Cytosensor techniques for examining signal transduction of neurohormones

Smart, Darren; Wood, Martyn

doi:10.1139/o00-019

Cited by 21 publications

(6 citation statements)

References 39 publications

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“…To analyze the effect of peptides on GPCR signal transduction, two distinct models were used. Initially, the use of a microphysiometer was employed to study signal transduction pathways, since activation or inhibition of a wide variety of effectors of transduction processes is known to affect the metabolic rate (53). Energy metabolism is tightly coupled to cellular ATP consumption, which generates acidic products.…”

Section: Discussionmentioning

confidence: 99%

Intracellular Peptides as Natural Regulators of Cell Signaling

Cunha

Berti

Ferreira

et al. 2008

Journal of Biological Chemistry

138

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Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20 -80 M) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including ␣-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

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Section: Discussionmentioning

confidence: 99%

Intracellular Peptides as Natural Regulators of Cell Signaling

Cunha

Berti

Ferreira

et al. 2008

Journal of Biological Chemistry

138

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“…The results of our experiments with BAPTA indicate that activation of P2X receptors stimulates cariporide‐sensitive acid efflux by a mechanism that requires elevation of cytosolic Ca 2+ , consistent with regulation of acid efflux by a P2X receptor‐Ca 2+ ‐NHE1 pathway. Cytosolic Ca 2+ regulates the rates of ATP synthesis and utilization, thereby affecting the formation of acid metabolites (e.g., lactate and H + ) (Smart and Wood, 2000; Balaban, 2002). Moreover, Ca 2+ may acidify the cytosol by displacing protons from common binding sites (Austin and Wray, 2000), thus increasing the number of protons available for efflux.…”

Section: Discussionmentioning

confidence: 99%

Activation of P2 nucleotide receptors stimulates acid efflux from astrocytes

Dixon

Panupinthu

et al. 2004

Glia

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Acidification of the extracellular fluid modulates neurotransmission and ischemic injury in brain. The purpose of the present study was to investigate the effects of purine and pyrimidine transmitters on acid efflux from brain astrocytes. Using RT-PCR, we detected transcripts for the following nucleotide receptors in rat primary astrocyte cultures: P2X1, P2X2, P2X3, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6. Adenosine 5'-triphosphate (ATP) was found to rapidly induce a biphasic increase in acid efflux, monitored by microphysiometry, consisting of an initial transient and a sustained plateau. Compared with ATP, the P2Y agonist uridine 5'-triphosphate (UTP) induced a much smaller initial response but an equal plateau. The poorly hydrolyzable ATP analogue ATPgammaS caused the same initial response as did ATP, but a much smaller plateau, suggesting that the latter phase was due to extracellular degradation of nucleotides. The P2 receptor antagonist, suramin, blocked stimulation of acid efflux by ATP. Removal of extracellular glucose or elevation of extracellular K+ decreased the basal rate of acid efflux but not the stimulation induced by ATP. Inhibition of Na+/H+ exchange by cariporide suppressed the initial phase of ATP-stimulated acid efflux. The intracellular Ca2+ chelator bisaminophenoxyethane- tetraacetic acid (BAPTA) lowered basal acid efflux and abolished the initial phase of the response to ATP. In conclusion, ATP acts through P2 nucleotide receptors on astrocytes to stimulate the Ca2+ -dependent efflux of protons, mediated in part by activation of Na+/H+ exchange. The resulting acidification of the extracellular fluid may serve as an intercellular signal in brain.

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“…Another technique used a light-addressable potentiometric sensor (LAPS) to determine acidification rates and has been commercialized as the Cytosensor Microphysiometer. This instrument has been used for a large number of cellular studies [8][9][10][14][15][16][17][18][19][20][21] where the change in pH in the extracellular environment is related to metabolic changes in the cells. However, most of these techniques and others provide only single-analyte measurements, whereas a multidimensional approach in which responses are obtained simultaneously from multiple analytes offers a more complete understanding of cellular metabolic pathways and changes in these pathways in response to various chemical and biological stimulations.…”

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confidence: 99%

A Microphysiometer for Simultaneous Measurement of Changes in Extracellular Glucose, Lactate, Oxygen, and Acidification Rate

et al. 2003

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A microphysiometer capable of measuring changes in extracellular glucose, lactate, oxygen, and acidification rate has been developed by incorporating modified electrodes into a standard Cytosensor Microphysiometer plunger. Glucose and lactate are measured indirectly at platinum electrodes by amperometric oxidation of hydrogen peroxide, which is produced from catalysis of glucose and lactate at films containing their respective entrapped oxidase. Oxygen is measured amperometrically at a platinum electrode coated with a Nafion film, while the acidification rate is measured potentiometrically by a Cytosensor Microphysiometer. Analytical information is obtained during the Cytosensor stop-flow cycles, where the electrodes measure changes in the extracellular medium corresponding to the consumption or production of the analyte by the cells. Modification of the Cytosensor plunger for multianalyte determination is described, and the operation of the technique is illustrated by the simultaneous measurement of all four analytes during the addition of fluoride and DNP to Chinese hamster ovary cells and fluoride and antimycin A to mouse fibroblast cells. Cell metabolic recovery and dynamics after exposure to agents can also be observed in specific cases.

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Cytosensor techniques for examining signal transduction of neurohormones

Cited by 21 publications

References 39 publications

Intracellular Peptides as Natural Regulators of Cell Signaling

Intracellular Peptides as Natural Regulators of Cell Signaling

Activation of P2 nucleotide receptors stimulates acid efflux from astrocytes

A Microphysiometer for Simultaneous Measurement of Changes in Extracellular Glucose, Lactate, Oxygen, and Acidification Rate

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