doi: 10.1161/CIRCRESAHA.108.176420
© 2008 American Heart Association, Inc.
Editorials |
Message Delivered
How Myocytes Control cAMP Signaling
From the Division of Cardiology, Department of Medicine, Johns Hopkins University Medical Institutions, Baltimore, Md.
Correspondence to David A. Kass, MD, Ross 858, Division of Cardiology, Johns Hopkins Medical Institutions, 720 Rutland Ave, Baltimore, MD 21205. E-mail dkass{at}jhmi.edu
See related article, pages 1091–1100
Key Words: Ca2+ channels • protein kinase A phosphorylation • phosphodiesterase inhibitor • cAMP • compartmentation
Despite the vast array of specific receptors and ligands through which cells receive information from their outside environment, the first line of messengers that relay this onto internal signaling cascades is more limited. Yet, somehow, the purpose of the original message is conveyed (ie, we know who knocked on the front door), and the message ends up influencing just the right proteins to produce the desired effect. A striking example of this selectivity is the cyclic nucleotides for which only 2 basic types exist; cAMP and cGMP. Despite this limited cast, these molecules precisely regulate many complex and disparate cellular activities. Their trick is to be in the right place at the right time and with the right amount of signal. It is now well established that cyclic nucleotides are spatially compartmentalized to enable different external triggers to vary the internal cellular response.1–5 This can be achieved by localizing the cyclases that synthesize cAMP or cGMP in different parts of the cell, parking the effector kinase protein kinase (PK)A in particular regions by means of anchoring proteins (AKAPs)6 (may apply to PKG though still unclear), and targeting phosphodiesterases to one area or another and/or providing them with hierarchical control.1 The latter is thought to be particularly important by limiting diffusion of a cyclic nucleotide from its site of generation to other parts of the cell. There are eleven 3',5'-phosphodiesterase (PDE) subfamily members with approximately 50 isoforms, targeting cAMP, cGMP, or in some instances both.7 Given that many of these PDEs can themselves be activated by cyclic nucleotides and/or their effector kinases, the system has substantial built-in complexity for localized regulation. Compartmental signaling was first reported and remains best characterized for cAMP2,4–6,8 and even PKA9 itself. Compartmentation was more recently revealed for cGMP as well,3,10,11 although imaging tools useful in myocytes remain under development12,13 and mechanisms are less well understood. The primary tools used to examine compartmentation of cyclic nucleotide signaling have been reporters such as olfactory cyclic nucleotide-stimulated ion channels that when expressed reside in the outer sarcolemmal membrane and probes based mostly on fluorescent energy transfer to reveal the presence of a cyclic nucleotide. Fluorescence resonance energy transfer (FRET)-based studies in neonatal myocytes first revealed that cAMP stimulated by β-adrenergic agonists appears in microdomains near T-tubules/junctional sarcoplasmic reticulum, and its diffusion is markedly limited by localized PDEs.2 This was further examined in adult myocytes using a mouse expressing a cAMP-sensitive FRET probe in these cells, which showed differential stimulation of cAMP pools that depended on whether β1 or β2 receptors were targeted.4 The β2 pool remained at the outer membrane, whereas the former started there but quickly diffused broadly into the cytosol. In another study using an outer membrane cAMP-reporter ion channel, β1-stimulated cAMP was detected, whereas β2-stimulated cAMP was not. The latter was greatly enhanced if both PDE3 and PDE4 (cAMP esterases) were coinhibited, whereas, for β-1, inhibition of either PDE enhanced the cAMP signal.14 Both studies found that between the 2 PDEs, PDE4 was more potent with respect to cAMP regulation. Importantly, these and essentially all prior studies examined sustained (tonic) stimulation responses and used a single probe (membrane channel or FRET) to detect spatial cAMP changes. They did not address the more rapid kinetics of the signaling and differential role of PDEs in such kinetics, features that could contribute to compartmentalization. They also did not accurately detect cAMP kinetics at different sites simultaneously.
In this issue of Circulation Research, Leroy et al15 start to fill in this gap, providing the first analysis of cardiac myocyte cAMP regulation kinetics simultaneously using an outer membrane channel sensor and cytosolic FRET probe. Importantly, both have rapid responses, the FRET probe being based on an exchange proteins directly activated by cAMP binding sequence. Following short exposure to the β-adrenergic receptor agonist isoproterenol, cAMP rose first at the outer membrane and slightly later in the cytosol, with the latter also showing slower decay kinetics. As a physiological readout, the investigators assessed L-type Ca2+ current density, showing this to peak after maximal cAMP and declined with a much longer time constant (well after cAMP levels had fallen to baseline).
The different kinetics suggested regulatory PDEs may be involved. To test this, selective PDE4 and PDE3 inhibitors were administered. PDE4 inhibition markedly increased the peak cAMP and Ca2+ current response and also prolonged the decay of the transient, particularly in the FRET-detected cytosolic cAMP signal. In striking contrast, PDE3 inhibition only slightly increased the peak cAMP at the membrane and had no detectable influence on the cytosolic cAMP response nor on Ca2+ current. However, when both PDEs were inhibited, the residual decay of both cytosolic cAMP and Ca2+ current was effectively prevented, and membrane localized cAMP decay markedly slowed. This indicates that PDE3 could regulate cAMP but only if PDE4 was already inhibited, supporting hierarchical control that could relate to differential activation and/or localization.
One way PDEs are activated is by phosphorylation by A and/or G kinases, PKA, in the case of PDE4 and PDE3. This feedback mechanism means that a rise in cAMP stimulates the relevant PDE (via PKA) to reduce the initiating signal. PKA appeared to particularly activate PDE4 in their study, having a smaller impact on PDE3. Indeed, inhibition of PDE4 with or without concomitant inhibition of PKA resulted in a similar cytosolic cAMP transient response, indicating that the primary PKA moderating target was PDE4. PKA activation is also presumed to explain the more prolonged ICa decay, although this was not directly examined. It may also explain why PDE3 inhibition influenced cAMP hydrolysis only after PDE4 was first inhibited. Inhibiting PDE4 would then be expected to further elevate PKA activity, enhancing PDE3 regulation. This would indicate yet another mechanism for selective regulation: hierarchical control based on relative catalytic activities controlled by feedback loops.
The findings in Leroy et al15 should be considered in the context of the specific experimental design and models used. For example, the dominance of PDE4 over PDE3 regulation may relate to the use of rat myocytes, which unlike dog, rabbit, or human, have far less PDE3 and more PDE4 regulating their cAMP hydrolysis.16 Mouse is similar to rat in this regard. In isolated human and rabbit myocytes, ICa amplitude increased with a PDE3 but not PDE4 inhibitor.17 PDE3 inhibition has been widely tested in humans as a positive inotropic agent,18 both by itself and with concomitant β-adrenergic receptor, where it was shown to augment contractility.19 Thus, whether the same cAMP kinetics and differential regulation by the 2 PDEs applies to other mammalian species remains unknown. Another issue relates to the short-time constant of isoproterenol exposure. In a prior study, the investigators found PDE3 inhibition could enhance outer membrane cAMP in rat adult myocytes when exposed to sustained β1 stimulation, albeit still less so than with PDE4 inhibition. Furthermore, both PDE3 and PDE4 inhibition augmented Ca2+ transients, sarcoplasmic reticulum Ca2+ content, and phospholamban phosphorylation in mice, with PDE4 modulation shown to phosphatidylinositol 3-kinase-
–dependent.20 Thus, the relative role of the two PDEs may also depend on the chronicity of the stimuli that may further recruit feedback loops yet identified. Both PDE3 and PDE4 are expressed as multiple genes and isoforms, and, certainly in the case of PDE4, this results in specific to subcellular targeting (ie, PDE4D3 with AKAP18 at the sarcoplasmic reticulum, PDE4D5 with β-arrestin, and PDE4D4 interacting with SH3 domains on tyrosine kinases such as Fyn and Src).21 The commonly used PDE4 inhibitor rolipram (used in the present study) does not confer isoform selectivity, so even more subcompartmentalized signaling is likely. In addition to these 2 PDEs, PDE2 has been shown to regulate a pool of cAMP coupled with NO synthase activation,5 and PDE8,7 also a cAMP-PDE, may also contribute in a manner that remains to be elucidated.
In the science of cyclic nucleotides and PDEs, compartmentalization is becoming a mantra, helping explain complex signaling behavior and targeting of the message. Much remains to be discovered, including whether disease disrupts localized targeting, so a message delivered can get garbled. Leroy et al have moved the field further, and it will be exciting to see where this leads.
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Acknowledgments |
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Sources of Funding
Supported by NIH grant HL-089297 and Abraham and a Virginia Weiss Professorship.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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 Top References |
|---|
1. Fischmeister R, Castro LR, Abi-Gerges A, Rochais F, Jurevicius J, Leroy J, Vandecasteele G. Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases. Circ Res. 2006; 99: 816–828.
2. Zaccolo M, Pozzan T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science. 2002; 295: 1711–1715.
3. Takimoto E, Belardi D, Tocchetti CG, Vahebi S, Cormaci G, Ketner EA, Moens AL, Champion HC, Kass DA. Compartmentalization of cardiac beta-adrenergic inotropy modulation by phosphodiesterase type 5. Circulation. 2007; 115: 2159–2167.
4. Nikolaev VO, Bunemann M, Schmitteckert E, Lohse MJ, Engelhardt S. Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined beta2-adrenergic receptor-mediated signaling. Circ Res. 2006; 99: 1084–1091.
5. Mongillo M, Tocchetti CG, Terrin A, Lissandron V, Cheung YF, Dostmann WR, Pozzan T, Kass DA, Paolocci N, Houslay MD, Zaccolo M. Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway. Circ Res. 2006; 98: 226–234.
6. Dodge-Kafka KL, Langeberg L, Scott JD. Compartmentation of cyclic nucleotide signaling in the heart: the role of A-kinase anchoring proteins. Circ Res. 2006; 98: 993–1001.
7. Conti M, Beavo J. Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem. 2007; 76: 481–511.[CrossRef][Medline] [Order article via Infotrieve]
8. DiPilato LM, Cheng X, Zhang J. Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc Natl Acad Sci U S A. 2004; 101: 16513–16518.
9. Zhang J, Ma Y, Taylor SS, Tsien RY. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci U S A. 2001; 98: 14997–15002.
10. Piggott LA, Hassell KA, Berkova Z, Morris AP, Silberbach M, Rich TC. Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments. J Gen Physiol. 2006; 128: 3–14.
11. Castro LR, Verde I, Cooper DM, Fischmeister R. Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes. Circulation. 2006; 113: 2221–2228.
12. Nikolaev VO, Gambaryan S, Lohse MJ. Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat Methods. 2006; 3: 23–25.[CrossRef][Medline] [Order article via Infotrieve]
13. Nausch LW, Ledoux J, Bonev AD, Nelson MT, Dostmann WR. Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors. Proc Natl Acad Sci U S A. 2008; 105: 365–370.
14. Rochais F, Abi-Gerges A, Horner K, Lefebvre F, Cooper DM, Conti M, Fischmeister R, Vandecasteele G. A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes. Circ Res. 2006; 98: 1081–1088.
15. Leroy J, Abi-Gerges A, Nikolaev VO, Richter W, Lechene P, Mazet J, Conti M, Fischmeister R. Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes. Role of phosphodiesterases. Circ Res. 2008; 102: 1091–1100.
16. Osadchii OE. Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease. Cardiovasc Drugs Ther. 2007; 21: 171–194.[CrossRef][Medline] [Order article via Infotrieve]
17. Kajimoto K, Hagiwara N, Kasanuki H, Hosoda S. Contribution of phosphodiesterase isozymes to the regulation of the L-type calcium current in human cardiac myocytes. Br J Pharmacol. 1997; 121: 1549–1556.[CrossRef][Medline] [Order article via Infotrieve]
18. Packer M, Carver JR, Rodeheffer RJ, Ivanhoe RJ, DiBianco R, Zeldis SM, Hendrix GH, Bommer WJ, Elkayam U, Kukin ML, Mallis GI, Sollano JA, Shannon J, Tandon PK, deMets DL. Effect of oral milrinone on mortality in severe chronic heart failure. The PROMISE Study Research Group. N Engl J Med. 1991; 325: 1468–1475.[Abstract]
19. Endoh M, Hori M. Acute heart failure: inotropic agents and their clinical uses. Expert Opin Pharmacother. 2006; 7: 2179–2202.[CrossRef][Medline] [Order article via Infotrieve]
20. Kerfant BG, Zhao D, Lorenzen-Schmidt I, Wilson LS, Cai S, Chen SR, Maurice DH, Backx PH. PI3Kgamma is required for PDE4, not PDE3, activity in subcellular microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes. Circ Res. 2007; 101: 400–408.
21. Houslay MD, Baillie GS, Maurice DH. cAMP-specific phosphodiesterase-4 enzymes in the cardiovascular system: a molecular toolbox for generating compartmentalized cAMP signaling. Circ Res. 2007; 100: 950–966.
Related Article:
Circ. Res. 2008 102: 1091-1100.
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