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.

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 Synergistic activation of the type I adenylyl cyclase by Ca2+ and Gs-coupled receptors in vivo.

1994 ◽  
Vol 269 (41) ◽  
pp. 25400-25405
Author(s):  
G A Wayman ◽  
S Impey ◽  
Z Wu ◽  
W Kindsvogel ◽  
L Prichard ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Type I ◽  
Synergistic Activation

scholarly journals Shift in collagen type as an early response to induction of the metanephric mesenchyme.

1981 ◽  
Vol 89 (2) ◽  
pp. 276-283 ◽  
Author(s):  
P Ekblom ◽  
E Lehtonen ◽  
L Saxén ◽  
R Timpl
Keyword(s):  
Basal Lamina ◽  
Type Iv Collagen ◽  
Early Response ◽  
Type I ◽  
Metanephric Mesenchyme ◽  
Type Iv ◽  
Interstitial Collagens ◽  
Membrane Components

Conversion of the nephrogenic mesenchyme into epithelial tubules requires an inductive stimulus from the ureter bud. Here we show with immunofluorescence techniques that the undifferentiated mesenchyme before induction expresses uniformly type I and type III collagens. Induction both in vivo and in vitro leads to a loss of these proteins and to the appearance of basement membrane components including type IV collagen. This change correlates both spatially and temporally with the determination of the mesenchyme and precedes and morphological events. During morphogenesis, type IV collagen concentrates at the borders of the developing tubular structures where, by electron microscopy, a thin, often discontinuous basal lamina was seen to cover the first pretubular cell aggregates. Subsequently, the differentiating tubules were surrounded by a well-developed basal lamina. No loss of the interstitial collagens was seen in the metanephric mesenchyme when brought into contact with noninducing tissues or when cultured alone. Similar observations were made with nonnephrogenic mesenchyme (salivary, lung) when exposed to various heterotypic tissues known to induce tubules in the nephrogenic mesenchyme. The sequential shift in the composition of the extracellular matrix from an interstitial, mesenchymal type to a differentiated, epithelial type is so far the first detectable response of the nephrogenic mesenchyme to the tubule-inducing signal.

Type I adenylyl cyclase functions as a coincidence detector for control of cyclic AMP response element-mediated transcription: synergistic regulation of transcription by Ca2+ and isoproterenol

1994 ◽  
Vol 14 (12) ◽  
pp. 8272-8281
Author(s):  
S Impey ◽  
G Wayman ◽  
Z Wu ◽  
D R Storm
Keyword(s):  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Long Term Potentiation ◽  
Regulation Of Transcription ◽  
Type I ◽  
Hek 293 ◽  
293 Cells ◽  
Term Potentiation ◽  
Stimulation Of

Studies carried out with mammals and invertebrates suggest that Ca(2+)-sensitive adenylyl cyclases may be important for neuroplasticity. Long-term potentiation in the hippocampus requires increases in intracellular Ca2+ which are accompanied by elevated cyclic AMP (cAMP). Furthermore, activation of cAMP-dependent protein kinase is required for the late stage of long-term potentiation in the CA1 region of the hippocampus, which is also sensitive to inhibitors of transcription. Therefore, some forms of synaptic plasticity may require coordinate regulation of transcription by Ca2+ and cAMP. In this study, we demonstrate that the expression of type I adenylyl cyclase in HEK-293 cells allows Ca2+ to stimulate reporter gene activity mediated through the cAMP response element. Furthermore, simultaneous activation by Ca2+ and isoproterenol caused synergistic stimulation of transcription in HEK-293 cells and cultured neurons. We propose that Ca2+ and neurotransmitter stimulation of type I adenylyl cyclase may play a role in synaptic plasticity by generating optimal cAMP signals for regulation of transcription.

Role Of Cyclic Amp In The Inhibition Of Human Platelet Functions By Quercetin, A Flav0N0Id That Potentiates PGI2 Effect

1981 ◽  
Author(s):  
J P Cazenave ◽  
A Beretz ◽  
A Stierlé ◽  
R Anton
Keyword(s):  
Maximum Level ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Pde Inhibitors ◽  
Pde Inhibition ◽  
Induced Aggregation ◽  
Camp Levels ◽  
Dose Dependent

Injury to the endothelium (END) and subsequent platelet (PLAT)interactions with the subEND are important steps in thrombosis and atherosclerosis. Thus,drugs that protect the END from injury and also inhibit PLAT function are of interest. It has been shown that some flavonoids(FLA), a group of compounds found in plants, prevent END desquamation in vivo, inhibit cyclic nucleotide phosphodiesterases(PDE)and inhibit PLAT function. We have studied the structure-activity relationships of 13 purified FLA on aggregation and secretion of 14c-5HT of prelabeled washed human PLAT induced by ADP, collagen(COLL) and thrombin(THR). All the FLA were inhibitors of the 3 agents tested. Quercetin(Q), was the second best after fisetin. It inhibited secretion and aggregation with I50 of 330µM against 0.1 U/ML.THR, 102µM against 5µM ADP and 40 µM against COLL. This inhibitory effect is in the range of that of other PDE inhibitors like dipyridamole or 3-isobutyl-l- methylxanthine. The aggregation induced by ADP, COLL and THR is at least mediated by 3 mechanisms that can be inhibited by increasing cAMP levels. We next investigated if Q, which is a PDE inhibitor of bovine aortic microsomes,raises PLAT cAMP levels. cAMP was measured by a protein-binding method. ADP- induced aggregation(5µM) was inhibited by PGI2 (0.1 and 0.5 nM) . Inhibition was further potentiated(l.7 and 3.3 times) by lOµM Q, which alone has no effect on aggregation. The basal level of cAMP(2.2 pmol/108PLAT) was not modified by Q (50 to 500µM). Using these concentrations of Q,the rise in cAMP caused by PGI2(0.1 and 0.5nM) was potentiated in a dose dependent manner. Q potentiated the effect of PGI2 on the maximum level of cAMP and retarded its breakdown. Thus Q and possibly other FLA could inhibit the interaction of PLAT with the components of the vessel wall by preventing END damage and by inhibiting PLAT function through a rise in cAMP secondary to PDE inhibition and potentiation of the effect of vascular PGI2 on PLAT adenylate cyclase.

scholarly journals Hypersensitivity Reactions to Monoclonal Antibodies in Children

Medicina ◽  
2020 ◽  
Vol 56 (5) ◽  
pp. 232
Author(s):  
Francesca Mori ◽  
Francesca Saretta ◽  
Annamaria Bianchi ◽  
Giuseppe Crisafulli ◽  
Silvia Caimmi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Therapeutic Option ◽  
Provocation Test ◽  
Type I ◽  
Hypersensitivity Reactions ◽  
Biologic Drugs ◽  
Type Iv ◽  
Drug Provocation Test

Biologic drugs are widely used in pediatric medicine. Monoclonal antibodies (mAbs) in particular are a therapeutic option for rheumatic, autoinflammatory and oncologic diseases. Adverse drug reactions and hypersensitivity reactions (HSR) to mAbs may occur in children. Clinical presentation of HSRs to mAbs can be classified according to phenotypes in infusion-related reactions, cytokine release syndrome, both alpha type reactions and type I (IgE/non-IgE), type III, and type IV reactions, all beta-type reactions. The aim of this review is to focus on HSRs associated with the most frequent mAbs in childhood, with particular attention to beta-type reactions. When a reaction to mAbs is suspected a diagnostic work-up including in-vivo and in-vitro testing should be performed. A drug provocation test is recommended only when no alternative drugs are available. In selected patients with immediate IgE-mediated drug allergy a desensitization protocol is indicated. Despite the heavy use of mAbs in childhood, studies evaluating the reliability of diagnostic test are lacking. Although desensitization may be effective in reducing the risk of reactions in children, standardized pediatric protocols are still not available.

scholarly journals Regulation of type I adenylyl cyclase by calmodulin kinase IV in vivo.

1996 ◽  
Vol 16 (11) ◽  
pp. 6075-6082 ◽  
Author(s):  
G A Wayman ◽  
J Wei ◽  
S Wong ◽  
D R Storm
Keyword(s):  
Adenylyl Cyclase ◽  
Long Term Potentiation ◽  
Type I ◽  
Cam Kinase ◽  
Regulatory Systems ◽  
Calmodulin Kinase ◽  
Term Potentiation ◽  
G Protein Coupled ◽  
Cam Kinase Iv

Type I adenylyl cyclase is a neurospecific enzyme that is stimulated by Ca2+ and calmodulin (CaM). This enzyme couples the Ca2+ and cyclic AMP (cAMP) regulatory systems in neurons, and it may play an important role for some forms of synaptic plasticity. Mutant mice lacking type I adenylyl cyclase show deficiencies in spatial memory and altered long-term potentiation (Z. Wu, S. A. Thomas, Z. Xia, E. C. Villacres, R. D. Palmiter, and D. R. Storm, Proc. Natl. Acad. Sci. USA 92:220-224, 1995). Although type I adenylyl cyclase is synergistically stimulated by Ca2+ and G-protein-coupled receptors in vivo, very little is known about mechanisms for inhibition of the enzyme. Here, we report that type I adenylyl cyclase is inhibited by CaM kinase IV in vivo. Expression of constitutively active or wild-type CaM kinase IV inhibited Ca2+ stimulation of adenylyl cyclase activity without affecting basal or forskolin-stimulated activity. Type I adenylyl cyclase has two CaM kinase IV consensus phosphorylation sequences near its CaM binding domain at Ser-545 and Ser-552. Conversion of either serine to alanine by mutagenesis abolished CaM kinase IV inhibition of adenylyl cyclase. This suggests that the activity of this enzyme may be directly inhibited by CaM kinase IV phosphorylation. Type VIII adenylyl cyclase, another enzyme stimulated by CaM, was not inhibited by CaM kinase II or IV. We propose that CaM kinase IV may function as a negative feedback regulator of type I adenylyl cyclase and that CaM kinases may regulate cAMP levels in some cells.

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 Prick, patch or blood test? A simple guide to allergy testing

2021 ◽  
Vol 16 (2) ◽  
pp. 19-26
Author(s):  
Leelavathi Muthupalaniappen ◽  
Adawiyah Jamil
Keyword(s):  
Test Method ◽  
Type I ◽  
In Vitro Test ◽  
Delayed Reaction ◽  
Allergy Testing ◽  
Prick Test ◽  
Type Iv ◽  
Allergy Test

This article provides information on allergy testing and serves as a simple guide for physicians who are considering using allergy testing as a step in patient management. Basic principles of allergy testing, indications for testing, and how and when to choose a suitable allergy test are discussed. Allergy testing in general refers to evaluation of either type I or type IV hypersensitivity reactions. The type I (immediate) reaction is evaluated using the skin prick test (in vivo) or serum IgE (in vitro) test methods, while the type IV (delayed) reaction is determined via the skin patch test method. The allergens responsible for a specific reaction can be identified from allergy testing, and this information is useful in administering avoidance measures. Appropriate treatment of allergic reactions along with allergen avoidance ensure a successful treatment outcome and prevent future reactions.

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