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.

cAMP, the classical second messenger, regulates many diverse cellular functions. The primary effector of cAMP signals, protein kinase A, differentially phosphorylates hundreds of cellular targets. Little is known, however, about the spatial and temporal nature of cAMP signals and their information content. Thus, it is largely unclear how cAMP, in response to different stimuli, orchestrates such a wide variety of cellular responses. Previously, we presented evidence that cAMP is produced in subcellular compartments near the plasma membrane, and that diffusion of cAMP from these compartments to the bulk cytosol is hindered. Here we report that a uniform extracellular stimulus initiates distinct cAMP signals within different cellular compartments. By using cyclic nucleotide-gated ion channels engineered as cAMP biosensors, we found that prostaglandin E 1 stimulation of human embryonic kidney cells caused a transient increase in cAMP concentration near the membrane. Interestingly, in the same time frame, the total cellular cAMP rose to a steady level. The decline in cAMP levels near the membrane was prevented by pretreatment with phosphodiesterase inhibitors. These data demonstrate that spatially and temporally distinct cAMP signals can coexist within simple cells.

No abstract

Ca2ϩ -sensitive adenylyl cyclases provide a key point for integration of signaling by [Ca 2ϩ ] i 1 and cAMP (1). Their likely contribution to cellular regulation is underscored by the fact that whether they are expressed heterologously or endogenously, these cyclases are regulated by physiological transitions in [Ca 2ϩ ] i (2-9). Somewhat unexpectedly, the Ca 2ϩ -sensitive adenylyl cyclases, whether they are expressed endogenously or heterologously, show a preference for regulation by Ca 2ϩ entering the cell over Ca 2ϩ released from intracellular stores (7, 10). Even more strikingly, Ca 2ϩ entry promoted by ionophore is unable to regulate transfected Ca 2ϩ -stimulable adenylyl cyclases (7). Consequently, we had proposed that Ca 2ϩ -stimulable adenylyl cyclases and capacitative Ca 2ϩ entry channels (I CRAC s) 2 were functionally colocalized (7). However, it is always conceivable that when they are transfected, adenylyl cyclases are expressed in discrete cellular domains, which reflects the response of the cell to overexpression of signaling molecules. It is therefore of considerable interest to determine whether similar colocalization is encountered with endogenously expressed adenylyl cyclase in continuous cell lines, which are more appropriate models of a normal signaling repertoire. Previous studies have established that the endogenous Ca 2ϩ -inhibitable adenylyl cyclase, which is the predominant form in C6 -2B glioma cells (11), is also regulated by the entry of Ca 2ϩ rather than release from intracellular stores, which was triggered by a variety of treatments (10). However, it is not known whether such endogenous adenylyl cyclases show a similar absolute dependence on capacitative versus any other form of entry. Such a requirement would predict a close association between entry sites and the cyclases. The potential intimacy of an endogenous Ca 2ϩ -inhibitable adenylyl cyclase and Ca 2ϩ entry channels is explored in the present study by assessing (i) the sensitivity of the cyclase to various types of [Ca 2ϩ ] i rise, (ii) whether this action can be differentially modulated by fast versus slow chelators of Ca 2ϩ , (iii) the role of the cytoskeleton, and (iv) whether the simple activity of the I CRAC , independent of the ion being transported, can modulate the enzyme. Cell Culture-C6 -2B rat glioma cells were maintained in 13 ml of F-10 medium (Life Technologies, Inc.) with 10% (v/v) bovine calf serum (Gemini) in 75-cm 2 flasks at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . Cells were plated at approximately 70% confluency in 24-well plates for cAMP accumulation experiments. EXPERIMENTAL PROCEDURES MaterialsMeasurement of cAMP Accumulation-cAMP accumulation in intact cells was measured according to the method of Evans et al. (12) as described previously (7) with some modifications. C6 -2B cells on 24-well plates were incubated in F-10 medium (60 min at 37°C) with [2-3 H] adenine (1.5 Ci/well) to label the ATP pool. The cells were then washed once and incubated with a nominally Ca 2ϩ -free Kre...

Endothelial nitric-oxide synthase (eNOS), a Ca2؉ /calmodulin-dependent enzyme, is critical for vascular homeostasis. While eNOS is membrane-associated through its N-myristoylation, the significance of membrane association in locating eNOS near sources of Ca 2؉ entry is uncertain. To assess the Ca 2؉ source required for eNOS activation, chimera containing the full-length eNOS cDNA and HA-tagged aequorin sequence (EHA), and MHA (myristoylation-deficient EHA) were generated and transfected into COS-7 cells. The EHA chimera was primarily targeted to the plasma membrane while MHA was located intracellularly. Both constructs retained enzymatic eNOS activity and aequorin-mediated Ca 2؉ sensitivity. The plasma membrane-associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca 2؉ concentration, demonstrating preferential sensitivity to Ca 2؉ originating from intracellular pools (MHA) or from capacitative Ca 2؉ entry (EHA). Measurements of eNOS activation in intact cells revealed that the eNOS enzymatic activity of EHA was more sensitive to Ca 2؉ influx via capacitative Ca 2؉ entry than intracellular release, whereas MHA eNOS activity was more responsive to intracellular Ca 2؉ release. When eNOS activation by CCE was compared with that generated by an equal rise in [Ca 2؉ ] i due to the Ca 2؉ ionophore ionomycin, a 10-fold greater increase in NO production was found in the former condition. These results demonstrate that EHA and MHA chimera are properly targeted and retain full functions of eNOS and aequorin, and that capacitative Ca 2؉ influx is the principle stimulus for sustained activation of eNOS on the plasma membrane in intact cells.