ABSTRACT
Experiments with fluorescent dyes showed that high concentrations of K(+) ions in the medium depolarize the membrane and enhance exo-endocytosis in nerve structures, which is accompanied by an increase in acetylcholine concentration in the somatic muscle of earthworm. In the presence of BAPTA and without Ca(2+) exo-endocytosis is sharply decelerated, the level of acetylcholine in the muscle decreases, but remains relatively high.
Subject(s)
Acetylcholine/analysis , Oligochaeta/chemistry , Presynaptic Terminals/chemistry , Animals , Calcium/analysis , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Endocytosis , Exocytosis , Fluorescent Dyes , Muscles/chemistry , Muscles/innervation , Nerve Tissue/chemistry , Potassium/analysis , Presynaptic Terminals/ultrastructure , Staining and LabelingABSTRACT
The somatic muscle of earthworm contains myoneural synapses forming clusters of "synaptic buttons". In these "buttons", the proteins syntaxin 1, synaptotagmin 1 and alpha 1B subunit of the Ca(2+)-channel of N-type were identified. We suppose that "synaptic buttons" contain a limited number of active zones due to their small size (1-2 microm) and the pattern of distribution of proteins of exo-endocytotic cycle. The postsynaptic membrane of cholinetgic synapses contains nicotinic acetylcholine receptors capable to bind alpha-bungarotoxin. The area of location of receptors on postsynaptic membrane is strictly limited to the region of synaptic contact.
Subject(s)
Calcium Channels, N-Type , Receptors, Nicotinic , Synaptotagmin I , Syntaxin 1 , Animals , Bungarotoxins/metabolism , Calcium Channels, N-Type/isolation & purification , Calcium Channels, N-Type/metabolism , Muscles/metabolism , Neuromuscular Junction/metabolism , Oligochaeta/metabolism , Receptors, Nicotinic/isolation & purification , Receptors, Nicotinic/metabolism , Synapses/metabolism , Synaptic Membranes/metabolism , Synaptotagmin I/isolation & purification , Synaptotagmin I/metabolism , Syntaxin 1/isolation & purification , Syntaxin 1/metabolismABSTRACT
Luminous spots with a diameter of 1-2 microm, which are clusters of "synaptic buds", were revealed in the muscular wall of the earthworm using endocytotic fluorescent dyes FM1-43, FM2-10 and FM4-64. Application of the membrane probe Dil that is capable of being subjected to anterograde axonal transport to abdominal ganglia of the nervous chain, and subsequent (in a day) staining of nerve formations by endocytotic dye FM4-64 showed complete imposition of the emission data of the dyes that fluoresce in different parts of the spectrum. Using fluorescent marker DiBAC4(3) showed an increased emission of neural elements with increasing concentration of K+ in the extracellular environment. Application of FM2-10 showed that the higher concentration of K+ in solution, and hence the depolarization of the nerve cells, the faster the upload of the dye, and vice versa, the process slowed down in the absence of K+ in the medium. The seizure and removal of FM2-10 were blocked in calcium-free solutions in the presence of Ca2+ buffers, BABTA or BABTA-AM, but only after a preliminary 40 min incubation. The processes of exo- and endocytosis occurred in the clusters of synaptic "buds" and were preserved in conditions of "rest". This vesicle cycle depends on membrane potential and concentration of K+ and Ca2+, and, it is very likely that the calcium sensor operates on the principle "all or nothing".
Subject(s)
Calcium/metabolism , Membrane Potentials/physiology , Motor Neurons/metabolism , Nerve Tissue/metabolism , Oligochaeta/physiology , Potassium/metabolism , Synaptic Vesicles/physiology , Animals , Barbiturates/analysis , Barbiturates/metabolism , Calcium/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/analysis , Egtazic Acid/metabolism , Endocytosis/drug effects , Endocytosis/physiology , Exocytosis/drug effects , Exocytosis/physiology , Fluorescent Dyes/analysis , Fluorescent Dyes/metabolism , Isoxazoles/analysis , Isoxazoles/metabolism , Membrane Potentials/drug effects , Microscopy, Fluorescence , Motor Neurons/cytology , Muscles/cytology , Muscles/metabolism , Nerve Tissue/cytology , Potassium/pharmacology , Pyridinium Compounds/analysis , Pyridinium Compounds/metabolism , Quaternary Ammonium Compounds/analysis , Quaternary Ammonium Compounds/metabolism , Synaptic Vesicles/drug effects , Synaptic Vesicles/ultrastructureABSTRACT
Replacement of Cl(-) for Br(-) in bathing solution did not reduce resting potential and had no effect on modulation of transmembrane potential in hyper- and hypoosmotic solutions. Under these conditions, baclofen, an agonist of GABAergic B-type receptors, failed to activate Na(+)/K(+)-pump in earthworm somatic muscle cells. It was hypothesized that the contribution of Cl(-) symport to osmotic homeostasis is not highly selective in respect to replacement of Cl(-) to Br(-) ions, whereas in case of activation of electrogenic ion pumps, this replacement is equivalent to removal of Cl(-) ions from the bathing solution.
Subject(s)
Chlorides/metabolism , Muscle Cells/metabolism , Oligochaeta/metabolism , Animals , Baclofen/pharmacology , Bromides/metabolism , Furosemide/pharmacology , GABA Agonists/pharmacology , Homeostasis/drug effects , Ion Transport/drug effects , Membrane Potentials/drug effects , Muscle Cells/drug effects , Oligochaeta/cytology , Osmosis/drug effects , Sodium Potassium Chloride Symporter Inhibitors/pharmacologyABSTRACT
Carbacholine reduced, while baclofen and norepinephrine increased resting membrane potential in earthworm somatic muscle cells. In the presence of carbacholine, neither norepinephrine, nor baclofen hyperpolarized the membrane. Ouabain decreased resting potential and abolished the effects of carbacholine, norepinephrine, and baclofen on membrane potential. It was hypothesized that carbacholine directly inhibited the ouabain-sensitive component of Na+/K+ pump and abolished the activating effect of norepinephrine and baclofen.
