ß2-Adrenergic Regulation of the Neuromuscular Transmission and Its Lipid-Dependent Switch.

ß2-Adrenoceptors (ß2-ARs) are the most abundant subtype of adrenergic receptors in skeletal muscles. Their activation via a stabilization of postsynaptic architecture has beneficial effects in certain models of neuromuscular disorders. However, the ability of ß2-ARs to regulate neuromuscular transmission at the presynaptic level is poorly understood. Using electrophysiological recordings and fluorescent FM dyes, we found that ß2-AR activation with fenoterol enhanced an involvement of synaptic vesicles in exocytosis and neurotransmitter release during intense activity at the neuromuscular junctions of mouse diaphragm. This was accompanied by an improvement of contractile responses to phrenic nerve stimulation (but not direct stimulation of the muscle fibers) at moderate-to-high frequencies. ß2-ARs mainly reside in lipid microdomains enriched with cholesterol and sphingomyelin. The latter is hydrolyzed by sphingomyelinases, whose upregulation occurs in many conditions characterized by muscle atrophy and sympathetic nerve hyperactivity. Sphingomyelinase treatment reversed the effects of ß2-AR agonist on the neurotransmitter release and synaptic vesicle recruitment to the exocytosis during intense activity. Inhibition of Gi protein with pertussis toxin completely prevented the sphingomyelinase-mediated inversion in the ß2-AR agonist action. Note that lipid raft disrupting enzyme cholesterol oxidase had the same effect on ß2-AR agonist-mediated changes in neurotransmission as sphingomyelinase. Thus, ß2-AR agonist fenoterol augmented recruitment and release of synaptic vesicles during intense activity in the diaphragm neuromuscular junctions. Sphingomyelin hydrolysis inversed the effects of ß2-AR agonist on neurotransmission probably via switching to Gi protein-dependent signaling. This phenomenon may reflect a dependence of the ß2-AR signaling on lipid raft integrity in the neuromuscular junctions.

Effects of cholesterol oxidase on neurotransmission and acetylcholine levels at the mice neuromuscular junctions.

Membrane cholesterol oxidation is a hallmark of redox and metabolic imbalance, and it may accompany neurodegenerative disorders. Using microelectrode recordings of postsynaptic responses as well as fluorescent dyes for monitoring synaptic vesicle cycling and membrane properties, the action of enzymatic cholesterol oxidation on neuromuscular transmission was studied in the mice diaphragms. Cholesterol oxidase (ChO) at low concentration disturbed lipid-ordering specifically in the synaptic membranes, but it did not change markedly spontaneous exocytosis and evoked release in response to single stimuli. At low external Ca2+ conditions, analysis of single exocytotic events revealed a decrease in minimal synaptic delay and the probability of exocytosis upon plasmalemmal cholesterol oxidation. At moderate- and high-frequency activity, ChO treatment enhanced both neurotransmitter and FM-dye release. Furthermore, it precluded a change in exocytotic mode from full-fusion to kiss-and-run during high-frequency stimulation. Accumulation of extracellular acetylcholine (without stimulation) dependent on vesamicol-sensitive transporters was suppressed by ChO. The effects of plasmalemmal cholesterol oxidation on both neurotransmitter/dye release at intense activity and external acetylcholine levels were reversed when synaptic vesicle membranes were also exposed to ChO (i.e., the enzyme treatment was combined with induction of exo-endocytotic cycling). Thus, we suggest that plasmalemmal cholesterol oxidation affects exocytotic machinery functioning, enhances synaptic vesicle recruitment to the exocytosis and decreases extracellular neurotransmitter levels at rest, whereas ChO acting on synaptic vesicle membranes suppresses the participation of the vesicles in the subsequent exocytosis and increases the neurotransmitter leakage. The mechanisms underlying ChO action can be related to the lipid raft disruption.

Sphingomyelinase modulates synaptic vesicle mobilization at the mice neuromuscular junctions.

AIMS: Sphingomyelin is an abundant component of the presynaptic membrane and an organizer of lipid rafts. In several pathological conditions, sphingomyelin is hydrolyzed due to an upregulation and release of secretory sphingomyelinases (SMases). Herein, the effects of SMase on exocytotic neurotransmitter release were studied in the diaphragm neuromuscular junctions of mice. MAIN METHODS: Microelectrode recordings of postsynaptic potentials and styryl (FM) dyes were used to estimate neuromuscular transmission. Membrane properties were assessed with fluorescent techniques. KEY FINDINGS: Application of SMase at a low concentration (0.01 U ml-1) led to a disruption of lipid-packing in the synaptic membranes. Neither spontaneous exocytosis nor evoked neurotransmitter release (in response to single stimuli) were affected by SMase treatment. However, SMase significantly increased neurotransmitter release and the rate of fluorescent FM-dye loss from the synaptic vesicles at 10, 20 and 70 Hz stimulation of the motor nerve. In addition, SMase treatment prevented a shift of the exocytotic mode from "full-collapse" fusion to "kiss-and-run" during high-frequency (70 Hz) activity. The potentiating effects of SMase on neurotransmitter release and FM-dye unloading were suppressed when synaptic vesicle membranes were also exposed to this enzyme (i.e., stimulation occurred during SMase treatment). SIGNIFICANCE: Thus, hydrolysis of the plasma membrane sphingomyelin can enhance mobilization of synaptic vesicles and facilitate full fusion mode of exocytosis, but SMase acting on vesicular membrane had a depressant effect on the neurotransmission. Partially, the effects of SMase can be related with the changes in synaptic membrane properties and intracellular signaling.

Frequency-Dependent Engagement of Synaptic Vesicle Pools in the Mice Motor Nerve Terminals.

Nerve terminals contain numerous synaptic vesicles (SVs) whose exo-endocytic cycling maintains neurotransmitter release. SVs may have different properties, thereby constituting separate pools. However, behavior of SV pools remains elusive in many synapses. To fill this gap, we studied the functioning of SV pools at both low- and higher-frequency stimulations utilizing microelectrode recording and dual-labeling of SVs with FM-dyes at the mice motor nerve terminals. It was found that higher-frequency stimulation caused exocytosis of different kinds of SVs. One type of SVs contributed to exocytosis exclusively at intense activities and their exocytotic rate was depended on the order in which these SVs were recovered by endocytosis. Another type of SVs can sustain the release in response to both low- and higher-frequency stimulations, but increasing activity did not lead to enhanced exocytotic rate of these SVs. In addition, depression of neurotransmitter release induced by 20 Hz stimulation occurred independent on previous episode of 10 Hz activity. We suggest that during prolonged stimulation at least two SV pools can operate. One termed "house-keeping" that would be active at different frequencies and the other termed "plug-in" that would respond to increasing activity.

KATP channels as ROS-dependent modulator of neurotransmitter release at the neuromuscular junctions.

AIMS: Neurotransmitter release requires high energy demands, making the nerve terminals metabolically fragile and susceptible to oxidative stress. ATP-sensitive potassium (KATP) channels can be an important regulator orchestrating the influence of metabolic-related signals on exocytosis. Here, the relevance of ROS in KATP channel-dependent control of neurotransmitter release at the frog neuromuscular junction was studied. METHODS: Microelectrode recordings of end plate potentials at the distal and proximal compartments of nerve terminals as well as fluorescent techniques were used. KEY FINDINGS: Activation of KATP channels in the proximal region suppressed evoked and spontaneous release in a lipid raft-dependent manner. Activation of KATP channels in the distal region reduced solely evoked release which was preserved after lipid raft disruption. Chelation of ROS potentiated the effects of KATP channel activation and unmasked the effects of KATP channel blocker on evoked exocytosis. Activation or inhibition of KATP channels suppressed or enhanced the depressant action of extracellular adenosine on evoked exocytosis. This was accompanied with an increase or decrease in adenosine-induced ROS production, respectively. KATP channel-dependent modulation of adenosine action was halted by antioxidant and NADPH-oxidase inhibitor. Also, activation of KATP channels led to an increase in ROS production suppressing the negative effects of extracellular ATP on evoked release in a ROS-dependent manner. SIGNIFICANCE: KATP channel-mediated modulation of release has specific features in distal and proximal compartments and depends on endogenous ROS levels and lipid raft integrity. Activation of KATP channels suppresses the action of extracellular adenosine and ATP on evoked release by increasing ROS production.