The role of cyclic nucleotides in modulation of crayfish neuromuscular junctions by a neuropeptide /

DF2, a heptapeptide, is a member of the family of FMRFamide-like peptides and has been shown to increase the amount of transmitter released at neuromuscular junctions of the crayfish, Procambarus clarkit Recent evidence has shown that protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII) and the cAMPdependent protein kinase (PKA) play a role in the neuromodulatory pathway of DF2. The involvement of these kinases led to the prediction that a G-protein-coupled receptor (GPCR) is activated by DF2 due to the role that each kinase plays in traditional GPCR pathways seen in other organisms and in other cells. G-proteins can also act on an enzyme that generates cyclic guanosine monophosphate (cGMP) which mediates its effects through a cGMP-dependent protein kinase (PKG). This thesis addresses the question of whether or not DF2's effects on synaptic transmission in crayfish are mediated by the cyclic nucleotides cAMP and cGMP. The effects of DF2 on synaptic transmission were examined using deep abdominal extensor muscles of the crayfish Procambarus clarkii. An identified motor neuron was stimulated, and excitatory post-synaptic potentials (EPSPs) were recorded in abdominal extensor muscle LI . A number of activators and inhibitors were used to determine whether or not cAMP, PKA, cGMP and PKG mediate the effect of this peptide. Chemicals that are known to activate PKA (Sp-cAMPS) and/or PKG (8-pCPTcGMP) mimic and potentiate DF2's effect by increasing EPSP amplitude. Inhibitors of either PKA (Rp-cAMPS) or PKG (Rp-8-pCPT-cGMPS) block a portion of the increase in EPSP amplitude induced by the peptide. When both kinase inhibitors are applied simultaneously, the entire effect of DF2 on EPSPs is blocked. The PKG inhibitor blocks the effects of a PKG activator but does not alter the effect of a PKA activator on EPSP amplitude. Thus, the PKG inhibitor appears to be relatively specific for PKG. A trend in the data suggests that the PKA inhibitor blocks a portion of the response elicited by the PKG activator. Thus, the PKA inhibitor may be less specific for PKA. Phosphodiesterase inhibitors, which are known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of the peptide. These results support the hypothesis that cAMP and cGMP, acting through their respective protein kinase enzymes, mediate the ability of DFi to increase transmitter output.

Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to Eag and cyclic nucleotide-gated channels

We have isolated a novel cDNA, that appears to represent a new class of ion channels, by using the yeast two-hybrid system and the SH3 domain of the neural form of Src (N-src) as a bait. The encoded polypeptide, BCNG-1, is distantly related to cyclic nucleotide-gated channels and the voltage-gated channels, Eag and H-erg. BCNG-1 is expressed exclusively in the brain, as a glycosylated protein of ≈132 kDa. Immunohistochemical analysis indicates that BCNG-1 is preferentially expressed in specific subsets of neurons in the neocortex, hippocampus, and cerebellum, in particular pyramidal neurons and basket cells. Within individual neurons, the BCNG-1 protein is localized to either the dendrites or the axon terminals depending on the cell type. Southern blot analysis shows that several other BCNG-related sequences are present in the mouse genome, indicating the emergence of an entire subfamily of ion channel coding genes. These findings suggest the existence of a new type of ion channel, which is potentially able to modulate membrane excitability in the brain and could respond to regulation by cyclic nucleotides.

Selective PDE4 inhibitors as potent anti-inflammatory drugs for the treatment of airway diseases

Phosphodiesterases (PDEs) are responsible for the breakdown of intracellular cyclic nucleotides, from which PDE4 are the major cyclic AMP metabolizing isoenzymes found in inflammatory and immune cells. This generated greatest interest on PDE4 as a potential target to treat lung inflammatory diseases. For example, cigarette smoke-induced neutrophilia in BAL was dose and time dependently reduced by cilomilast. Beside the undesired side effects associated with the first generation of PDE4 inhibitors, the second generation of selective inhibitors such as cilomilast and roflumilast showed clinical efficacy in asthma and chronic obstrutive pulmonary diseases trials, thus re-enhancing the interest on these classes of compounds. However, the ability of PDE4 inhibitors to prevent or modulate the airway remodelling remains relatively unexplored. We demonstrated that selective PDE4 inhibitor RP 73-401 reduced matrix metalloproteinase (MMP)-9 activity and TGF-beta1 release during LPS-induced lung injury in mice and that CI-1044 inhibited the production of MMP-1 and MMP-2 from human lung fibroblasts stimulated by pro-inflammatory cytokines. Since inflammatory diseases of the bronchial airways are associated with destruction of normal tissue structure, our data suggest a therapeutic benefit for PDE4 inhibitors in tissue remodelling associated with chronic lung diseases.

Expression of steroidogenic proteins and genes in bovine placenta from conventional and somatic cell nuclear transfer (SCNT) gestations

Pendant la grossesse, les hormones stéroïdes jouent un rôle indispensable dans la régulation des principales manifestations physiologiques telles que la reconnaissance maternelle de la gestation, la réceptivité de l'endomètre, le début du développement embryonnaire ainsi que le maintien de la gestation. Cependant, on sait très peu sur la production de ces hormones et les principaux facteurs des voies intracellulaires impliqués dans le processus de stéroïdogenèse dans le placenta bovin pendant les stades initiaux et plus avancés de la gestation. Par ailleurs, certaines anomalies du placenta chez les bovins suite à une mauvaise production de stéroïdes n'ont pas encore été démontrées. Les objectifs de cette thèse étaient donc de : 1) déterminer la présence et la localisation des principales protéines stéroïdiennes dans le placenta de bovins provenant de gestations de 50 à 120 jours, 2) comparer l'expression placentaire d'une série de gènes et de protéines stéroïdiennes entre une gestation impliquant un transfert de noyaux de cellules somatiques (SCNT) et une gestation non-clonale; 3) étudier l'impact des hormones trophiques et des seconds messagers sur la stéroïdogenèse dans le placenta bovin à 140 +10 jours de gestation. L’utilisation de techniques d’immunohistochimie, d’immunobuvardage et de PCR quantitatif nous a permis d’évaluer la présence d'un large éventail de gènes stéroïdiens (STAR, CYP11A1, HSD3B1, CYP17A1 et SCARB1) qui participent au transport du cholestérol et dans la production de différents types de stéroïdes. Dans cette thèse, nous avons démontré la capacité du placenta bovin d’initier la stéroïdogenèse au début de la gestation et nous avons également déterminé les principales cellules impliquées dans ce processus. Nous avons constaté que les tissus maternels expriment les principaux marqueurs de stéroïdogenèse suggérant une plus grande capacité stéroïdogénique que les tissus fœtaux. En outre, un modèle d'expression des protéines complémentaires stéroïdogéniques entre la caroncule et le cotylédon a été observé, indiquant que la stéroïdogenèse placentaire exige une communication cellule à cellule entre les cellules de la mère et du fœtus. Après avoir démontré les principales cellules impliquées dans la synthèse des hormones stéroïdiennes dans le placenta bovin en début de gestation, nous avons ensuite étudié les modifications possibles de la stéroïdogenèse dans les tissus SCNT cotylédonaires à 40 jours de gestation. Nous avons identifié d'importantes modifications dans l'expression des gènes STAR, CYP11A1, HSD3B1, CYP17A1, et SULT1E1. Conséquemment, nous postulons que l'expression réduite des gènes stéroïdiens peut provoquer une insuffisance de la biosynthèse des hormones stéroïdiennes, ce qui pourrait contribuer à un développement anormal du placenta et du fœtus dans les gestations SCNT à court ou long terme. Finalement, nous avons développé un modèle efficace de culture d’explants de placentome qui nous a permis d'explorer les mécanismes sous-jacents spécifiques à la stéroïdogenèse placentaire. Nous avons exploré l'effet stimulant des hormones trophiques et différents messagers secondaires sur l'expression de différentes protéines stéroïdogéniques ainsi que le taux de progestérone (P4) dans les explants de placentome. En utilisant les techniques de RIA et de PCR quantitatif, nous avons constaté que même si les analogues de l'hormone lutéinisante (hCG) ont un effet stimulant sur plusieurs gènes stéroïdiens, le calcium ionophore est le principal modulateur dans la synthèse de la P4. Ces résultats suggèrent que dans le placenta bovin, la synthèse de la P4 est modulée principalement par l'afflux de calcium intracellulaire, et apparemment les nucléotides cycliques ne semblent pas contrôler ce processus. En conclusion, cette étude contribue de manière significative à une meilleure compréhension des mécanismes d'entraînement de la synthèse des stéroïdes placentaires au début de la gestation et permet aussi d’apporter de nouveaux éclairages sur l'importance des stéroïdes placentaires dans la régulation du développement du placenta et du fœtus.

Physiological analysis of central and peripheral insect circadian pacemaker neurons

Alle bisher untersuchten Lebewesen besitzen (circadiane) innere Uhren, die eine endogene Perioden-länge von ungefähr 24 Stunden generieren. Eine innere Uhr kann über Zeitgeber mit der Umwelt synchronisiert werden und ermöglicht dem Organismus, rhythmische Umweltveränderungen vorweg zu nehmen. Neben einem zentralen Schrittmacher, der Physiologie und Verhalten des Organismus steuert, gibt es in unterschiedlichen Organen auch periphere Uhren, die die zeitlichen Abläufe in der spezifischen Funktion dieser Organe steuern. In dieser Arbeit sollten zentrale und periphere Schrittmacherneurone von Insekten physiologisch untersucht und verglichen werden. Die Neurone der akzessorischen Medulla (AME) von Rhyparobia maderae dienten als Modellsystem für zentrale Schrittmacher, während olfaktorische Rezeptorneurone (ORNs) von Manduca sexta als Modellsystem für periphere Schrittmacher dienten. Die zentralen Schrittmacherneurone wurden in extrazellulären Ableitungen an der isolierten AME (Netzwerkebene) und in Patch-Clamp Experimenten an primären AME Zellkulturen (Einzelzellebene) untersucht. Auf Netzwerkebene zeigten sich zwei charakteristische Aktivitätsmuster: regelmäßige Aktivität und Wechsel zwischen hoher und niedriger Aktivität (Oszillationen). Es wurde gezeigt, dass Glutamat ein Neurotransmitter der weitverbreiteten inhibitorischen Synapsen der AME ist, und dass in geringem Maße auch exzitatorische Synapsen vorkommen. Das Neuropeptid pigment-dispersing factor (PDF), das von nur wenigen AME Neuronen exprimiert wird und ein wichtiger Kopplungsfaktor im circadianen System ist, führte zu Hemmungen, Aktivierungen oder Oszillationen. Die Effekte waren transient oder langanhaltend und wurden wahrscheinlich durch den sekundären Botenstoff cAMP vermittelt. Ein Zielmolekül von cAMP war vermutlich exchange protein directly activated by cAMP (EPAC). Auf Einzelzellebene wurde gezeigt, dass die meisten AME Neurone depolarisiert waren und deshalb nicht feuerten. Die Analyse von Strom-Spannungs-Kennlinien und pharmakologische Experimente ergaben, dass unterschiedliche Ionenkanäle vorhanden waren (Ca2+, Cl-, K+, Na+ Kanäle sowie nicht-spezifische Kationenkanäle). Starke, bei hohen Spannungen aktivierende Ca2+ Ströme (ICa) könnten eine wichtige Rolle bei Ca2+-abhängiger Neurotransmitter-Ausschüttung, Oszillationen, und Aktionspotentialen spielen. PDF hemmte unterschiedliche Ströme (ICa, IK und INa) und aktivierte nicht-spezifische Kationenströme (Ih). Es wurde angenommen, dass simultane PDF-abhängige Hyper- und Depolarisationen rhythmische Membranpotential-Oszillationen verursachen. Dieser Mechanismus könnte eine Rolle bei PDF-abhängigen Synchronisationen spielen. Die Analyse peripherer Schrittmacherneurone konzentrierte sich auf die Charakterisierung des olfaktorischen Corezeptors von M. sexta (MsexORCO). In anderen Insekten ist ORCO für die Membran-Insertion von olfaktorischen Rezeptoren (ORs) erforderlich. ORCO bildet Komplexe mit den ORs, die in heterologen Expressionssystemen als Ionenkanäle fungieren und Duft-Antworten vermitteln. Es wurde die Hypothese aufgestellt, dass MsexORCO in pheromonsensitiven ORNs in vivo nicht als Teil eines ionotropen Rezeptors sondern als Schrittmacherkanal fungiert, der unterschwellige Membranpotential-Oszillationen generiert. MsexORCO wurde mit vermeintlichen Pheromonrezeptoren in human embryonic kidney (HEK 293) Zellen coexprimiert. Immuncytochemie und Ca2+ Imaging Experimente zeigten sehr schwache Expressionsraten. Trotzdem war es möglich zu zeigen, dass MsexORCO wahrscheinlich ein spontan-aktiver, Ca2+-permeabler Ionenkanal ist, der durch den ORCO-Agonisten VUAA1 und cyclische Nucleotide aktiviert wird. Außerdem wiesen die Experimente darauf hin, dass MsexOR-1 offensichtlich der Bombykal-Rezeptor ist. Eine weitere Charakterisierung von MsexORCO in primären M. sexta ORN Zellkulturen konnte nicht vollendet werden, weil die ORNs nicht signifikant auf ORCO-Agonisten oder -Antagonisten reagierten.

Structural insights into the allosteric activation mechanism of cGMP-dependent protein kinase I

Cyclic GMP-dependent protein kinase (PKG) is a key transducer in the NO-cGMP signaling pathway. In this line, PKG has been considered an important drug target for treating hypertensive cardiovascular and pulmonary diseases. However, the investigation of PKG’s allosteric activation mechanism has been hampered by a lack of structural information. One of the fundamental questions on the cGMP-dependent activation of PKG is how the enzyme can distinguish cGMP over cAMP and selectively respond to cGMP. To ensure proper signaling, PKG must have developed unique features to ensure its activation upon the right activation signal. In this thesis, the cGMP-selective activation mechanism of PKG was studied through determining crystal structures of three truncated constructs of the regulatory domain [CNB-A (92-227), CNB-B (271-369), and CNB-A/B (92-351)] of PKG Iβ in the absence or presence of cyclic nucleotides. Herein, two individual CNB domain structures with biochemical data revealed that the C-terminal CNB domain (CNB-B) is responsible for cGMP selectivity, while the N-terminal CNB-domain (CNB-A) has a higher binding affinity for both cGMP and cAMP without showing any selectivity. Based on these crystal structures, mutagenesis studies were performed in which the critical residues for cyclic nucleotide selectivity and activation were identified. Furthermore, we discovered that the conformational changes of the C-terminal helix of the CNB-B that bridges between the regulatory and catalytic domains including the hydrophobic capping interaction are crucial for PKG activation. In addition, to observe the global conformation of the activated R-domain, I solved a co-crystal structure of the CNB-A/B with cGMP. Although a monomeric construct was crystallized, the structure displays a dimer. Strikingly, the CNB-A domain and its bound cGMP provide a key interface for this dimeric interaction. Using small angle X-ray scattering (SAXS), the existence of the cGMP-mediated dimeric interface within the CNB domains was confirmed. Furthermore, measuring cGMP-binding affinities (EC50) of the dimeric interface mutants as well as determining activation constants (Ka) revealed that the interface formation is important for PKG activation. To conclude, this thesis study provides a new mechanistic insight in PKG activation along with a newly found interface that can be targeted for designing PKG-specific activity modulators.

Sistema NPPC-NPR2 na espécie bovina: identificação em folículos antrais e modulação na concentração de nucleotídeos cíclicos e na expressão gênica de PDE3 em complexos cumulus-oócito

Pós-graduação em Biociências - FCLAS

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments.

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca2+]cyt response. Cyclic-nucleotide-mediated elevations in [Ca2+]cyt involved both internal and external Ca2+ stores. Both cAMP- and cGMP-mediated [Ca2+]cyt elevations could be inhibited by the Ca2+-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca2+]cyt. Addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca2+]cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca2+ signaling.

Primary structure and functional expression of a cGMP-gated potassium channel.

Cyclic nucleotides modulate potassium (K) channel activity in many cells and are thought to act indirectly by inducing channel protein phosphorylation. Herein we report the isolation from rabbit of a gene encoding a K channel (Kcn1) that is specifically activated by cGMP and not by cAMP. Analysis of the deduced amino acid sequence (725 amino acids) indicates that, in addition to a core region that is highly homologous to Shaker K channels, Kcn1 also contains a cysteine-rich region similar to that of ligand-gated ion channels and a cyclic nucleotide-binding region. Northern blot analysis detects gene expression in kidney, aorta, and brain. Kcn1 represents a class of K channels that may be specifically regulated by cGMP and could play an important role in mediating the effects of substances, such as nitric oxide, that increase intracellular cGMP.

Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells.

Whole-cell patch-clamp recordings and single-cell Ca2+ measurements were used to study the control of Ca2+ entry through the Ca2+ release-activated Ca2+ influx pathway (ICRAC) in rat basophilic leukemia cells. When intracellular inositol 1,4,5-trisphosphate (InsP3)-sensitive stores were depleted by dialyzing cells with high concentrations of InsP3, ICRAC inactivated only slightly in the absence of ATP. Inclusion of ATP accelerated inactivation 2-fold. The inactivation was increased further by the ATP analogue adenosine 5'-[gamma-thio]triphosphate, which is readily used by protein kinases, but not by 5'-adenylyl imidodiphosphate, another ATP analogue that is not used by kinases. Neither cyclic nucleotides nor inhibition of calmodulin or tyrosine kinase prevented the inactivation. Staurosporine and bisindolylmaleimide, protein kinase C inhibitors, reduced inactivation of ICRAC, whereas phorbol ester accelerated inactivation of the current. These results demonstrate that a protein kinase-mediated phosphorylation, probably through protein kinase C, inactivates ICRAC. Activation of the adenosine receptor (A3 type) in RBL cells did not evoke much Ca2+ influx or systematic activation of ICRAC. After protein kinase C was blocked, however, large ICRAC was observed in all cells and this was accompanied by large Ca2+ influx. The ability of a receptor to evoke Ca2+ entry is determined, at least in part, by protein kinase C. Antigen stimulation, which triggers secretion through a process that requires Ca2+ influx, activated ICRAC. The regulation of ICRAC by protein kinase will therefore have important consequences on cell functioning.

The control of mitosis in mammalian tissues

The question of which factors are central in determining whether a cell will undertake a new round of mitosis or will decycle has been examined in the isolated thymic lymphocyte model. Such cell populations possess both in vivo and in vitro a subpopulation of quiescent lymphoblasts which may be induced to reinitiate their mitotic programme. In the intact animal the major determinant of proliferative activity is the plasma ionised calcium concentration. However it has been established in culture that a variety of hormones, ions, cyclic nucleotides, plant lectins and ionophores may like calcium elicit a mitogenic response. These agents do not appear however to initiate DNA synthesis in an identical fashion. Rather there are two distinct intracellular mitogenic axes. The first axis includes a number of adenylate cyclase stimulants, cyclic AMP, phosphodiesterase inhibitors and magnesium ions. It was found that all these mitogens required extracellular magnesium ions to exhibit their stimulatory capacity. This dichotomy in mitogenic activity was further emphasised by the observation that these mitogens are all inhibited by testosterone, whilst the magnesium-independent mitogens were insensitive to this androgen. Indeed this second group of stimulatory factors required the presence of calcium ions in the extracellular milieu for activity, and were, in contrast to the magnesium-dependent mitogens inhibited by the presence of oestradiol in the culture. By examining the interrelationships between these various mitogens and inhibitors it has been possible to propose a mechanism to describe the activation process in the thymocyte. Studies of the metabolism of cyclic nucleotides, membrane potential and transmembrane ion fluxes indicate that there may be a complex relationship between membrane fluidity, ion balance and cyclic nucleotide levels which may individually or in concert promote the initiation of DNA synthesis. A number of possible mechanisms are discussed to account for these observations.

Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets

Background: Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbβ3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.

Methods: Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.

Results: Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) PKA-mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.

Conclusions: We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

Phosphodiesterase 6 generates intracellular cGMP microdomains in the native endothelium

Endothelial cells (EC) are essential regulator of vascular homeostasis through the generation and release of various bioactive agents, including nitric oxide (NO). NO modulates several vascular functions such as vascular tone and permeability, through the stimulation of soluble guanylate cyclase (sGC) leading to the production of cGMP. Conversely, phosphodiesterases (PDEs) are enzymes metabolizing cyclic nucleotides (cGMP and cAMP) and are therefore major regulatory players for cGMP and cAMP signalling pathways. Although ECs are the main source of NO, little is known on the endothelial NO-cGMP signalling pathway and cellular outcomes. It was then hypothesized that a specific population of cGMP-phosphodiesterases allows ECs to stabilize cGMP levels despite the elevated production of NO. Expression of cGMP-phosphodiesterases was initially studied in resistance mesenteric arteries from mice. PDE5 and PDE6 were both found at mRNA and protein levels in native arteries but PDE6 is not found in cultured ECs. Interestingly, subcellular distributions of both enzymes were distinct. PDE5 appeared to be homogeneously distributed whilst PDE6 catalytic subunits (PDE6 and PDE6) showed a preferential staining in the perinuclear region. These results suggest that PDE6 might be involved in the regulation of cGMP microdomains. Based on these findings, a mathematical model was developed. Simulations of dynamic cGMP levels in ECs support the notion of cGMP microdomains dependent on PDE6 expression and localization. In the absence of PDE6, application of NO either as a single bolus or repetitive pulses led to a homogeneous increase in cGMP levels in ECs despite PDE5 homogeneous distribution. However, PDE6 subcellular targeting to the perinuclear membrane generated a cGMP-depleted perinuclear space. The findings from this study provide the first evidence of the expression and specific intracellular distribution of PDE6 in native endothelial cells that strongly support their involvement in the generation of cGMP microdomains