1,720,968 research outputs found
Construction, theory, and practical considerations for using self-referencing of Ca2+-selective microelectrodes for monitoring extracellular Ca2+ gradients
Full text linkAuthor Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Methods in Cell Biology 99 (2010): 91-111, doi:10.1016/B978-0-12-374841-6.00004-9.Ca2+ signaling in the extra- and intracellular domains is linked to Ca2+ transport across the plasma membrane. Non-invasive monitoring of these resulting extracellular Ca2+ gradients with self-referencing of Ca2+-selective microelectrodes is used for studying Ca2+ signaling across Kingdoms. The quantitated Ca2+ flux enables comparison with changes to intracellular [Ca2+] measured with other methods and determination of Ca2+ transport stoichiometry. Here we review the construction of Ca2+-selective microelectrodes, their physical characteristics and their use in self-referencing mode to calculate Ca2+ flux. We also discuss potential complications when using them to measure Ca2+ gradients near the boundary layers of single cells and tissues.NIH:NCRR grant P41 RR00139
Characterization of optimized Na+ and Cl? liquid membranes for use with extracellular, self-referencing microelectrodes
No full textSelf-referencing with ion-selective microelectrodes (ISMs) is a useful approach for monitoring near-real-time ion flux near single cells and across epithelia. While ISMs for H+, Ca2+, and K+ have been optimized for use with self-referencing, ISMs for two other primary inorganic ions, Na+ and Cl-, have not. In this study, we have characterized ISMs based on three Na+ ionophores (I, VI, and X) and one Cl- ionophore to assess their suitability for use with self-referencing. ISMs constructed with Na+ ionophore VI have short response times (approximately 100 ms) but possess nearly an order of magnitude less selectivity for Na+ over K+ than ISMs constructed with Na+ ionophore X. The Na+ ionophore X mixture was enhanced to give it a shorter response time while not compromising its selectivity. A Cl(-)-selective microelectrode was constructed and characterized with superior anionic selectivity compared with previously reported Cl- ISMs used with self-referencing. This Cl(-)-selective microelectrode, however, has a relatively slow response time (approximately 3 s), thus requiring changes to the self-referencing protocol. Self-referencing with these ISMs will enable near-real-time ion flux measurements for Na+ and Cl-
Windows to cell function and dysfunction: signatures written in the boundary layers
Full text linkThe medium surrounding cells either in culture or in tissues contains a chemical mix varying with cell state. As solutes move in and out of the cytoplasmic compartment they set up characteristic signatures in the cellular boundary layers. These layers are complex physical and chemical environments the profiles of which reflect cell physiology and provide conduits for intercellular messaging. Here we review some of the most relevant characteristics of the extracellular/intercellular space. Our initial focus is primarily on cultured cells but we extend our consideration to the far more complex environment of tissues, and discuss how chemical signatures in the boundary layer can or may affect cell function. Critical to the entire essay are the methods used, or being developed, to monitor chemical profiles in the boundary layers. We review recent developments in ultramicro electrochemical sensors and tailored optical reporters suitable for the task in hand
Ion trapping with fast-response ion-selective microelectrodes enhances detection of extracellular ion channel gradients
Full text linkPreviously, functional mapping of channels has been achieved by measuring the passage of net charge and of specific ions with electrophysiological and intracellular fluorescence imaging techniques. However, functional mapping of ion channels using extracellular ion-selective microelectrodes has distinct advantages over the former methods. We have developed this method through measurement of extracellular K+ gradients caused by efflux through Ca2+-activated K+ channels expressed in Chinese hamster ovary cells. We report that electrodes constructed with short columns of a mechanically stable K+-selective liquid membrane respond quickly and measure changes in local [K+] consistent with a diffusion model. When used in close proximity to the plasma membrane (<4 µm), the ISMs pose a barrier to simple diffusion, creating an ion trap. The ion trap amplifies the local change in [K+] without dramatically changing the rise or fall time of the [K+] profile. Measurement of extracellular K+ gradients from activated rSlo channels shows that rapid events, 10–55 ms, can be characterized. This method provides a noninvasive means for functional mapping of channel location and density as well as for characterizing the properties of ion channels in the plasma membrane
Measuring extracellular ion gradients from single channels with ion-selective microelectrodes
No full textUnder many different conditions activated plasma membrane ion channels give rise to changes in the extracellular concentration of the permeant ion(s). The magnitude and duration of these changes are dependent on the electrochemical driving force(s) on the permeant ion(s) as well as conductance, open time, and channel density. We have modeled the change in the extracellular [K+] due to efflux through Ca2+-activated K+ channels, mSlo, to determine the range of parameters that would give rise to measurable signals in the surrounding media. Subsequently we have used extracellular, K+-selective microelectrodes to monitor localized changes in [K+]ext due to efflux through mSlo channels expressed in Xenopus oocytes. The rapid changes in [K+] show a close fit with the predicted model when the time response of the ion-selective microelectrode is taken into account, providing proof of the concept. Measurement of the change in extracellular ion concentration with ion-selective microelectrodes provides a noninvasive means for functional mapping of channel location and density, as well as characterizing the properties of ion channels in the plasma membran
Fast response, noninvasive, potentiometric microelectrodes resolve single potassium channel activity in the diffusive boundary layer of a single cell
No full textIon transport across the plasma membrane of cells involves an inevitable chemical modification of the solutions on both sides. Noninvasive, real-time detection of these subtle ionic signatures is most easily achieved in the extracellular diffusive boundary layer with electrochemical detection. In order to perform these measurements near single cells it is critical to use a device that enables high spatial and temporal resolution. While ion-selective microelectrodes (ISMs) provide the high spatial resolution, they give rise to orders of magnitude increase in the time constant and response time of the microsensors when compared to larger sensors. By constructing and using fast response ISMs biological events as brief as 10ms have been resolved. The signal-to-noise ratio is enhanced with self-referencing and signal processing techniques, enabling long-term monitoring of small magnitude, steady ion gradients and rapid ionic transients that reflect the physiological and metabolic activity of single cell
Development and characterization of a self-referencing glutamate-selective micro-biosensor
No full textFrom genes to genomes: beyond biodiversity in Spain's Rio Tinto
No full textSpain's Rio Tinto, or Red River, an example of an extremely acidic (pH 1.7-2.5) environment with a high metal content, teems with prokaryotic and eukaryotic microbial life. Our recent studies based on small-subunit rRNA genes reveal an unexpectedly high eukaryotic phylogenetic diversity in the river when compared to the relatively low prokaryotic diversity. Protists can therefore thrive in and dominate extremely acidic, heavy-metal-laden environments. Further, because we have discovered protistan acidophiles closely related to neutrophiles, we can hypothesize that the transition from neutral to acidic environments occurs rapidly over geological time scales. How have these organisms adapted to such environments? We are currently exploring the alterations in physiological mechanisms that might allow for growth of eukaryotic microbes at acid extremes. To this end, we are isolating phylogenetically diverse protists in order to characterize and compare ion-transporting ATPases from cultured acidophiles with those from neutrophilic counterparts. We predict that special properties of these ion transporters allow protists to survive in the Rio Tint
Synergistic amplification of beta-amyloid-and interferon-?-induced microglial neurotoxic response by the senile plaque component chromogranin A
No full textActivation of the microglial neurotoxic response by components of the senile plaque plays a critical role in the pathophysiology of Alzheimer's disease (AD). Microglia induce neurodegeneration primarily by secreting nitric oxide (NO), tumor necrosis factor-? (TNF?), and hydrogen peroxide. Central to the activation of microglia is the membrane receptor CD40, which is the target of costimulators such as interferon-? (IFN?). Chromogranin A (CGA) is a recently identified endogenous component of the neurodegenerative plaques of AD and Parkinson's disease. CGA stimulates microglial secretion of NO and TNF?, resulting in both neuronal and microglial apoptosis. Using electrochemical recording from primary rat microglial cells in culture, we have shown in the present study that CGA alone induces a fast-initiating oxidative burst in microglia. We compared the potency of CGA with that of {beta}-amyloid ({beta}A) under identical conditions and found that CGA induces 5–7 times greater NO and TNF? secretion. Coapplication of CGA with {beta}A or with IFN? resulted in a synergistic effect on NO and TNF? secretion. CD40 expression was induced by CGA and was further increased when {beta}A or IFN? was added in combination. Tyrphostin A1 (TyrA1), which inhibits the CD40 cascade, exerted a dose-dependent inhibition of the CGA effect alone and in combination with IFN? and {beta}A. Furthermore, CGA-induced mitochondrial depolarization, which precedes microglial apoptosis, was fully blocked in the presence of TyrA1. Our results demonstrate the involvement of CGA with other components of the senile plaque and raise the possibility that a narrowly acting agent such as TyrA1 attenuates plaque formatio
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