Detection of Cell-Affecting Agents with a Silicon Biosensor

Parce, J W; Owicki, John C.; Kercso, K.M.; Sigal, GB; Wada, H G; Muir, V.C.; Bousse, L.; Ross, K.; Sikic, BI; McConnell, H. M.

doi:10.1126/science.2799384

Cited by 288 publications

(165 citation statements)

References 26 publications

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“…We then investigated the role of PKC in the PTH-induced increase in ECAR. Treatment of cells with the PKC inhibitor AMG-C 16 significantly reduced the PTH-stimulated increase in ECAR. In control experiments, AMG-C 16 inhibited the PMA-induced increase in ECAR, while it did not decrease basal ECAR or affect stimulation of ECAR by isoproterenol, indicating that AMG-C 16 was inhibiting PKC and was not nonspecifically toxic to the cells.…”

Section: Role Of Na ϩ /H ϩ Exchange In the Pth-induced Increase In Ecar-mentioning

confidence: 99%

“…(n ϭ 4). The asterisk indicates a significant decrease (p Ͻ 0.05) of PTH-stimulated ECAR in the presence of AMG-C 16 .…”

Section: Role Of Na ϩ /H ϩ Exchange In the Pth-induced Increase In Ecar-mentioning

confidence: 99%

“…We next examined the effect of AMG-C 16 , a PKC inhibitor that has been shown to have little effect on PKA or Ca 2ϩ / calmodulin-dependent protein kinase (37), on the PTH-induced increase in ECAR. AMG-C 16 (200 M) reduced the increase in ECAR elicited by 20 nM PMA to 35% of the control PMA response (data not shown) and reduced the response to 100 nM hPTH-(1-34) to 37% of the control PTH response, while the increase in ECAR stimulated by 1 mM (Ϫ)-isoproterenol was unaffected (Fig.…”

Section: Fig 6 Pth-induced Increases In Ecar Inmentioning

confidence: 99%

“…Microphysiometry is a novel method for monitoring cellular metabolism (16) and has been effectively adapted to monitor acid secretion and metabolic rates in small populations of cultured cells in real time (17,18). Because local pH homeostasis is physiologically important in mineral ion metabolism, and PTH-stimulated bone resorption has been linked to acid production by osteoclasts (13), we utilized microphysiometry to monitor the effect of PTH on extracellular acidification in osteoblasts, the primary target cell for PTH in bone.…”

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A New Action of Parathyroid Hormone

Barrett

Belinsky

Tashjian

1997

Journal of Biological Chemistry

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The major physiological function of parathyroid hormone (PTH) is the maintenance of Ca 2؉ /P i homeostasis via the parathyroid hormone/parathyroid hormone-related protein receptor (PTHR) in kidney and bone. An important consequence of PTHR activation in bone is enhanced local acidification of the extracellular space. Agonist activation of some seven transmembrane-domain receptors increases the extracellular acidification rate (ECAR). We utilized microphysiometry to investigate PTH-stimulated, receptor-mediated increases in ECAR in human osteoblast-like SaOS-2 cells. PTH-(1-34) elicited a large, acute, dose-dependent increase in ECAR with an EC 50 of about 2 nM. The PTH-induced increase in ECAR was specific to cells expressing the PTHR and was inhibited by PTHR antagonists. Rapid, partial, homologous desensitization of the PTH-induced increase in ECAR was observed. Incubation of SaOS-2 cells with 8-bromo-cyclic AMP neither mimicked nor abrogated the PTH effect, and PTH stimulated an acute increase in ECAR in cAMP-resistant SaOS-2 Ca#4A cells. Stimulation of ECAR by PTH was independent of transient increases in cytosolic free calcium. Both inhibition and down-regulation of PKC reduced the PTH-induced increase in ECAR. Inhibition of Na ؉ /H ؉ exchange did not affect the PTH-induced ECAR response. We conclude that PTH caused a receptor-mediated, concentration-dependent, increase in ECAR, which was not dependent on the cAMP/PKA signaling pathway or the Na ؉ /H ؉ exchanger but involved the action of PKC. Thus, acid production in bone, a physiologically important action of PTH, is not confined to osteoclasts as previously considered but is also mediated by osteoblasts.The parathyroid hormone/parathyroid hormone-related protein receptor (PTHR) 1 is a member of the G protein-coupled, seven-transmembrane domain superfamily of receptors (1). The principal physiological action of PTH is on the regulation of Ca 2ϩ /P i homeostasis, which is mediated by the PTHR in kidney and bone. The biochemical and molecular mechanisms of the receptor-mediated actions of PTH are not completely understood. PTH stimulates osteoclast-mediated bone resorption indirectly via the activation of the PTHR on osteoblasts (2); however, the mechanism coupling these events is not known. Additionally, PTH has an anabolic effect on bone when administered intermittently (3). It has been proposed that the diverse actions of PTH may be a consequence of activation of both the cAMP/PKA and inositol lipid/Ca 2ϩ /PKC signaling pathways by the PTHR in osteoblasts (4). Receptor regulation is also important in the action of PTH on osteoblasts (4 -9). Homologous desensitization of PTH-stimulated increases in intracellular cAMP accumulation ([cAMP] i ) and in cytosolic free calcium concentration ([Ca 2ϩ ] i ) and down-regulation of the PTHR have been described in several target cell systems (4 -9).Previous investigations of the physiological actions of PTH have largely used animal and whole bone experimental systems (10 -14), while studies of PTHR signaling have co...

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Section: Role Of Na ϩ /H ϩ Exchange In the Pth-induced Increase In Ecar-mentioning

confidence: 99%

“…(n ϭ 4). The asterisk indicates a significant decrease (p Ͻ 0.05) of PTH-stimulated ECAR in the presence of AMG-C 16 .…”

Section: Role Of Na ϩ /H ϩ Exchange In the Pth-induced Increase In Ecar-mentioning

confidence: 99%

Section: Fig 6 Pth-induced Increases In Ecar Inmentioning

confidence: 99%

mentioning

confidence: 99%

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A New Action of Parathyroid Hormone

Barrett

Belinsky

Tashjian

1997

Journal of Biological Chemistry

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“…Arrays are used to effectively record, amplify and condition extracellular signals as well to perform extracellular stimulation (Eversmann et al, 2003;Lambacher et al, 2004). Nowadays microelectrode arrays are considered as a basic platform for the development of cell-based sensors (Parce et al, 1989;DeBusschere & Kovacs, 2001;Yeung et al, 2001;Pancrazio et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Extracellular electrical signals in a neuron-surface junction: model of heterogeneous membrane conductivity

et al. 2012

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Signals recorded from neurons with extracellular planar sensors have a wide range of waveforms and amplitudes. This variety is a result of different physical conditions affecting the ion currents through a cellular membrane. The transmembrane currents are often considered by macroscopic membrane models as essentially a homogeneous process. However, this assumption is doubtful, since ions move through ion channels, which are scattered within the membrane. Accounting for this fact, the present work proposes a theoretical model of heterogeneous membrane conductivity. The model is based on the hypothesis that both potential and charge are distributed inhomogeneously on the membrane surface, concentrated near channel pores, as the direct consequence of the inhomogeneous transmembrane current. A system of continuity equations having non-stationary and quasi-stationary forms expresses this fact mathematically. The present work performs mathematical analysis of the proposed equations, following by the synthesis of the equivalent electric element of a heterogeneous membrane current. This element is further used to construct a model of the cell-surface electric junction in a form of the equivalent electrical circuit. After that a study of how the heterogeneous membrane conductivity affects parameters of the extracellular electrical signal is performed. As the result it was found that variation of the passive characteristics of the cell-surface junction, conductivity of the cleft and the cleft height, could lead to different shapes of the extracellular signals.

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Duration and magnitude of nerve growth factor signaling depend on the ratio of p75LNTR to TrkA

Twiss¹,

Wada²,

Fok³

et al. 1998

J. Neurosci. Res.

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The role of the low affinity neurotrophin receptor p75LNTR in neurotrophin signal transduction remains open. Recent reports show that this receptor generates intracellular signals independent of Trk activity, and others imply that it collaborates with Trk(s) to enhance cellular responses to low neurotrophin concentrations. We have used the Cytosensor microphysiometer as a direct marker of intracellular metabolic activity to address the physiologic role of p75LNTR in nerve growth factor (NGF) signal transduction. NGF treatment of PC12 or TrkA-transfected Chinese hamster ovary (CHO) cells results in a rapid, transient increase in the extracellular acidification rate as measured by the Cytosensor; in both cell types, p75LNTR enhances this response. p75LNTR affects both the magnitude of and the duration of the extracellular acidification response to NGF. Moreover, it is not merely the presence of p75LNTR, but also the ratio of p75LNTR:TrkA which determines cellular responsiveness to NGF. In transiently transfected CHO cells, a 5:1 ratio of p75LNTR:trkA cDNAs produced the greatest change in NGF-induced acid secretion. Pretreatment of PC12 cells with anti-p75LNTR antibodies decreased the responsiveness to NGF. However, long-term NGF exposure to PC12 cells in which p75LNTR expression was decreased to approximately 10% of wild-type levels showed a longer duration of acid secretion compared to wild-type PC12 cells. Together, these data suggest that p75LNTR may play a dual role in modulating NGF signal transduction by enhancing and extending cellular responses to short-term ligand exposures while attenuating the metabolic response to long-term ligand exposures. With regard to potential Trk-independent p75LNTR signal transduction mechanisms, we detected no change in extracellular acidification response in 75LNTR-transfected CHO cells, PCNA-15 fibroblasts, or Schwann cells, all of which express large amounts of p75LNTR and no Trk. Thus, p75LNTR cannot produce any signal detected by microphysiometry in the absence of TrkA.

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Detection of Cell-Affecting Agents with a Silicon Biosensor

Cited by 288 publications

References 26 publications

A New Action of Parathyroid Hormone

A New Action of Parathyroid Hormone

Extracellular electrical signals in a neuron-surface junction: model of heterogeneous membrane conductivity

Duration and magnitude of nerve growth factor signaling depend on the ratio of p75LNTR to TrkA

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