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.

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.

Mapping the Heart

2010 ◽  
Vol 18 (4) ◽  
pp. 6-8
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
G Protein Coupled Receptors ◽  
Receptor Subtypes ◽  
Cardiac Muscle Cells ◽  
Guanine Nucleotide Binding Protein ◽  
The Common ◽  
Guanine Nucleotide Binding ◽  
First Time ◽  
G Protein Coupled

Some of the receptors on the surface of cardiac muscle cells (cardiomyocytes) mediate the response of these cells to catecholamines by causing the production of the common second messenger cyclic adenosine monophosphate (cAMP). An example of such receptors are the β1- and β2-adrenergic receptors (βARs) that are heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors. Selective stimulation of these two receptor subtypes leads to distinct physiological and pathophysiological responses, but their precise location on the surface of cardiomyocytes has not been correlated with these responses. In an ingenious combination of techniques, Viacheslav Nikolaev, Alexey Moshkov, Alexander Lyon, Michele Miragoli, Pavel Novak, Helen Paur, Martin Lohse, Yuri Korchev, Sian Harding, and Julia Gorelik have mapped the function of these receptors for the first time.

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.

scholarly journals Differential requirements for platelet aggregation and inhibition of adenylate cyclase by epinephrine. Studies of a familial platelet alpha 2-adrenergic receptor defect

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 494-501 ◽  
Author(s):  
AK Rao ◽  
J Willis ◽  
MA Kowalska ◽  
YT Wachtfogel ◽  
RW Colman
Keyword(s):  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Camp Levels ◽  
Epinephrine Concentration

Abstract We describe a family whose members have impaired platelet aggregation and secretion responses to epinephrine with normal responses to adenosine diphosphate and collagen. Platelet alpha 2-adrenergic receptors (measured using 3H methyl-yohimbine) were diminished in the propositus (78 sites per platelet), his two sisters (70 and 27 sites per platelet), and parents (37 and 63 sites per platelet), but not in two maternal aunts (12 normal subjects, 214 +/- 18 sites per platelet; mean +/- SE). However, the inhibition of cyclic adenosine monophosphate (cAMP) levels by epinephrine in platelets exposed to 400 nmol/L PGI2 was similar in the patients and five normal subjects (epinephrine concentration for 50% inhibition, 0.04 +/- 0.01 mumol/L v 0.03 +/- 0.01 mumol/L; P greater than .05). In normal platelets, the concentration of yohimbine (0.18 mumol/L) required for half maximal inhibition of aggregation induced by 2 mumol/L epinephrine was lower than that for inhibition of its effect on adenylate cyclase (1.6 mumol/L). In quin2 loaded platelets, thrombin (0.1 U/mL) stimulated rise in cytoplasmic Ca2+ concentration, [Ca2+]i, was normal in the two patients studied. The PGI2 analog ZK 36,374 completely inhibited thrombin-induced rise in [Ca2+]i; the reversal of this inhibition by epinephrine was normal in the two patients. Thus, despite the impaired aggregation response to epinephrine, platelets from these patients have normal ability to inhibit PGI2-stimulated cAMP levels. These patients with an inherited receptor defect provide evidence that fewer platelet alpha 2-adrenergic receptors are required for epinephrine-induced inhibition of adenylate cyclase than for aggregation.

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