ABSTACT Cell surface adenosine receptors mediate either stimulation or inhibition of adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], and the receptors that mediate these different responses can be discriminated with selected adenosine analogs. 5'-N-Ethylcarboxamideadenosine is a more potent agonist at stimulatory receptors (Ra) than is N8-phenylisopropyladenosine, whereas the reverse potency order is seen with inhibitory receptors (Ri). The potency of adenosine is intermediate between the potencies of these two analogs. The relative potencies of adenosine receptor agonists are maintained in physiological responses in intact cells, such as steroidogenesis and inhibition of lipolysis. As with adrenergic receptors, subelasses of adenosine receptors differ functionally and pharmacologically. Adenosine modifies the physiological function and cyclic AMP concentration in a large variety of cell types by interacting with external receptors (1-3), the basic properties of which were described by Sattin and Rall (4). In plasma membrane preparations from many different cell types, adenosine and several purine-modified analogs stimulate adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (1-5), whereas in adipocyte membranes the adenosine receptor mediates decreased activity (1,3,6). Previously published studies suggested that, despite their superficial similarities, stimulatory and inhibitory adenosine receptors might differ. Thus, whereas N6-phenylisopropyladenosine (PIA) and N6-methyladenosine were equipotent in stimulating the Leydig tumor cell adenylate cyclase (5), the former analog was far more potent that N6-methyladenosine in inhibiting the adipocyte adenylate cyclase (7). However, the fat cell studies were performed in the presence of adenosine deaminase, added to metabolize adenosine resulting from breakdown of the substrate, ATP. In this case, the analogs might have acted either at the receptor or by inhibiting the adenosine deaminase, which has a rather broad substrate specificity (8). Another method to circumvent interference from "intrinsic" adenosine is the use of dATP as the cyclase substrate (9). Metabolism of this substrate yields 2'-deoxyadenosine, which has little activity at adenosine receptors. In this report we present a pharmacological investigation of stimulatory and inhibitory receptors associated with adenylate cyclases, using dATP as substrate in the absence of adenosine deaminase. From a screening of numerous adenosine analogs we have selected two that demonstrate the existence of subclasses of adenosine receptors: PIA and 5'-N-ethylcarboxamideadenosine (NECA). The relative potencies of the analogs in the adenylate cyclase studies are maintained in physiological studies in intact cells.* Fig. 1 presents a comparison of the concentration dependencies of adenosine, PIA, and NECA in their actions on three adenylate cyclase systems: liver and 1-10 Leydig cell enzymes, which are activated by adenosine, and rat adipocyte enzyme, which is inhibit...

Adenylyl cyclase is the prototypical second messenger generator. Nearly all of the eight cloned adenylyl cyclases are regulated by one or other arm of the phospholipase C pathway. Functional and ultrastructural investigations have shown that adenylyl cyclases are intimately associated with sites of calcium ion entry into the cell. Oscillations in cellular cyclic AMP levels are predicted to arise because of feedback inhibition of adenylyl cyclase by Ca2+. Such findings inextricably intertwine cellular signalling by cAMP and internal Ca2+ and extend the known regulatory modes available to cAMP.

The adenylyl cyclases are variously regulated by G protein subunits, a number of serine/threonine and tyrosine protein kinases, and Ca(2+). In some physiological situations, this regulation can be readily incorporated into a hormonal cascade, controlling processes such as cardiac contractility or neurotransmitter release. However, the significance of some modes of regulation is obscure and is likely only to be apparent in explicit cellular contexts (or stages of the cell cycle). The regulation of many of the ACs by the ubiquitous second messenger Ca(2+) provides an overarching mechanism for integrating the activities of these two major signaling systems. Elaborate devices have been evolved to ensure that this interaction occurs, to guarantee the fidelity of the interaction, and to insulate the microenvironment in which it occurs. Subcellular targeting, as well as a variety of scaffolding devices, is used to promote interaction of the ACs with specific signaling proteins and regulatory factors to generate privileged domains for cAMP signaling. A direct consequence of this organization is that cAMP will exhibit distinct kinetics in discrete cellular domains. A variety of means are now available to study cAMP in these domains and to dissect their components in real time in live cells. These topics are explored within the present review.

Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca2+, cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotide–gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory α subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotide–gated channels colocalize with adenylyl cyclase in microdomains, and diffusion of cAMP between these domains and the bulk cytosol is significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 22–56 s for substances that monitor exchange with the cytosol). Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotide–gated channels, unless the entire cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotide–gated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signals should also facilitate the differential regulation of cellular targets.

Adenylyl cyclases are a critically important family of multiply regulated signalling molecules. Their susceptibility to many modes of regulation allows them to integrate the activities of a variety of signalling pathways. However, this property brings with it the problem of imparting specificity and discrimination. Recent studies are revealing the range of strategies utilized by the cyclases to solve this problem. Microdomains are a consequence of these solutions, in which cAMP dynamics may differ from the broad cytosol. Currently evolving methodologies are beginning to reveal cAMP fluctuations in these various compartments.