Download The interplay of multiple molecular and cellular components is

Am J Physiol Cell Physiol 302: C837–C838, 2012;
doi:10.1152/ajpcell.00012.2012.
Editorial Focus
The interplay of multiple molecular and cellular components is necessary
for compartmentalization of cAMP. Focus on “Assessment of cellular
mechanisms contributing to cAMP compartmentalization in pulmonary
microvascular endothelial cells”
Fiona Murray
Department of Medicine, University of California San Diego, La Jolla, California
Address for reprint requests and other correspondence: F. Murray, Dept. of
Medicine, Univ. of California San Diego, La Jolla, CA 92093 (e-mail:
[email protected]).
http://www.ajpcell.org
cAMP gradients and functional responses (1, 8). Fully defining
the mechanisms that are responsible for cAMP compartmentalization should uncover how signaling specificity is achieved
and likely reveal new pharmacological targets for physiologically important events.
In this current issue of American Journal of Physiology-Cell
Physiology, Feinstein et al. (3) use mathematical modeling
together with experimental measurements to further elucidate
how cAMP is compartmentalized in cultured confluent monolayers and “idealized” pulmonary microvascular endothelial
cells (PMVECs, geometry based on measurements of a series
of image stacks by confocal microscopy). Using the Virtual
Cell modeling environment (www.vcell.org), the authors evaluate the contribution of the localization and activities of AC,
PDE (limited to physiological levels measured experimentally), and cAMP buffering (e.g., cAMP binding to PKA bound
to AKAPs), structural impediments, the shape of the PMVECs,
and other factors that lower the effective cAMP diffusion
coefficient in the formation of cAMP gradients. The data show
that altering the activity of PDEs or ACs or concentrating them
in discrete subcellular domains (subplasmalemmal, cytosolic,
or perinuclear region) has little effect in generating intracellular cAMP gradients, unless the effective diffusion coefficient is
Fig. 1. The key determinants in the spatial regulation of cAMP. Subcellular
compartmentalization of cAMP is controlled by the interaction between
adenylyl cyclase (AC) and phosphodiesterase (PDE) localization and activity,
cell shape and cAMP buffering (protein binding), cytosolic viscosity, and
structural impediments that reduce cAMP diffusion coefficient. AKAP, A-kinase anchoring protein; EPAC, exchange protein directly activated by cAMP;
CNG, cyclic nucleotide-gated channels.
0363-6143/12 Copyright © 2012 the American Physiological Society
C837
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THE SECOND MESSENGER cyclic AMP (cAMP) regulates a wide
number of cellular responses, which include gene transcription,
metabolism, and cell death, and tissue-specific functions such
as the integrity of the endothelial barrier in the lung. cAMP
signaling is triggered by binding of hormones and neurotransmitters to G protein-coupled receptors (GPCRs) via the stimulatory G␣ subunit, leading to the stimulation of adenylyl
cyclase (AC) and the subsequent activation of downstream
effectors such as protein kinase A (PKA), exchange protein
directly activated by cAMP (EPAC), and cyclic nucleotidegated (CNG) channels: cyclic nucleotide phosphodiesterases
(PDEs) hydrolyze cAMP and terminate its actions. It is currently accepted that cAMP signaling is not merely a linear
cascade but more complex, since each enzymatic component
involved in its generation and action is expressed as different
isoforms and cAMP signaling is organized in discrete subcellular domains (2, 10). Compartmentalization of cAMP, by
bringing cAMP close to specific targets, is important in shaping the differential physiological responses of various agonists
and has become the standard model for numerous other signaling pathways.
Evidence for the spatial and temporal regulation of cAMP
has been directly shown with the development of live-cell
biosensors, such as CNG-channel sensors and fluorescence
resonance energy transfer (FRET)-based sensors for PKA or
EPAC (4); however, the exact mechanisms that shape the
intracellular gradients of cAMP are not fully understood. In
particular, subcellular anchoring of components of the cAMP
pathway and diffusional barriers, primarily provided by PDEs,
play a key role in the subcellular localization and effect of
cAMP signaling. A-kinase anchoring proteins (AKAP) sequester cAMP effectors, such as PKA, EPAC, and PDEs, thereby
forming intracellular-specific signaling complexes in close
proximity to cAMP targets (2): disruption of specific AKAPs
in subcellular domains can remove PKA from a specific target,
thus altering the cellular effects of cAMP (7). In parallel, PDEs
provide an enzymatic barrier to locally degrade cAMP and
generate multiple subcellular “pools” with different cAMP
concentrations, thereby controlling the level and persistence of
a cAMP signal and activation of downstream effects (10). For
example, dominant-negative and knockdown strategies have
shown that individual PDE4 isoforms control cAMP levels in
defined subcellular compartments and couple to specific GPCR
signaling cascades, and are thereby important for generating
Editorial Focus
C838
ple, Feinstein et al. found that disruption of the F-actin cortical
rim with cytochalasin D caused cell rounding and decreased
PDE and AC activities; the authors were unable to determine
the effects of each parameter on cAMP compartmentalization
(3). Even so, this study provides data that yield deeper understanding of cAMP signaling and how it could be manipulated
to control PMVEC function in normal and pathological conditions. Fig. 1.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author.
AUTHOR CONTRIBUTIONS
F.M. prepared the figures; drafted the manuscript; and approved the final
version of the manuscript.
REFERENCES
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3. Feinstein WP, Zhu B, Leavesley SJ, Sayner SL, Rich TC. Assessment
of cellular mechanisms contributing to cAMP compartmentalization in
pulmonary microvascular endothelial cells. Am J Physiol Cell Physiol
(November 23, 2011). doi:10.1152/ajpcell.00361.2011.
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SM, Alberini CM, Schaff JC, Blitzer RD, Moraru II, Iyengar R. Cell
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Zaccolo M, Blackwell KT. The role of type 4 phosphodiesterases in
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reduced from 300 ␮m2/s to ⬍3 ␮m2/s. Altered cellular geometry and hindered diffusion appear necessary for PDEs, ACs, or
cAMP buffering to generate physiologically meaningful cAMP
gradients in PMVECs, thus adding new complexity to ideas
about compartmentation of cAMP signaling.
Previous mathematical modeling in human embryonic kidney-293 (HEK-293) cells showed, however, that reduced
cAMP diffusion is not required for its compartmentalization
and in fact that PDEs and ACs alone could generate relevant
intracellular cAMP gradients (5, 6). The apparent discrepancies
between these data appear to be based on the estimates of AC
and PDE activities used in the modeling by Oliveira et al. (6),
which were higher than physiologically relevant levels [500- to
1,000-fold greater than that measured experimentally in HEK293 cells in vitro (3)]. Thus, Feinstein et al. highlight the
importance of estimating parameters experimentally in the
specific cell type that is to be studied by mathematical modeling so that the conditions used are as close to the natural
environment as possible. The results also imply that cellspecific regulation of cAMP compartmentalization may occur;
for example, in cells with high PDE activity, such as hippocampal neurons, this alone may be sufficient to generate
gradients—in contrast with what Feinstein et al. observed in
PMVECs (3, 5).
The subcellular location of cAMP has been shown to be of
direct physiological relevance in PMVECs since it can have
opposing effects on barrier function in the lung: cAMP at the
plasma membrane enhances endothelial barrier function (PKAmediated phosphorylation of myosin light chain), whereas
cAMP in the cytosol disrupts barrier integrity (PKA-mediated
phosphorylation of tau serine 214), thereby increasing lung
permeability (9). It would be of interest in future studies to
investigate whether such mathematical modeling can aid in
studies that seek to define how changes in PDEs, ACs, cell
shape, and other parameters discussed in the article alter cAMP
compartmentalization and contribute to the development of
pulmonary pathology, such as in acute lung injury. For example, if cell structure is altered with disease then even small
changes in PDEs might have a large impact on cAMP gradients
and physiological response.
The current article provides a good example of how mathematical modeling together with detailed experimental studies
can be used to identify components that contribute to cAMP
gradients (or potentially that of other second messengers);
however, one limitation that arises from such data is the
difficulty in confirming such ideas in vivo since changing one
parameter would undoubtedly directly affect others: for exam-