Mechanisms of secretion studied with two-photon excitation imaging and uncaging

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Exocytosis and fluid-secretion in epithelial cells and synaptic functions were studied with two-photon excitation imaging and uncaging of caged-compounds.1) We have developed functional mapping of Ca^<2+>-activated channels using two-photon uncaging of caged-Ca^<2+> compound, which has an extraordinary spatial resolution because of the Ca^<2+> domain effect caused by caged-Ca^<2+> itself. In mouse pancreatic acinar preparations, we found that Ca^<2+>-activated Cl^- channel distributed both in the apical and basal membranes, but was absent in the lateral membrane. This distribution prevents the reuptake of Cl^- secreted into the lumen to the lateral membrane, and allows effective unidirectional transport of Cl^-from the basal membrane into the lumen. The lack of functional channels or transporters in the lateral membrane might be utilized in other epithelial transports, and supports the push-pull mechanism of fluid secretion in rodent pancreas.2) We have also developed a new imaging app … More roach to visualize exocytosis in intact tissue based on two-photon imaging of extracellular polar tracer (TEP imaging). TEP imaging revealed the sequential exocytosis in exocrine pancreas, where Ω-shaped profiles of individual vesicles were stably maintained by rapid coating of the vesicles with F-actin. Impairment of F-actin coating resulted in vacuole formation, which is an early sign of acute pancreatitis.3) We have found that sequential exocytosis also occurred in the guinea pig nasal gland, but with a substantial delay from the onset of fluid secretion.4) TEP imaging visualized exocytosis of individual insulin vesicles in the intact islets of Langerhans, and suggested that the fusion pore of insulin vesicles is made of lipid from its very beginning.5) We have developed an optical approach to stimulate neurons with submicron spatial resolution using two-photon uncaging of a caged-glutamate compound. We found that spine-head volumes tightly correlated with glutamate sensitivity, and enlargement of spine head underlay long-term potentiation. Less