Af luorescent sensor for catecholamines,N S510, is presented. The sensor is based on aq uinolone fluorophore incorporating ab oronic acid recognition element that gives it high affinity for catecholamines and at urn-on response to norepinephrine.T he sensor results in punctate staining of norepinephrine-enriched chromaffin cells visualizedu sing confocal microscopyi ndicating that it stains the norepinephrine in secretory vesicles.A mperometry in conjunction with total internal reflection fluorescence (TIRF) microscopy demonstrates that the sensor can be used to observe destaining of individual chromaffin granules upon exocytosis.N S510 is the highest affinity fluorescent norepinephrine sensor currently available and can be used for measuring catecholamines in live-cell assays.
Carbon-fiber electrodes (CFEs) are the gold standard for quantifying the release of oxidizable neurotransmitters from single vesicles and single cells. Over the last 15 years, microfabricated devices have emerged as alternatives to CFEs that offer the possibility of higher throughput, subcellular spatial resolution of exocytosis, and integration with other techniques for probing exocytosis including microfluidic cell handling and solution exchange, optical imaging and stimulation, and electrophysiological recording and stimulation. Here we review progress in developing electrochemical electrode devices capable of resolving quantal exocytosis that are fabricated using photolithography.
A fluorescent sensor for catecholamines, NS510, is presented. The sensor is based on a quinolone fluorophore incorporating a boronic acid recognition element that gives it high affinity for catecholamines and a turn‐on response to norepinephrine. The sensor results in punctate staining of norepinephrine‐enriched chromaffin cells visualized using confocal microscopy indicating that it stains the norepinephrine in secretory vesicles. Amperometry in conjunction with total internal reflection fluorescence (TIRF) microscopy demonstrates that the sensor can be used to observe destaining of individual chromaffin granules upon exocytosis. NS510 is the highest affinity fluorescent norepinephrine sensor currently available and can be used for measuring catecholamines in live‐cell assays.
Duchenne muscular dystrophy (DMD), a genetic disorder caused by mutations in the dystrophin gene, is characterized by progressive muscle degeneration/ weakness. Enhanced store-operated Ca 2þ entry (SOCE), a Ca 2þ influx mechanism coordinated by STIM1 and Orai1, is proposed to potentiate Ca 2þ -mediated damage in DMD. To determine the role of SOCE in promoting muscular dystrophy, we crossed mdx mice with tamoxifen-inducible, musclespecific Orai1 knockout mice (mdx-Orai1 KO). Analysis of transcript levels revealed that Orai1 expression was $1.5x higher in muscle from mdx mice compared to WT mice, while nearly undetectable in muscle from mdx-Orai1 KO mice. Levels of Orai1 transcript correlated with SOCE activity measured using Mn 2þ quench of fura-2 fluorescence in single flexor digitorum brevis muscle fibers. Specifically, SOCE was increased in mdx fibers and absent in fibers from mdx-Orai1 KO mice. Moreover, compared to WT fibers, mdx fibers exhibited: i) increased myoplasmic Ca 2þ levels; ii) reduced Ca 2þ store content; and iii) a significant prolongation in the rate of electrically-evoked Ca 2þ transient decay. All of these effects were normalized in fibers from mdx-Orai1 KO mice. Ex vivo contractility experiments revealed that extensor digitorum longus muscles from mdx mice displayed a reduction in maximal tetanic specific force (150.353.5 mN/mm 2 ) compared to WT muscles (199.6511.1 mN/mm 2 ), a parameter ameliorated in muscles from mdx-Orai1 KO mice (188.1510.9 mN/mm 2 ). When exposed to 10 consecutive eccentric contractions, mdx muscles exhibited a higher damage-induced force reduction than WT muscles (residual force after the 10 th eccentric contraction was 35.553.8% and 61.851.9%, respectively). Interestingly, Orai1 ablation partially protected muscles from damage in mdx-Orai1 KO mice (residual force was 46.152.6% after 10 contractions). Our results indicate that enhanced Orai1dependent SOCE exacerbates the dystrophic phenotype and Orai1 deficiency promotes sarcolemmal integrity/stability during eccentric contraction. Adrenal chromaffin cells (ACCs) comprise the neuroendocrine arm of the sympathetic nervous system and secrete catecholamines to coordinate the physiological response to stress. Although SERT is prominently expressed in ACCs, its role there has remained unclear. Our data show that SERT interacts with 5-HT 1A recep-
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.