ReviewCompartmentalisation of cAMP and Ca2+ signals
Introduction
The term ‘second messenger’ was coined with the discovery of cAMP. It was meant to identify a small molecule generated intracellularly upon cell stimulation and capable of regulating downstream cellular events. Strictly speaking, the term applies only to a few molecules (i.e. cAMP and cGMP, inositol 1,4,5-trisphosphate [InsP3], diacyl glycerol [DAG] and Ca2+). These are ubiquitous molecules, capable of controlling a myriad of cellular process. In each case, the mode of action is deceptively simple: a stimulus triggers an increase in the second messenger level, which in turn, upon binding to effector molecules, modifies their activity and eventually elicits a specific response. This apparent simplicity masks complex mechanisms that enable these small molecules to exert tight control over many different (and sometimes opposite) functions. The parameters that enable this remarkable versatility include the kinetics of the second messenger increases, their amplitude and their spatio-temporal patterns. In this review, we will concentrate on this latter aspect, as it concerns the two best-studied second messengers — Ca2+ and cAMP — discussing primarily data published in the very recent past. We also will give a brief overview of the current state of the art in this field.
The idea that the cell cytoplasm is not a homogeneous container of saline solution is an established fact. Not only is the cell full of different organelles, but the cytoplasm itself is highly structured because of the presence of an intricate network of filamentous proteins. Moreover, some second messengers have the intrinsic capacity of self-regeneration (e.g. the so-called Ca2+-induced Ca2+ release), whereas others can be attacked and degraded by enzymes. Essentially, theoretically three parameters are involved in the formation of local second-messenger gradients: the localised site of generation; of degradation-accumulation; and the diffusion rates of the molecule within the cytosol. Regarding Ca2+, all three parameters are well suited to allow the generation of local domains. In fact, this ion enters the cytoplasm through channels distributed non-homogeneously in the cell. Moreover, Ca2+ is sequestered or extruded via specifically located pumps and exchangers [1]. Finally, the diffusion rate of Ca2+ within the cytosol is slow (10–50 μm2 s−1), owing to the large concentration of relatively immobile binding sites [2]. The situation is substantially different when it comes to cAMP: it is generated exclusively at the plasma membrane, its degradation can occur throughout the cytoplasm, and its diffusion rate is quite high (∼500 μm2 s−1) 3., 4..
Section snippets
Ca2+ measurements in living cells
With respect to Ca2+, methods to monitor local domains have been available for quite some time — intracellularly trapped fluorescent indicators associated with high-speed microscopic imaging [5]. Additional tools to monitor the heterogeneity of intracellular Ca2+ are the recombinant targeted Ca2+ probes — chimeric aequorins and various families of calcium-sensing probes engineered from mutants of green fluorescent protein (GFP), named cameleons, camgaroos and pericams 6., 7., 8., 9., 10., 11.,
cAMP measurements in living cells
Unlike Ca2+, methodologies for measuring cAMP levels in single living cells have been lacking for a long time. Determination has relied on indirect measurements (i.e. recording the activation of reactions that depend on cAMP concentration), or on radio-immunoassays carried out on cell extracts. The first tool used to monitor cAMP directly, introduced in 1991, is based on cAMP-sensitive fluorescence resonance energy transfer (FRET), and involves micro-injecting chemically labelled subunits of
Functional role of Ca2+ heterogeneity
The existence of local gradients is undisputed with respect to Ca2+, and evidence is rapidly accumulating for cAMP. The functional role of such micro-heterogeneity, however, is still a matter for debate. Regarding Ca2+, sparks and puffs are considered to represent unitary events (due to local activation of single or small numbers of channels) that either merge, leading to a global cell response [14], or individually control, specific responses [23]. Individual Ca2+ release events are at the
Functional role of cAMP heterogeneity
Although it is now well documented that cAMP signalling relies on the organisation of macromolecular complexes, and that proximity of receptors to their ultimate targets can guarantee velocity of response and contribute to specificity, the picture in this field is murkier than in the case of Ca2+. The hypothesis of spatially restricted domains of cAMP was formulated over 20 years ago, to explain experimental data obtained in cardiac myocytes. In these cells, β-adrenergic effects correlate with
Local domains of cAMP in heart cells
PKA — the main effector of cAMP — is a key regulator of excitation/contraction coupling in muscles cells. In the heart, sympathetic control of the frequency and strength of contraction is exerted by β-adrenergic receptor stimulation, activation of G proteins and, in turn, activation of adenylyl cyclase (AC) and synthesis of cAMP. The second messenger activates PKA, which, by phosphorylating L-type Ca2+ channels (dihydropyridine receptor [DHPR]) and the ryanodine receptor, increases the amount
What limits cAMP diffusion?
The mechanisms that limit cAMP diffusion remain elusive. Suggestions put forth so far include a physical barrier, possibly formed by elements of the endoplasmic reticulum, localised beneath the plasma membrane [22], or a molecular ‘channelling’ of cAMP directly from AC to PKA [53], similarly to what happens to the product of certain metabolic enzymes [54]. However, evidence is accumulating rapidly that suggests an important role in cAMP diffusional restriction for phosphodiesterases (PDEs), the
Conclusions
Unravelling the complexity of second messenger compartmentalisation has undergone important advances in recent times. Most importantly, issues that had remained hitherto in the realm of pure speculation are beginning to be addressed experimentally. This is largely owing to the emerging armamentarium available to signal transduction biologists. Indeed, the development of new tools and technology not only has enabled pertinent practical questions to be posed, but important answers have already
Acknowledgements
The original work in the authors’ laboratory was supported by grants from Telethon-Italy, from the Italian Association for Cancer Research (AIRC), from the European Community, from the Italian Minister of University and Scientific Research (MURST), from the CNR (Target Project Biotechnology), from the Agenzia Spaziale Italiana (ASI) and from the Armenise Harvard Foundation. M.Z. is an Assistant Telethon Scientist.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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