University of California, San Diego, and Veterans Affairs San Diego Healthcare System, San Diego, California
FLUORESCENCE IMAGING of cAMP in single cells was first described in Roger Tsien's laboratory in 1991 (1). The ability to perform real-time imaging of signaling pathways in living cells has been a major advance in cell biology and physiology. The standard approach has been microinjection of fluorophore pairs. The fluorescence donor, cyan fluorescent protein (CFP) and fluorescence acceptor, yellow fluorescent protein (YFP), are often used because of their superior imaging characteristics. In the special case of assessing cAMP, CFP is fused to the catalytic subunit of protein kinase A (PKA), and YFP to the regulatory subunit (RII). In the inactive state (low cAMP levels), the catalytic subunit and RII are closely associated, and fluorescent resonance energy transfer (FRET) occurs, which can be detected by imaging techniques (11). An increase in cAMP results in binding of cAMP to RII and disassociation of the catalytic subunit, which increases the distance between CFP and YFP and cessation of FRET. Fluorophore pairs must be in close proximity (within 6 nm) for FRET to occur; if this distance is exceeded, FRET ceases.
Image analysis provides a ratio of emissions (donor/acceptor), which is correlated to changes in catalytic and RII association and reflects cAMP levels. This enables one to obtain data regarding the dynamic formation of cAMP over small increments in time (in seconds), temporal resolution that is superior to alternative methods. FRET-based microscopy has a spatial resolution of 46 nm, far better than the 200 nm provided by standard light microscopy (10). The unique features of FRET enable visualization of protein-protein interactions and other molecular events in living cells as they happen (3, 9).
In this issue of American Journal of PhysiologyCell Physiology, Warrier and colleagues (8) have used biosensor imaging of FRET to assess cAMP levels in cardiac myocytes. Recombinant fluorescent-labeled PKA subunits were expressed in cultured cardiac myocytes using adenovirus vectors. This study is important because of the use of adenovirus vectors rather than microinjection to exploit biosensor imaging, and because it demonstrates the value of methods that allow preservation of cell integrity and that provide temporal resolution of intracellular events. The authors have used a reductionist approach with a twist: namely, taking the heart apart but keeping the cardiac myocyte intact, so that all of its parts still function in an integrated and appropriately organized manner.
By using adenovirus vectors encoding fluorescent protein-labeled RII and catalytic subunits of PKA to infect cultured adult guinea pig cardiac myocytes, Warrier and colleagues (8) demonstrated that the fluorescent-labeled recombinant PKA subunits accurately reflected cAMP levels through a physiological range of -adrenergic receptor stimulation and responded appropriately to cyclic nucleotide phosphodiesterase inhibition. Patch clamping was used to measure L-type Ca2+ channel function. Through a physiological range of
-adrenergic receptor stimulation, the authors determined that there was a high correlation between cAMP activity assessed by FRET and L-type Ca2+ channel function, suggesting that channel function was not affected adversely by the adenovirus vector. Finally, Warrier and colleagues demonstrated that muscarinic cholinergic receptor stimulation with acetylcholine provided a 50% reduction in
-adrenergic receptor-stimulated cAMP. The authors collected data that provides quantitative information on the ability of parasympathetic input to alter sympathetic stimulation in intact cardiac myocytes. In doing so, because of the temporal resolution afforded by biosensor imaging, they were able to document a late increase in cAMP activity after cessation of muscarinic cholinergic receptor stimulation, a phenomenon of substantial importance. By applying biosensor imaging on intact cardiac myocytes, Warrier and colleagues have collected data that answer an important question that has eluded us because of techniques that ultimately were too insensitive and crude for the job.
An example may serve to highlight the consequences of attempting to study signaling in membrane preparations. A standard approach used to assess -adrenergic receptor-stimulated cAMP production in the heart involves assessment of cAMP levels using membrane preparations derived from left ventricular homogenates. When isoproterenol-stimulated cAMP production is measured in preparations of left ventricular homogenates from mice, one observes a 1.5-fold increase above basal levels in cAMP production (unpublished data from our laboratory). The same measurements made on intact adult murine cardiac myocytes yields a fourfold increase (2). The absence of proper
-adrenergic receptor-Gs-adenylyl cyclase alignment probably contributes to this apparent decrement of cAMP generating capacity that results from disruption of the cardiac myocyte (6). The consequence of this approach is an erosion of the accuracy of our assessment of
-adrenergic receptor signaling.
Even if the cardiac myocyte remains intact, with -adrenergic receptor-Gs-adenylyl cyclase (and other transmembrane and intracellular pathways) in proper alignment, when agonist stimulation is followed by cell lysing and measurement of cAMP using standard methods, one loses ground. Such an approach presupposes that all portions of the cell membrane and interior are equally engaged and uniformly responsive to stimulation of adenylyl cyclase. Over the past several years, studies (4, 7, 9) have indicated that this is simply not the case. The most accurate assessment of cAMP production may require intact cells and methods with high temporal and spatial resolution. Because compartments within the cell membrane and interior are not uniform in their responsiveness to stimulation, cAMP production is not increased or decreased homogeneously throughout the cell. By focusing on a specific subcellular compartment rather than settling for a "total cell average," our measurements of signaling events will have more precision and context.
As previously stated, biosensor imaging of cAMP has most often been accomplished by microinjection of fluorescent-labeled fluorophores linked to PKA subunits. If CFP and YFP could instead be targeted to endogenous PKA subunits, FRET would occur from fluorophores that are aligned with endogenous PKA. Such an approach would circumvent the possibility that exogenous PKA subunits might alter the intracellular environment. An added benefit of targeting the endogenous machinery of the cell is the ability to garner information about the function of endogenous PKA per se. Indeed, this approach has been used by employing gene transfer methods to evaluate endogenous PKA phosphorylation (9). Although this approach provides qualitatively different information than that provided by the standard methods used to assess cAMP employing FRET, the two techniques are complementary: one providing data regarding the subcellular distribution of cAMP and the other providing data regarding PKA function.
FRET imaging, potentially, provides a temporally and spatially sensitive means to assess signaling in subcellular compartments of interest. In the present study, Warrier and colleagues did not examine subcompartments in the cell for variations in cAMP activity resulting from alterations in -adrenergic receptor and muscarinic cholinergic receptor stimulation. Instead, the authors acquired data from the entire cell or single large areas of the cell. It will be of interest to see whether the methods described in this study, when applied to physiological and pathophysiological states in which cAMP generation is altered, are able to elucidate mechanisms for desensitization and better define the alterations of cAMP activity in specific subcompartments of the cardiac myocyte. For example, in desensitization of
-adrenergic receptor function, are cAMP levels homogeneously decreased, or are there specific compartments that exhibit preserved cAMP activity, whereas others show marked diminution? Does one see a different pattern of cAMP distribution or a change in the time course of cAMP production when A-kinase anchoring proteins are altered by pharmacological or genetic means (5, 9)?
If biosensor imaging is so powerful and potentially definitive, then why has it not been used more universally? After all, the method has been available since 1991 (1), ample time to be assimilated widely. The technique necessitates moderately sophisticated imaging and analysis techniques, and this would be predicted to limit widespread application of the method. However, the key impediment to more prevalent use of biosensor imaging of FRET lies in the fact that the fluorophores that provide FRET must be delivered into living cells. This has been accomplished mainly by microinjection, a challenging technique that requires both special equipment and talent. Warrier and colleagues circumvent this impediment by using an approach (constructing adenovirus vectors encoding fluorescent-labeled catalytic and RII subunits of PKA) that is more readily available to more scientists. This increased availability should open the door for the examination of signal transduction involving key pathways in cardiac myocytes and other cells, and more importantly, in physiologically relevant contexts. Warrier and colleagues describe a method that will be useful for studying a variety of cell types with activity that is regulated by hormones, neurotransmitters, and drugs that modulate cAMP levels.
GRANTS
H. K. Hammond is supported by National Heart, Lung, and Blood Institute Grant 1P01-HL-66941 and a Merit Grant from the Department of Veterans Affairs.
FOOTNOTES
Address for reprint requests and other correspondence: H. K. Hammond, VA San Diego Healthcare System (111A), 3350 La Jolla Village Dr., San Diego, CA 92161 (e-mail: khammond{at}ucsd.edu)
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