Cyclic AMP (3′,5′-cyclic AMP, cAMP( 

Nucleotides play a universal role in life, as components of nucleic acids, as forms in which chemical free energy is stored, and as regulators of gene expression or enzyme activity. Cyclic adenosine 3′,5′-monophosphate (cAMP) plays a universal role in the control of gene expression as well as in the integration of metabolic functions. It is present both in eucaryotes and in procaryotes (see 1 for early recognition of cAMP presence in bacterial genera). cAMP seems to be absent only from archaebacteria. Its presence was controversial in plants, but a work from the J. Schell laboratory investigating opine catabolism in plants resulted in the cloning of a gene that appeared to specify cAMP synthesis in plants. It was, however, later reported that this work was a fake, so that it is now admitted that plants do not make cAMP (2). Cyclic AMP has been reported to exist in cyanobacteria and in algae. This ubiquity explains the major interest displayed in its mode of synthesis, and the vast amount of literature devoted to the enzymes that produce cAMP from ATP, the adenylate cyclases (E. C. 4.6.1.1). Because cAMP is a regulatory molecule, it must be either excreted in the environment or inactivated in order not to accumulate. This is performed by 3′,5′-cyclic-nucleotide phosphodiesterases (E. C. 3.1.4.17). These enzymes are generally specific for cyclic nucleotides (namely cAMP and cGMP) and sometimes specific for cAMP or cGMP alone. A variety of natural inhibitors modulate their activity (nucleoside triphosphates, pyrophosphate, and especially methylated xanthines, such as theophylline) by a variety of processes involving protein phosphorylation or calcium. Adenylate cyclases (see entry) form four independent classes of enzymes, and this raises the question of the origin of cyclic nucleotides as regulatory molecules, as well as their universal implication in regulatory networks. Because it is very polar and negatively

charged, cAMP does not permeate easily into cells (unless through specific transporters, generally unknown at present). More lipophilic analogs such as N6,O2′-dibutyryladenosine-3′,5′ monophosphate are, therefore, used to modulate its concentration and mimic its effect in cell cultures ex vivo, but in vivo inhibitors of phosphodiesterase or specific mediators (neuromediators in particular) are used for therapeutic purposes where cAMP concentration must be altered.

 Cyclic AMP was discovered in 1958 by E. Sutherland, who obtained a Nobel prize in 1971 for this and other discoveries on hormone action. As he has himself written, it is within the scope of molecular biology that cAMP was discovered: “When I first entered the study of hormone action, some 25 years ago, there was a widespread feeling among biologists that hormone action could not be studied meaningfully in the absence of organized cell structure. However, as I reflected upon the history of biochemistry, it seemed to me there was a real possibility that hormones might act at the molecular level”. Sutherland built up a cell-free system where well-known hormones could control glycolysis in vitro. Using this system he isolated a small thermostable molecule that was able to activate glycogen phosphorylase. Chemical analysis of the molecule permitted its identification as adenosine 3′-5′ cyclic monophosphate. Synthesis of cAMP was shown to be the result of the action of an enzyme, adenylate cyclase, that generated cAMP and PPi from ATP, when activated by adrenaline (3). Since this pioneering work, the study of cAMP-mediated effects required the identification of the structure, function, and regulation of adenylate cyclases, the cAMP synthesizing enzymes. And, contrary to expectation, this did not yield a unifying picture of the role of cAMP, but, rather, demonstrated that this molecule has been used over and over again by living organisms for very different functions.

 At the time of cAMP discovery, the aphorism of Jacques Monod, “what is true for Escherichia coli is true for the elephant,” induced biochemists to try bacterial systems to unravel cAMP function. After the discovery of cAMP by Sutherland in 1958, Mackman and Sutherland (4) demonstrated that glucose-starved E. coli cells accumulated cAMP. Ullmann and Monod (5) later established that part of the catabolite repression phenomenon  was controlled by cAMP. This discovery raised hopes that the study of this mediator in bacteria would help to understand what happens in eucaryotes (even perhaps in higher eucaryotes). However, it soon became clear that cAMP in eucaryotes was generally, as found by Sutherland, a “second messenger” that was used as an intracellular relay molecule to the action of extracellular hormones, while it acted directly on transcription via its receptor, the Catabolite Activator Protein in E. coli (6). Study of the slime mold Dictyostelium discoïdeum revealed another function of cAMP, phylogenetically linked to is hormone-mediated action in higher eucaryotes, namely a pulsatile synthesis and degradation used by bacteria as a signal to control their aggregation properties as a differentiating multicellular organism (7).

The universal role of cAMP in controlling such diverse metabolic processes is puzzling because the enzymes needed for its synthesis, adenylate cyclases, are extremely varied and submitted to a wide variety of regulations. Why does this result in the synthesis of the same molecular species, cAMP? Is not all the regulatory process lost in this way? How can the cAMP signal generated by one enzyme type be distinguished from another? Compartmentalization is often invoked in this process, but, while this is relatively easily accounted for in the case of macromolecules, this is difficult to see in the case of small molecules such as cAMP. Another usual answer is to say that it is the combination of hormonal receptors of differing types and cAMP—and not cAMP alone—that is required for specificity. But would not cAMP synthesized from different sources also be recognized? Another answer is to remark that cAMP is known to be only one among many second messengers: cGMP has been added to the list as well as inositol phosphates, phosphatidyldiglycerides, calcium, etc. This certainly permits generation of a combinatorial control of activities, but would be very sensitive to accidental synthesis of cAMP. Cyclic AMP is not synthesized in a steady state way (even in bacteria). It is, therefore, important not to consider cAMP as such, with some average concentration, but to consider the shape of its time-dependent variation in concentration. In fact, observations are accumulating that strongly suggest that cAMP does not have the same effect when it is delivered in a steady state fashion, rather than in a pulse (or a series of pulses) (7).

The motile and aggregating amoeba D. discoïdeum has been used as a paradigm for cell differentiation because undifferentiated cells start to differentiate into specific tissues sometime after starvation. Secreted in the external medium, cAMP is necessary for aggregation. The genetics, biochemistry, cellular biology, and physiology of phenomena involving cAMP have been investigated in detail in this organism, where it controls, as in higher eucaryotes, a protein phosphorylation cascade, initiated after a regulatory cAMP-binding subunit of a protein kinase detaches from its target enzyme. This cascade is necessary not only for chemotaxis and aggregation but also for the triggering of genes involved in differentiation. The regulation of cAMP pulsatile concentration is mediated by two sets of enzymes adenylate cyclases and phosphodiesterases. In contrast with the situation with higher eucaryotes, however, cAMP and phosphodiesterase control operates not from the interior of the cell but from the external medium. This requires specific membrane receptors for cAMP and a process of signal transduction. In D. discoïdeum the pulses are generated by an appropriate coupling between adenylate cyclase activity, phosphodiesterase activity, and diffusion. The main observation is that variation in the cAMP pulse frequency changes the response of the cell. Many biochemical models can account for such cAMP pulses. These models require simple enzyme properties (in particular standard nonlinear features, such as self-activation and desensitization after saturating activity). They do not require the existence of many gene products, but only a specific behavior of enzymes (appropriate Vm and Km of biosynthetic and degradative enzymes). Assuming that cAMP concentration modulation in time is the control event is, therefore, not a biochemical paradox.

 In bacteria, Utsumi et al (8) have investigated cyclic AMP synthesis during the cell cycle of E. coli on synchronized cells, and they have given an unambiguous demonstration that there was a strong correlation of cAMP synthesis and replication or cell division, suggesting that the molecule may play some role in the cell cycle. This is also correlated with the position of the adenylate cyclase gene near the chromosome's origin of replication and with its very low level of expression, suggesting that expression is strongly coupled to DNA replication. This observation has long been overlooked because cells deficient for adenylate cyclase or CAP are viable, suggesting that cAMP is dispensible. Specific time-dependent variation of the concentration of cAMP for fine coordination of replication and division in E. coli is achieved by excretion of the nucleotide, rather than coupling to the activity of a phosphodiesterase (6). In this respect it is interesting that high concentrations of cAMP produced by foreign genes in E. coli are not toxic until they reach a very high level (at least 10-fold the normal concentration), whereas much lower concentration of cAMP produced by the endogeneous adenylate cyclase are toxic (9). Cyclic AMP has been formally linked to catabolite repression, but there are many catabolite sensitive operons that do not respond to cAMP. In addition cAMP synthesis is very strong in E. coli when cells enter the stationary phase of growth, suggesting that it could be a cell-to-cell signal as it is in D. discoïdeum. This may be one of its function in other bacteria (such as Rhizobium species), where it is clearly not linked to catabolite repression.

 In the same way, intracellular and extracellular levels of cAMP vary during the cell cycle of Saccharomyces cerevisiae. Using centrifugal elutriation, Smith et al (10) showed that the intracellular cAMP concentration followed the stages of the cell cycle, being highest during the division cycle and lowest immediately before or just after cell separation; at the same time the external cAMP concentration did not vary. Therefore, in yeast, as in E. coli, it appears that the role of the external medium is to behave as a sink. These observations substantiate the demonstration that, under normal conditions, appropriate enzyme systems can generate a specific time-dependent pattern of cAMP concentration. As in the case of E. coli, it is known that in S. cerevisiae adenylate cyclase is dispensible in mutants of the cAMP receptor, and in S. pombe adenylate cyclase is dispensible during vegetative growth (11). But, as in this former case, cells that carry the mutation and are deficient in adenylate cyclase have several growth defects. In this respect, the function of the time-dependent cAMP pattern could be optimization of transient processes, in particular cell division and chromosome segregation.

In all these cases cAMP is recognized on the cell surface by a specific receptor. It is, therefore, interesting to identify cases where membrane targets of cAMP have been demonstrated (D. discoïdeum aside). Nerve cells typically generate and are sensitive to transient signals; they also have very involved patterns of adenylate and guanulate cyclase regulation. In this respect it is important to observe that cGMP (but also cAMP) has been shown to be involved as a central molecule in vision, taste, and olfaction. In particular, in addition to their role as second messengers in protein phosphorylation cascades, cyclic nucleotides are involved at the membrane surface, but intracellularly, in gating ion channels in olfactory and taste neurons. This certainly permits generation of a variety of time-dependent patterns for cAMP regulation, as a function of environmental inputs as well as of the fine molecular structure of the enzyme or its subunits (12, 13). Because ion channels are involved in the main functions of neurons (firing patterns), this makes cyclic nucleotides important in learning processes.

 Indeed, many experiments have demonstrated that cAMP is involved as a mediator of learning and memory in invertebrates [Aplysia (14) and Drosophila melanogaster (15)], as well as vertebrates (16, 17). The study of mutants of D. melanogaster that are defective in learning or memory has been of major importance in our understanding of the physiology, biochemistry, and anatomy underlying conditioned behaviors. D. melanogaster learning mutants have been separated into two general classes: those with structural defects in the brain and those without obvious brain alterations. From studies of mutants affected in the brain structure, two areas have been found to be involved in conditioned behavior: the mushroom bodies and the central complex. Analysis of the mutants has shown that many types of molecules are involved in learning, but the cAMP-mediated phosphorylation cascade has emerged as especially important. During learning, time-dependent processes are involved in the stabilization of synapses, a general view being that they are created during growth as transient entities that can either regress or be stabilized. In this process, the evolution of the synaptic pattern is dependent on the pattern of neurotransmitter delivery. Analysis of the minimal requirements for synapses stabilization suggests that neurotransmitter release must be coupled to some other transient metabolic process in a retrograde manner in order to yield a stable geometry (18). In the cases where cAMP is involved, one can, therefore, speculate that the role of this mediator is to trigger an appropriate biochemical process when the proper time-dependent control of its synthesis is at work (19). Accordingly, once again, it is not the cAMP concentration that is important, but, rather, the time-variation of its concentration. In the process of learning, the regulation of adenylate cyclase activity would, therefore, be exquisitely tuned to permit delivery of the molecule in the proper time-dependent manner.

In D. melanogaster, five different genes have proven important for normal learning: dunce (a cAMP phosophodiesterase), rutabaga (an adenylyl cyclase), amnesiac (a product similar to adenylate cyclase activating peptides), DCO (protein kinase A), and dCREB2 (a cAMP-response element binding protein). The products of many of these learning mutants are enriched in mushroom bodies. A process involving control of transcription by the cAMP response element binding protein (CREB)-responsive plays a central role in the formation of long-term memory in D. melanogaster, Aplysia and mammals. This is one of the examples where cAMP is involved in the control of transcription in eucaryotes, as it is in eubacteria, although through a different chain of events. Agents that prevent CREB activity interfere with the formation of long-term memory, whereas agents that increase the amount or activity of the transcription factor accelerate the process, thus indicating that CREB is essential for the switch from short-term memory to long-term memory (protein synthesis dependent)  (20) . Further work involving inbred mice strains as well as knock-out mutants affecting the hippocampal region demonstrated that both the genetic background and the temporal pattern of synaptic activity affects the cAMP-dependent synaptic plasticity (21).

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