Imaging cytoplasmic cAMP in mouse brainstem neurons

Mironov, SL; Skorova, E.; Taschenberger, Grit; Hartelt, N.; Vo, Nikolaev; Lohse, Martin J.; Kügler, Sebastian

doi:10.1186/1471-2202-10-29

Cited by 40 publications

(31 citation statements)

References 39 publications

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“…2A to C). Using a cytosolic biosensor for cAMP levels, Epac1-camps (16,18), we observed effects of PDE inhibition on cAMP levels similar to those that were observed on PKA activity, specifically that IBMX and milrinone generate similar levels of cAMP accumulation while rolipram generates less cAMP (Fig. 2D).…”

Section: Resultsmentioning

confidence: 74%

Spatiotemporally Regulated Protein Kinase A Activity Is a Critical Regulator of Growth Factor-Stimulated Extracellular Signal-Regulated Kinase Signaling in PC12 Cells

Herbst

Allen

Zhang

2011

Molecular and Cellular Biology

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PC12 cells exhibit precise temporal control of growth factor signaling in which stimulation with epidermal growth factor (EGF) leads to transient extracellular signal-regulated kinase (ERK) activity and cell proliferation, whereas nerve growth factor (NGF) stimulation leads to sustained ERK activity and differentiation. While cyclic AMP (cAMP)-mediated signaling has been shown to be important in conferring the sustained ERK activity achieved by NGF, little is known about the regulation of cAMP and cAMP-dependent protein kinase (PKA) in these cells. Using fluorescence resonance energy transfer (FRET)-based biosensors localized to discrete subcellular locations, we showed that both NGF and EGF potently activate PKA at the plasma membrane, although they generate temporally distinct activity patterns. We further show that both stimuli fail to induce cytosolic PKA activity and identify phosphodiesterase 3 (PDE3) as a critical regulator in maintaining this spatial compartmentalization. Importantly, inhibition of PDE3, and thus perturbation of the spatiotemporal regulation of PKA activity, dramatically increases the duration of EGF-stimulated nuclear ERK activity in a PKA-dependent manner. Together, these findings identify EGF and NGF as potent activators of PKA activity specifically at the plasma membrane and reveal a novel regulatory mechanism contributing to the growth factor signaling specificity achieved by NGF and EGF in PC12 cells.

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Section: Resultsmentioning

confidence: 74%

Spatiotemporally Regulated Protein Kinase A Activity Is a Critical Regulator of Growth Factor-Stimulated Extracellular Signal-Regulated Kinase Signaling in PC12 Cells

Herbst

Allen

Zhang

2011

Molecular and Cellular Biology

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“…To enable real-time cAMP measurements in intact living cells and tissues, genetically encoded FRET-biosensors have been developed (8 -12). It has also been demonstrated that changes in FRET can be calibrated to obtain absolute cAMP values from intact cells (20)(21)(22).…”

Section: Discussionmentioning

confidence: 99%

Real-Time Monitoring of Somatostatin Receptor-cAMP Signaling in Live Pituitary

et al. 2010

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Fluorescence resonance energy transfer using genetically encoded biosensors has proven to be a powerful technique to monitor the spatiotemporal dynamics of cAMP signals stimulated by Gs-coupled receptors in living cells. In contrast, real-time imaging of Gi-mediated cAMP signals under native conditions remains challenging. Here, we describe the use of transgenic mice for cAMP imaging in living pituitary slices and primary pituitary cells. This technique can be widely used to assess the contribution of various pituitary receptors, including individual Gi protein-coupled somatostatin receptors, to the regulation of cAMP levels under physiologically relevant settings.

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“…Although it has been possible to generate images of primary cells or of functional units such as thyroid follicles or pancreatic islets isolated from mice transgenically expressing second-messenger sensors Calebiro et al, 2009;Mironov et al, 2009;von Hayn et al, 2010;Werthmann et al, 2009Werthmann et al, , 2011, the high degree of background fluorescence calls for several improvements for in vivo microscopy, including red-shifted sensors and FRET analysis by multiphoton and second harmonic generation microscopy (Provenzano et al, 2009). In addition, BRET studies have been performed in cells isolated from transgenic mice expressing luciferaselabeled ␤ 2 -adrenergic receptors and GFP2-labeled ␤-arrestin2 (Audet et al, 2010).…”

Section: Discussionmentioning

confidence: 99%