Regulation Of cAMP Levels Research Articles

Dopamine D1, D2 and mu-opioid receptors are co-expressed with adenylyl cyclase 5 and phosphodiesterase 7B mRNAs in striatal rat cells

Intracellular cAMP levels are regulated by cAMP synthesis and degradation rate. Nine isoforms of cAMP-synthesizing enzymes called adenylyl-cyclases (ACs) and eleven phosphodiesterases (PDEs) that degrade cyclic nucleotides have been identified. Both types of enzymes exhibit variations not only in their expression pattern distribution throughout the brain, but also in their regulatory characteristics. Different isoforms of ACs and PDEs may be co-expressed in a single cell, thus a gradient of cAMP intracellular levels is formed, which accounts for the diversity of cell responses. Among these isoforms, AC5 and PDE7B are highly expressed in striatum, where the cAMP pathway is implicated in diverse behavioural functions. Striatal AC5 is involved in drug reinforcing actions and motor activity. Less is known about the role of the PDE7B isoenzyme. We performed a double in situ hybridization analysis of the co-expression patterns of AC5 and PDE7B with μ-opioid-receptor (MOR), D1- and D2-receptor mRNAs to contribute to a better understanding in the regulation of cAMP levels under dopamine or opioidergic pathway activation in striatum. We found co-expression of AC5 and PDE7B mRNAs in caudate-putamen and nucleus accumbens; we also encountered that more than 50% of MOR, D2- and D1-expressing cells contained AC5 and PDE7B mRNAs. The presence of AC5 and PDE7B mRNAs in D1- and D2-containing cells suggests the participation of these enzymes in striatal functions involving dopaminergic pathways. Co-localization of both isoenzyme mRNAs with MOR expressing cells suggests their involvement in opioid reinforcing effects.

Radial Spoke Protein 3 Is a Mammalian Protein Kinase A-anchoring Protein That Binds ERK1/2

Initially identified in Chlamydomonas, RSP3 (radial spoke protein 3) is 1 of more than 20 identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be characterized. We found mammalian RSP3 to bind to the MAPK ERK2 through a yeast two-hybrid screen designed to identify interacting proteins that have a higher affinity for the phosphorylated, active form of the protein kinase. Consistent with the screening result, the human homolog, RSPH3, interacts with and is a substrate for ERK1/2. Moreover, RSPH3 is a protein kinase A-anchoring protein (AKAP) that scaffolds the cAMP-dependent protein kinase holoenzyme. The binding of RSPH3 to the regulatory subunits of cAMP-dependent protein kinase, RIIalpha and RIIbeta, is regulated by ERK1/2 activity and phosphorylation. Here we describe an ERK1/2-interacting AKAP and suggest a mechanism by which cAMP-dependent protein kinase-AKAP binding can be modulated by the activity of other enzymes.

An Adenylyl Cyclase-mAKAPβ Signaling Complex Regulates cAMP Levels in Cardiac Myocytes

Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein beta (mAKAPbeta) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAPbeta selectively binds type 5 AC in the heart and that mAKAPbeta-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAPbeta-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245-340), and expression of this peptide disrupts endogenous mAKAPbeta-AC association. Accordingly, disruption of mAKAPbeta-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAPbeta complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy.

Cell death mechanisms: life in the balance

This report summarizes a 1-day meeting on Cell Death Mechanisms organized by the European Tissue Culture Society (UK branch) held at University of Bristol on 23 April 2008. The meeting brought together scientists interested in mechanisms of neuronal and glial survival and degeneration with those interested in reactivating or mimicking cell death signals to treat cancer. David Rubinsztein (Cambridge) described how upregulating autophagy protects against neurodegeneration in diseases caused by aggregate-prone mutant proteins. Macroautophagy is a bulk degradation process involved in the clearance of long-lived proteins, protein complexes and organelles, in which autophagosomes containing these constituents fuse with lysosomes where the contents are degraded by acidic hydrolases. Huntington’s disease is caused by a polyglutamine-expansion mutation in the huntingtin protein that makes it toxic and aggregate prone. Of the two main pathways to protein clearance in mammalian cells – the ubiquitin-proteasome and the autophagy-lysosome pathways, the latter is thought to be the better therapeutic target, as the proteasome has a narrow opening and cannot accommodate polymers. As proof of principle, the mTOR inhibitor rapamycin activates autophagy and enhances the clearance of polyglutamine proteins in cell culture and in Drosophila eye models, protecting against cytotoxicity. A rapamycin ester is favoured over rapamycin for clinical use, and in mouse models this resulted in much smaller and fewer aggregates in the brain than a placebo control and a marked improvement in a range of behavioural correlates of disease. Autophagy protects against other aggregate proteins such as ataxin 3 (mutated in spinocerebellar ataxia type 3) and tau mutants that cause temporal dementia/tauopathy. Therefore it appears that autophagy is important for clearing protein aggregate-prone proteins, and upregulating autophagy is helpful in these diseases. Recently, from drug screening experiments to identify mTOR-independent autophagy-inducing drugs, L-type Ca2þ -channel antagonists such as verapamil, the KþATPchannel opener minoxidil, and the G(i) signalling activator clonidine have been shown to induce autophagy. These drugs act to decrease intracytosolic calcium or cAMP levels and impact on a pathway, where myo-inositol-1,4,5-triphosphate (InsP3) releases Ca 2þ from the endoplasmic reticulum, activating calpains, which in turn, cleave and activate G(s)a (thus increasing cAMP levels) and inhibit autophagy. A further potential feed forward loop occurs in which cAMP regulates InsP3 levels, releasing endoplasmic reticulum (ER) Ca 2þ and enhancing calpain activity, activating G(s)a, which in turn regulates cAMP levels. So, insults that elevate cytosolic Ca2þ inhibit autophagy, thus retarding clearance of aggregateprone proteins. This may lead to an array of new candidate drugs for many incurable neurodegenerative diseases. Alexei Verkhratsky (Manchester) reminded us that the glial cells form 90% of the human brain, whereas interestingly they are much less numerous in other species. Glial cells are central in maintaining brain homeostasis and are represented by astrocytes, oligodendrocytes and microglial cells. Each of these subtypes expresses a variety of neurotransmitter receptors, many of which, when activated by neural activity, initiate glial Ca2þ responses, which produce interglial Ca2þ waves, that in turn activate release of ‘glio’ transmitters (which include glutamate, ATP, taurine, D-serine and probably many others) that can signal back to neurones, thus functionally integrating neuronal and glial circuitries. Astrocytes can form an extended physically connected syncytium. In brain pathologies, glial scars serve to fence off damaged areas. Within these areas, glutamate release from damaged cells is cytotoxic, and in stroke, death signals from the infarction lead to a propagated wave of cell death. It is believed that the glial cells deal with the damage by sealing and eliminating the damaged area to protect the rest of the brain. Atrophy of the glia is a feature of Alzheimer’s disease and other dementias and can be important in driving disease progression. Pierluigi Nicotera (Leicester) took up the story. Although Ca2þ signals are necessary for cell communication and survival, abnormal cellular Ca2þ load can trigger different cell death programs depending on variety of factors, such as energy requirement, signalling molecules, differentiation status or the intensity of the insult. During excitotoxicity, local glutamate-driven Ca2þ overloads at the postsynaptic areas

Identification and molecular characterization of a novel PDE4D11 cAMP-specific phosphodiesterase isoform

Here we report the cloning and characterization of a novel PDE4D isoform (PDE4D11) identified in mouse brain cDNA. This novel isoform has a unique isoform-specific 5′-UTR and N-terminal sequence, whereas, downstream regulatory N-terminal and catalytic C-terminal regions are homologous to other long PDE4D isoforms (Ex2-15). In silico analysis of PDE4D11 cDNA transcript identified the predicted translational start site and the use of a different transcriptional start site compared to other PDE4D isoforms. This isoform is ubiquitously expressed in different mouse tissues, particularly in the brain, liver and spleen. In the brain, PDE4D11 expression levels increased in the cerebellum, but decreased in the hippocampus with progressive age, highlighting a potential role for this isoform in the development of the brain. When transfected in vitro into murine neuroblastoma cells PDE4D11_EGFP expression is cytosolic, consistent with other long PDE4D isoforms. The appearance of cytosolic protein aggregates in discrete microdomains with this isoform, however, may represent a method of compartmentalizing PDE4D11 activity. The novel 5′-sequence of PDE4D11 is conserved among higher vertebrates including human, monkey, dog, horse and rat. Identification of this new isoform highlights the mutliplicity of unique PDE4D isoforms and their potential importance in regulating cAMP levels through compartmentalization and cell-specific expression and underscores the importance of understanding the functional role of each isoform in the development of specific drugs for the treatment of memory disorders.

Molecular mechanisms of stress-induced prefrontal cortical impairment: Implications for mental illness

The symptoms of mental illness often involve weakened regulation of thought, emotion, and behavior by the prefrontal cortex. Exposure to stress exacerbates symptoms of mental illness and causes marked prefrontal cortical dysfunction. Studies in animals have revealed the intracellular signaling pathways activated by stress exposure that induce profound prefrontal cortical impairment: Excessive dopamine stimulation of D1 receptors impairs prefrontal function via cAMP intracellular signaling, leading to disconnection of prefrontal networks, while excessive norepinephrine stimulation of alpha1 receptors impairs prefrontal function via phosphatidylinositol-protein kinase C intracellular signaling. Genetic studies indicate that the genes disrupted in serious mental illness (bipolar disorder and schizophrenia) often encode for the intracellular proteins that serve as brakes on the intracellular stress pathways. For example, disrupted in schizophrenia 1 (DISC1) normally regulates cAMP levels, while regulator of G protein signaling 4 (RGS4) and diacylglycerol kinase (DGKH)-the molecule most associated with bipolar disorder- normally serve to inhibit phosphatidylinositol-protein kinase C intracellular signaling. Patients with mutations resulting in loss of adequate function of these genes likely have weaker endogenous regulation of these stress pathways. This may account for the vulnerability to stress and the severe loss of PFC regulation of behavior, thought, and affect in these illnesses. This review highlights the signaling pathways onto which genetic vulnerability and stress converge to impair PFC function and induce debilitating symptoms such as thought disorder, disinhibition, and impaired working memory.

Enhanced long‐term potentiation and impaired learning in phosphodiesterase 4D‐knockout (PDE4D−/−) mice

Elevation of intracellular cyclic adenosine monophosphate (cAMP) concentrations and subsequent regulation of downstream target gene expression through phosphorylation of cAMP-responsive element binding protein (CREB) is hypothesized to underlie the mechanism(s) of long-term memory (LTM) formation. The phosphodiesterase 4 (PDE4) enzyme family is believed to play a key role in LTM by regulating cAMP levels. Thus far, four PDE4 isoforms have been identified (PDE4A, B, C and D); however, the requisite involvement of each of these isoforms in mediating LTM has yet to be elucidated. In the present study, genetic knockout mice were used to investigate the involvement of the PDE4D isoform in both in vitro and in vivo models of learning and memory. Hippocampal synaptic transmission measured electrophysiologically in CA1 slice preparations was similar between wild-type and PDE4D (-/-) mice yet, relative to wild-type controls, knockout mice displayed enhanced early long-term potentiation (LTP) following multiple induction protocols. Interestingly, the PDE4D (-/-) animals exhibited significant behavioral deficits in associative learning using a conditioned fear paradigm as compared with control littermates. The impairment in fear conditioning observed in the PDE4D (-/-) mice could not be attributed to differences in acquisition of the task, alterations in locomotor activity or effects on shock sensitivity. Overall, the in vitro and in vivo alterations in synaptic plasticity observed in the PDE4D (-/-) mice may be explained by adaptive responses occurring throughout development, and suggest that the PDE4D isoform may be an important mediator of LTM formation.

Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP

Increases in the second messenger cAMP are associated with receptor-mediated ATP release from erythrocytes. In other signaling pathways, cAMP-specific phosphodiesterases (PDEs) hydrolyze this second messenger and thereby limit its biological actions. Although rabbit and human erythrocytes possess adenylyl cyclase and synthesize cAMP, their PDE activity is poorly characterized. It was reported previously that the prostacyclin analog iloprost stimulated receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the PDEs that hydrolyze erythrocyte cAMP synthesized in response to iloprost were not identified. PDE3 inhibitors were reported to augment increases in cAMP stimulated by prostacyclin analogs in platelets and pulmonary artery smooth muscle cells. Additionally, PDE3 activity was identified in embryonic avian erythrocytes, but the presence of this PDE in mammalian erythrocytes has not been investigated. Here, using Western blot analysis, we determined that PDE3B is a component of rabbit and human erythrocyte membranes. In addition, we report that the preincubation of rabbit and human erythrocytes with the PDE3 inhibitors milrinone and cilostazol potentiates iloprost-induced increases in cAMP. In addition, cilostamide, the parent compound of cilostazol, potentiated iloprost-induced increases in cAMP in human erythrocytes. These findings demonstrate that PDE3B is present in rabbit and human erythrocytes and are consistent with the hypothesis that PDE3 activity regulates cAMP levels associated with a signaling pathway activated by iloprost in these cells.

The cyclic AMP-dependent catabolite repression system of Serratia marcescens mediates biofilm formation through regulation of type 1 fimbriae.

The mechanisms by which environmental carbon sources regulate biofilm formation are poorly understood. This study investigates the roles of glucose and the catabolite repression system in Serratia marcescens biofilm formation. The abilities of this opportunistic pathogen to proliferate in a wide range of environments, to cause disease, and to resist antimicrobials are linked to its ability to form biofilms. We observed that growth of S. marcescens in glucose-rich medium strongly stimulated biofilm formation, which contrasts with previous studies showing that biofilm formation is inhibited by glucose in Escherichia coli and other enteric bacteria. Glucose uptake is known to inversely mediate intracellular cyclic AMP (cAMP) synthesis through regulation of adenylate cyclase (cyaA) activity, which in turn controls fundamental processes such as motility, carbon utilization and storage, pathogenesis, and cell division in many bacteria. Here, we demonstrate that mutation of catabolite repression genes that regulate cAMP levels (crr and cyaA) or the ability to respond to cAMP (crp) confers a large increase in biofilm formation. Suppressor analysis revealed that phenotypes of a cAMP receptor protein (crp) mutant require the fimABCD operon, which is responsible for type 1 fimbria production. Consistently, fimA transcription and fimbria production were determined to be upregulated in a cyaA mutant background by using quantitative real-time reverse transcription-PCR and transmission electron microscopy analysis. The regulatory pathway by which environmental carbon sources influence cAMP concentrations to alter production of type 1 fimbrial adhesins establishes a novel mechanism by which bacteria control biofilm development.

Human PDE4A8, a novel brain-expressed PDE4 cAMP-specific phosphodiesterase that has undergone rapid evolutionary change

We have isolated cDNAs encoding PDE4A8 (phosphodiesterase 4 isoform A8), a new human cAMP-specific PDE4 isoform encoded by the PDE4A gene. PDE4A8 has a novel N-terminal region of 85 amino acids that differs from those of the related 'long' PDE4A4, PDE4A10 and PDE4A11 isoforms. The human PDE4A8 N-terminal region has diverged substantially from the corresponding isoforms in the rat and other mammals, consistent with rapid evolutionary change in this region of the protein. When expressed in COS-7 cells, PDE4A8 localized predominantly in the cytosol, but approx. 20% of the enzyme was associated with membrane fractions. Cytosolic PDE4A8 was exquisitely sensitive to inhibition by the prototypical PDE4 inhibitor rolipram (IC(50) of 11+/-1 nM compared with 1600 nM for PDE4A4), but was less sensitive to inhibition by cilomilast (IC(50) of 101+/-7 nM compared with 61 nM for PDE4A4). PDE4A8 mRNA was found to be expressed predominantly in skeletal muscle and brain, a pattern that differs from the tissue expression of other human PDE4 isoforms and also from that of rat PDE4A8. Immunohistochemical analysis showed that PDE4A8 could be detected in discrete regions of human brain, including the cerebellum, spinal cord and cerebral cortex. The unique tissue distribution of PDE4A8, combined with the evolutionary divergence of its N-terminus, suggest that this isoform may have a specific function in regulating cAMP levels in human skeletal muscle and brain.

Novel targets for Huntington's disease in an mTOR-independent autophagy pathway

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the G(i) signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating G(s)alpha, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.

Administration of phosphodiesterase inhibitors and an adenosine A1 receptor antagonist induces phrenic nerve recovery in high cervical spinal cord injured rats

High cervical spinal cord hemisection interrupts the descending respiratory drive from the medulla to the ipsilateral phrenic motoneurons, consequently leading to the paralysis of the ipsilateral hemidiaphragm. Previous studies have shown that chronic oral administration of theophylline, a phosphodiesterase inhibitor and an adenosine receptor antagonist, can restore function to the quiescent phrenic nerve and hemidiaphragm ipsilateral to hemisection. Both of these actions of theophylline result in an increase in 3′-5′-cyclic adenosine monophosphate (cAMP). Furthermore, the chronic theophylline-mediated respiratory recovery persists long after the animals have been weaned from the drug. To date, the precise cellular mechanisms underlying the recovery induced by theophylline are still not known. Since theophylline has two modes of action, in the present study we tested whether chronic administration of pentoxifylline, a non-selective phosphodiesterase inhibitor, rolipram, a phosphodiesterase-4 specific inhibitor, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1 receptor antagonist, would induce recovery similar to that induced by theophylline in male Sprague–Dawley rats following a left C2 spinal cord lesion. Recovery of left phrenic nerve activity was assessed at 5 or 10 days after the last drug administrations to assess the persistent nature of the recovery. Pentoxifylline, rolipram and DPCPX, all capable of modulating 3′,5′-cyclic monophosphate (cAMP) levels, brought about long-term respiratory recovery in the phrenic nerve ipsilateral to the left C2 lesion at 5 and 10 days after the last drug administration. Therefore, these results suggest that compounds capable of regulating cAMP levels may be therapeutically useful in promoting functional recovery following spinal cord injury.

IGF-1 regulates cAMP levels in astrocytes through a β2-adrenergic receptor-dependant mechanism

We have recently demonstrated that neonatal astrocytes derived from mice lacking beta-2 adrenergic receptors (beta(2)AR) possess higher proliferation rates, as compared to wild-type cells, an attribute that was shown to involve insulin-like growth factor (IGF) signaling. In the present study, we demonstrate that basal cAMP levels in beta(2)AR knockout astrocytes were significantly lower than in wild type cells. Furthermore, treatment with IGF-1 reduced intracellular cAMP levels in wild type astrocytes, yet had no effects on cAMP levels in beta(2)AR deficient astrocytes. Our data suggests that IGF-1 treatment influences cAMP production through a beta(2)AR-dependant mechanism in astrocytes. A deficit of beta(2)AR on astrocytes, as previously reported in multiple sclerosis, may influence cell proliferation, an action which could have implications in processes involved in astrogliosis.

PI3Kγ Is Required for PDE4, not PDE3, Activity in Subcellular Microdomains Containing the Sarcoplasmic Reticular Calcium ATPase in Cardiomyocytes

We recently showed that phosphoinositide-3-kinase-gamma-deficient (PI3Kgamma(-/-)) mice have enhanced cardiac contractility attributable to cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca(2+) content and release but not L-type Ca(2+) current (I(Ca,L)), demonstrating PI3Kgamma locally regulates cAMP levels in cardiomyocytes. Because phosphodiesterases (PDEs) can contribute to cAMP compartmentation, we examined whether the PDE activity was altered by PI3Kgamma ablation. Selective inhibition of PDE3 or PDE4 in wild-type (WT) cardiomyocytes elevated Ca(2+) transients, SR Ca(2+) content, and phospholamban phosphorylation (PLN-PO(4)) by similar amounts to levels observed in untreated PI3Kgamma(-/-) myocytes. Combined PDE3 and PDE4 inhibition caused no further increases in SR function. By contrast, only PDE3 inhibition affected Ca(2+) transients, SR Ca(2+) loads, and PLN-PO(4) levels in PI3Kgamma(-/-) myocytes. On the other hand, inhibition of PDE3 or PDE4 alone did not affect I(Ca,L) in either PI3Kgamma(-/-) or WT cardiomyocytes, whereas simultaneous PDE3 and PDE4 inhibition elevated I(Ca,L) in both groups. Ryanodine receptor (RyR(2)) phosphorylation levels were not different in basal conditions between PI3Kgamma(-/-) and WT myocytes and increased in both groups with PDE inhibition. Our results establish that L-type Ca(2+) channels, RyR(2), and SR Ca(2+) pumps are regulated differently in distinct subcellular compartments by PDE3 and PDE4. In addition, the loss of PI3Kgamma selectively abolishes PDE4 activity, not PDE3, in subcellular compartments containing the SR Ca(2+)-ATPase but not RyR(2) or L-type Ca(2+) channels.

Distinct Functional Domains of Neurofibromatosis Type 1 Regulate Immediate versus Long-Term Memory Formation

Neurofibromatosis type 1 (NF1) is a dominant genetic disorder that causes tumors of the peripheral nervous system. In addition, >40% of afflicted children have learning difficulties. The NF1 protein contains a highly conserved GTPase-activating protein domain that inhibits Ras activity, and the C-terminal region regulates cAMP levels via G-protein-dependent activation of adenylyl cyclase. Behavioral analysis indicates that learning is disrupted in both Drosophila and mouse NF1 models. Our previous work has shown that defective cAMP signaling leads to the learning phenotype in Drosophila Nf1 mutants. In the present report, our experiments showed that in addition to learning, long-term memory was also abolished in Nf1 mutants. However, altered NF1-regulated Ras activity is responsible for this defect rather than altered cAMP levels. Furthermore, by expressing clinically relevant human NF1 mutations and deletions in Drosophila Nf1-null mutants, we demonstrated that the GAP-related domain of NF1 was necessary and sufficient for long-term memory, whereas the C-terminal domain of NF1 was essential for immediate memory. Thus, we show that two separate functional domains of the same protein can participate independently in the formation of two distinct memory components.

The Drosophila DCO mutation suppresses age-related memory impairment without affecting lifespan

The study of age-related memory impairment (AMI) has been hindered by a lack of AMI-specific mutants. In a screen for such mutants in Drosophila melanogaster, we found that heterozygous mutations of DCO (DCO/+), which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), delay AMI more than twofold without affecting lifespan or memory at early ages. AMI is restored when a DCO transgene is expressed in mushroom bodies, structures important for olfactory memory formation. Furthermore, increasing cAMP and PKA activity in mushroom bodies causes premature AMI, whereas reducing activity suppresses AMI. In Drosophila AMI consists of a specific reduction in memory dependent on the amnesiac (amn) gene. amn encodes putative neuropeptides that have been proposed to regulate cAMP levels in mushroom bodies. Notably, both the memory and AMI defects of amn mutants are restored in amn;DCO/+ double mutants, suggesting that AMI is caused by an age-related disruption of amn-dependent memory via PKA activity in mushroom bodies.

Altered role of C-type natriuretic peptide-activated pGC–cGMP–PDE3–cAMP signaling in hyperthyroid beating rabbit atria

The role of C-type natriuretic peptide (CNP) in the pathophysiology of atrial function in hyperthyroidism has not been defined. This study was to define the role of CNP-activated particulate (p) guanylyl cyclase (GC)-cGMP-phosphodiesterase (PDE)3 signaling in the regulation of cAMP levels and contractile and secretory functions in the atria from hyperthyroid rabbits. Experiments were performed in perfused beating rabbit atria. CNP was used to activate pGC. In euthyroid atria from sham-treated rabbits, CNP (100 nM) increased cGMP and cAMP efflux by 176.7 ± 17.7 and 55.3 ± 10.0%, respectively. CNP decreased stroke volume and pulse pressure and ANP release by 51 ± 7 and 41 ± 2 and 60.4 ± 3.2%, respectively. Pretreatment with milrinone blocked the CNP-induced increase of cAMP but without significant changes in decrease of atrial dynamics and ANP release. In hyperthyroid atria, CNP-induced increase of cGMP levels was accentuated, while CNP-induced increase of cAMP was attenuated. The gain of cAMP, i.e., change in cAMP efflux concentration in terms of cGMP was attenuated in the hyperthyroid compared to euthyroid atria. CNP rather increased atrial dynamics in hyperthyroid atria instead of decrease. CNP-induced decrease in atrial ANP release was attenuated. Pretreatment with milrinone blocked the CNP-induced increase of cAMP levels concomitantly with a decrease of atrial dynamics. The present study demonstrates that altered role of CNP-activated pGC–cGMP–PDE3–cAMP signaling is involved in the pathophysiology of hyperthyroid heart.

Phosphodiesterase 3B Regulates cAMP Levels in Human Erythrocytes

It was reported that cAMP participates in a signaling pathway that couples activation of Gs with ATP release from erythrocytes (RBCs). cAMP levels are the product of adenylyl cyclase (AC) activity and degradation by phosphodiesterases (PDEs). RBCs possess AC activity, however, their PDE activity is poorly characterized. Insulin, a PDE3 activator, decreases cAMP levels in RBCs. Here, we investigate the hypothesis that PDE3B is present and active in RBCs. PDE3B was identified in RBC membranes by Western analysis. RBCs (50% HCT) were incubated with the PGI2 analog, iloprost (ILO, 1 μM), in the absence and presence of selective PDE3 inhibitors. ILO alone produced a 90.3±20.6% increase in cAMP (P<0.05). In the presence of cilostazol (CILO, 30 or 100 μM) or its parent compound, cilostamide (CMDE, 30 μM), basal cAMP levels were unchanged. However, in the presence of 30 and 100 μM CILO, ILO–induced cAMP levels increased from 3.0±0.5 to 5.4±1.1 (n=6, P<0.05) and 3.9±0.6 to 10.2±2.7 (n=7, P<0.05) pmole/1010 RBCs, respectively. In addition, CMDE also potentiated ILO-induced cAMP levels from 3.0±0.7 to 5.1±1.4 pmole/1010 RBCs (n=4, P<0.05). Here we demonstrate that PDE3 is present in RBC membranes and is important in the regulation of ILO-induced increases in cAMP. Inhibition of PDE3B could facilitate ATP release from RBCs in response to physiological stimuli. Funded by NIH grant HL-64180 and ADA grant RA-133.

G Protein Signaling Mediates Developmental Processes and Pathogenesis of Alternaria alternata

A G protein alpha subunit gene (AGA1) has been cloned and characterized from a toxigenic and necrotrophic Alternaria alternata pathogen. Targeted disruption of AGA1 in the apple pathotype of A. alternata gave rise to mutants that differed in colony and conidial morphology as well as sporulation. The conidia of wild type and deltaAGA1 mutants showed equal germination on cellulose membranes. However, wild-type germ tubes formed readily from different points around the conidia, grew randomly, and were often branched, whereas those of the mutants formed only at one or both ends of the conidia and tended to grow in straight paths. Targeted disruption of AGA1 also resulted in reduction of pathogenicity on apple leaves, although the mutant produced host-specific AM-toxin, a fungal secondary metabolite associated with pathogenicity of the pathogen, at levels similar to the wild-type strain. Measurement of the intracellular cAMP levels of the mutant revealed that it was consistently higher than that of the wild type, indicating that AGA1 negatively regulates cAMP levels similar to mammalian Galphai systems. These results indicate that the signal transduction pathway represented by AGA1 appears to be involved in developmental pathways leading to sporulation and pathogenesis of A. alternata.

Soluble adenylyl cyclase is required for netrin-1 signaling in nerve growth cones

Growth cones at the tips of nascent and regenerating axons direct axon elongation. Netrin-1, a secreted molecule that promotes axon outgrowth and regulates axon pathfinding, elevates cyclic AMP (cAMP) levels in growth cones and regulates growth cone morphology and axonal outgrowth. These morphological effects depend on the intracellular levels of cAMP. However, the specific pathways that regulate cAMP levels in response to netrin-1 signaling are unclear. Here we show that 'soluble' adenylyl cyclase (sAC), an atypical calcium-regulated cAMP-generating enzyme previously implicated in sperm maturation, is expressed in developing rat axons and generates cAMP in response to netrin-1. Overexpression of sAC results in axonal outgrowth and growth cone elaboration, whereas inhibition of sAC blocks netrin-1-induced axon outgrowth and growth cone elaboration. Taken together, these results indicate that netrin-1 signals through sAC-generated cAMP, and identify a fundamental role for sAC in axonal development.