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Recent evidence indicates that ethanol modulates the function of specific intracellular signaling cascades, including those that contain cyclic adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase A (PKA), protein kinase C (PKC), the tyrosine kinase Fyn, and phospholipase D (PLD). In some cases, the specific components of these cascades appear to mediate the effects of ethanol, whereas other components indirectly modify responses to ethanol. Studies utilizing selective inhibitors and genetically modified mice have identified specific isoforms of proteins involved in responses to ethanol. The effects of ethanol on neuronal signaling appear restricted to certain brain regions, partly due to the restricted distribution of these proteins. This likely contributes specificity to ethanol's actions on behavior. This review summarizes recent work on ethanol and intracellular signal transduction, emphasizing studies that have identified specific molecular events that underlie behavioral responses to ethanol.

The past decade has produced an explosion of new information on the development, neuroanatomy, and possible functions of the mushroom bodies. This review provides a concise, contemporary overview of the structure of the mushroom bodies. Two topics are highlighted: the volume plasticity of mushroom body neuropils evident in the brains of some adult insects and a possible essential role for the gamma lobe in olfactory memory.

Objectives: Most dental resinous materials contain the diluent monomer triethyleneglycol dimethacrylate (TEGDMA), which has been reported to be bioactive. Previously, it was demonstrated that TEGDMA induces vasorelaxation. The present study examines the mechanism(s) of the TEGDMA-induced vasorelaxation by measuring vascular nitrite and prostacyclin levels., Methods: Nitrite and prostacyclin levels were assayed in rat aortic tissues in response to TEGDMA. The involvement of guanylyl and adenylyl cyclases in TEGDMA-induced aortic vasorelaxation was determined using the enzyme inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), respectively., Results: TEGDMA enhanced the levels of nitrites in endothelium-intact and that of protacyclin in both endothelium-intact and denuded rat aortas. The increase in nitrites was associated with endothelium-dependent aortic relaxation mediated via the activation of guanylyl cyclase, while the increase in prostacyclin was associated with both endothelium-dependent and independent relaxation linked to adenylyl cyclase stimulation., Significance: Data from the present investigation can be relevant to dental practice employing materials containing TEGDMA by providing insights into the vasorelaxant effect of the monomer following placement of the materials in the oral cavity. Additional studies that are more relevant to the clinical situation are required to confirm these initial results and further explore their implications.

Receptor- and ion channel-coupled signal transduction mechanisms are downstream communication processes used by regulatory molecules to modulate the essential cell processes of growth, differentiation and survival. Knowledge of signal transduction processes has dramatically increased in the past decade, and the basic principles of intracellular signaling are now quite well established. Cell signaling in higher organisms is a major, highly complex, phenomena that occupies a central position in current biomedical research. The complex machinery of intracellular signaling also has the potential to provide a wealth of novel drug discovery targets, from protein kinases, adaptor proteins, lipases, and cytoskeletal proteins, to nuclear effectors. This overview describes common features of cellular signaling pathways, including their interactions and responses to environmental stimuli. In particular, the overview focuses on the regulation of signaling pathways by protein functional-domain interactions as well as the intracellular proteins that mediate signal transduction.

Vascular complications, including impaired contractility and increased cell proliferation, are the most common complications with diabetes. Chronic hyperglycemia seems to be an important contributing factor in this process. Various signaling pathways are implicated in diabetes/hyperglycemia-induced impaired vascular functions. Nonenzymatic glycation, enhanced production of diacylglycerol, increased activity of membranous protein kinase C (PKC), and increased oxidative stress have been proposed to explain the adverse effects of hyperglycemia on vascular smooth muscle cells. Hyperglycemia-induced stimulation of L-type Ca2+ channel via G protein-coupled adenylyl cyclase/cAMP and phospholipase C/PKC pathways also has been shown. In addition, hyperglycemia has been reported to decrease the availability of nitric oxide in humans, which may contribute to all the hemodynamic and physiological changes occurring in diabetes. G protein-adenylyl cyclase signaling that plays an important role in the regulation of cardiovascular functions also has been reported to be impaired in diabetes and under hyperglycemic conditions. In this review article, various G protein-linked cell signaling and functions in diabetes and hyperglycemia are discussed.

Memories can have different strengths, largely dependent on the intensity of reinforcers encountered. The relationship between reinforcement and memory strength is evident in asymptotic memory curves, with the level of the asymptote related to the intensity of the reinforcer. Although this is likely a fundamental property of memory formation, relatively little is known of how memory strength is determined. Memory performance at different levels in Drosophila can be measured in an operant heat-box conditioning paradigm. In this spatial learning paradigm, flies learn and remember to avoid one-half of a dark chamber associated with a temperature outside of the preferred range. The reinforcement temperature has a strong effect on the level of learning in wild-type flies, with higher temperatures inducing stronger memories. Additionally, two mutations alter memory-acquisition curves, either changing acquisition rate or asymptotic memory level. The rutabaga mutation, affecting a type-1 adenylyl cyclase, decreases the acquisition rate. In contrast, the white mutation, modifying an ABC transporter, limits asymptotic memory. The white mutation does not negatively affect classical olfactory conditioning but actually improves performance at low reinforcement levels. Thus, memory acquisition/memory strength and classical olfactory/operant spatial memories can be genetically dissociated. A conceptual model of operant conditioning and the levels at which rutabaga and white influence conditioning is proposed.

Spinal NMDA receptor (NMDAR), protein kinase C (PKC), and glucocorticoid receptor (GR) have all been implicated in the mechanisms of morphine tolerance; however, how these cellular elements interact after chronic morphine exposure remains unclear. Here we show that the expression of spinal NMDAR and PKCgamma after chronic morphine is regulated by spinal GR through a cAMP response element-binding protein (CREB)-dependent pathway. Chronic morphine (10 microg, i.t.; twice daily for 6 d) induced a time-dependent upregulation of GR, the NR1 subunit of NMDAR, and PKCgamma within the rat's spinal cord dorsal horn. This NR1 and PKCgamma upregulation was significantly diminished by intrathecal coadministration of morphine with the GR antagonist RU38486 or a GR antisense oligodeoxynucleotide. Intrathecal coadministration of morphine with an adenylyl cyclase inhibitor (2',5'-dideoxyadenosine) or a protein kinase A inhibitor (H89) also significantly attenuated morphine-induced NR1 and PKCgamma expression, whereas intrathecal treatment with an adenylyl cyclase activator (forskolin) alone mimicked morphine-induced expression of GR, NR1, and PKCgamma. Moreover, the expression of phosphorylated CREB was upregulated within the spinal cord dorsal horn after chronic morphine, and a CREB antisense oligodeoxynucleotide coadministered intrathecally with morphine prevented the upregulation of GR, NR1, and PKCgamma. These results indicate that spinal GR through the cAMP-CREB pathway played a significant role in NMDAR and PKCgamma expression after chronic morphine exposure. The data suggest that genomic interaction among spinal GR, NMDAR, and PKCgamma may be an important mechanism that contributes to the development of morphine tolerance.

The rhythmic nocturnal production of melatonin in pineal glands is controlled by the periodic release of norepinephrine from the superior cervical ganglion. Norepinephrine binds to the beta-adrenergic receptor and stimulates an increase in intracellular cAMP levels, leading to the transcriptional activation of serotonin N-acetyltransferase, which in turn promotes melatonin production. In the present study, we report that bradykinin inhibits basal- and forskolin-stimulated adenylyl cyclase activity, norepinephrine-induced cAMP generation, and N-acetyltransferase expression in a calcium-dependent manner. These effects were blocked by pretreatment with U73122 (a selective phospholipase C inhibitor), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (a Ca(2+) chelator), but not pertussis toxin. The calcium ionophore, ionomycin, inhibited isoproterenol-mediated cAMP generation, similar to bradykinin. Interestingly, the inhibitory effect of bradykinin was evident only during the daytime. At midday, bradykinin inhibited the cAMP level by approximately 50% but markedly stimulated cAMP production (by approximately 50%) at night. Northern blotting and immunoblotting data disclosed circadian expression of calcium-inhibitable adenylyl cyclase type 6. Expression of adenylyl cyclase type 6 was maximal at Zeitgeber Time (ZT) 01 and very low at ZT 13. Our results suggest that bradykinin-induced calcium inhibits melatonin synthesis through the mediation of adenylyl cyclase type 6 expression.

The binding between an enzyme and its substrate is highly specific, despite the fact that many different enzymes show significant sequence and structure similarity. There must be, then, substrate specificity-determining residues that enable different enzymes to recognize their unique substrates. We reason that a coordinated, not independent, action of both conserved and non-conserved residues determine enzymatic activity and specificity. Here, we present a surface patch ranking (SPR) method for in silico discovery of substrate specificity-determining residue clusters by exploring both sequence conservation and correlated mutations. As case studies we apply SPR to several highly homologous enzymatic protein pairs, such as guanylyl versus adenylyl cyclases, lactate versus malate dehydrogenases, and trypsin versus chymotrypsin. Without using experimental data, we predict several single and multi-residue clusters that are consistent with previous mutagenesis experimental results. Most single-residue clusters are directly involved in enzyme-substrate interactions, whereas multi-residue clusters are vital for domain-domain and regulator-enzyme interactions, indicating their complementary role in specificity determination. These results demonstrate that SPR may help the selection of target residues for mutagenesis experiments and, thus, focus rational drug design, protein engineering, and functional annotation to the relevant regions of a protein.

In several cases of immunodeficiency and autoimmunity, the dysfunctional immune system is associated with either hypo- or hyperactive T and B cells. In autoimmune conditions such as systemic lupus erythematosus (SLE) and immunodeficiencies such as acquired immunodeficiency syndrome (AIDS), it has been demonstrated that the regulatory effect of the signaling pathway of cyclic 3', 5' adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) is abrogated. PKA is well-known as a key regulator of immune responses in that it inhibits both early and late phases of antigen induced T and B cell activation. Here we will discuss a potential useful strategy for therapeutic interventions of dysfunctional T cells associated with SLE and HIV by modulation of the cAMP-PKA pathway. Therefore, we will describe the components and architecture of the cAMP-PKA signaling pathway in T cells in order to point out one or several steps which potentially may serve as targets for therapeutic intervention.

We demonstrated changes in the sensitivity of the adenylate cyclase signaling system to biogenic amines (adrenoceptor agonists and serotonin) underwent a change in skeletal muscles of rats with 30-day streptozotocin-induced diabetes. Isoproterenol had a less significant stimulatory effect on adenylate cyclase in diabetic rats. Hormonal signals via Gi proteins were suppressed in animals with diabetes, which determined a greater stimulatory effect of norepinephrine and serotonin on adenylate cyclase. Hormones less significantly increased guanosine triphosphate-binding activity of G proteins in diabetic rats, which reflects the impairment of their functional coupling with receptors.

Physiological activation of kainate receptors and GABA(B) receptors within the pedunculopontine tegmentum (PPT) is involved in regulation of rapid-eye-movement (REM) sleep. Because these two types of receptors may also directly and/or indirectly activate the intracellular cyclic adenosine monophosphate (cAMP) signaling pathway, we hypothesized that this signaling pathway may be involved in the PPT to regulate spontaneous REM sleep. To test this hypothesis, four different doses (0.25, 0.50, 0.75, and 1.0 nmol) of a specific adenylyl cyclase (AC) inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), were microinjected bilaterally (100 nl/site) into the PPT, and the effects on REM sleep in freely moving chronically instrumented rats were quantified. By comparing alterations in the patterns of REM sleep after control injections of vehicle or one of the four different doses of SQ22536, the contributions made by each dose of SQ22536 to REM sleep were evaluated. The results demonstrated that the local microinjection of AC inhibitor SQ22536 into the PPT decreased the total amount of REM sleep for 3 h and increased slow-wave sleep (SWS) for 2 h in a dose-dependent manner. This reduction in REM sleep was due to increased latency and decreased frequency of REM sleep episodes. These results provide evidence that inhibition of AC within the PPT can successfully reduce REM sleep. These findings suggest that activation of the cAMP-signaling pathway within the cholinergic cell compartment of the PPT is an intracellular biochemical/molecular step for generating REM sleep in the freely moving rat.

Oedema factor (OF) and protective antigen (PA) are secreted by Bacillus anthracis, and their binary combination yields oedema toxin (OT). Following PA-mediated delivery to the cytosol, OF functions as an adenylate cyclase generating high levels of cAMP. To assess OT as a possible cause of tissue damage and cell death, a novel approach was developed, which utilized a developing zebrafish embryo model to study toxin activity. Zebrafish embryos incubated with OT exhibited marked necrosis of the liver, cranium and gastrointestinal tract, as well as reduced swim bladder inflation. The OT-treated embryos survived after all stages of development but succumbed to the toxin within 7 days. Additional analysis of specific cell lines, including macrophage and non-macrophage, showed OT-induced cell death is cell type-specific. There was no discernible correlation between levels of OF-generated cAMP and cell death. Depending on the type of cell analysed, cell death could be detected in low levels of cAMP, and, conversely, cell survival was observed in one cell line in which high levels of cAMP were found following treatment with OT. Collectively, these data suggest OT is cytotoxic in a cell-dependent manner and may contribute to disease through direct cell killing leading to tissue necrosis.

Activity-dependent long-term potentiation (LTP) in the CNS is thought to be important in learning, memory, development, and persistent pain. Here, we report that NMDA receptor-dependent LTP is the major form of long-term plasticity in the anterior cingulate cortex (ACC). In addition to N-methyl-D-aspartate (NMDA) receptors, L-type voltage-gated calcium channels are also required for inducing LTP. Activation of calcium-stimulated adenylyl cyclase subtype 1 (AC1) is essential for the induction of LTP in ACC neurons, while AC8 subunit partially contributes to forskolin-induced potentiation. Our results suggest that calcium-stimulated cAMP-dependent signaling pathways play a critical role in cingulate LTP.

We identified the human ortholog of soluble adenylyl cyclase (hsAC) in a locus linked to familial absorptive hypercalciuria and cloned it from a human cDNA library. hsAC transcripts were expressed in multiple tissues using RT-PCR and RNA blotting. RNA blot analysis revealed a predominant 5.1-kb band in a multiple human tissue blot, but three splice transcript variants were detected using RT-PCR and confirmed by performing sequence analysis. Immunoblot analysis showed 190- and 80-kDa bands in multiple human cell lines from gut, renal, and bone origins in both cytosol and membrane fractions, including Caco-2 colorectal adenocarcinomas, HEK-293 cells, HOS cells, and primary human osteoblasts, as well as in vitro induced osteoclast-like cells. The specificity of the antiserum was verified by peptide blocking and reduction using sequence-specific small interfering RNA. Confocal immunofluorescence cytochemistry localized hsAC primarily in cytoplasm, but some labeling was observed in the nucleus and the plasma membrane. Cytoplasmic hsAC colocalized with microtubules but not with microfilaments. To test the function of hsAC, four constructs containing catalytic domains I and II (aa 1-802), catalytic domain II (aa 231-802), noncatalytic domain (aa 648-1,610), and full-length protein (aa 1-1,610) were expressed in Sf9 insect cells. Only catalytic domains I and II or full-length proteins showed adenylyl cyclase activity. Mg(2+), Mn(2+), and Ca(2+) all increased adenylyl cyclase activity in a dose-dependent manner. While hsAC had a minimal response to HCO(3)(-) in the absence of divalent cations, HCO(3)(-) robustly stimulated Mg(2+)-bound hsAC but inhibited Mn(2+)-bound hsAC in a dose-dependent manner. In summary, hsAC is a divalent cation and HCO(3)(-) sensor, and its HCO(3)(-) sensitivity is modulated by divalent cations.

We previously reported that p38 MAP kinase takes part in thrombin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether Akt is involved in the phosphorylation of HSP27 and the role of adenylyl cyclase-cAMP system. Thrombin time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) and Akt in aortic smooth muscle A10 cells. SB203580, a p38 MAP kinase inhibitor, significantly suppressed the thrombin-induced phosphorylation of Akt and the Akt inhibitor suppressed the phosphorylation of HSP27. Furthermore, the thrombin-induced phosphorylation of HSP27, p38 MAP kinase and Akt were decreased by dibutyryl-cAMP (DBcAMP). These results strongly suggest that Akt functions the thrombin-induced phosphorylation of HSP27 at a point downstream from p38 MAP kinase in aortic smooth muscle cells and the adenylyl cyclase-cAMP system is upstream regulator of the HSP27 phosphorylation in these cells.

The present paper studied the effect and mechanism of neurosteroid pregnenolone sulfate (PREGS) on spontaneous glutamate release using electrophysiological and biochemical methods combined with a pharmacological approach. The results suggested that PREGS had a selective enhancing effect on spontaneous glutamate release in the prelimbic cortex and the hippocampus but not in the striatum. The effect of PREGS in the prelimbic cortex appeared to be via modulation of alpha1-adrenergic and sigma1 receptors, but in the hippocampus it might be dependent on sigma1 receptors only. The activation of alpha1-adrenergic receptors synergized sigma1 receptor activation in the prelimbic cortex. Intracellular calcium released from the endoplasmic reticulum, protein kinase C, adenylyl cyclase and protein kinase A played a key role in the effect of PREGS. Intracellular calcium, protein kinase C and adenylyl cyclase might be upstream events in the activation of protein kinase A after PREGS.

Store-operated calcium (SOC) entry is sufficient to disrupt the extra-alveolar, but not the alveolar, endothelial cell barrier. Mechanism(s) underlying such insensitivity to transitions in cytosolic calcium ([Ca2+]i) in microvascular endothelial cells are unknown. Depletion of stored Ca2+ activates a larger SOC entry response in extra-alveolar (pulmonary artery; PAECs) than alveolar (pulmonary microvascular; PMVECs) endothelial cells. In vivo permeation studies revealed that Ca2+ store depletion activates similar nonselective cationic conductances in PAECs and PMVECs, while only PAECs possess the calcium-selective, store-operated Ca2+ entry current, I(SOC). Pretreatment with the type 4 phosphodiesterase inhibitor, rolipram, abolished thapsigargin-activated I(SOC) in PAECs, and revealed I(SOC) in PMVECs. Rolipram pretreatment shifted the thapsigargin-induced fluid leak site from extra-alveolar to alveolar vessels in the intact pulmonary circulation. Thus, our results indicate I(SOC) provides a [Ca2+]i source that is needed to disrupt the endothelial cell barrier, and demonstrate that intracellular events controlling I(SOC) activation coordinate the site-specific vascular response to inflammation.

The discovery of the adenylyl cyclase signaling mechanism (ACSM) of insulin and related peptides allows for advancing the hypothesis of its participation in regulatory action on cell growth, apoptosis, and metabolism. The following evidences were obtained: (1) The order of efficiency of insulin and IGF-I growth-promoting action and their antiapoptotic effect coincides with that of the peptide-activating action on AC. (2) The growth-promoting and antiapoptotic effects of insulin and IGF-I are distinctly reproduced by cAMP. (3) The anabolic effects of insulin and IGF-I activation on glycogen synthesis and the pentosophosphate pathway are inhibited by cAMP.

For the first time, the adenylyl cyclase system (ACS) sensitive to biogenic amines in the mollusk Anodonta cygnea ganglions was revealed and characterized. Serotonin and isoproterenol stimulated AC activity and GTP-binding activity of heterotrimeric G-proteins. The AC-stimulating action of serotonin and isoproterenol was blocked by cyproheptadine and adrenergic antagonists, respectively. Using synthetic C-terminal peptides of G-protein alpha-subunit, it was shown that the biogenic amines realized their action on the ACS through different G-protein types: serotonin and isoproterenol activate G(s)-protein, while adrenaline preferably activates G(i)-protein.

We previously demonstrated that TNF-alpha-dependent activation of p65 nuclear factor kappaB in rat thyroid FRTL-5 cells requires TSH. In the present study, we investigated the mechanism of this TSH action. Western blot analysis revealed that, in both the presence and absence of TSH, degradation of a cytosolic kappaB inhibitor (IkappaBalpha) occurred in response to TNF-alpha, resulting in nuclear translocation of p65 in both conditions. However, no DNA binding of p65 was detected in the absence of TSH, suggesting that posttranslational modification of p65 by TSH is required for its binding. Treatment of the cells cultured in the presence of TSH with a protein kinase A (PKA) inhibitor, H89, markedly reduced p65 binding and its transcriptional activity. However, transient block of TSH/cAMP-dependent activation of PKA catalytic subunit (PKAc) by adenylate cyclase inhibitor, SQ22536, had no effects on the p65 activation. Interestingly, it was found that PKAc formed a complex with IkappaBalpha and beta only in the presence of TSH, and this PKAc could be activated by TNF-alpha. TNF-alpha-dependent p65 activation was temporally associated with PKAc/IkappaBalpha complex formation. More than 3 h exposure of TSH was required for the complex formation and p65 activation. These results demonstrate that TSH induces the formation of PKAc/IkappaB complex in FRTL-5 cells and that this PKAc bound with IkappaB plays a critical role in TNF-alpha-dependent activation of p65.

We previously reported that acute agonist activation of G(i/o)-coupled receptors inhibits adenylate cyclase (AC) type VIII activity, whereas agonist withdrawal following chronic activation of these receptors induces AC-VIII superactivation. Three splice variants of AC-VIII have been identified, which are called AC-VIII-A, -B and -C (with AC-VIII-B missing the glycosylation domain and AC-VIII-C lacking most of the C1b area). We report here that AC-VIII-A and -B, but not -C, are inhibited by acute mu-opioid and dopaminergic type D2 receptor activation, indicating that the C1b area of AC-VIII has an important role in AC inhibition by G(i/o)-coupled receptor activation. On the other hand the glycosylation sites in AC-VIII did not play a role in AC-VIII regulation. Although AC-VIII-A and -C differed in their capacity to be inhibited by acute agonist exposure, agonist withdrawal after prolonged treatment led to a similar superactivation of all three splice variants, with no significant change in AC-VIII expression. AC-VIII superactivation was not affected by pre-incubation with a cell permeable cAMP analogue, indicating that the superactivation does not depend on the agonist-induced reduction in cAMP levels. The superactivated AC-VIII-A, -B and -C were similarly re-inhibited by re-application of agonist (morphine or quinpirole), returning the activity to control levels. These results demonstrate marked differences in the agonist inhibition of the AC-VIII splice variants before, but not after, superactivation.

The tandem GAF domains from the cyanobacterium Anabaena PCC7120 cyaB2 adenylyl cyclase form an antiparallel dimer with cAMP bound to all four binding sites. cAMP binding causes highly cooperative allosteric enzyme activation (>500-fold; EC(50) = 1 microM; Hill coefficient >2.0). The cyaB2 GAF domains, like those of the cyclic nucleotide phosphodiesterases (PDEs), contain conserved NKFDE motifs that when mutated in the PDEs abrogate cyclic nucleotide binding. We mutated the aspartic acids within this motif in cyaB2 to determine which domains were required for signaling. Constructs containing an Asp/Ala mutation in either GAF domain still showed positive cooperative cAMP stimulation but with reduced Hill coefficients. The cyaB2 GAF domain NKFDE motifs contain inserts of 14 (GAF-A) and 19 (GAF-B) amino acids not present in PDE2 or cyaB1. Constructs having these inserts deleted could still be activated by cAMP (23- to 100-fold) but lost all positive cooperative activation, suggesting that the inserts play an important role in domain interaction and/or stabilization of the cAMP-binding pockets. In the shortened constructs, even those with a single Asp/Ala mutation in the NKFDE motifs could still be activated by cAMP. However, in a double Asp/Ala mutant of the shortened construct, stimulation by cAMP was almost completely lost, and the EC(50) shifted far to the right. Overall, the data suggest that in GAF domains without these inserts, only the canonical lysine:aspartate salt bridge keeps the alpha4-helix and the alpha4-beta5 linker that close over the cyclic nucleotide properly oriented, thereby stabilizing the binding pocket. The cyaB2 GAF ensemble appears to be an evolutionary intermediate where both GAF domains still participate in allosteric activation by cAMP.

Neuroglial interactions are most profound during development or damage of nerve tissue. We studied the responses of crayfish stretch receptor neurons (SRN) and satellite glial cells to photosensitization with sulfonated aluminum phthalocyanine Photosens. Although Photosens was localized mainly in the glial envelope, neurons were very sensitive to photodynamic treatment. Photosensitization gradually inhibited and then abolished neuron activity. Neuronal and glial nuclei shrank. Some neurons and glial cells lost the integrity of the plasma membrane and died through necrosis after the treatment. The nuclei of other glial cells but not neurons become fragmented, indicating apoptosis. The number of glial nuclei around neuron soma increased, probably indicating proliferation for enhanced neuron protection. Adenylate cyclase (AC) inhibition by MDL-12330A, or tyrosine kinase (TK) inhibition by genistein, shortened neuron lifetime, whereas AC activation by forskolin or protein tyrosine phosphatases (PTP) inhibition by sodium orthovanadate prolonged neuronal activity. Therefore, cAMP and phosphotyrosines produced by AC and TK, respectively, protected SRN against photoinactivation. AC inhibition reduced photodamage of the plasma membrane and subsequent necrosis in neuronal and glial cells. AC activation prevented apoptosis in photosensitized glial cells and stimulated glial proliferation. TK inhibition protected neurons but not glia against photoinduced membrane permeabilization and subsequent necrosis whereas PTP inhibition more strongly protected glial cells. Therefore, both signaling pathways involving cAMP and phosphotyrosines might contribute to the maintenance of neuronal activity and the integrity of the neuronal and glial plasma membranes. Adenylate cyclase but not phosphotyrosine signaling pathways modulated glial apoptosis and proliferation under photooxidative stress., (Copyright 2004 Wiley-Liss, Inc.)

Chronic exposure of cells to mu-opioid agonists leads to tolerance which can be measured by a reduced ability to activate signaling pathways in the cell. Cell signaling through inhibitory G proteins is negatively regulated by RGS (regulator of G protein signaling) proteins. Here we examine the hypothesis that the GTPase accelerating activity of RGS proteins, by altering the lifetime of Galpha and Gbetagamma, plays a role in the development of cellular tolerance to mu-opioids. C6 glioma cells were stably transfected with mu-opioid receptor and pertussis toxin (PTX)-insensitive Galpha(o) that was either sensitive or insensitive to endogenous RGS proteins. Cells were treated with PTX to uncouple endogenous Galpha proteins followed by exposure to the mu-opioid agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) or morphine. Receptor desensitization as measured by agonist-stimulated [(35)S]GTPgammaS binding and receptor down-regulation as measured by [(3)H]diprenorphine binding were increased in cells expressing RGS-insensitive Galpha(o). Exposure to high concentrations of morphine or the peptidic mu-opioid agonist DAMGO led to a tolerance to inhibit adenylyl cyclase activity in both cell types with a rapid (30 min) and a slower component. Using a submaximal concentration of DAMGO to induce a reduced level of tolerance, a shift in the concentration-effect curve for DAMGO to inhibit adenylyl cyclase activity was seen in the cells expressing RGS-insensitive Galpha(o), but not in the cells expressing RGS-sensitive Galpha(o), which can be partly explained by an increased supersensitization of the adenylyl cyclase response. The results show that RGS proteins endogenously expressed in C6 cells reduce agonist-induced mu-opioid receptor desensitization, down-regulation, and sensitivity to tolerance to inhibit adenylyl cyclase activity.

cAMP-mediated signaling potentiates glucocorticoid-mediated apoptosis in lymphoid cells, but an effective means by which to take advantage of this observation in the treatment of lymphoid malignancies has not been identified. The primary objective of the current study was to determine whether PDE4 inhibitors, a class of compounds in late clinical development that raise intracellular cAMP levels by inhibiting type 4 cyclic nucleotide phosphodiesterases (PDE4), increase the efficacy of glucocorticoid-mediated apoptosis in leukemic cells from patients with B cell chronic lymphocytic leukemia (B-CLL). Rolipram, a prototypic PDE4 inhibitor, synergized with glucocorticoids in inducing B-CLL but not T cell apoptosis. Rolipram also augmented glucocorticoid receptor element (GRE) transactivation in B-CLL cells. In contrast, inhibition of protein kinase A (PKA) with the cAMP antagonist Rp-8Br-cAMPS reversed both glucocorticoid-induced apoptosis and GRE transactivation. CCRF-CEM cells, a well-studied model of glucocorticoid and cAMP-induced apoptosis, differed from B-CLL cells in that stimulation of adenylyl cyclase with the diterpene forskolin was required to increase both glucocorticoid-mediated apoptosis and GRE activation, while PDE4 inhibition had no effect. Consistent with these results, inhibition of PDE4 induced cAMP elevation in B-CLL but not CCRF-CEM cells, while forskolin augmented cAMP levels in CCRF-CEM but not B-CLL cells. While rolipram treatment up-regulated PDE4B in B-CLL, forskolin treatment up-regulated PDE4D in CCRF-CEM cells. These studies suggest that PKA is required for and enhances glucocorticoid-induced apoptosis in B-CLL by modulating glucocorticoid receptor signal transduction. Clinical trials that examine whether PDE4 inhibitors enhance the efficacy of glucocorticoid-containing chemotherapy regimens in B-CLL are indicated.

Oligomerization plays an important role in endoplasmic reticulum processing and membrane insertion (and ultimately in regulation of function) of a number of transmembrane spanning proteins. Furthermore, it is known that adenylyl cyclases (ACs), critical regulators of cellular functions, associate into higher order (dimeric) forms. However, the importance of these higher order aggregates in regulating adenylyl cyclase activity or trafficking to the cell membrane is unclear. Therefore, we examined the potential role of oligomerization in the membrane trafficking of adenylyl cyclase. For this purpose, the ability of full-length adenylyl cyclase and various truncation mutants to self-assemble and to be targeted to the cell membrane was assessed. A truncation mutant comprised of the initial six transmembrane spanning domains and half of the C1 catalytic domain coimmunoprecipitated with full-length AC VI. Using both biotinylation assays and assessment of enzyme distribution using sucrose density gradients, we demonstrate that expression of this mutant in human embryonic kidney 293 cells impaired the ability of AC VI to traffic to the plasma membrane. Furthermore, mutant expression resulted in a significant reduction in adenylyl cyclase activity. The decrease in AC VI membrane expression was not caused by alterations in enzyme transcription. The effect of the mutant was specific for the AC V and VI isoforms and expression of the transmembrane M1 domain but not the C1a domain was required for the mutant to affect adenylyl cyclase activity. In aggregate, these data suggest that alterations in the ability of adenylyl cyclases to form higher order forms regulate both enzyme trafficking and enzyme activity.

The level of adhesion molecules expressed at the endothelial cell surface is critical in the control of inflammation. Adenylate cyclase (AC) activity allowing cyclic adenosine monophosphate (cAMP) production can modulate the inflammatory process. We investigated the AC-dependent modulation of ICAM-1 surface expression in human umbilical venous endothelial cells (HUVEC). Pretreatment of HUVEC with forskolin significantly upregulated tumor necrosis factor alpha (TNF-alpha)- and interleukin-1 alpha (IL1-alpha)-induced ICAM-1 surface expression exclusively after a prolonged time of incubation with forskolin (at least 7-8 h). A poorly metabolizable analog of cAMP, dibutyryl-cAMP, mimicked forskolin effect on ICAM-1 surface expression. Protein kinase A (PKA) inhibitor H89 prevented forskolin effect on ICAM-1 surface expression. Upregulation of ICAM-1 surface level occurred through the increase in its mRNA levels and also to a subsequent activation of ICAM-1 intracellular trafficking towards cell surface. Stimulation by agonists of beta-adrenergic receptors did not alter the TNF-alpha-induced ICAM-1 surface expression. Pretreatment of HUVEC with pertussis toxin, which is known to activate AC through Gialpha inhibition, upregulated mRNA levels and surface expression of ICAM-1 induced by TNF-alpha. This effect was serum-dependent, since ICAM-1 expression was no more upregulated by pertussis toxin in cells cultured in 1% instead of 20% serum-enriched medium. However, forskolin treatment of HUVEC did not modify their overall adhesive properties. In conclusion, a persistent cAMP level elevation activating PKA is able to enhance ICAM-1 expression at the cell surface of endothelial cells placed under pro-inflammatory conditions. Combination of activation of gene transcription and membrane targeting may account for this augmentation., (2004 Wiley-Liss, Inc.)

Cyclic nucleotide levels are controlled through their synthesis from nucleotide triphosphates by cyclases and their degradation to 5'-monophosphates by phosphodiesterases (PDEs). Components controlling cyclic AMP-induced relaxation in the urinary tract include receptors, inhibitory and stimulatory G-proteins, isoforms of adenylyl cyclase and PDEs. The responsiveness of PDEs to a variety of physiological challenges is related to the presence of multiple families of isoenzymes with specific localization within tissues and within cells. At least 11 families of PDEs encode more than 50 PDE proteins produced in mammalian cells. In the urinary tract, characterization of PDE isoforms has lagged behind other systems and much of the literature was published prior to identification of PDE7, 8, 9, 10, 11. Specific PDE inhibitors regulate smooth muscle function in the bladder, urethra, prostate and ureter. The pharmacological potential of these inhibitors may include treatment of urge incontinence and the low compliance bladder, and treatment of prostate cancer. G-proteins also regulate cyclic AMP production. Changes in specific G- protein isoforms with aging, most prominently Gialpha2, cause decreased relaxation response in the aging bladder. As we have seen here with aging and certainly in other disease processes, levels of the components of adenylyl cyclase/phosphodiesterase/protein kinase can change and thus affect the relaxation response. By exploitation of differences in PDE expression in disease, such as the overexpression of PDEs in cancer, treatment options may present themselves.

We have previously shown that lobster olfactory receptor neurons (ORNs) express an odorant-suppressible Cl(-) conductance that modulates the output of the cells. Here, we develop a more complete pharmacological profile of this conductance, showing it is blockable by the Cl(-) channel blockers DIDS, 9-AC and flufenamic acid, but not by niflumic acid. We then show that a conductance with this pharmacological profile is mediated by cyclic nucleotide signaling. These findings further our understanding of the cellular mechanisms through which odorants can modulate the output of lobster ORNs.

Transformation of fibroblasts to myofibroblasts, characterized by expression of alpha-smooth muscle actin (alpha-SMA) and production of extracellular matrix (ECM) components, is a key event in connective tissue remodeling. Approaches to inhibit this transformation are needed in tissues, such as the heart, where excessive ECM production by cardiac fibroblasts (CFs) causes fibrosis, myocardial stiffening, and cardiac dysfunction. We tested whether adenylyl cyclase (AC) activation (increased cAMP levels) modulates the transformation of adult rat CF to myofibroblasts, as assessed by immunofluorescent microscopy, immunoblotting, and collagen synthesis. A 24-h incubation of CF with TGF-beta or angiotensin II increased alpha-SMA expression, which was inhibited by the AC agonist forskolin and a cAMP analog that activates protein kinase A. Treatment with forskolin blunted serum-, TGF-beta-, and angiotensin II-stimulated collagen synthesis. CFs engineered to overexpress type 6 AC had enhanced forskolin-promoted cAMP formation, greater inhibition by forskolin of TGF-beta-stimulated alpha-SMA expression, and a decrease in the EC(50) of forskolin to reduce serum-stimulated collagen synthesis. The AC stimulatory agonist adrenomedullin inhibited collagen synthesis in CF that overexpressed AC6 but not in controls. Thus, AC stimulation blunts collagen synthesis and, in parallel, the transformation of adult rat CF to myofibroblasts. AC overexpression enhances these effects, "uncovering" an inhibition by adrenomedullin. These findings implicate cAMP as an inhibitor of ECM formation by means of blockade of the transformation of CF to myofibroblasts and suggest that increasing AC expression, thereby enhancing cAMP generation through stimulation of receptors expressed on CF, could provide a means to attenuate and prevent cardiac fibrosis and its sequelae.

Adenosine A1-receptor-mediated inhibition of exocytotic norepinephrine (NE) release from sympathetic nerve endings has been implicated as an endogenous cardioprotective mechanism. So far, the intraneuronal signal transduction underlying the adenosine A1-receptor-elicited inhibition of NE release is not known. In the present study, we determined in isolated Langendorff-perfused rat hearts the role of inhibitory G-proteins and of adenylyl cyclase (AC) on NE release after pharmacologic adenosine A1-receptor activation. NE release was induced by electrical field stimulation and was assessed in the coronary effluent by high-performance liquid chromatography. Adenosine A1-receptor activation with 2-chloro-N6-cyclopentyladenosine (CCPA) decreased NE release by approximately 50% in hearts from both untreated and pertussis toxin-pretreated rats. In hearts from untreated rats, suppression of NE release in response to CCPA was completely abolished by the cell-permeable AC inhibitor 9-(tetrahydro-2'-furyl)adenine (SQ 22536). Direct activation of AC with forskolin increased NE release by approximately 20%. In the presence of forskolin, stimulation of adenosine A1-receptors with CCPA or inhibition of AC with SQ 22536 decreased NE release to baseline. These findings suggest a Gi-protein-independent but AC-dependent inhibition of NE release following adenosine A1-receptor activation.

Vasopressin, a hypothalamic hormone, acts on its target tissues via three different G protein coupled receptors. The vasopressin V1a and V1b receptors, associated to Gq protein and phospholipase C, are responsible for vasoconstriction and regulation of the corticotroph axis respectively. The V2 vasopressin receptor is coupled to Gs protein and adenylyl cyclase and is responsible for water reabsorption in the renal collecting duct. Mutations of the V2 receptor are involved in diabetes insipidus and most of these mutations result in an endoplasmic reticulum (ER) retention of the mutated receptor. With the V1b receptor model, we have identified a proximal sequence of the C-terminal segment, which is crucial for ER export. Mutations in this short domain result in ER accumulation and degradation of the receptor. SSR 149415, a nonpeptide antagonist of V1bR, which is permeable to cell membrane, is able to rescue the mutant phenotype and acts as a pharmacological chaperone.

ATP and ADP activate functionally distinct G protein-coupled purinergic (P2Y) receptors. We determined the expression and function of adenine nucleotide-specific P2Y receptors on cord blood-derived human mast cells (hMCs). Human MCs expressed mRNA encoding the ADP-specific P2Y1, P2Y12, and P2Y13 receptors; the ATP/UTP-specific P2Y2 receptor; and the ATP-selective P2Y11 receptor. ADP (0.05-50 muM) induced calcium flux that was completely blocked by a P2Y1 receptor-selective antagonist and was not cross-desensitized by ATP. Low doses of ADP induced strong phosphorylation of ERK and p38 MAPKs; higher doses stimulated eicosanoid production and exocytosis. Although MAPK phosphorylation was blocked by a combination of P2Y1- and P2Y12-selective antagonists, neither interfered with secretion responses. Unexpectedly, both ADP and ATP inhibited the generation of TNF-alpha in response to the TLR2 ligand, peptidoglycan, and blocked the production of TNF-alpha, IL-8, and MIP-1beta in response to leukotriene D(4). These effects were mimicked by two ATP analogues, adenosine 5'-O-(3-thiotriphosphate) and 2',3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), but not by adenosine. ADP, ATP, adenosine 5'-O-(3-thiotriphosphate), and 2',3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate each induced cAMP accumulation, stimulated the phosphorylation of CREB, and up-regulated the expression of inducible cAMP early repressor, a CREB-dependent inhibitor of cytokine transcription. Human MCs thus express several ADP-selective P2Y receptors and at least one G(s)-coupled ADP/ATP receptor. Nucleotides could therefore contribute to MC-dependent microvascular leakage in atherosclerosis, tissue injury, and innate immunity while simultaneously limiting the extent of subsequent inflammation by attenuating the generation of inducible cytokines by MCs.

Development of the retino-collicular pathway has served as an important model system for examining the cellular mechanisms responsible for the establishment of neuronal maps of the sensory periphery. A consensus has emerged that molecular or chemical cues are responsible for the initial establishment of gross topography in this map, and that activity dependent factors sharpen this initial rough topography into precision. However, there is little evidence available concerning the biochemical signaling mechanisms that are responsible for topographic map refinement in the retino-collicular system. Using a combination of anatomical and biochemical techniques in normal and mutant mice, we provide evidence that Ca2+/Calmodulin regulated Adenylate Cyclase 1 (AC1), which is strongly expressed in the superficial layers of the colliculus, is an important downstream signaling agent for activity dependent map refinement in the superior colliculus.

Arterial ATP-sensitive K+ (K(ATP)) channels are critical regulators of vascular tone, forming a focal point for signaling by many vasoactive transmitters that alter smooth muscle contractility and so blood flow. Clinically, these channels form the target of antianginal and antihypertensive drugs, and their genetic disruption leads to hypertension and sudden cardiac death through coronary vasospasm. However, whereas the biochemical basis of K(ATP) channel modulation is well-studied, little is known about the structural or spatial organization of the signaling pathways that converge on these channels. In this study, we use discontinuous sucrose density gradients and Western blot analysis to show that K(ATP) channels localize with an upstream signaling partner, adenylyl cyclase, to smooth muscle membrane fractions containing caveolin, a protein found exclusively in cholesterol and sphingolipid-enriched membrane invaginations known as caveolae. Furthermore, we show that an antibody against the K(ATP) pore-forming subunit, Kir6.1 co-immunoprecipitates caveolin from arterial homogenates, suggesting that Kir6.1 and caveolin exist together in a complex. To assess whether the colocalization of K(ATP) channels and adenylyl cyclase to smooth muscle caveolae has functional significance, we disrupt caveolae with the cholesterol-depleting agent, methyl-beta-cyclodextrin. This reduces the cAMP-dependent protein kinase A-sensitive component of whole-cell K(ATP) current, indicating that the integrity of caveolae is important for adenylyl cyclase-mediated channel modulation. These results suggest that to be susceptible to protein kinase A-dependent activation, arterial K(ATP) channels need to be localized in the same lipid compartment as adenylyl cyclase; the results also provide the first indication of the spatial organization of signaling pathways that regulate K(ATP) channel activity.

Previous research has indicated that volatile odorants are detected through the main olfactory epithelium (MOE), whereas pheromones are detected via the vomeronasal organ (VNO). Gene disruption studies have established that olfactory signaling through the MOE is mediated through receptor stimulation of type 3 adenylyl cyclase (AC3). Mice lacking AC3 cannot detect odorants through the MOE. Recently, it was discovered using olfactory-based behavioral assays that AC3 mutant mice can detect some volatile odorants. An analysis of these mutant mice led to the surprising discovery that some odorants are detected through the VNO.

The autacoid and neurotransmitter histamine activates the H(1) G protein-coupled receptor (GPCR) to stimulate predominantly phospholipase C (PLC)/inositol phosphate (IP) signaling and, to a lesser extent, adenylyl cyclase (AC)/cAMP signaling in a variety of mammalian cells and tissues, as well as H(1)-transfected clonal cell lines. This study reports that two novel H(1) receptor ligands developed in our laboratory, (-)-trans-1-phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (trans-PAT) and (+/-)-cis-5-phenyl-7-dimethylamino-5,6,7,8-tetrahydro-9H-benzocycloheptane (cis-PAB), activate H(1) receptors to selectively stimulate AC/cAMP formation and PLC/IP formation, respectively, in Chinese hamster ovary cells transfected with guinea pig H(1) receptor cDNA. trans-PAT and cis-PAB also are shown to be functionally selective antagonists of H(1)-linked PLC/IP and AC/cAMP signaling, respectively. Whereas cis-PAB H(1) receptor activity is shown to be typically competitive, trans-PAT displays a complex interaction with the H(1) receptor that is not competitive regarding antagonism of saturation binding by the standard H(1) antagonist radioligand [(3)H]mepyramine or H(1)/PLC/IP functional activation by histamine. trans-PAT, however, does competitively block H(1)/PLC/IP functional activation by cis-PAB. Molecular determinants for trans-PAT versus cis-PAB differential binding to H(1) receptors, which presumably leads to differential activation of AC/cAMP versus PLC/IP signaling, likely involves stereochemical factors as well as more subtle steric influences. Results suggest the trans-PAT and cis-PAB probes will be useful to study molecular mechanisms of ligand-directed GPCR multifunctional signaling. Moreover, because most untoward cardiovascular-, respiratory-, and gastrointestinal H(1) receptor-mediated effects proceed via the PLC/IP pathway, PAT-type agonists that selectively enhance H(1)-mediated AC/cAMP signaling provide a mechanistic basis for exploiting H(1) receptor activation for drug design purposes.

In addition to the somatodendritic region, myenteric motoneuron terminals are endowed with nicotinic autoreceptors. We aimed at investigating the effect of nicotinic receptor (nAChR) activation on [3H]-acetylcholine ([3H]-ACh) release from longitudinal muscle-myenteric plexus of the rat ileum and to evaluate whether this could be modulated by adenosine, an endogenous neuromodulator typically operating changes in intracellular cyclic AMP. The nAChR agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 1-30 microM, 3 min) increased [3H]-ACh release in a concentration-dependent manner. DMPP (30 microM)-induced [3H]-ACh outflow was attenuated by hexamethonium (0.1-1 mM), tubocurarine (1-5 microM), or by removing external Ca2+ (plus EGTA, 1 mM). In contrast to veratridine (0.2-10 microM)-induced [3H]-ACh release, the DMPP (30 microM)-induced outflow was resistant to tetrodotoxin (1 microM) and cadmium (0.5 mM). Pretreatment with adenosine deaminase (0.5 U/mL) or with the adenosine A(2A)-receptor antagonist, ZM 241385 (50 nM), enhanced nAChR-induced transmitter release. Activation of A(2A) receptors with CGS 21680C (3 nM) reduced the DMPP-induced release of [3H]-ACh. CGS 21680C (3 nM) inhibition was prevented by MDL 12,330A (10 microM, an adenylate cyclase inhibitor) and by H-89 (10 microM, an inhibitor of protein kinase A), but was potentiated by rolipram (300 microM, a phosphodiesterase inhibitor). DMPP-induced transmitter release was decreased by 8-bromo-cyclic AMP (1 mM, a protein kinase A activator), rolipram (300 microM), and forskolin (3 microM, an activator of adenylate cyclase). Both MDL 12,330A (10 microM) and H-89 (10 microM) facilitated DMPP-induced release of [3H]-ACh. The results indicate that nAChR-induced [3H]-ACh release is triggered by the influx of Ca2+, independent of voltage-sensitive calcium channels, presumably directly through nAChRs located on myenteric axon terminals. It was also shown that endogenous adenosine, activating A(2A) receptors coupled to the adenylate cyclase/cyclic AMP transducing system, is tonically downregulating this nAChR-mediated control of [3H]-ACh release.

Nucleotidyl cyclases synthesize cAMP or cGMP in response to various forms of sensory input. Their regulation and function in mammalian hormone action and neurotransmission has been well characterized over the past 40 years. More recently a much broader repertoire of adenylyl- and guanylyl cyclase forms is emerging from studies of more basal eukaryotes and prokaryotes. These cyclases play crucial roles in the development, physiology and pathogenicity of these organisms. This series of reviews highlights both unique and highly conserved aspects of the activation mechanisms of these enzymes, as compared to those of their mammalian descendants.

Completion of several malaria parasite genome sequences and advances in Plasmodium gene manipulation technology, will lead to significant advances in our knowledge of the biology of these organisms. Biochemical analysis of the cyclic nucleotide signalling pathways of P. falciparum has provided important information on malaria parasite development. The Plasmodium purine nucleotide cyclase enzymes have extremely unusual structures and the regulatory mechanisms controlling parasite enzyme activity are distinct from those operating on the analogous host molecules. Study of these enzymes could therefore lead to novel strategies for anti-malarial intervention in addition to providing unique insights into the intriguing biology of the parasite.

Cyclic AMP metabolism is essential for the survival of the social amoebae Dictyostelium discoideum. Three distinct adenylyl cyclases are expressed and required for the normal development of this simple eukaryote. The adenylyl cyclase expressed during aggregation, ACA, is related to the mammalian and Drosophila G protein-coupled enzymes and is responsible for the synthesis of cAMP that is required for cell-cell signaling in early development. ACB harbors histidine kinase and response-regulator domains and is required for terminal differentiation. Finally, the adenylyl cyclase expressed during germination, ACG, acts as an osmosensor and is involved in controlling spore germination. Together, these enzymes generate the various levels of cAMP that are required for D. discoideum to transition from uni- to multi-cellularity. This review will highlight the properties of these enzymes and describe the signaling cascades that lead to their activation.

Retrograde signaling is an essential component of synaptic development and physiology. Previous studies show that bone morphogenetic protein (BMP)-dependent retrograde signaling is required for the proper development of the neuromuscular junction (NMJ) in Drosophila. These studies, moreover, raised the significant possibility that the development of central motor circuitry might similarly be reliant on such signaling. To test this hypothesis, retrograde signaling between postsynaptic motoneurons and their presynaptic interneurons is examined. Postsynaptic expression of an adenylate cyclase encoded by rutabaga (rut), is sufficient to strengthen synaptic transmission at these identified central synapses. Results are presented to show that the underlying mechanism is dependent on BMP retrograde signaling. Thus, presynaptic expression of an activated TGF-beta receptor, thickvien (tkv), or postsynaptic expression of a TGF-beta ligand, glass-bottom boat (gbb), is sufficient to phenocopy strengthening of synaptic transmission. In the absence of gbb, endogenous synaptic transmission is significantly weakened and, moreover, postsynaptic overexpression of rut is unable to potentiate synaptic function. Potentiation of presynaptic neurotransmitter release, mediated by increased postsynaptic expression of gbb, is dependent on normal cholinergic activity, indicative that either the secretion of this retrograde signal, or its transduction, is activity dependent. Thus, in addition to the development of the NMJ and expression of myoactive FMRFamide-like peptides in specific central neurons, the results of the present study indicate that this retrograde signaling cascade also integrates the development and function of central motor circuitry that controls movement in Drosophila larvae.

Bordetella pertussis, the causative agent of whooping cough, secretes among other virulence factors an adenylate cyclase (AC) toxin that is able to enter into eukaryotic cells where it is activated upon binding to endogenous calmodulin (CaM) and synthesizes supraphysiological cAMP levels. In vivo, the AC toxin, through its specific interaction with the CD11b/CD18 integrin, primarily targets phagocytic cells such as neutrophils and macrophages. Because neutrophil priming and activation result in the production of reactive oxygen species that may cause intracellular oxidation, we have examined the biological consequences of the oxidation of CaM methionines upon its interaction with AC. We show here that the interaction of CaM with AC is dependent on the reduced state of methionines, because oxidation of all methionine residues of CaM dramatically decreases its affinity for AC. Peptide methionine sulfoxide reductases A (MsrA) and B (MsrB) were able to partially reduce the oxidized CaM, and these partially "repaired" forms could interact with AC nearly as efficiently as the native protein. We further showed that the CaM.AC complex is resistant to oxidation with tert-butylhydroperoxide, and we identified methionine residues 109, 124, and 145 as critical for binding to AC. The resistance of the AC.CaM complex to oxidation and the ability of AC to be efficiently activated by partially oxidized CaM molecules should allow the toxin to exert its cytotoxic effects on activated neutrophils and contribute to the host colonization.

Two types of consolidated memory have been described in Drosophila, anesthesia-resistant memory (ARM), a shorter-lived form, and stabilized long-term memory (LTM). Until now, it has been thought that ARM and LTM coexist. On the contrary, we show that LTM formation leads to the extinction of ARM. Flies devoid of mushroom body vertical lobes cannot form LTM, but spaced conditioning can still erase their ARM, resulting in a remarkable situation: The more these flies are trained, the less they remember. We propose that ARM acts as a gating mechanism that ensures that LTM is formed only after repetitive and spaced training.

Opioids are the drugs of choice for the treatment of moderate to severe acute and chronic pain. However, clinical evidence suggests that opioids can elicit increased sensitivity to noxious stimuli suggesting that administration of opioids can activate both pain inhibitory and pain facilitatory systems. Acute receptor desensitization via uncoupling of the receptor from G-proteins, up-regulation of the cAMP pathway, activation of the N-methyl-D-aspartate (NMDA) receptor system, as well as descending facilitation, have been proposed as potential mechanisms underlying opioid-induced hyperalgesia. Numerous reports exist demonstrating that opioid-induced hyperalgesia is observed both in animal and human experimental models. Brief exposures to micro-receptor agonists induce long-lasting hyperalgesic effects for days, which might by reflected by clinical observations that large doses of intraoperative micro-receptor agonists increased postoperative pain and morphine consumption. Furthermore, the prolonged use of opioids in patients often requires increasing doses and may be accompanied by the development of abnormal pain. Successful strategies that may decrease or prevent opioid-induced hyperalgesia include the concomitant administration of drugs like NMDA-antagonists, alpha(2)-agonists, or non-steroidal anti-inflammatory drugs (NSAIDs), opioid rotation or combinations of opioids with different receptor selectivity.

The biophysical properties of neuron R15 in Aplysia endow it with the ability to express multiple modes of oscillatory electrical activity, such as beating and bursting. Previous modeling studies examined the ways in which membrane conductances contribute to the electrical activity of R15 and the ways in which extrinsic modulatory inputs alter the membrane conductances by biochemical cascades and influence the electrical activity. The goals of the present study were to examine the ways in which electrical activity influences the biochemical cascades and what dynamical properties emerge from the ongoing interactions between electrical activity and these cascades. The model proposed by Butera et al. in 1995 was extended to include equations for the binding of Ca(2+) to calmodulin (CaM) and the actions of Ca(2+)/CaM on both adenylyl cyclase and phosphodiesterase. Simulations indicated that levels of cAMP oscillated during bursting and that these oscillations were approximately antiphasic to the oscillations of Ca(2+). In the presence of cAMP oscillations, brief perturbations could switch the electrical activity between bursting and beating (bistability). Compared with a constant-cAMP model, oscillations of cAMP substantially expanded the range of bistability. Moreover, the integrated electrical/biochemical model simulated some early experimental results such as activity-dependent inactivation of the anomalous rectifier. The results of the present study suggest that the endogenous activity of R15 depends, in part, on interactions between electrical activity and biochemical cascades.

The two stimulatory G protein alpha subunits, Galphas and Galphaolf, activate adenylyl cyclase in a similar way. We examined whether type 5 adenylyl cyclase knockout, the major striatal isoform, can differentially and/or developmentally change the expression of these G proteins in the striatum. Galphas and Galphaolf expressions at birth were unaffected in knockouts, which, however, demonstrated a blunted developmental increase in Galphaolf, but not Galphas. Adenylyl cyclase activity was unaffected at birth, but subsequently became lower in knockouts. These findings suggest that type 5 adenylyl cyclase does not contribute to striatal cAMP signaling at birth. However, it may play an important role in developmental changes in the expression of Galphaolf, but not Galphas.

The pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide of the vasoactive intestinal peptide/secretin/glucagon superfamily. Studies in two related patients with a partial trisomy 18p revealed three copies of the PACAP gene and elevated PACAP concentrations in plasma. The patients suffer from severe mental retardation and have a bleeding tendency with mild thrombocytopenia, and their fibroblasts show increased PACAP mRNA levels. The PACAP receptor (vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor 1 [VPAC1]) in platelets and fibroblasts is coupled to adenylyl cyclase activation. Accordingly, we found increased basal cAMP levels in patients' platelets and fibroblasts, providing a basis for the reduced platelet aggregation in these patients. Megakaryocyte-specific transgenic overexpression of PACAP in mice correspondingly increased PACAP release from platelets, reduced platelet activation, and prolonged the tail bleeding time. In contrast, the PACAP antagonist PACAP(6-38) or a monoclonal PACAP antibody enhanced the collagen-induced aggregation of normal human platelets, and in PACAP knockout mice, an increased platelet sensitivity toward collagen was found. Thus, we found that PACAP modulates platelet function and demonstrated what we believe to be the first hemostatic defect associated with PACAP overexpression; our study suggests the therapeutic potential to manage arterial thrombosis or bleeding by administration of PACAP mimetics or inhibitors, respectively.