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The role of cAMP/cAMP-dependent protein kinase (PKA) on the late phase of exocytosis has been studied by amperometry on Ba(2+)-stimulated single bovine chromaffin cells. We conclude that UV photolysis of caged Ca2+ is a suitable stimulation technique for higher-throughput studies of Ca2+-dependent exocytosis on transparent electrochemical microelectrode arrays. We observed a burst of amperometric spikes upon rapid elevation of i and a “priming” effect of sub-stimulatory i on the response of cells to subsequent i elevation, similar to previous reports using different techniques. Upon flash photorelease of caged Ca2+, a uniform rise of i within the target cell leads to quantal release of oxidizable catecholamines measured amperometrically by the underlying ITO electrode. We loaded bovine chromaffin cells with acetoxymethyl (AM) ester derivatives of the Ca2+ cage NP-EGTA and Ca2+ indicator dye Fura-4F, then transferred these cells onto the working ITO electrodes for amperometric recordings. A 110 nm – thick transparent Indium-Tin-Oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (~20 μm by 20 μm). Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca2+ together with electrochemical detection of quantal catecholamine secretion from individual cells or cell arrays as a step towards developing high-throughput experimental devices. Photorelease of caged Ca2+ is a uniquely powerful tool to study the dynamics of Ca2+-triggered exocytosis from individual cells. Use Green Fluorescent Protein as a reporter of gene expression could benefit from the transparent device. Screening of drugs and toxins that target exocytosis. This biochip device has potential applications for higherthroughput Have designed and microfabricated a novel biochip device to integrate photolysis of caged Ca2+, ITOĪmperometry, and fluorescence photometry together for higher-throughput on-chip stimulation andĭetection of Ca2+-dependent exocytosis. Using photolithography and other microfabrication techniques, we Triggered exocytosis from individual cells. Photorelease of caged Ca2+ is a uniquely powerful tool to study the dynamics of Ca2+. Neurotransmitters into the extracellular space in response to an increase in intracellular Ca2+Ĭoncentration. In which intracellular vesicles fuse with the cell membrane and release their contents of hormones and Neurons and neuroendocrine cells secrete signaling molecules via Ca2+-dependent exocytosis, a process
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Strontium possesses both an intermediate permeability and an intermediate ability to induce secretion. Although Ca2+ is less permeable through its own channels, it is more efficient in triggering exocytosis. These results are compatible with the following sequence of events: Ba2+ blocks KCa channels from both the outside and inside of the cell, causing membrane depolarization that, in turn, opens voltage-sensitive Ca2+ channels and favors the entry of Ca2+ and Ba2+. Intracellular Ba2+ dialysis provokes exocytosis at concentrations 100-fold higher than those of Ca2+, whereas Sr2+ exhibits an intermediate sensitivity. Conversely, amperometric recordings of permeabilized cells show that Ca2+ promotes the longest lasting secretion, as Ba2+ only provokes secretion while it is present and Sr2+ induces intermediate-lasting secretion. Extracellular Ba2+ increases cytosolic Ca2+ concentrations in Fura-2-loaded intact cells, and it induces long-lasting catecholamine release. Voltage-clamp monitoring of Ca2+-activated K+ channels (KCa) shows that Ba2+ reduces outward currents, which were enhanced by Sr2+. When applied intracellularly, only Ba2+ provokes action potentials. Current-clamp recordings show that extracellular Sr2+ and Ba2+ cause membrane depolarization and action potentials, which are not blocked by Cd2+ but that can be mimicked by tetra-ethyl-ammonium. The extent to which secretion is elicited by the cations in intact depolarized cells was Ba2+ > Sr2+ ≥ Ca2+, contrasting with that elicited by these cations in permeabilized cells (Ca2+ > Sr2+ > Ba2+). Three divalent cations can elicit secretory responses in most neuroendocrine cells, including chromaffin cells.