We previously demonstrated that the [Ca2+]i response to PTH is heterogeneous in single UMR-106-01 osteogenic sarcoma cells. To verify whether response heterogeneity is a universal feature of PTH signal transduction, cAMP production was monitored in monolayer cultures of UMR-106-01 cells and human trabecular bone osteoblasts (HOB) using the cAMP-sensitive fluorescent ... More
VIP modulates neuronal nicotinic acetylcholine receptor function by a cyclic AMP-dependent mechanism.
AuthorsGurantz D, Harootunian AT, Tsien RY, Dionne VE, Margiotta JF
JournalJ Neurosci
PubMed ID8207470
Neuronal nicotinic ACh receptors (AChRs) mediate synaptic transmission throughout the nervous system, and are regulated by cellular processes and interactions that include second messenger signaling pathways. In the case of chick ciliary ganglion neurons, activation of the cAMP-dependent signaling pathway with cAMP analogs enhances ACh sensitivity in a manner consistent ... More
Confocal imaging of cAMP signals in Aplysia neurons.
AuthorsAdams SR, Bacskai BJ, Hochner B, Kaang BK, Kandel ER, Mahaut-Smith M, Tsien RY
JournalJpn J Physiol
PubMed ID8271521
Serotonin suppresses the slow afterhyperpolarization in rat intralaminar and midline thalamic neurones by activating 5-HT(7) receptors.
AuthorsGoaillard JM, Vincent P
JournalJ Physiol
PubMed ID12042351
While the highest expression level of 5-HT(7) receptors in the brain is observed in intralaminar and midline thalamic neurones, the physiological role of these receptors in this structure is unknown. In vivo recordings have shown that stimulation of the serotonergic raphe nuclei can alter the response of these neurones to ... More
Dynamic interactions of cyclic AMP transients and spontaneous Ca(2+) spikes.
AuthorsGorbunova YV, Spitzer NC
JournalNature
PubMed ID12097913
Transient increases of intracellular Ca(2+) drive many cellular processes, ranging from membrane channel kinetics to transcriptional regulation, and links of Ca(2+) to other second messengers should activate signalling networks. However, real-time kinetic interactions have been difficult to investigate. Here we report observations of spontaneous increases in concentration of cyclic AMP ... More
Cyclic AMP imaging in neurones in brain slice preparations.
AuthorsVincent P, Brusciano D
JournalJ Neurosci Methods
PubMed ID11478978
The second messenger cascade of cyclic AMP (cAMP) plays an important physiological role in neurones, modulating neuronal excitability and synaptic transmission. The fluorescent probe FlCRhR allows real time ratiometric imaging of cAMP changes inside cells (Nature 349 (1991) 694). Until now, the only way to introduce FlCRhR into cells was ... More
Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons.
AuthorsBacskai BJ, Hochner B, Mahaut-Smith M, Adams SR, Kaang BK, Kandel ER, Tsien RY
JournalScience
PubMed ID7682336
'Cyclic adenosine monophosphate (cAMP)-dependent protein kinase, labeled with fluorescein and rhodamine on the catalytic and regulatory subunits, respectively, was injected into Aplysia sensory neurons either in culture or in intact cell clusters. Energy transfer between the subunits, a measure of cytosolic cAMP concentration ([cAMP]), and compartmentation of the dissociated subunits ... More
Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging.
AuthorsDeBernardi MA, Brooker G
JournalProc Natl Acad Sci U S A
PubMed ID8643446
'The spatial and temporal dynamics of two intracellular second messengers, cAMP and Ca2+, were simultaneously monitored in living cells by digital fluorescence ratio imaging using FlCRhR, a single-excitation dual-emission cAMP indicator, and fura-2, a dual-excitation single-emission Ca2+ probe. In single C6-2B glioma cells, isoproterenol- or forskolin-evoked cAMP accumulation (measured in ... More
cAMP acts as a second messenger in pollen tube growth and reorientation.
Authors Moutinho A; Hussey P J; Trewavas A J; Malhó R;
JournalProc Natl Acad Sci U S A
PubMed ID11517303
'Pollen tube growth and reorientation is a prerequisite for fertilization and seed formation. Here we report imaging of cAMP distribution in living pollen tubes microinjected with the protein kinase A-derived fluorosensor. Growing tubes revealed a uniform distribution of cAMP with a resting concentration of approximately 100-150 nM. Modulators of adenylyl ... More
Acetoxymethyl esters of phosphates, enhancement of the permeability and potency of cAMP.
AuthorsSchultz C, Vajanaphanich M, Harootunian AT, Sammak PJ, Barrett KE, Tsien RY
JournalJ Biol Chem
PubMed ID8384207
'Acetoxymethyl esters of alkyl or aryl phosphates can be prepared by reacting their trialkylammonium or silver salts with acetoxymethyl bromide. Because acetoxymethyl esters are rapidly cleaved intracellularly, they facilitate the delivery of organophosphates into the cytoplasm without puncturing or disruption of the plasma membrane. In addition, acylation of free hydroxyls, ... More
Micro-perfusion flow cell for imaging cultured cells.
AuthorsAgnihotri N, Kisaalita WS, Keith CH
JournalBiotechniques
PubMed ID10524313
We present a unique design for a flow cell with a small working volume that allows rapid displacement of media viewed under high power and short working distance objectives. The flow cell has a small internal depth (ca. 0.033 cm) and volume (ca. 0.05 mL) and is easy to handle. ... More
Fluorescence ratio imaging of cyclic AMP in single cells.
AuthorsAdams SR, Harootunian AT, Buechler YJ, Taylor SS, Tsien RY
JournalNature
PubMed ID1847505
Fluorescence imaging is perhaps the most powerful technique currently available for continuously observing the dynamic intracellular biochemistry of single living cells. However, fluorescent indicator dyes have been available only for simple inorganic ions such as Ca2+, H+, Na+, K+, Mg2+ and Cl-. We now report a fluorescent indicator for the ... More
Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion.
AuthorsHarootunian AT, Adams SR, Wen W, Meinkoth JL, Taylor SS, Tsien RY
JournalMol Biol Cell
PubMed ID8298196
The catalytic (C) subunit of cyclic AMP (cAMP) dependent protein kinase (PKA) has previously been shown to enter and exit the nucleus of cells when intracellular cAMP is raised and lowered, respectively. To determine the mechanism of nuclear translocation, fluorescently labeled C subunit was injected into living REF52 fibroblasts either ... More
Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A.
AuthorsHagiwara M, Brindle P, Harootunian A, Armstrong R, Rivier J, Vale W, Tsien R, Montminy MR
JournalMol Cell Biol
PubMed ID8336722
Cyclic AMP (cAMP) regulates a number of eukaryotic genes by mediating the protein kinase A (PKA)-dependent phosphorylation of the CREB transcription factor at Ser-133. In this study, we test the hypothesis that the stoichiometry and kinetics of CREB phosphorylation are determined by the liberation and subsequent translocation of PKA catalytic ... More
Simultaneous measurements of intracellular cAMP and L-type Ca2+ current in single frog ventricular myocytes.
AuthorsGoaillard JM, Vincent PV, Fischmeister R
JournalJ Physiol
PubMed ID11136860
The cAMP fluorescent probe FlCRhR was used to monitor changes in intracellular cAMP concentration ([cAMP]i) in isolated frog ventricular myocytes. The probe was introduced into the cell through a patch pipette which allowed simultaneous recording of the whole-cell L-type Ca2+ current (ICa). Ratiometric imaging was used to monitor [cAMP]i changes ... More
Intracellular cyclic AMP not calcium, determines the direction of vesicle movement in melanophores: direct measurement by fluorescence ratio imaging.
AuthorsSammak PJ, Adams SR, Harootunian AT, Schliwa M, Tsien RY
JournalJ Cell Biol
PubMed ID1348251
Intracellular movement of vesiculated pigment granules in angelfish melanophores is regulated by a signalling pathway that triggers kinesin and dyneinlike microtubule motor proteins. We have tested the relative importance of intracellular Ca2+ ([Ca2+]i) vs cAMP ([cAMP]i) in the control of such motility by adrenergic agonists, using fluorescence ratio imaging and ... More