ABSTRACT
The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.
Subject(s)
Animals , Humans , Rats , Brain , Metabolism , Calcium , Metabolism , Cells, Cultured , Cerebellum , Cell Biology , Fluorescence Resonance Energy Transfer , HEK293 Cells , Nitric Oxide , Metabolism , Nitric Oxide Synthase Type I , Metabolism , PDZ Domains , Plasma Membrane Calcium-Transporting ATPases , Metabolism , Protein Interaction Maps , Protein Isoforms , Metabolism , Rats, Sprague-DawleyABSTRACT
OBJECTIVE: To explore the resource and solution of the expired drugs in citizen family through investigating its constituents and expired time.METHODS: By a random sampling,the records of the reclaimed drugs of a total of 1 000 cases collected from 6 drugstores were analyzed statistically.RESULTS: The main expired drugs reclaimed from citizen family were cardiovascular and cerebral vessels drugs,drugs for respiratory disease,and reinforcing and regulating drugs,and most of which were overdue 50 months ago.CONCLUSIONS: The situation of expired drugs in citizen family is serious,thus measures such as reclaiming expired drugs and popularizing knowledge about taking drugs should be taken to tackle the problem of expired drugs.