Robert H. Chow

Kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2

The kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+ were studied by capacitance (Cm) measurements with millisecond time resolution. After elevation of the internal Ca2+ concentration ([Ca2+]i), Cm rises rapidly with one or more exponentials. The time constant of the fastest component decreases for higher [Ca2+]i (range 3-600 microM) over three orders of magnitude before it saturates at approximately 1 ms. The corresponding maximal rates of secretion can be as fast as 100,000 fF/s or 40,000 vesicles/s. There is a Ca(2+)-dependent delay before Cm rises, which may reflect the kinetics of multiple Ca2+ ions binding to the secretory apparatus. The initial rise in Cm is described by models containing a sequence of two to four single Ca(2+)-binding steps followed by a rate-limiting exocytosis step. The predicted Ca2+ dissociation constant (Kd) of a single Ca(2+)-binding site is between 7 and 21 microM. At [Ca2+]i > 30 microM clear indications of a fast endocytotic process complicate the analysis of the secretory response.

Functional and spatial segregation of secretory vesicle pools according to vesicle age

Synaptic terminals and neuroendocrine cells are packed with secretory vesicles, only a few of which are docked at the plasma membrane and readily releasable. The remainder are thought to constitute a large cytoplasmic reserve pool awaiting recruitment into the readily releasable pool (RRP) for exocytosis. How vesicles are prioritized in recruitment is still unknown: the choice could be random, or else the oldest or the newest ones might be favoured. Here we show, using a fluorescent cargo protein that changes colour with time, that vesicles in bovine adrenal chromaffin cells segregate into distinct populations, based on age. Newly assembled vesicles are immobile (morphologically docked) at the plasma membrane shortly after biogenesis, whereas older vesicles are mobile and located deeper in the cell. Different secretagogues selectively release vesicles from the RRP or, surprisingly, selectively from the deeper cytoplasmic pool. Thus, far from being equal, vesicles are segregated functionally and spatially according to age.

A two-step model of secretion control in neuroendocrine cells

Recent experiments on a variety of neuroendocrine cells indicate that intense stimuli readily depress the secretory response. The most likely explanation for this depression is that a pool of release-ready granules is depleted. We present a two-step model of secretion that allows one to simulate the dynamics of such a pool for different time courses of free intracellular Ca concentration [Ca2+]i. We derive rate constants of the model from two types of experiment and find that, for the simplest type of model, not only the rate of consumption (exocytosis) but also the rate of vesicle supply to the pool of release-ready granules must be made Ca-dependent. Given these functional dependences a variety of results from the literature can be simulated. In particular, the model predicts the occurrence of secretory depression and augmentation under appropriate conditions.

Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I

Synaptotagmin I is a synaptic vesicle-associated protein essential for synchronous neurotransmission. We investigated its impact on the intracellular Ca2+-dependence of large dense-core vesicle (LDCV) exocytosis by combining Ca2+-uncaging and membrane capacitance measurements in adrenal slices from mouse synaptotagmin I null mutants. Synaptotagmin I-deficient chromaffin cells displayed prolonged exocytic delays and slow, yet Ca2+-dependent fusion rates, resulting in strongly reduced LDCV release in response to short depolarizations. Vesicle recruitment, the shape of individual amperometric events, and endocytosis appeared unaffected. These findings demonstrate that synaptotagmin I is required for rapid, highly Ca2+-sensitive LDCV exocytosis and indicate that it regulates the equilibrium between a slowly releasable and a readily releasable state of the fusion machinery. Alternatively, synaptotagmin I could function as calcium sensor for the readily releasable pool, leading to the destabilization of the pool in its absence.

Extrasynaptic Vesicular Transmitter Release from the Somata of Substantia Nigra Neurons in Rat Midbrain Slices

Substantia nigra neurons release dopamine from their somatodendritic regions. A long-unresolved question is whether this release occurs by exocytosis or by a nonvesicular mechanism. We used carbon fiber microelectrodes in a brainstem slice to assay secretion from single cell bodies that had been cleared of connective tissue. Amperometry at the carbon fiber microelectrodes revealed unitary events in ∼90% of cells in resting conditions. These events had charge integrals ranging from a few femtocoulombs to several hundred femtocoulombs (fC). Local glutamate application enhanced the event frequency by 3.5-fold on average and up to 10-fold in highly responsive cells, although the mean charge integral was not modified. Local application of a high K+-containing saline had effects similar to those of glutamate. The frequency of resting and stimulated amperometric events was much lower at 21–22°C than at 32–35°C. The addition of Cd2+ (50 μm), a blocker of voltage-dependent Ca2+ channels, to the bath solution blocked the stimulatory effects of glutamate. These results suggest that dopamine is released from the somata of substantia nigra neurons by exocytosis and that this mechanism is regulated by neuronal electrical activity. More generally, this study demonstrates the applicability of carbon fiber microelectrodes to the measurement of quantal monoamine secretion in brain slices.

Mechanisms Determining the Time Course of Secretion in Neuroendocrine Cells

Transmitter release from chromaffin cells differs from that in synapses in that it persists for a longer time after Ca2+ entry has stopped. This prolonged secretion is not due to a delay between vesicle fusion and transmitter release, nor to slow detection of released substance: step increases in capacitance due to single vesicle fusion precede the release detected by amperometry by only a few milliseconds. The persistence of secretion after a depolarization is reduced by addition of mobile calcium buffer. This suggests that most of the delay is due to diffusion of Ca2+ between channels and release sites, implying that Ca2+ channels and secretory vesicles are not colocalized in chromaffin cells, in contrast to presynaptic active zones.

Electrochemical Detection of Secretion from Single Cells

Electrochemical methods based on the oxidation or reduction of specific transmitters enable exquisitely sensitive measurements of secretion from single cells. We start with a brief review of the history of their application to single cells. Then we focus on basic principles. The remainder of the chapter concerns how to make measurements and how to analyze the results.

Calcium-activated endoplasmic reticulum stress as a major component of tumor cell death induced by 2,5-dimethyl-celecoxib, a non-coxib analogue of celecoxib

A drawback of extensive coxib use for antitumor purposes is the risk of life-threatening side effects that are thought to be a class effect and probably due to the resulting imbalance of eicosanoid levels. 2,5-Dimethyl-celecoxib (DMC) is a close structural analogue of the selective cyclooxygenase-2 inhibitor celecoxib that lacks cyclooxygenase-2–inhibitory function but that nonetheless is able to potently mimic the antitumor effects of celecoxib in vitro and in vivo. To further establish the potential usefulness of DMC as an anticancer agent, we compared DMC and various coxibs and nonsteroidal anti-inflammatory drugs with regard to their ability to stimulate the endoplasmic reticulum (ER) stress response (ESR) and subsequent apoptotic cell death. We show that DMC increases intracellular free calcium levels and potently triggers the ESR in various tumor cell lines, as indicated by transient inhibition of protein synthesis, activation of ER stress–associated proteins GRP78/BiP, CHOP/GADD153, and caspase-4, and subsequent tumor cell death. Small interfering RNA–mediated knockdown of the protective chaperone GRP78 further sensitizes tumor cells to killing by DMC, whereas inhibition of caspase-4 prevents drug-induced apoptosis. In comparison, celecoxib less potently replicates these effects of DMC, whereas none of the other tested coxibs (rofecoxib and valdecoxib) or traditional nonsteroidal anti-inflammatory drugs (flurbiprofen, indomethacin, and sulindac) trigger the ESR or cause apoptosis at comparable concentrations. The effects of DMC are not restricted to in vitro conditions, as this drug also generates ER stress in xenografted tumor cells in vivo, concomitant with increased apoptosis and reduced tumor growth. We propose that it might be worthwhile to further evaluate the potential of DMC as a non-coxib alternative to celecoxib for anticancer purposes. [Mol Cancer Ther 2007;6(4):1262–75]

A simple method for insulating carbon-fiber microelectrodes using anodic electrophoretic deposition of paint.

We describe a simple method for the insulation of carbon-fiber microelectrodes (CFMEs). Using the technique of anodic electrophoretic deposition of paint (EDP), we deposited thin and uniform films of electrodeposition paint onto 10-μm-diameter carbon fibers. The polymer films were then heat cured, leading to an electrically insulating coat. The insulated carbon fibers were transected perpendicular to their axes to expose a 10-μm carbon disk and characterized using conventional electrochemical methods and scanning electron microscopy. As expected, cyclic voltammograms measured with electropainted CFMEs in solutions containing ferricyanide displayed a sigmoidal response without hysteresis. The insulating films had a low dielectric constant, resulting in low capacitance. In addition, the film thickness could be controlled simply by varying such deposition parameters as the applied voltage and the duration of treatment. Electrical noise of the transected fibers was determined principally by the cut surface of the fiber, not by the exposed insulated surfaces. Compared to electropolymerization of phenol compounds, another electrochemical method for insulating CFMEs, insulation by anodic EDP has the very significant advantages of greater simplicity, much shorter processing time, and much lower toxicity of the reagents involved, important considerations for those who work with the procedure on a daily basis. Furthermore, electrodeposition paints are commercially available and have long shelf lives.

Multiple stimulation-dependent processes regulate the size of the releasable pool of vesicles

Abstract In neuroendocrine cells and neurones, changes in the size of a limited pool of readily releasable vesicles contribute to the plasticity of secretion. We have studied the dynamic alterations in the size of a near-membrane pool of vesicles in living neuroendocrine cells. Using evanescent wave microscopy we monitored the behaviour of individual secretory vesicles at the plasma membrane. Vesicles undergo sequential transitions between several states of differing fluorescence intensity and mobility. The transitions are reversible, except for the fusion step, and even in nonstimulated conditions the vesicles change states in a dynamic equilibrium. Stimulation selectively speeds up the three forward transitions leading towards exocytosis. Vesicles lose mobility in all three dimensions upon approach of the plasma membrane. Their movement is directed and targeted to the docking fusion sites. Sites of vesicle docking and exocytosis are distributed non-uniformly over the studied “footprint” region of the cell. While some areas are the sites of repeated vesicle docking and fusion, others are completely devoid of spots. Vesicular mobility at the membrane is confined, as if the vesicle were imprisoned in a cage or tethered to a binding site.