A. L. Zefirov

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.

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.

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.

Cholesterol and Lipid Rafts in the Plasma Membrane of Nerve Terminal and Membrane of Synaptic Vesicles

In this study, using neuromusclar preparations of frog cutaneous pectoris muscle and mouse diaphragm we have shown that the membranes of nerve terminals (NT) contain nearly two times more cholesterol than the plasma membrane of muscle fibers. Using the fluorescent B subunit of cholera toxin (CT-B), we identified the areas in the NT membranes with high concentrations of GM1 ganglioside, the marker molecule for cholesterol- and sphingolipid-enriched membrane microdomains, so-called lipid rafts. Intense fluorescent spots appear in the NT when lipid rafts were identified during high-frequency stimulation, which cause massive exocytosis of synaptic vesicles followed by endocytosis. The double staining of neuromusclar preparations with CT-B and FM1-43, a dye that is taken up into synaptic vesicles during endocytosis, has demonstrated good colabeling by these dyes. This indicates the presence of large amounts of cholesterol and lipid rafts in membranes of synaptic vesicles. Thus, cholesterol is present in NT membranes at the high concentration required for the organization of lipid rafts, which are common in the plasma membranes of the NT and the membranes of synaptic vesicles. The role of cholesterol and lipid rafts in the processes of exo- and endocytosis is discussed.

Sensitivity of Intracellular Calcium-Binding Sites for Exo- and Endocytosis of Synaptic Vesicles to Sr, Ba, and Mg Ions

Experiments on frog cutaneous-thoracic muscle preparations using electrophysiological (intra- and extracellular recording of postsynaptic signals) and optical (confocal microscopy with the fluorescent endocytic stain FM 1-43) methods were performed to study neurotransmitter secretion and the processes of exo- and endocytosis of synaptic vesicles in motor nerve endings on substitution of extracellular Ca ions with other alkaline earth metals (Sr, Ba, or Mg). Massive asynchronous exocytosis was induced by highpotassium solution, while synchronous exocytosis was induced by prolonged high-frequency stimulation of the motor nerve. The calcium-binding site for asynchronous exocytosis was found to be sensitive to Sr, Ba, and Mg ions, while the site for synchronous exocytosis was only sensitive to Sr ions. During stimulation of both asynchronous and synchronous exocytosis, the calcium-binding site for endocytosis was sensitive to Sr and Ba ions and had the lowest affinity for Sr ions. These experiments led to the conclusion that different intracellular calcium-binding sites exist for the exocytosis and endocytosis of synaptic vesicles and that they have different sensitivities for alkaline earth metals.

The role of membrane cholesterol in effects of β2 adrenoreceptors activation of the mouse atrium

1 In our previous studies we discovered the positive inotropic effects of activation of β2 adrenergic recepp tors (AR) in the mouse atrium by the selective agonist fenoterol. Low doses of fenoterol (5 μM) increased the amplitude of contractions within 15–20 min after administration of the agonist; high doses (50 μM) increased the amplitude immediately [1, 2, 8]. Activaa tion of β2 AR by 5 μM agonist triggered two signal cass cades that had opposite effects on atrium contractility: the first pathway increased Caasignals, the second cass cade stimulated NO production. Therefore, the delay in positive inotropic effect of fenoterol was observed. After NO production decreased while Ca signals still had a high amplitude, atrium contractions increased [1, 2]. When 50 μM β2 AR agonist was administered, the signal cascade that increased Ca signals was actii vated faster, while the increase in NO production was delayed. Thus, the positive inotropic effect of the agoo nist appeared immediately [8]. Cholesterol is one of the main components of plasma membranes; it regulates membrane thickness and fluidity and forms specialized microdomains or lipid rafts together with sphingolipids [3, 11]. Most cardiac β2 ARs are accumulated in lipid rafts. In this study, we investigated the role of membrane choles terol in the effects of β2 AR activation. For this purr pose, we remove membrane cholesterol using methyll βcyclodextrin (MCD) [7, 9, 12]. Experiments were conducted on an isolated mouse atrium. Standard Krebs solution saturated with oxyy gen was used. All drugs were purchased from Sigma (United States), except for Fluo44AM and DAFFFM diacetate (Molecular Probes, United States). The atrium was stimulated with electric discharges with an amplitude exceeding the threshold and a frequency of 1 Hz. Atrium contractions were recorded using a pressure sensor (AD Instruments) and a PowerLab system (the Chart program). To study the changes in the intracellular Ca 2+ concentration, Fluoo4AM was used. Changes in Ca indicator fluorescence were observed (Caasignals) when cytoplasmic Ca concenn tration increased due to the influx of extracellular Ca 2+ via voltageedependent Ca channels on the plasmatic membrane and Ca 2+ release from sarcoplasmic reticuu lum via ryanodine receptors [4]. To estimate the amplitude of Caasignals, the minimum diastolic fluoo rescence values were subtracted from the maximum systolic fluorescence values. NO production was detected using DAFFFM acetate, a membrane permee able marker. This marker has a moderate NO sensitivv ity; hence, mild changes in DAFFFM fluorescence …

Lipids in the Processes of Exo- and Endocytosis of Synaptic Vesicles

The phenomenon of synaptic transmission is underlain by the processes of exo- and endocytosis of synaptic vesicles, which involve complex protein mechanisms. The proteins forming the SNARE complex (synaptobrevin, syntaxin, SNAP-25), synaptotagmin, Munc-13, Munc-18, NSF, and α-SNAP, are involved in exocytosis, while other proteins (clathrin, AP-2, epsin, endophilin, amphiphysin, dynamin, synaptojanin, Hsc70, and others) mediate the endocytosis of synaptic vesicles. Recent years have seen the accumulation of data on the critical roles of various lipids in exo- and endocytosis. Particularly interesting results were obtained in relation to the significance of cholesterol, sphingomyelins, phosphoinositides, and phosphatidic and polyunsaturated fatty acids for the exo-endocytic cycle. The involvement of lipid rafts in the recycling of synaptic vesicles is discussed. We present contemporary data, including our own results, on the role of lipids and lipid-modifying enzymes in the processes of exo- and endocytosis.