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Bioanalytical Applications of Real-Time ATP Imaging via Bioluminescence.

DE2004822057

Publication Date	2003
Personal Author	Gruenhagen, J. A.
Page Count	152
Abstract	The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G(sub q)-type protein to initiate ATP release from HUVECs. Ca(sup 2+) imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca(sup 2+) signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K(sup+) and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol functionalized CdS monocrystals. Aggregates of nanospheres were bathed in imaging solution, and ATP bioluminescence was monitored to investigated the release kinetics of the nanosphere drug delivery systems. Addition of disulfide bond-cleaving molecules induced uncapping of the nanospheres and subsequently, the release of ATP. Increasing the concentration of the uncapping molecule decreased the temporal maximum and increased the magnitude of release of encapsulated ATP from the nanospheres. Furthermore, the release kinetics from the nanospheres varied with the size of the particle aggregates.
Keywords	Bioluminescence Adenosine Real time Nerve cells Endothelial cells Monitoring Central nervous system Imaging Nanospheres Microscopy Monocrystals Enzymes Proteins Theses Adenosine 5'-triphosphate
Source Agency	Technical Information Center Oak Ridge Tennessee
Corporate Authors	Iowa State Univ., Ames.; Department of Energy, Washington, DC.
Supplemental Notes	Sponsored by Department of Energy, Washington, DC.
Document Type	Technical Report
NTIS Issue Number	200422

Bioanalytical Applications of Real-Time ATP Imaging via Bioluminescence.

Bioanalytical Applications of Real-Time ATP Imaging via Bioluminescence.
DE2004822057

Publication Date	2003
Personal Author	Gruenhagen, J. A.
Page Count	152
Abstract	The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G(sub q)-type protein to initiate ATP release from HUVECs. Ca(sup 2+) imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca(sup 2+) signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K(sup+) and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol functionalized CdS monocrystals. Aggregates of nanospheres were bathed in imaging solution, and ATP bioluminescence was monitored to investigated the release kinetics of the nanosphere drug delivery systems. Addition of disulfide bond-cleaving molecules induced uncapping of the nanospheres and subsequently, the release of ATP. Increasing the concentration of the uncapping molecule decreased the temporal maximum and increased the magnitude of release of encapsulated ATP from the nanospheres. Furthermore, the release kinetics from the nanospheres varied with the size of the particle aggregates.
Keywords	Bioluminescence Adenosine Real time Nerve cells Endothelial cells Monitoring Central nervous system Imaging Nanospheres Microscopy Monocrystals Enzymes Proteins Theses Adenosine 5'-triphosphate
Source Agency	Technical Information Center Oak Ridge Tennessee
Corporate Authors	Iowa State Univ., Ames.; Department of Energy, Washington, DC.
Supplemental Notes	Sponsored by Department of Energy, Washington, DC.
Document Type	Technical Report
NTIS Issue Number	200422