in control muscles but was ineffective in suspended muscles, which showed an initial loss of α2 Na-K-ATPase activity. Lipid rafts were able to recover with cholesterol supplementation, suggesting that disturbance results from cholesterol loss. Repetitive nerve stimulation also restores lipid rafts, specifically in the junctional sarcolemma region. Disuse locally lowered the resting intracellular Ca2+ concentration only near the neuromuscular junction of muscle fibers. Our results provide evidence to suggest that the ordering of lipid rafts strongly depends on motor nerve input and may involve interactions with the α2 Na-K-ATPase. Lipid raft disturbance, accompanied by intracellular Ca2+ dysregulation, is among the earliest remodeling events induced by skeletal muscle disuse.

Similar oxysterols may lead to opposite effects on synaptic transmission: Olesoxime versus 5α-cholestan-3-one at the frog neuromuscular junction.

Cholesterol oxidation products frequently have a high biological activity. In the present study, we have used microelectrode recording of end plate currents and FM-based optical detection of synaptic vesicle exo-endocytosis to investigate the effects of two structurally similar oxysterols, olesoxime (cholest-4-en-3-one, oxime) and 5ɑ-cholestan-3-one (5ɑCh3), on neurotransmission at the frog neuromuscular junction. Olesoxime is an exogenous, potentially neuroprotective, substance and 5ɑCh3 is an intermediate product in cholesterol metabolism, which is elevated in the case of cerebrotendinous xanthomatosis. We found that olesoxime slightly increased evoked neurotransmitter release in response to a single stimulus and significantly reduced synaptic depression during high frequency activity. The last effect was due to an increase in both the number of synaptic vesicles involved in exo-endocytosis and the rate of synaptic vesicle recycling. In contrast, 5ɑCh3 reduced evoked neurotransmitter release during the low- and high frequency synaptic activities. The depressant action of 5ɑCh3 was associated with a reduction in the number of synaptic vesicles participating in exo- and endocytosis during high frequency stimulation, without a change in rate of the synaptic vesicle recycling. Of note, olesoxime increased the staining of synaptic membranes with the B-subunit of cholera toxin and the formation of fluorescent ganglioside GM1 clusters, and decreased the fluorescence of 22-NBD-cholesterol, while 5ɑCh3 had the opposite effects, suggesting that the two oxysterols have different effects on lipid raft stability. Taken together, these data show that these two structurally similar oxysterols induce marked different changes in neuromuscular transmission which are related with the alteration in synaptic vesicle cycle.

Effects of Oxidation of Membrane Cholesterol on the Vesicle Cycle in Motor Nerve Terminals in the Frog Rana Ridibunda

Experiments on neuromuscular preparations from the frog Rana ridibunda assessed the effects of oxidation of membrane cholesterol on the presynaptic vesicle cycle. Application of cholesterol oxidase (1 activity unit) for 30 min oxidized about 0.007 mg of cholesterol per g of preparation and decreased the stability of lipid rafts in nerve terminals. Electrophysiological studies demonstrated that oxidation of cholesterol decreased evoked transmitter release. Experiments using fluorescent FM dyes demonstrated suppression of synaptic vesicle exo- and endocytosis processes, along with dispersal of clusters of synaptic vesicles. Comparative analysis of electrophysiological and optical data, along with experiments using a dye quencher, demonstrated that transmitter could be released from some synaptic vesicles through transient fusion pores (the kiss-and-run mechanism). It is suggested that oxidation of cholesterol suppresses evoked exocytosis and delivery of synaptic vesicles of the reserve pool to the exocytosis site, thus interfering with their clustering. Vesicles of the recycling pool release transmitter by the “kiss-and-run” mechanism.