Abstract 464: Two Different Microdomains of β 1 -adrenoreceptor Signaling Revealed by Live Cell Imaging

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Alexander Froese ◽  
Viacheslav O Nikolaev
Keyword(s):  
Plasma Membrane ◽  
Cardiac Contractility ◽  
Pressure Overload ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Calcium Handling ◽  
Membrane Structures ◽  
T Tubules

Background: 3’,5’-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger and a crucial regulator of cardiac function and disease. In cardiomyocytes, it is produced predominantly after activation of β 1 -adrenergic receptors (β 1 -ARs) by catecholamines and acts intracellularly in discrete functionally relevant microdomains formed, for example, around calcium-handling proteins. Previously, we reported that β 1 -ARs are distributed across various cardiomyocyte membrane areas, including transverse (T)-tubules and cell crests. However, it is unknown whether these two β 1 -AR pools contribute differentially to the regulation of cardiac contractility and gene expression. Methods and Results: To directly visualize receptor-microdomain communication in cardiomyocytes, we established a combination of scanning ion conductance microscopy (SICM) with transgenically expressed targeted Förster resonance energy transfer (FRET)-based biosensors. Using this approach, we measured local cAMP responses in distinct microdomains of mouse ventricular cardiomyocytes (such as plasma membrane, cytosol and nucleus) after localized stimulation of β 1 -AR on different membrane structures of healthy and diseased cardiomyocytes. Using a plasma membrane targeted cAMP biosensor, we found that β 1 -AR stimulation at the crest induced stronger cAMP signals compared to β 1 -AR stimulated in the T-tubuli where cAMP was highly confined by PDE3. This difference was abolished in a pressure overload hypertrophy model due to submembrane redistribution of PDEs. Interestingly, crest β 1 -AR signals could propagate deeper inside the cell, inducing higher nuclear cAMP responses than recorded from receptors stimulated in the T-tubules. Conclusions: in the present study, we have demonstrated that β 1 -ARs located in T-tubuli and cell crests form two differentially regulated cAMP microdomains, each having its typical PDE repertoire and generating distinct second messenger signals. More detailed understanding of these two microdomains at different subsarcolemmal locations may contribute to new therapeutic strategies including more specific β-blockers.

scholarly journals Cardiac Hypertrophy Changes Compartmentation of cAMP in Non-Raft Membrane Microdomains

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 535
Author(s):  
Nikoleta Pavlaki ◽  
Kirstie A. De Jong ◽  
Birgit Geertz ◽  
Viacheslav O. Nikolaev ◽  
Alexander Froese
Keyword(s):  
Cardiac Hypertrophy ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Pressure Overload ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Differential Regulation ◽  
Membrane Microdomains ◽  
Transgenic Mouse Line

3′,5′-Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which plays critical roles in cardiac function and disease. In adult mouse ventricular myocytes (AMVMs), several distinct functionally relevant microdomains with tightly compartmentalized cAMP signaling have been described. At least two types of microdomains reside in AMVM plasma membrane which are associated with caveolin-rich raft and non-raft sarcolemma, each with distinct cAMP dynamics and their differential regulation by receptors and cAMP degrading enzymes phosphodiesterases (PDEs). However, it is still unclear how cardiac disease such as hypertrophy leading to heart failure affects cAMP signals specifically in the non-raft membrane microdomains. To answer this question, we generated a novel transgenic mouse line expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor E1-CAAX targeted to non-lipid raft membrane microdomains of AMVMs and subjected these mice to pressure overload induced cardiac hypertrophy. We could detect specific changes in PDE3-dependent compartmentation of β-adrenergic receptor induced cAMP in non-raft membrane microdomains which were clearly different from those occurring in caveolin-rich sarcolemma. This indicates differential regulation and distinct responses of these membrane microdomains to cardiac remodeling.

scholarly journals Visualization of β-adrenergic receptor dynamics and differential localization in cardiomyocytes

2021 ◽  
Vol 118 (23) ◽  
pp. e2101119118
Author(s):  
Marc Bathe-Peters ◽  
Philipp Gmach ◽  
Horst-Holger Boltz ◽  
Jürgen Einsiedel ◽  
Michael Gotthardt ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Adrenergic Receptor ◽  
Cardiac Contractility ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Receptor Subtypes ◽  
Specific Cell ◽  
Adult Cardiomyocytes ◽  
Outer Plasma Membrane

A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two β‐adrenergic receptor (β‐AR) subtypes, β1 and β2, in cardiomyocytes. They are both coupled to stimulatory Gs proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for β1‐AR but not for β2-AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent β‐AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the β2‐AR is confined to and diffuses within the T-tubular network, as opposed to the β1‐AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the β2‐AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the β2‐AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.

scholarly journals BacMam System for FRET-Based cAMP Sensor Expression in Studies of Melanocortin MC1 Receptor Activation

2012 ◽  
Vol 17 (8) ◽  
pp. 1096-1101 ◽  
Author(s):  
Olga Mazina ◽  
Reet Reinart-Okugbeni ◽  
Sergei Kopanchuk ◽  
Ago Rinken
Keyword(s):  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Endogenous Expression ◽  
Mammalian Cell Lines

Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors (GPCRs) and a useful readout molecule to estimate the biological activity of various GPCR-specific agents. Here we report the development and use of a Förster resonance energy transfer (FRET) biosensor for cAMP (Epac2-camps) combined with a baculovirus-based BacMam transduction system. The constructed BacMam-Epac2-camps viral transduction system is a simple and robust tool for ligand screening at the second-messenger level in a variety of mammalian cell lines. The level of biosensor protein expression can easily be adjusted in a dose-dependent manner depending on the multiplicity of viral infection. For setting up the assay, we used a B16F10 murine melanoma cell line with endogenous expression of melanocortin-1 receptor (MC1R). The receptor activation was characterized by a set of MC1R full and partial agonists. Bivalent ions Ca2+ as well as Mg2+ modulated ligand potencies, whereas the effect was ligand and ion specific. Results obtained for MC1R indicate that the BacMam-Epac2-camps system may also be applicable for studying the activation of other GPCRs and may be implemented in routine analysis as well as in high-throughput screening.

scholarly journals Increased intracellular cyclic adenosine monophosphate inhibits T lymphocyte-mediated cytolysis by two distinct mechanisms.

1988 ◽  
Vol 167 (6) ◽  
pp. 1963-1968 ◽  
Author(s):  
L S Gray ◽  
J Gnarra ◽  
E L Hewlett ◽  
V H Engelhard
Keyword(s):  
Cholera Toxin ◽  
T Lymphocyte ◽  
Pertussis Toxin ◽  
Target Cell ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Stimulation Of

Cholera toxin (CT), but not pertussis toxin (PT), treatment of cloned murine CTL inhibited target cell lysis in a dose-dependent fashion. The effects of CT were mimicked by forskolin and cyclic adenosine monophosphate (cAMP) analogues. Inhibition of cytotoxicity by CT and cAMP analogs was mediated in part by attenuation of conjugate formation. Additionally, both CT and cAMP analogs blocked the increase in intracellular Ca2+ induced by stimulation of the TCR complex by mAbs. These findings indicate that cAMP inhibits the activity of CTL by two distinct mechanisms and suggests a role for this second messenger in CTL-mediated cytolysis.

scholarly journals Heart failure leads to altered β2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain

2018 ◽  
Vol 115 (3) ◽  
pp. 546-555 ◽  
Author(s):  
Zeynep Bastug-Özel ◽  
Peter T Wright ◽  
Axel E Kraft ◽  
Davor Pavlovic ◽  
Jacqueline Howie ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adult Rat ◽  
C Terminus ◽  
Complex Forms ◽  
Cardiac Excitation

Abstract Aims Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation–contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time cAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium–potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of cAMP compartmentation in heart failure. Methods and results A novel Förster resonance energy transfer (FRET)-based biosensor termed phospholemman (PLM)-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of PLM. Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain-associated cAMP levels by β2-adrenoceptors (β2-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific β2-AR/cAMP signals and β2-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects. Conclusions NKA/PLM complex forms a distinct cAMP microdomain which is directly regulated by β2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted β2-AR signalling to cAMP in the vicinity of PLM.

Secondary Messenger Systems in PTSD

2016 ◽  
Author(s):  
Ulrike Schmidt
Keyword(s):  
Second Messengers ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Brain Regions ◽  
Guanosine Monophosphate ◽  
Intracellular Camp ◽  
Post Traumatic Stress ◽  
Secondary Messenger

Second messengers such as cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositoltriphosphate, and diacylglycerol (DAG) are a prerequisite for the signal transduction of extracellular receptors. The latter are central for cellular function and thus are implicated in the pathobiology of a variety of disorders, such as schizophrenia, bipolar disorder, major depression, and post-traumatic stress disorder (PTSD). This chapter focuses on the involvement of second messenger molecules and their regulators as direct targets in human and animal PTSD and aims to stimulate the underdeveloped research in this field. The synthesis of literature reveals that second messengers clearly play a central role in PTSD-associated brain regions and processes. In particular, pituitary adenylate cyclase-activating polypeptide (PACAP), an important regulator of intracellular cAMP levels, as well as protein kinase c, the major target of DAG, belong to the hitherto most promising PTSD candidate molecules directly involved in second messenger signaling.

scholarly journals Effects of integrin-mediated cell adhesion on plasma membrane lipid raft components and signaling

2011 ◽  
Vol 22 (18) ◽  
pp. 3456-3464 ◽  
Author(s):  
Andrés Norambuena ◽  
Martin A. Schwartz
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Lipid Raft ◽  
Rho Gtpases ◽  
Cancer Metastasis ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Membrane Lipid ◽  
Cell Detachment ◽  
Suspended Cells

Anchorage dependence of cell growth, which is mediated by multiple integrin-regulated signaling pathways, is a key defense against cancer metastasis. Detachment of cells from the extracellular matrix triggers caveolin-1–dependent internalization of lipid raft components, which mediates suppression of Rho GTPases, Erk, and phosphatidylinositol 3-kinase in suspended cells. Elevation of cyclic adenosine monophosphate (cAMP) following cell detachment is also implicated in termination of growth signaling in suspended cells. Studies of integrins and lipid rafts, however, examined mainly ganglioside GM1 and glycosylphosphatidylinositol-linked proteins as lipid raft markers. In this study, we examine a wider range of lipid raft components. Whereas many raft components internalized with GM1 following cell detachment, flotillin2, connexin43, and Gαs remained in the plasma membrane. Loss of cell adhesion caused movement of many components from the lipid raft to the nonraft fractions on sucrose gradients, although flotillin2, connexin43, and H-Ras were resistant. Gαs lost its raft association, concomitant with cAMP production. Modification of the lipid tail of Gαs to increase its association with ordered domains blocked the detachment-induced increase in cAMP. These data define the effects of that integrin-mediated adhesion on the localization and behavior of a variety of lipid raft components and reveal the mechanism of the previously described elevation of cAMP after cell detachment.

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