cAMP, the classical second messenger, regulates many diverse cellular functions. The primary effector of cAMP signals, protein kinase A, differentially phosphorylates hundreds targets. Little is known, however, about spatial and temporal nature signals their information content. Thus, it largely unclear how in response to different stimuli, orchestrates such a wide variety responses. Previously, we presented evidence that produced subcellular compartments near plasma membrane, diffusion from...

Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca2+, signals are simple contain little information. Here we show development of adenovirus-expressed cyclic nucleotide–gated channels as sensors cAMP. Homomultimeric composed olfactory α subunit responded rapidly jumps in concentration, their sensitivity was measured calibrate...

Phosphodiesterases (PDEs) catalyze the hydrolysis of second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside specialized cells, there are few methods with appropriate spatial temporal resolution to measure concentrations. We have previously demonstrated that adenovirus-expressed, olfactory nucleotide–gated channels provide real-time sensors for produced subcellular compartments restricted diffusion near...

Abstract Standard fluorescence microscopy approaches rely on measurements at single excitation and emission bands to identify specific fluorophores the setting of thresholds quantify fluorophore intensity. This is often insufficient reliably resolve fluorescent labels in tissues due high autofluorescence. Here we describe use hyperspectral analysis techniques fluorescently labeled cells highly autofluorescent lung tissue. approach allowed accurate detection green protein (GFP) spectra, even...

Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA historically accepted effector, although assumption made on basis of associative not direct evidence. Recent studies have asserted that exchange protein activated cAMP (Epac), PKA, mediates relaxation airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify role prorelaxant effects ASM. Inhibition...

FÖrster resonance energy transfer (FRET) is a non-radiative process that has been extensively applied for probing molecular events involved in range of cellular and biological processes 1. FRET provides means measuring intermolecular spacings on the order ∼1–10 nm 2, an ability allowed measurement such as protein folding, substrate interactions, enzyme kinetics, intercellular signaling molecules. The advent wide fluorescent proteins from which to construct reporters enabled variety live-cell...

Optical spectroscopy and hyperspectral imaging have shown the potential to discriminate between cancerous noncancerous tissue with high sensitivity specificity. However, date, these techniques not been effectively translated real-time endoscope platforms. Hyperspectral of fluorescence excitation spectrum represents new technology that may be well suited for endoscopic implementation. feasibility detecting differences normal mucosa using excitation-scanning has evaluated. The goal this study...

Cyclic nucleotide-gated (CNG) channels are a family of ion activated by the binding cyclic nucleotides. Endogenous have been used to measure nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we investigated subcellular localization cGMP monitoring CNG channel activity response agonists that activate either particulate or soluble guanylyl cyclase. were heterologously expressed human embryonic kidney (HEK)-293 cells stably overexpress cyclase (HEK-NPRA...

Abstract Förster resonance energy transfer (FRET) techniques have proven invaluable for probing the complex nature of protein–protein interactions, protein folding, and intracellular signaling events. These traditionally been implemented with use one or more fluorescence band‐pass filters, either as microscopy filter cubes, dichroic mirrors filters in flow cytometry. In addition, new approaches measuring FRET, such lifetime acceptor photobleaching, developed. Hyperspectral imaging cytometry...

Hyperspectral imaging is a versatile tool that has recently been applied to variety of biomedical applications, notably live-cell and whole-tissue signaling. Traditional hyperspectral approaches filter the fluorescence emission over broad wavelength range while exciting at single band. However, these emission-scanning have shown reduced sensitivity due light attenuation from spectral filtering. Consequently, scanning limited applicability for time-sensitive studies photosensitive...

Cyclic AMP signals encode information required to differentially regulate a wide variety of cellular responses; yet it is not well understood how encrypted within these signals. An emerging concept that compartmentalization underlies specificity the cAMP signaling pathway. This based on series observations indicating levels are distinct in different regions cell. One such observation production at plasma membrane increases pulmonary microvascular endothelial barrier integrity, whereas...

Cyclic nucleotide-gated (CNG) channels are the primary targets of light- and odorant-induced signaling in photoreceptors olfactory sensory neurons. Compartmentalized cyclic nucleotide is necessary to ensure rapid efficient activation these nonselective cation channels. However, relatively little known about subcellular localization CNG or mechanisms their membrane partitioning. Lipid raft domains specialized microdomains rich cholesterol sphingolipids that have been implicated organization...

Signaling through cAMP regulates most cellular functions. The spatiotemporal control of is, therefore, crucial for differential regulation specific targets. Here we investigated the consequences PDE4B or PDE4D gene ablation on signaling at a subcellular level using mouse embryonic fibroblasts. had no effect global bulk cytosol accumulation but increased both basal and hormone-dependent in near-membrane pool. Conversely, enhanced agonist-induced as well plasma membrane. Both significantly...

Autofluorescence, the endogenous fluorescence present in cells and tissues, has historically been considered a nuisance biomedical imaging. Many fluorophores, specifically, collagen, elastin, nicotinamide adenine dinucleotide, flavin dinucleotide (FAD), are found throughout human body. In imaging scenarios, these signals can be prohibitive as they outcompete introduced for diagnostic purposes. However, autofluorescence also contains information that value. Recent advances hyperspectral have...

In the last 20 years there have been remarkable advances in our ability to track movement and activities of proteins within cells. This is largely due improved chemical probes fluorescent proteins, technical microscopy. A remaining challenge real-time multiplexed imaging. Excitation scan-based hyperspectral imaging (HSI) approaches are well suited for However, excitation HSI has not widely adopted, part a lack protocols selection combinations labels determining range wavelengths dichroic...

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. The gold standard for diagnosis tissue biopsy during colonoscopy and subsequent histopathology. Limitations current techniques include turnaround time required histopathology limited ability to detect flat lesions due inadequate contrast provided by traditional white light endoscopy (WLE). focus this work was assess detection accuracy differentiating CRC noncancerous tissues using excitation-scanning...

We have previously used cyclic nucleotide-gated (CNG) channels as sensors to measure cAMP signals in human embryonic kidney (HEK)-293 cells. found that prostaglandin E(1) (PGE(1)) triggered transient increases concentration near the plasma membrane, whereas total levels rose a steady plateau over same time course. In addition, we presented evidence decline near-membrane was due primarily PGE(1)-induced stimulation of phosphodiesterase (PDE) activity, and differences between were largely...

An important focus in cell biology is understanding how different feedback mechanisms regulate G protein–coupled receptor systems. Toward this end we investigated the regulation of endogenous β2 adrenergic receptors (β2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals single HEK-293 cells. We monitored using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure cells 1 μM isoproterenol...

A-kinase anchoring proteins (AKAPs) have emerged as important regulatory molecules that can compartmentalize cAMP signaling transduced by β2-adrenergic receptors (β2ARs); such compartmentalization ensures speed and fidelity of effects on cell function. This study aimed to assess the role AKAPs in regulating global compartmentalized β2AR human airway smooth muscle (ASM). Transcriptome proteomic analyses were used characterize AKAP expression ASM. Stable or injection peptides AKAP-IS Ht31 was...

Hyperspectral imaging and analysis approaches offer accurate detection quantification of fluorescently-labeled proteins cells in highly autofluorescent tissues. However, selecting optimum acquisition settings for hyperspectral is often a daunting task. In this study, we compared two systems-a widefield system with acoustic optical tunable filter (AOTF) charge coupled device (CCD) camera, confocal diffraction gratings photomultiplier tube (PMT) array. We measured the effects parameters on...

In addition to xenobiotics and several other endogenous metabolites, multidrug-resistance proteins (MRPs) extrude the second-messenger cAMP from various cells. Pharmacological and/or genetic inactivation of MRPs has been shown augment intracellular signaling, an effect assumed be a direct consequence blockade extrusion. Here we provide evidence that augmented levels are not due exclusively prevention efflux because MRP is also associated with reduced degradation by phosphodiesterases (PDEs)....

A(2B) adenosine receptors protect against ischaemia/reperfusion injury by activating survival kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). However, the underlying mechanism(s) signalling pathway(s) remain undefined.

Second messengers such as Ca2+, cGMP, and cAMP are known to regulate diverse cellular functions including excitability, contraction, movement, proliferation, gene expression. Our understanding of how Ca2+ signals orchestrate has increased dramatically over the