Thomas C. Rich

A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell

cAMP, the classical second messenger, regulates many diverse cellular functions. The primary effector of cAMP signals, protein kinase A, differentially phosphorylates hundreds of cellular targets. Little is known, however, about the spatial and temporal nature of cAMP signals and their information content. Thus, it is largely unclear how cAMP, in response to different stimuli, orchestrates such a wide variety of cellular responses. Previously, we presented evidence that cAMP is produced in subcellular compartments near the plasma membrane, and that diffusion of cAMP from these compartments to the bulk cytosol is hindered. Here we report that a uniform extracellular stimulus initiates distinct cAMP signals within different cellular compartments. By using cyclic nucleotide-gated ion channels engineered as cAMP biosensors, we found that prostaglandin E1 stimulation of human embryonic kidney cells caused a transient increase in cAMP concentration near the membrane. Interestingly, in the same time frame, the total cellular cAMP rose to a steady level. The decline in cAMP levels near the membrane was prevented by pretreatment with phosphodiesterase inhibitors. These data demonstrate that spatially and temporally distinct cAMP signals can coexist within simple cells.

PDE4D and PDE4B Function in Distinct Subcellular Compartments in Mouse Embryonic Fibroblasts

Signaling through cAMP regulates most cellular functions. The spatiotemporal control of cAMP is, therefore, crucial for differential regulation of specific cellular targets. Here we investigated the consequences of PDE4B or PDE4D gene ablation on cAMP signaling at a subcellular level using mouse embryonic fibroblasts. PDE4B ablation had no effect on the global or bulk cytosol accumulation of cAMP but increased both basal and hormone-dependent cAMP in a near-membrane pool. Conversely, PDE4D ablation enhanced agonist-induced cAMP accumulation in the bulk cytosol as well as at the plasma membrane. Both PDE4B and PDE4D ablation significantly modified the time course and the level of isoproterenol-induced phosphorylation of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component. A second membrane response through Toll-like receptor signaling, however, was only affected by PDE4B ablation. PDE4D but not PDE4B ablation significantly prolonged cAMP-response element-binding protein-mediated transcription. These findings demonstrate that PDE4D and PDE4B have specialized functions in mouse embryonic fibroblasts with PDE4B controlling cAMP in a discrete subdomain near the plasma membrane.

Roles of GRK and PDE4 Activities in the Regulation of β2 Adrenergic Signaling

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 in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 μM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 μM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 μM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 μM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of β2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of β2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system.

Assessment of cellular mechanisms contributing to cAMP compartmentalization in pulmonary microvascular endothelial cells

Cyclic AMP signals encode information required to differentially regulate a wide variety of cellular responses; yet it is not well understood how information is encrypted within these signals. An emerging concept is that compartmentalization underlies specificity within the cAMP signaling pathway. This concept is based on a series of observations indicating that cAMP levels are distinct in different regions of the cell. One such observation is that cAMP production at the plasma membrane increases pulmonary microvascular endothelial barrier integrity, whereas cAMP production in the cytosol disrupts barrier integrity. To better understand how cAMP signals might be compartmentalized, we have developed mathematical models in which cellular geometry as well as total adenylyl cyclase and phosphodiesterase activities were constrained to approximate values measured in pulmonary microvascular endothelial cells. These simulations suggest that the subcellular localizations of adenylyl cyclase and phosphodiesterase activities are by themselves insufficient to generate physiologically relevant cAMP gradients. Thus, the assembly of adenylyl cyclase, phosphodiesterase, and protein kinase A onto protein scaffolds is by itself unlikely to ensure signal specificity. Rather, our simulations suggest that reductions in the effective cAMP diffusion coefficient may facilitate the formation of substantial cAMP gradients. We conclude that reductions in the effective rate of cAMP diffusion due to buffers, structural impediments, and local changes in viscosity greatly facilitate the ability of signaling complexes to impart specificity within the cAMP signaling pathway.

β-Agonist-mediated Relaxation of Airway Smooth Muscle Is Protein Kinase A-dependent

Background: Mechanisms by which β-2-adrenoreceptor agonists effect bronchorelaxation remain unestablished. Results: Direct inhibition of PKA via molecular approaches reversed β-agonist-mediated antagonism of procontractile signaling and relaxation of contracted airway smooth muscle (ASM) despite augmenting intracellular cAMP. Conclusion: PKA is the primary mechanism by which β-agonists relax ASM. Significance: PKA-dependent signaling and functions should guide the development of bronchodilator drugs. Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of β-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased β-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, β-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which β-agonists exert their relaxant effects.

Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently-labeled cells in highly autofluorescent tissue

Standard fluorescence microscopy approaches rely on measurements at single excitation and emission bands to identify specific fluorophores and the setting of thresholds to quantify fluorophore intensity. This is often insufficient to reliably resolve and quantify fluorescent labels in tissues due to high autofluorescence. Here we describe the use of hyperspectral analysis techniques to resolve and quantify fluorescently labeled cells in highly autofluorescent lung tissue. This approach allowed accurate detection of green fluorescent protein (GFP) emission spectra, even when GFP intensity was as little as 15% of the autofluorescence intensity. GFP-expressing cells were readily quantified with zero false positives detected. In contrast, when the same images were analyzed using standard (single-band) thresholding approaches, either few GFP cells (high thresholds) or substantial false positives (intermediate and low thresholds) were detected. These results demonstrate that hyperspectral analysis approaches uniquely offer accurate and precise detection and quantification of fluorescence signals in highly autofluorescent tissues.

Inactivation of Multidrug Resistance Proteins Disrupts Both Cellular Extrusion and Intracellular Degradation of cAMP

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 to augment intracellular cAMP signaling, an effect assumed to be a direct consequence of the blockade of cAMP extrusion. Here we provide evidence that the augmented intracellular cAMP levels are not due exclusively to the prevention of cAMP efflux because MRP inactivation is also associated with reduced cAMP degradation by phosphodiesterases (PDEs). Several prototypical MRP inhibitors block PDE activity at concentrations widely used to inhibit MRPs. Their dose-dependent effects in several paradigms of cAMP signaling are more consistent with their potency in inhibiting PDEs than MRPs. Moreover, genetic manipulation of MRP expression results in concomitant changes in PDE activity and protein levels, thus affecting cAMP degradation in parallel with cAMP efflux. These findings suggest that the effects of MRP inactivation on intracellular cAMP levels reported previously may be due in part to reduced degradation by PDEs and identify MRP-dependent transport mechanisms as novel regulators of cellular PDE expression levels. Mathematical simulations of cAMP signaling predict that selective ablation of MRP-dependent cAMP efflux per se does not affect bulk cytosolic cAMP levels, but may control cAMP levels in restricted submembrane compartments that are defined by small volume, high MRP activity, limited PDE activity, and limited exchange of cAMP with the bulk-cytosolic cAMP pool. Whether this regulation occurs in cells remains to be confirmed experimentally under conditions that do not affect PDE activity.

A-kinase anchoring proteins regulate compartmentalized cAMP signaling in airway smooth muscle

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 cAMP signaling and effects on cell function. This study aimed to assess the role of AKAPs in regulating global and compartmentalized β2AR signaling in human airway smooth muscle (ASM). Transcriptome and proteomic analyses were used to characterize AKAP expression in ASM. Stable expression or injection of peptides AKAP‐IS or Ht31 was used to disrupt AKAP‐PKA interactions, and global and compartmentalized cAMP accumulation stimulated by β‐agonist was assessed by radioimmunoassay and membrane‐delineated flow through cyclic nucleotide‐gated channels, respectively. ASM expresses multiple AKAP family members, with gravin and ezrin among the most readily detected. AKAP‐PKA disruption had minimal effects on whole‐cell cAMP accumulation stimulated by β‐agonist (EC50 and Bmax) concentrations, but significantly increased the duration of plasma membrane‐delineated cAMP (τ=251±51 s for scrambled peptide control vs. 399±79 s for Ht31). Direct PKA inhibition eliminated decay of membrane‐delineated cAMP levels. AKAPs coordinate compartmentalized cAMP signaling in ASM cells by regulating multiple elements of β2AR‐mediated cAMP accumulation, thereby representing a novel target for manipulating β2AR signaling and function in ASM.—Horvat, S. J., Deshpande, D. A., Yan, H., Panettieri, R. A., Codina, J., DuBose Jr., T. D., Xin, W., Rich, T. C., Penn, R. B. A‐kinase anchoring proteins regulate compartmentalized cAMP signaling in airway smooth muscle. FASEB J. 26, 3670–3679 (2012). www.fasebj.org

Excitation-scanning hyperspectral imaging microscope

Abstract. Hyperspectral imaging is a versatile tool that has recently been applied to a variety of biomedical applications, notably live-cell and whole-tissue signaling. Traditional hyperspectral imaging approaches filter the fluorescence emission over a broad wavelength range while exciting at a single band. However, these emission-scanning approaches have shown reduced sensitivity due to light attenuation from spectral filtering. Consequently, emission scanning has limited applicability for time-sensitive studies and photosensitive applications. In this work, we have developed an excitation-scanning hyperspectral imaging microscope that overcomes these limitations by providing high transmission with short acquisition times. This is achieved by filtering the fluorescence excitation rather than the emission. We tested the efficacy of the excitation-scanning microscope in a side-by-side comparison with emission scanning for detection of green fluorescent protein (GFP)-expressing endothelial cells in highly autofluorescent lung tissue. Excitation scanning provided higher signal-to-noise characteristics, as well as shorter acquisition times (300  ms/wavelength band with excitation scanning versus 3  s/wavelength band with emission scanning). Excitation scanning also provided higher delineation of nuclear and cell borders, and increased identification of GFP regions in highly autofluorescent tissue. These results demonstrate excitation scanning has utility in a wide range of time-dependent and photosensitive applications.

A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin

BACKGROUND AND PURPOSE A2B 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) and signalling pathway(s) remain undefined.