Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction
Acetazolamide (AZ), a molecule frequently used to treat different neurological syndromes, is an inhibitor of the carbonic anhydrase (CA), an enzyme that regulates pH inside and outside cells. We combined fluorescent FM styryl dyes and electrophysiological techniques at ex vivo levator auris longus n...
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2017
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_08874476_v71_n12_p_Bertone http://hdl.handle.net/20.500.12110/paper_08874476_v71_n12_p_Bertone |
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paper:paper_08874476_v71_n12_p_Bertone2023-06-08T15:46:51Z Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction bromophenol endocytosis endplate potentials exocytosis FM styryl dyes myosin light chain kinase synaptophysin-pHluorin transmitter release acetazolamide bromophenol blue dye myosin light chain kinase acetazolamide carbonate dehydratase inhibitor cardiac myosin muscle relaxant agent myosin light chain myosin light chain 2 myosin light chain kinase acidification animal cell animal tissue Article controlled study endplate potential enzyme activation enzyme phosphorylation evoked response ex vivo study exocytosis fluorescence analysis immunohistochemistry male motor end plate mouse neuromuscular junction neurotransmitter release nonhuman priority journal synapse vesicle synaptic transmission animal C57BL mouse cytology cytosol drug effects membrane potential metabolism neuromuscular junction pH phosphorylation physiology synapse vesicle transgenic mouse Acetazolamide Animals Carbonic Anhydrase Inhibitors Cardiac Myosins Cytosol Exocytosis Hydrogen-Ion Concentration Male Membrane Potentials Mice, Inbred C57BL Mice, Transgenic Myosin Light Chains Myosin-Light-Chain Kinase Neuromuscular Agents Neuromuscular Junction Phosphorylation Synaptic Vesicles Acetazolamide (AZ), a molecule frequently used to treat different neurological syndromes, is an inhibitor of the carbonic anhydrase (CA), an enzyme that regulates pH inside and outside cells. We combined fluorescent FM styryl dyes and electrophysiological techniques at ex vivo levator auris longus neuromuscular junctions (NMJs) from mice to investigate the modulation of synaptic transmission and vesicle recycling by AZ. Transmitter release was minimally affected by AZ, as evidenced by evoked and spontaneous end-plate potential measurements. However, optical evaluation with FM-styryl dyes of vesicle exocytosis elicited by 50 Hz stimuli showed a strong reduction in fluorescence loss in AZ treated NMJ, an effect that was abolished by bathing the NMJ in Hepes. The remaining dye was quenched by bromophenol, a small molecule capable of diffusing inside vesicles. Furthermore, in transgenic mice expressing Synaptophysin-pHluorin (SypHy), the fluorescence responses of motor nerve terminals to a 50 Hz train of stimuli was decrease to a 50% of controls in the presence of AZ. Immunohistochemistry experiments to evaluate the state of the Myosin light chain kinase (MLCK), an enzyme involved in vesicle recycling, demonstrated that MLCK phosphorylation was much stronger in the presence than AZ than in its absence in 50 Hz stimulated NMJs. We postulate that AZ, via cytosol acidification and activation of MLCK, shifts synaptic vesicle recycling to a fast (kiss-and-run) mode, which changes synaptic performance. These changes may contribute to the therapeutic action reported in many neurological syndromes like ataxia, epilepsy, and migraine. © 2017 Wiley Periodicals, Inc. 2017 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_08874476_v71_n12_p_Bertone http://hdl.handle.net/20.500.12110/paper_08874476_v71_n12_p_Bertone |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
bromophenol endocytosis endplate potentials exocytosis FM styryl dyes myosin light chain kinase synaptophysin-pHluorin transmitter release acetazolamide bromophenol blue dye myosin light chain kinase acetazolamide carbonate dehydratase inhibitor cardiac myosin muscle relaxant agent myosin light chain myosin light chain 2 myosin light chain kinase acidification animal cell animal tissue Article controlled study endplate potential enzyme activation enzyme phosphorylation evoked response ex vivo study exocytosis fluorescence analysis immunohistochemistry male motor end plate mouse neuromuscular junction neurotransmitter release nonhuman priority journal synapse vesicle synaptic transmission animal C57BL mouse cytology cytosol drug effects membrane potential metabolism neuromuscular junction pH phosphorylation physiology synapse vesicle transgenic mouse Acetazolamide Animals Carbonic Anhydrase Inhibitors Cardiac Myosins Cytosol Exocytosis Hydrogen-Ion Concentration Male Membrane Potentials Mice, Inbred C57BL Mice, Transgenic Myosin Light Chains Myosin-Light-Chain Kinase Neuromuscular Agents Neuromuscular Junction Phosphorylation Synaptic Vesicles |
| spellingShingle |
bromophenol endocytosis endplate potentials exocytosis FM styryl dyes myosin light chain kinase synaptophysin-pHluorin transmitter release acetazolamide bromophenol blue dye myosin light chain kinase acetazolamide carbonate dehydratase inhibitor cardiac myosin muscle relaxant agent myosin light chain myosin light chain 2 myosin light chain kinase acidification animal cell animal tissue Article controlled study endplate potential enzyme activation enzyme phosphorylation evoked response ex vivo study exocytosis fluorescence analysis immunohistochemistry male motor end plate mouse neuromuscular junction neurotransmitter release nonhuman priority journal synapse vesicle synaptic transmission animal C57BL mouse cytology cytosol drug effects membrane potential metabolism neuromuscular junction pH phosphorylation physiology synapse vesicle transgenic mouse Acetazolamide Animals Carbonic Anhydrase Inhibitors Cardiac Myosins Cytosol Exocytosis Hydrogen-Ion Concentration Male Membrane Potentials Mice, Inbred C57BL Mice, Transgenic Myosin Light Chains Myosin-Light-Chain Kinase Neuromuscular Agents Neuromuscular Junction Phosphorylation Synaptic Vesicles Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction |
| topic_facet |
bromophenol endocytosis endplate potentials exocytosis FM styryl dyes myosin light chain kinase synaptophysin-pHluorin transmitter release acetazolamide bromophenol blue dye myosin light chain kinase acetazolamide carbonate dehydratase inhibitor cardiac myosin muscle relaxant agent myosin light chain myosin light chain 2 myosin light chain kinase acidification animal cell animal tissue Article controlled study endplate potential enzyme activation enzyme phosphorylation evoked response ex vivo study exocytosis fluorescence analysis immunohistochemistry male motor end plate mouse neuromuscular junction neurotransmitter release nonhuman priority journal synapse vesicle synaptic transmission animal C57BL mouse cytology cytosol drug effects membrane potential metabolism neuromuscular junction pH phosphorylation physiology synapse vesicle transgenic mouse Acetazolamide Animals Carbonic Anhydrase Inhibitors Cardiac Myosins Cytosol Exocytosis Hydrogen-Ion Concentration Male Membrane Potentials Mice, Inbred C57BL Mice, Transgenic Myosin Light Chains Myosin-Light-Chain Kinase Neuromuscular Agents Neuromuscular Junction Phosphorylation Synaptic Vesicles |
| description |
Acetazolamide (AZ), a molecule frequently used to treat different neurological syndromes, is an inhibitor of the carbonic anhydrase (CA), an enzyme that regulates pH inside and outside cells. We combined fluorescent FM styryl dyes and electrophysiological techniques at ex vivo levator auris longus neuromuscular junctions (NMJs) from mice to investigate the modulation of synaptic transmission and vesicle recycling by AZ. Transmitter release was minimally affected by AZ, as evidenced by evoked and spontaneous end-plate potential measurements. However, optical evaluation with FM-styryl dyes of vesicle exocytosis elicited by 50 Hz stimuli showed a strong reduction in fluorescence loss in AZ treated NMJ, an effect that was abolished by bathing the NMJ in Hepes. The remaining dye was quenched by bromophenol, a small molecule capable of diffusing inside vesicles. Furthermore, in transgenic mice expressing Synaptophysin-pHluorin (SypHy), the fluorescence responses of motor nerve terminals to a 50 Hz train of stimuli was decrease to a 50% of controls in the presence of AZ. Immunohistochemistry experiments to evaluate the state of the Myosin light chain kinase (MLCK), an enzyme involved in vesicle recycling, demonstrated that MLCK phosphorylation was much stronger in the presence than AZ than in its absence in 50 Hz stimulated NMJs. We postulate that AZ, via cytosol acidification and activation of MLCK, shifts synaptic vesicle recycling to a fast (kiss-and-run) mode, which changes synaptic performance. These changes may contribute to the therapeutic action reported in many neurological syndromes like ataxia, epilepsy, and migraine. © 2017 Wiley Periodicals, Inc. |
| title |
Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction |
| title_short |
Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction |
| title_full |
Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction |
| title_fullStr |
Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction |
| title_full_unstemmed |
Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction |
| title_sort |
carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction |
| publishDate |
2017 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_08874476_v71_n12_p_Bertone http://hdl.handle.net/20.500.12110/paper_08874476_v71_n12_p_Bertone |
| _version_ |
1768546215282081792 |