In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors.

Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside of specialized cells, there are few methods with the appropriate spatial and temporal resolution to measure cyclic nucleotide concentrations. We have previously demonstrated that adenovirus-expressed, olfactory cyclic nucleotide-gated channels provide real-time sensors for cAMP produced in subcellular compartments of restricted diffusion near the plasma membrane (Rich, T.C., K.A. Fagan, H. Nakata, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 2000. J. Gen. Physiol. 116:147-161). To increase the utility of this method, we have modified the channel, increasing both its cAMP sensitivity and specificity, as well as removing regulation by Ca(2)+-calmodulin. We verified the increased sensitivity of these constructs in excised membrane patches, and in vivo by monitoring cAMP-induced Ca(2)+ influx through the channels in cell populations. The improved cAMP sensors were used to monitor changes in local cAMP concentration induced by adenylyl cyclase activators in the presence and absence of PDE inhibitors. This approach allowed us to identify localized PDE types in both nonexcitable HEK-293 and excitable GH4C1 cells. We have also developed a quantitative framework for estimating the K(I) of PDE inhibitors in vivo. The results indicate that PDE type IV regulates local cAMP levels in HEK-293 cells. In GH4C1 cells, inhibitors specific to PDE types I and IV increased local cAMP levels. The results suggest that in these cells PDE type IV has a high K(m) for cAMP, whereas PDE type I has a low K(m) for cAMP. Furthermore, in GH4C1 cells, basal adenylyl cyclase activity was readily observable after application of PDE type I inhibitors, indicating that there is a constant synthesis and hydrolysis of cAMP in subcellular compartments near the plasma membrane. Modulation of constitutively active adenylyl cyclase and PDE would allow for rapid control of cAMP-regulated processes such as cellular excitability.     

An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics

The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities.     

The Role of Type 4 Phosphodiesterases in Generating Microdomains of cAMP: Large Scale Stochastic Simulations

Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons.     

Cyclic nucleotide signaling changes associated with normal aging and age-related diseases of the brain

Deficits in brain function that are associated with aging and age-related diseases benefit very little from currently available therapies, suggesting a better understanding of the underlying molecular mechanisms is needed to develop improved drugs. Here, we review the literature to test the hypothesis that a break down in cyclic nucleotide signaling at the level of synthesis, execution, and/or degradation may contribute to these deficits. A number of findings have been reported in both the human and animal model literature that point to brain region-specific changes in Galphas (a.k.a. Gαs or Gsα), adenylyl cyclase, 3′,5′-adenosine monophosphate (cAMP) levels, protein kinase A (PKA), cAMP response element binding protein (CREB), exchange protein activated by cAMP (Epac), hyperpolarization-activated cyclic nucleotide-gated ion channels (HCNs), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), soluble and particulate guanylyl cyclase, 3′,5′-guanosine monophosphate (cGMP), protein kinase G (PKG) and phosphodiesterases (PDEs). Among the most reproducible findings are 1) elevated circulating ANP and BNP levels being associated with cognitive dysfunction or dementia independent of cardiovascular effects, 2) reduced basal and/or NMDA-stimulated cGMP levels in brain with aging or Alzheimer's disease (AD), 3) reduced adenylyl cyclase activity in hippocampus and specific cortical regions with aging or AD, 4) reduced expression/activity of PKA in temporal cortex and hippocampus with AD, 5) reduced phosphorylation of CREB in hippocampus with aging or AD, 6) reduced expression/activity of the PDE4 family in brain with aging, 7) reduced expression of PDE10A in the striatum with Huntington's disease (HD) or Parkinson's disease, and 8) beneficial effects of select PDE inhibitors, particularly PDE10 inhibitors in HD models and PDE4 and PDE5 inhibitors in aging and AD models. Although these findings generally point to a reduction in cyclic nucleotide signaling being associated with aging and age-related diseases, there are exceptions. In particular, there is evidence for increased cAMP signaling specifically in aged prefrontal cortex, AD cerebral vessels, and PD hippocampus. Thus, if cyclic nucleotide signaling is going to be targeted effectively for therapeutic gain, it will have to be manipulated in a brain region-specific manner.     

Assessment of cellular mechanisms contributing to cAMP compartmentalization in pulmonary microvascular endothelial cells

Cyclic AMP signals encode information required to differentially regulate a wide variety of cellular responses; yet it is not well understood how information is encrypted within these signals. An emerging concept is that compartmentalization underlies specificity within the cAMP signaling pathway. This concept is based on a series of observations indicating that cAMP levels are distinct in different regions of the cell. One such observation is that cAMP production at the plasma membrane increases pulmonary microvascular endothelial barrier integrity, whereas cAMP production in the cytosol disrupts barrier integrity. To better understand how cAMP signals might be compartmentalized, we have developed mathematical models in which cellular geometry as well as total adenylyl cyclase and phosphodiesterase activities were constrained to approximate values measured in pulmonary microvascular endothelial cells. These simulations suggest that the subcellular localizations of adenylyl cyclase and phosphodiesterase activities are by themselves insufficient to generate physiologically relevant cAMP gradients. Thus, the assembly of adenylyl cyclase, phosphodiesterase, and protein kinase A onto protein scaffolds is by itself unlikely to ensure signal specificity. Rather, our simulations suggest that reductions in the effective cAMP diffusion coefficient may facilitate the formation of substantial cAMP gradients. We conclude that reductions in the effective rate of cAMP diffusion due to buffers, structural impediments, and local changes in viscosity greatly facilitate the ability of signaling complexes to impart specificity within the cAMP signaling pathway.     

Cyclic AMP control measured in two compartments in HEK293 cells: phosphodiesterase K(M) is more important than phosphodiesterase localization

The intracellular second messenger cyclic AMP (cAMP) is degraded by phosphodiesterases (PDE). The knowledge of individual families and subtypes of PDEs is considerable, but how the different PDEs collaborate in the cell to control a cAMP signal is still not fully understood. In order to investigate compartmentalized cAMP signaling, we have generated a membrane-targeted variant of the cAMP Bioluminiscence Resonance Energy Transfer (BRET) sensor CAMYEL and have compared intracellular cAMP measurements with it to measurements with the cytosolic BRET sensor CAMYEL in HEK293 cells. With these sensors we observed a slightly higher cAMP response to adenylyl cyclase activation at the plasma membrane compared to the cytosol, which is in accordance with earlier results from Fluorescence Resonance Energy Transfer (FRET) sensors. We have analyzed PDE activity in fractionated lysates from HEK293 cells using selective PDE inhibitors and have identified PDE3 and PDE10A as the major membrane-bound PDEs and PDE4 as the major cytosolic PDE. Inhibition of membrane-bound or cytosolic PDEs can potentiate the cAMP response to adenylyl cyclase activation, but we see no significant difference between the potentiation of the cAMP response at the plasma membrane and in cytosol when membrane-bound and cytosolic PDEs are inhibited. When different levels of stimulation were tested, we found that PDEs 3 and 10 are mainly responsible for cAMP degradation at low intracellular cAMP concentrations, whereas PDE4 is more important for control of cAMP at higher concentrations.     

Search for new cyclic AMP-binding proteins

Today, there is evidence that the cAMP-dependent kinases (PKA) are not the only intracellular receptors involved in intracellular cAMP signalling in eukaryotes. Other cAMP-binding proteins have been recently identified, including some cyclic nucleotide-gated channels and Epac (exchange protein directly activated by cAMP) proteins. All these proteins bind cAMP through conserved cyclic nucleotide monophosphate-binding domains. However, all putative cAMP-binding proteins having such domains, as revealed by computer analysis, do not necessarily bind cAMP, indicating that their presence is not a sufficient criteria to predict cAMP-binding property for a protein.     

Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP

Understanding how specific cyclic AMP (cAMP) signals are organized and relayed to their effectors in different compartments of the cell to achieve functional specificity requires molecular tools that allow precise manipulation of cAMP in these compartments. Here we characterize a new method using bicarbonate-activatable and genetically targetable soluble adenylyl cyclase to control the location, kinetics and magnitude of the cAMP signal. Using this live-cell cAMP manipulation in conjunction with fluorescence imaging and mechanistic modeling, we uncovered the activation of a resident pool of protein kinase A (PKA) holoenzyme in the nuclei of HEK-293 cells, modifying the existing dogma of cAMP-PKA signaling in the nucleus. Furthermore, we show that phosphodiesterases and A-kinase anchoring proteins (AKAPs) are critical in shaping nuclear PKA responses. Collectively, our data suggest a new model in which AKAP-localized phosphodiesterases tune an activation threshold for nuclear PKA holoenzyme, thereby converting spatially distinct second messenger signals to temporally controlled nuclear kinase activity.     

Cellular mechanisms underlying prostaglandin-induced transient cAMP signals near the plasma membrane of HEK-293 cells

We have previously used cyclic nucleotide-gated (CNG) channels as sensors to measure cAMP signals in human embryonic kidney (HEK)-293 cells. We found that prostaglandin E₁ (PGE₁) triggered transient increases in cAMP concentration near the plasma membrane, whereas total cAMP levels rose to a steady plateau over the same time course. In addition, we presented evidence that the decline in the near-membrane cAMP levels was due primarily to a PGE₁-induced stimulation of phosphodiesterase (PDE) activity, and that the differences between near-membrane and total cAMP levels were largely due to diffusional barriers and differential PDE activity. Here, we examine the mechanisms regulating transient, near-membrane cAMP signals. We observed that 5-min stimulation of HEK-293 cells with prostaglandins triggered a two- to threefold increase in PDE4 activity. Extracellular application of H89 (a PKA inhibitor) inhibited stimulation of PDE4 activity. Similarly, when we used CNG channels to monitor cAMP signals we found that both extracellular and intracellular (via the whole-cell patch pipette) application of H89, or the highly selective PKA inhibitor, PKI, prevented the decline in prostaglandin-induced responses. Following pretreatment with rolipram (a PDE4 inhibitor), H89 had little or no effect on near-membrane or total cAMP levels. Furthermore, disrupting the subcellular localization of PKA with the A-kinase anchoring protein (AKAP) disruptor Ht31 prevented the decline in the transient response. Based on these data we developed a plausible kinetic model that describes prostaglandin-induced cAMP signals. This model has allowed us to quantitatively demonstrate the importance of PKA-mediated stimulation of PDE4 activity in shaping near-membrane cAMP signals. G protein signaling; protein kinase A; phosphodiesterase; A-kinase anchoring protein; CNG channel     

Selective permeability of different connexin channels to the second messenger cyclic AMP

Gap junctions are intercellular conduits that are formed in vertebrates by connexin proteins and allow diffusion exchange of intracellular ions and small molecules. At least 20 different connexin genes in the human and mouse genome are cell-type specifically expressed with overlapping expression patterns. A possible explanation for this diversity could be different permeability of biologically important molecules, such as second messenger molecules. We have recently demonstrated that cyclic nucleotide-gated channels can be used to quantify gap junction-mediated diffusion of cyclic AMP. Using this method we have compared the relative permeability of gap junction channels composed of connexin 26, 32, 36, 43, 45, or 47 proteins toward the second messenger cAMP. Here we show that cAMP permeates through the investigated connexin channels with up to 30-fold different efficacy. Our results suggest that intercellular cAMP signaling in different cell types can be affected by the connexin expression pattern.     

Colocalization of protein kinase A with adenylyl cyclase enhances protein kinase A activity during induction of long-lasting long-term-potentiation

The ability of neurons to differentially respond to specific temporal and spatial input patterns underlies information storage in neural circuits. One means of achieving spatial specificity is to restrict signaling molecules to particular subcellular compartments using anchoring molecules such as A-Kinase Anchoring Proteins (AKAPs). Disruption of protein kinase A (PKA) anchoring to AKAPs impairs a PKA-dependent form of long term potentiation (LTP) in the hippocampus. To investigate the role of localized PKA signaling in LTP, we developed a stochastic reaction-diffusion model of the signaling pathways leading to PKA activation in CA1 pyramidal neurons. Simulations investigated whether the role of anchoring is to locate kinases near molecules that activate them, or near their target molecules. The results show that anchoring PKA with adenylyl cyclase (which produces cAMP that activates PKA) produces significantly greater PKA activity, and phosphorylation of both inhibitor-1 and AMPA receptor GluR1 subunit on S845, than when PKA is anchored apart from adenylyl cyclase. The spatial microdomain of cAMP was smaller than that of PKA suggesting that anchoring PKA near its source of cAMP is critical because inactivation by phosphodiesterase limits diffusion of cAMP. The prediction that the role of anchoring is to colocalize PKA near adenylyl cyclase was confirmed by experimentally rescuing the deficit in LTP produced by disruption of PKA anchoring using phosphodiesterase inhibitors. Additional experiments confirm the model prediction that disruption of anchoring impairs S845 phosphorylation produced by forskolin-induced synaptic potentiation. Collectively, these results show that locating PKA near adenylyl cyclase is a critical function of anchoring.     

Cyclic AMP Levels,Adenylyl Cyclase Activity,and Their Stimulation by Serotonin Quantif?ied in Intact Neurons

In molluscan central neurons that express cAMP-gated Na⁺ current (I_Na,cAMP), estimates of the cAMP binding affinity of the channels have suggested that effective native intracellular cAMP concentrations should be much higher than characteristic of most cells. Using neurons of the marine opisthobranch snail Pleurobranchaea californica, we applied theory and conventional voltage clamp techniques to use I_Na,cAMP to report basal levels of endogenous cAMP and adenylyl cyclase, and their stimulation by serotonin. Measurements were calibrated to iontophoretic cAMP injection currents to enable expression of the data in molar terms. In 30 neurons, serotonin stimulated on average a 23-fold increase in submembrane [cAMP], effected largely by an 18-fold increase in adenylyl cyclase activity. Serotonin stimulation of adenylyl cyclase and [cAMP] was inversely proportional to cells'' resting adenylyl cyclase activity. Average cAMP concentration at the membrane rose from 3.6 to 27.6 μM, levels consistent with the expected cAMP dissociation constants of the I_Na,cAMP channels. These measures confirm the functional character of I_Na,cAMP in the context of high levels of native cAMP. Methods similar to those employed here might be used to establish critical characters of cyclic nucleotide metabolism in the many cells of invertebrates and vertebrates that are being found to express ion currents gated by direct binding of cyclic nucleotides.     

Roles of GRK and PDE4 activities in the regulation of beta2 adrenergic signaling

An important focus in cell biology is understanding how different feedback mechanisms regulate G protein-coupled receptor systems. Toward this end we investigated the regulation of endogenous beta(2) adrenergic receptors (beta2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 muM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 muM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 muM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 muM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of beta2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of beta2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system.     

Two domains are critical for the nuclear localization of soluble adenylyl cyclase

Soluble adenylyl cyclase (sAC) is a newly identified source of cyclic adenosine 3',5'-monophosphate (cAMP). Unlike the well-known transmembrane adenylyl cyclases (tmACs), sAC locates to the nucleus, mitochondria and microtubules. For most cAMP-signaling microdomains, there is always an AC nearby, for example tmAC. But it was until the discovery of sAC that there was not known cAMP resource in the nucleus. sAC associates with nuclear cAMP-signaling microdomains, which were once considered to depend on the diffusion of cAMP produced by tmAC. In this report, we focus on the truncated soluble adenylyl cyclase (tsAC), the most common existence form of sAC in tissues. Two domains (145-200 aa and 257-318 aa) related with sAC nuclear localization were present here. The findings provide evidence that these two domains are critical for the nuclear localization of sAC and they collocated with the catalytic domains.     

Requirements for the adenylyl cyclases in the development of Dictyostelium

It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high.     

Trafficking of cholera toxin-ganglioside GM1 complex into Golgi and induction of toxicity depend on actin cytoskeleton

Intestinal epithelial lipid rafts contain ganglioside G_M1 that is the receptor for cholera toxin (CT). The ganglioside binds CT at the plasma membrane (PM) and carries the toxin through the trans-Golgi network (TGN) to the endoplasmic reticulum (ER). In the ER, a portion of the toxin unfolds and translocates to the cytosol to activate adenylyl cyclase. Activation of the cyclase leads to an increase in intracellular cAMP, which results in apical chloride secretion. Here, we find that an intact actin cytoskeleton is necessary for the efficient transport of CT to the Golgi and for subsequent activation of adenylyl cyclase. CT bound to G_M1 on the cell membrane fractionates with a heterogeneous population of lipid rafts, a portion of which is enriched in actin and other cytoskeletal proteins. In this actin-rich fraction of lipid rafts, CT and actin colocalize on the same membrane microdomains, suggesting a possible functional association. Depolymerization or stabilization of actin filaments interferes with transport of CT from the PM to the Golgi and reduces the levels of cAMP generated in the cytosol. Depletion of membrane cholesterol, which also inhibits CT trafficking to the TGN, causes displacement of actin from the lipid rafts while CT remains stably raft associated. On the basis of these observations, we propose that the CT-G_M1 complex is associated with the actin cytoskeleton via the lipid rafts and that the actin cytoskeleton plays a role in trafficking of CT from the PM to the Golgi/ER and the subsequent activation of adenylyl cyclase. membrane microdomains; membrane lipids; bacterial toxins; endocytosis; intestinal mucosa     

Genome-wide expression analyses of gene regulation during early development of Dictyostelium discoideum

Using genome-wide microarrays, we recognized 172 genes that are highly expressed at one stage or another during multicellular development of Dictyostelium discoideum. When developed in shaken suspension, 125 of these genes were expressed if the cells were treated with cyclic AMP (cAMP) pulses at 6-min intervals between 2 and 6 h of development followed by high levels of exogenous cAMP. In the absence of cAMP treatment, only three genes, carA, gbaB, and pdsA, were consistently expressed. Surprisingly, 14 other genes were induced by cAMP treatment of mutant cells lacking the activatable adenylyl cyclase, ACA. However, these genes were not cAMP induced if both of the developmental adenylyl cyclases, ACA and ACR, were disrupted, showing that they depend on an internal source of cAMP. Constitutive activity of the cAMP-dependent protein kinase PKA was found to bypass the requirement of these genes for adenylyl cyclase and cAMP pulses, demonstrating the critical role of PKA in transducing the cAMP signal to early gene expression. In the absence of constitutive PKA activity, expression of later genes was strictly dependent on ACA in pulsed cells.     

Role of Membrane Microdomains in Compartmentation of cAMP Signaling

Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.     

Regulation of adenylyl cyclase, ERK1/2, and CREB by Gz following acute and chronic activation of the delta-opioid receptor

Opioid tolerance and physical dependence in mammals can be rapidly induced by chronic exposure to opioid agonists. Recently, opioid receptors have been shown to interact with the pertussis toxin (PTX)-insensitive Gz (a member of the Gi subfamily), which inhibits adenylyl cyclase and stimulates mitogen-activated protein kinases (MAPKs). Here, we established stable human embryonic kidney 293 cell lines expressing delta-opioid receptors with or without Gz to examine the role of Gz in opioid receptor-regulated signaling systems. Each cell line was acutely or chronically treated with [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta-selective agonist, in the absence or presence of PTX. Subsequently, the activities of adenylyl cyclase, cyclic AMP (cAMP)-dependent response element-binding proteins (CREBs), and MAPKs were measured by determining cAMP accumulation and phosphorylation of CREBs and the extracellular signal-regulated protein kinases (ERKs) 1 and 2. In cells coexpressing Gz, DPDPE inhibited forskolin-stimulated cAMP accumulation in a PTX-insensitive manner, but Gz could not replace Gi to mediate adenylyl cyclase supersensitization upon chronic opioid treatment. DPDPE-induced adenylyl cyclase supersensitization was not associated with an increase in the phosphorylation of CREBs. Both Gi and Gz mediated DPDPE-induced activation of ERK1/2, but these responses were abolished by chronic opioid treatment. Collectively, our results show that although Gz mediated opioid-induced inhibition of adenylyl cyclase and activation of ERK1/2, Gz alone was insufficient to mediate opioid-induced adenylyl cyclase supersensitization.