scholarly journals Estimating the magnitude of near-membrane PDE4 activity in living cells

2015 ◽  
Vol 309 (6) ◽  
pp. C415-C424 ◽  
Author(s):  
Wenkuan Xin ◽  
Wei P. Feinstein ◽  
Andrea L. Britain ◽  
Cristhiaan D. Ochoa ◽  
Bing Zhu ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Cyclic Nucleotide ◽  
Single Cells ◽  
Cellular Functions ◽  
Spatial Spread ◽  
Cyclic Nucleotide Gated Channels ◽  
Subcellular Compartments ◽  
Camp Hydrolysis ◽  
Camp Levels

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments.

scholarly journals Cyclic Nucleotide–Gated Channels

1999 ◽  
Vol 114 (3) ◽  
pp. 377-392 ◽  
Author(s):  
Andrea Becchetti ◽  
Katia Gamel ◽  
Vincent Torre
Keyword(s):  
Plasma Membrane ◽  
Cyclic Nucleotide ◽  
Ion Selectivity ◽  
Xenopus Laevis Oocytes ◽  
Gating Currents ◽  
Α Subunit ◽  
Channel Pore ◽  
Cyclic Nucleotide Gated Channels ◽  
Internal Side ◽  
Inside Out

In voltage- and cyclic nucleotide–gated ion channels, the amino-acid loop that connects the S5 and S6 transmembrane domains, is a major component of the channel pore. It determines ion selectivity and participates in gating. In the α subunit of cyclic nucleotide–gated channels from bovine rod, the pore loop is formed by the residues R345–S371, here called R1-S27. These 24 residues were mutated one by one into a cysteine. Mutant channels were expressed in Xenopus laevis oocytes and currents were recorded from excised membrane patches. The accessibility of the substituted cysteines from both sides of the plasma membrane was tested with the thiol-specific reagents 2-aminoethyl methanethiosulfonate (MTSEA) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Residues V4C, T20C, and P22C were accessible to MTSET only from the external side of the plasma membrane, and to MTSEA from both sides of the plasma membrane. The effect of MTSEA applied to the inner side of T20C and P22C was prevented by adding 10 mM cysteine to the external side of the plasma membrane. W9C was accessible to MTSET from the internal side only. L7C residue was accessible to internal MTSET, but the inhibition was partial, ∼50% when the MTS compound was applied in the absence of cGMP and 25% when it was applied in the presence of cGMP, suggesting that this residue is not located inside the pore lumen and that it changes its position during gating. Currents from T15C and T16C mutants were rapidly potentiated by intracellular MTSET. In T16C, a slower partial inhibition took place after the initial potentiation. Current from I17C progressively decayed in inside-out patches. The rundown was accelerated by inwardly applied MTSET. The accessibility results of MTSET indicate a well-defined topology of the channel pore in which residues between L7 and I17 are inwardly accessible, residue G18 and E19 form the narrowest section of the pore, and T20, P21, P22 and V4 are outwardly accessible.

scholarly journals Cyclic Nucleotide–Gated Channels Colocalize with Adenylyl Cyclase in Regions of Restricted Camp Diffusion

2000 ◽  
Vol 116 (2) ◽  
pp. 147-162 ◽  
Author(s):  
Thomas C. Rich ◽  
Kent A. Fagan ◽  
Hiroko Nakata ◽  
Jerome Schaack ◽  
Dermot M.F. Cooper ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Cyclic Nucleotide ◽  
Single Cells ◽  
Compartment Model ◽  
Camp Concentration ◽  
Α Subunit ◽  
Whole Cell ◽  
Hek 293 ◽  
Cellular Targets ◽  
Cyclic Nucleotide Gated Channels

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+, cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotide–gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory α subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotide–gated channels colocalize with adenylyl cyclase in microdomains, and diffusion of cAMP between these domains and the bulk cytosol is significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 22–56 s for substances that monitor exchange with the cytosol). Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotide–gated channels, unless the entire cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotide–gated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signals should also facilitate the differential regulation of cellular targets.

scholarly journals In Vivo Assessment of Local Phosphodiesterase Activity Using Tailored Cyclic Nucleotide–Gated Channels as Camp Sensors

2001 ◽  
Vol 118 (1) ◽  
pp. 63-78 ◽  
Author(s):  
Thomas C. Rich ◽  
Tonia E. Tse ◽  
Joyce G. Rohan ◽  
Jerome Schaack ◽  
Jeffrey W. Karpen
Keyword(s):  
Adenylyl Cyclase ◽  
Cyclic Nucleotide ◽  
Type I ◽  
Hek 293 ◽  
Cyclic Nucleotide Gated Channels ◽  
Type Iv ◽  
Pde Inhibitors ◽  
Camp Levels ◽  
Hydrolysis Of

Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside of specialized cells, there are few methods with the appropriate spatial and temporal resolution to measure cyclic nucleotide concentrations. We have previously demonstrated that adenovirus-expressed, olfactory cyclic nucleotide–gated channels provide real-time sensors for cAMP produced in subcellular compartments of restricted diffusion near the plasma membrane (Rich, T.C., K.A. Fagan, H. Nakata, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 2000. J. Gen. Physiol. 116:147–161). To increase the utility of this method, we have modified the channel, increasing both its cAMP sensitivity and specificity, as well as removing regulation by Ca2+-calmodulin. We verified the increased sensitivity of these constructs in excised membrane patches, and in vivo by monitoring cAMP-induced Ca2+ influx through the channels in cell populations. The improved cAMP sensors were used to monitor changes in local cAMP concentration induced by adenylyl cyclase activators in the presence and absence of PDE inhibitors. This approach allowed us to identify localized PDE types in both nonexcitable HEK-293 and excitable GH4C1 cells. We have also developed a quantitative framework for estimating the KI of PDE inhibitors in vivo. The results indicate that PDE type IV regulates local cAMP levels in HEK-293 cells. In GH4C1 cells, inhibitors specific to PDE types I and IV increased local cAMP levels. The results suggest that in these cells PDE type IV has a high Km for cAMP, whereas PDE type I has a low Km for cAMP. Furthermore, in GH4C1 cells, basal adenylyl cyclase activity was readily observable after application of PDE type I inhibitors, indicating that there is a constant synthesis and hydrolysis of cAMP in subcellular compartments near the plasma membrane. Modulation of constitutively active adenylyl cyclase and PDE would allow for rapid control of cAMP-regulated processes such as cellular excitability.

Ticlopidine and Clopidogrel (SR 25990C) Selectively Neutralize ADP Inhibition of PGE1-Activated Platelet Adenylate Cyclase in Rats and Rabbits

1991 ◽  
Vol 65 (02) ◽  
pp. 186-190 ◽  
Author(s):  
G Defreyn ◽  
C Gachet ◽  
P Savi ◽  
F Driot ◽  
J P Cazenave ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Direct Effect ◽  
Cyclic Nucleotide ◽  
Camp Accumulation ◽  
Rabbit Platelets ◽  
Camp Levels ◽  
Kinetics Of ◽  
Camp Elevation ◽  
Rat Platelets

SummaryTiclopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGEj-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGEr stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGEX-induced cAMP elevation but this effect seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGEr activated platelet adenylate cyclase in rats and rabbits.

Exterior and Interior Events in Early Stage of Thrombin-induced Platelet Aggregation

1977 ◽  
Vol 38 (03) ◽  
pp. 0630-0639 ◽  
Author(s):  
Shuichi Hashimoto ◽  
Sachiko Shibata ◽  
Bonro Kobayashi
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Early Stage ◽  
Adenyl Cyclase ◽  
Cellular Component ◽  
Primary Event ◽  
Rabbit Platelets ◽  
Membrane Bound

SummaryTreatment of washed rabbit platelets with 1 u/ml of thrombin at 37° C resulted in a disappearance from platelets of a protein with 250,000 dalton molecular weight which was shown to be originated from plasma membrane. Parallel loss of adenyl cyclase was noted, and both reactions were complete within 30 sec. From the patterns of disc electrophoretograms, the importance of quick suppression of thrombin action in demonstrating the primary event was stressed.Thrombin induced an apparent activation of membrane bound phosphodiesterase. This reaction was also complete within 30 sec. The cellular component which contained the enzyme activity was distinct from plasma membrane. Soluble phosphodiesterase was not influenced by thrombin at all.These reactions required intact platelet cells to react with thrombin, and no reaction was detected when subcellular preparations were treated with thrombin.Possibility of collaboration of changes in externally located synthetic enzyme with those in internally located degrading enzyme in the early phase of thrombin action on platelets was suggested.

Multimodal Atomic Force Imaging of Open Hemichannelinduced Modulation of Cell Volume and Viscoelastic Properties

1999 ◽  
Vol 5 (S2) ◽  
pp. 998-999
Author(s):  
Seung K. Rhee ◽  
Arjan P. Quist ◽  
Hai Lin ◽  
Nils Almqvist ◽  
Ratneshx Lai
Keyword(s):  
Plasma Membrane ◽  
Cell Volume ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Dye Uptake ◽  
Aortic Endothelial Cells ◽  
Atomic Force ◽  
Cell Plasma ◽  
Uptake Assay ◽  
Gap Junctional

Hemichannels from two single cells can join upon contact between these cells to form gap junctions - an intercellular pathway for the direct exchange of ions and small metabolites. Using techniques of fluorescent dye-uptake assay, laser confocal fluorescence imaging and atomic force microscopy (AFM), we have examined the role of hemichannels, present in the non-junctional regions of single cell plasma membrane, in the modulation of cell volume.Antibodies against a gap junctional protein connexin43, were immunolocalized to nonjunctional plasma membrane regions of single BICR-MlRk k (breast tumor epithelial) cells, KOM-1 (bovine aortic endothelial) cells, and GM04260 (AD-free human) fibroblast cells. In the absence of extracellular calcium, cytoplasmic uptake of Lucifer yellow (LY) but not of dextran-conjugated LY was observed in single cells. Dye uptake was prevented by gap junctional inhibitors, ẞ-glycyrrhetinic acid (ẞGCA) and oleamide.

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