G Protein. Rat Drg Neurons Mediated By The Body To Swallow Study

Endocytosis is a fundamental cellular event in membrane retrieval followingexocytosis. In contrast to the well-studied depolarization-induced exocytosis,little is known about the timing of ligand-induced endocytosis.Ligand-receptor binding (LRB) is the initial step of signal transduction in cells.Majority of membrane receptors, including G-protein-coupled receptors(GPCRs) and tyrosine receptors have a silent "LRB" without causing amembrane current or intracellular calcium transients. Thus, it was difficult tostudy the dynamic effect of those LRB. In this thesis, I described fourindependent techniques for real time recording/imaging of LRB-inducedendocytosis as a rapid bioassay of LRB in single living cells: (1) measurementof membrane capacitance (Cm);(2) confocol FM-imaging, which provideshigh resolution in spatial distribution and high sensitivity measurement ofendocytosis;(3) time-resolved confocol FM-imaging, termed FII-recording;(4)vesicle age-resolved confocol FM-imaging. With these newly developedtechniques, in freshly isolated single DRG neurons, we showed thatADP-induced endocytosis/P2Y-receptor internalization had a time constant of1.7 s, which is 100 times faster than that were previously estimated.Furthermore, we revealed that ligand-and depolarization-induced endocytoticvesicles have distinct spatial distribution patterns, as visualized by multi-colorFM imaging. Following 3 min stimulation, ADP-induced endocytotic vesicleswere distributed evenly in the cytoplasm, while depolarization-inducedendocytotic vesicles were close to the cell surface. However, both ADP anddepolarization-induced endocytosis in somata required dynein for subcellulartranslocation of internalized vesicles. Neural growth factor (NGF)-TrkAbinding also induced endocytosis. These realtime endocytosis assays maybecome a powerful tool for both basic research and GPCR-targeting drugdiscovery with high-speed, high-sensitivity and high-throughput.Receptor internalization is the initial step in signal transduction. It hasbeen found by our laboratory that mAChRs significantly inhibit thenAChRs-induced current in rat superior cervical ganglion (SCG) neurons,which is called"M-inhibition". In this work, I also studied the mechanism ofM-inhibition. Norepinephrine and bradykinin but not somatostatin couldproduce an analogous M-inhibition, which indicates that a kind of G proteinmay participate in the M-inhibition. U73122, a phospholipase C (PLC)inhibitor, could partly block the M-inhibition;phorbol myristoyl acetate(PMA), a protein kinase C (PKC) agonist, had a similar M-inhibition asmAChR did. Furthermore, bisindolylmaleimide (Bis), a PKC inhibitor, couldremove the M-inhibition induced by mAChR or PMA, indicating thatPLC-PKC pathways are involved in M-inhibition. On the other hand,8-Br-cAMP and forskolin, two different protein kinase A (PKA) agonists,could also induce a similar M-inhibition. Moreover, H89, a PKA inhibitor,could completely remove M-inhibition induced by mAChR or 8-Br-cAMP,indicating that PKA pathway is also involved in M-inhibition. These resultsindicate that the M-inhibition is mediated by Gq protein and the increase inintracellular PKC and PKA is responsible for the M-inhibition.In addition to mAChR, other GPCRs can also modulate ion channels andsecretion. We found, ATP receptor P2Y inhibits voltage-gated Ca channels inDRG neurons. P2Y can also modulate quantal size of vesicle release byreducing the fusion pore open time in adrenal chromaffin cells (Chen et al,Nature Neuroscience, in press).