**2. the c-AMP signaling pathway**

#### **2.1. Overview**

The c-AMP signalling pathway begins with the release of cAMP into the cell which is mostly initiated by the activation of G-protein coupled receptors (GPCRs) by several different hormones and neurotransmitters. The ligand-bound GPCR catalyzes the exchange of GDP for GTP on the α-subunit of the coupled heterotrimeric G protein, which results in the activation of the α-subunit and its dissociation from the βγ dimer. Both the α and the βγ subunits can then activate or inhibit distinct intracellular signalling cascades. The αs of the Gs subtype activates adenylyl cyclases (AC) witch catalyzes the synthesis of cAMP from ATP. Increased levels of cAMP are translated into cellular responses by cAMP effectors. The best known is the c-AMP dependant protein kinase A (PKA), but also include cyclic-nucleotide gated ion channels (CNGCs) and the recently discovered Rap1-guanine nucleotide exchange factor (Epac), three effectors known to mediate a multitude of cAMP signalling pathways. (Figure 2). The end of cAMP signalling is achieved by its decomposition into AMP catalyzed by phosphodiesterases (PDEs) and its active efflux through transporters of the multidrug resistance-assocuated protein (MRP) family [4,5]. One particularity of the cAMP signalling pathway is its high degree of compartmentalization. Multiprotein complexes organize the location of the different cAMP effectors to specific subcellular locations and allow cAMP to propagate a plethora of cell responses in a spatio-temporal manner [3]. These multiprotein complexes are at the foundation of cAMP compartmentalization, they involve AC, the scaffolding proteins AKAPs and PDEs.

contractile proteins, maintenance of a low proliferation rate and can stimulate or inhibit apoptosis (Figure 1). The diversity of cAMP effects in VSMC (and in cells in general), is due to the ability of this second messenger to transduce extracellular signals in a compartmentalized manner, allowing individual stimuli to produce distinct pools of cAMP localized in discrete subcellular regions. These pools of cAMP are produced near a subset of cAMP effectors, themselves located near their substrates and engage specific cell responses according to the cellular context [3]. Adenylyl cyclases (AC), phosphodiesterases (PDE) and the scaffolding proteins A kinase anchored proteins (AKAPs) play a determinant role in cAMP compartmen‐ talization. Final cAMP effect depends on which isoforms of these proteins are expressed. During the VSMC trans-differentiation process, important changes in the expressions of such proteins occur, allowing a re-organization of the cAMP signalling compartmentalization, therefore giving VSMC the ability to acquire properties specific to the trans-differentiated state. After a presentation of the cAMP signalling pathway, this chapter discusses data demonstrat‐

ing the diversity of roles of cAMP in differentiated and transdifferentiated VSMCs.

**Figure 1.** Roles of cAMP (3'-5' adenosine monophosphate) in differenciated and trans-differentiated vascular smooth

The c-AMP signalling pathway begins with the release of cAMP into the cell which is mostly initiated by the activation of G-protein coupled receptors (GPCRs) by several different hormones and neurotransmitters. The ligand-bound GPCR catalyzes the exchange of GDP for GTP on the α-subunit of the coupled heterotrimeric G protein, which results in the activation of the α-subunit and its dissociation from the βγ dimer. Both the α and the βγ subunits can

muscle cells (VSMC); AC8: adenylyl cyclase 8.

**2.1. Overview**

122 Current Trends in Atherogenesis

**2. the c-AMP signaling pathway**

**Figure 2.** Cyclic adenosine 3', 5'-monophosphate (cAMP) is produced from ATP by adenylyl cyclase (AC) upon activa‐ tion of Gs-protein coupled receptors. The local concentration and distribution of cAMP gradients is limited by phos‐ phodiesterases (PDE) which generate localized pools of cAMP throughout the cell. The increase in cAMP is translated to cellular responses by the cAMP effectors protein kinase A (PKA), EPAC (exchange protein activated by cAMP) and cyclic nucleotide -gated ion channels (CNGCs). A kinase anchored proteins (AKAPs) target cAMP effectors to distinct cell compartments. They also intract with AC, PDE, cAMP effectors substrates and further scaffolding proteins, provid‐ ing spatial and temporal specificity of the cAMP pathway.

#### **2.2. Components of the c-AMP signalling pathway**

#### *2.2.1. Formation of c-AMP is regulated by adenylyl cyclases*

In mammals, cAMP is synthesized from ATP by members of the Class-III AC (Adenylyl Cy‐ clase)/ADCY family (E.C 4.6.1.1)1 [6]. This class is comprised of nine trans-membrane (tm) AC enzymes and one soluble AC (sAC). tmAC are grouped into three major sub-families: group 1: AC1, AC3, AC8; group 2: AC2, AC4, AC7; and group 3: AC5, AC6. All nine tmAC can be activated by GTP-bound Gαs and, with the exception of AC9, by the plant diterpen forskolin. Nevertheless, each isoform has a specific pattern of regulation by G proteins, calci‐ um/calmodulin, and proteine kinases [7-9]. For example, differences in patterns of regula‐ tion by G proteins have been associated with isoform-specific differences in AC activation. Whereas AC1, AC5, AC6 and AC8 are inhibited by Gαi, AC2, AC4, AC7 are not. Further‐ more, whereas Gβγ subunits inhibit isoforms AC1 and AC8, they stimulate AC2, AC4 and AC7. GTP-bound Gαs, the activator of all tmAC, is the result of the exchange of GDP for GTP on the α-subunit of G protein and its subsequent dissociation from the βγ dimer. This activation can be a consequence of the binding of GPCR by several different hormones or neurotransmitters (e.g., β-adrenergic, H2-histamine, EP2-prostaglandin, α2a adrenergic and M2-muscarinic receptor), making GPCRs guanine nucleotide exchange factors (GEFs) for Gα subunits. The exchange of GDP for GTP can also be mediated independently from con‐ ventional GPCR/G protein signalling. This way involves entities called "non-GPCR GEFs", such as the recently identified cholinesterase -8a (Ric8a), a cytosolic protein reported to bind to and act as a GEF for numerous Gα in mammalian cells [10]. Signal de-activation is ach‐ ieved by Gα-mediated GTP hydrolysis (endogenous GTPase activity) allowing return of the G*α* subunit to the inactive GDP-bound and its association with G*βγ* dimer to form a G*α βγ*heterotrimeric complex.

Differentiated VSMC have been shown to express different isoforms of AC [18,19]. AC3-5-6 are clearly the most highly expressed isoenzymes in VSMCs, while Type 8 AC (AC8) is undetectable in differentiated VSMCs and is strongly induced in trans-differenti‐

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Phosphodiesterases (PDE) comprise a large superfamily of enzymes; 11 families (PDE1- PDE11) have been characterized on the basis of their amino acid sequences, substrate specif‐ icity, allosteric regulatory characteristics and pharmacological properties [22,23]. In total, the superfamily of PDEs encompasses 25 genes in mammals giving rise to 200 reported distinct gene products corresponding to different splice variants that are often expressed in a tissuespecific manner. The substrate specificity of PDEs includes cAMP-specific, cGMP specific, and dual-specific PDE. PDE 4-7-8 are highly specific for the hydroysis of cAMP, PDE5, 6, 9 are cGMP specific and PDE1, -2, -3, -10, -11 hydrolyse both cAMP and cGMP. There are four major PDE families found in VSMCs: PDE1, PDE3, and PDE4 PDE5 [24]. PDE3 and PDE4 have been shown to account for the majority of cAMP hydrolysis, whereas PDE1 and PDE5 are mainly responsible for cGMP-hydrolysis [25,26]. PDE1A and -1B, are expressed in differentiated VSMC. PDE1A has the particularity to be localized in different cell compartments according to the VSMC phenotype; it is predominantly cytoplasmic in medial contractile VSMC and becomes nuclear in neointimal synthetic VSMC [27]. PDE1C is specifically induced in transdifferentiated VSMC [28]. PDE3A, the main isoform expressed in arterial tissue, platelets and cardiac tissue is found is VSMCs as well as PDE3B. The largest PDE family to date, the cAMP specific PDE4 family, is expressed in numerous tissues, notably in vascular tissue. Four genes (PDE4A/B/C/D) encode over 20 distinct PDE4 isoforms as a result of mRNA splicing and the use of distinct promoters [29]. It was reported that two PDE4 "long forms", PDE4D3 and PDE4D5 are expressed in rat and human VSMC [30,31] and that the two "short forms" PDE4D1 and PDE4D2 are specifically expressed in trans-differentiated VSMC [32]. PDE5A is the major

The first intracellular target of cAMP identified is the well characterized PKA holoenzyme. cAMP-PKA-mediated signalling is known to affect numerous intracellular targets in response to a wide variety of molecular signals. Numerous studies over the past 40 years have identified hundreds of PKA substrates in the plasma membrane, nucleus, and cytoplasm of cells. The PKA holoenzyme is a tetramere consisting of two catalytic subunits (C) that are maintained in an inactive conformation by a regulatory (R) subunit dimer [35]. Binding of two cAMP molecules on each R subunit leads to a conformational change and dissociation of two catalycally active C monomers, which phosphorylate serine and threonine residues on specific substrate proteins. Molecular cloning identified 4 R subunits and 4 C subunits called respec‐ tively RIα, RIβ, RIIα, RΙΙβ, Cα, Cβ, Cγ, and PRKX (the human X chromosome-encoded protein

*2.2.2. Degradation of cAMP is regulated by the cyclic nucleotide phosphodiesterases*

cGMP hydrolyzing PDE expressed in arterial tissues[33,34].

*2.2.3. Effectors of cAMP action*

*2.2.3.1. PKA*

ated VSMC [20,21].

Beyond their synthase activity, ACs can function as scaffolds, and therefore contribute to the cAMP signalling compartmentalization. Indeed, several works have shown that specific AC isoforms have the capacity to interact with several proteins/enzymes on their N-terminus allowing an isoform selective coupling with specific downstream signalling cascades [11,12]. AC isoforms are themselves confined in several structural specific cellular compartments. The best characterized is their association with caveolar, lipid-rafts and the anchoring proteins AKAP [13,14]. Selective adenylyl cyclase isoform localization, regulation and coupling with specific downstream targets provide adenylyl cyclase isoform-selective patterns of signalling, that links specific AC isoforms to distinct cell processes [15,16]. For example, alteration of the AC population expressed in DDT1-MF2 cells (derived from hamster vas deferens smooth muscle) changes the processing of stimulatory and inhibitory input [17] and differential expression of AC isoforms in two VSMC models account for opposite effect of isoprenaline on cAMP production [18].

<sup>1</sup> Adenylyl cyclases (ACs) are currently grouped in six classes based on their primary amino acid sequences. Class I ACs have been found exclusively in γ -proteobacteria. Class II ACs are toxins secreted by Bacillus anthracis, Bordetella pertussis and Pseudomonas aeruginosa. Only few members of class IV, V and VI ACs have been described to date and consists in bacterial enzymes. Class III ACs is universal. Class III ACs is found in metazoa, protozoa, fungi, eubacteria, some archaebacteria and certain green algae. Neither class III ACs nor any other type of AC has ever been conclusively identified in higher plants (Embryophyta).

Differentiated VSMC have been shown to express different isoforms of AC [18,19]. AC3-5-6 are clearly the most highly expressed isoenzymes in VSMCs, while Type 8 AC (AC8) is undetectable in differentiated VSMCs and is strongly induced in trans-differenti‐ ated VSMC [20,21].

#### *2.2.2. Degradation of cAMP is regulated by the cyclic nucleotide phosphodiesterases*

Phosphodiesterases (PDE) comprise a large superfamily of enzymes; 11 families (PDE1- PDE11) have been characterized on the basis of their amino acid sequences, substrate specif‐ icity, allosteric regulatory characteristics and pharmacological properties [22,23]. In total, the superfamily of PDEs encompasses 25 genes in mammals giving rise to 200 reported distinct gene products corresponding to different splice variants that are often expressed in a tissuespecific manner. The substrate specificity of PDEs includes cAMP-specific, cGMP specific, and dual-specific PDE. PDE 4-7-8 are highly specific for the hydroysis of cAMP, PDE5, 6, 9 are cGMP specific and PDE1, -2, -3, -10, -11 hydrolyse both cAMP and cGMP. There are four major PDE families found in VSMCs: PDE1, PDE3, and PDE4 PDE5 [24]. PDE3 and PDE4 have been shown to account for the majority of cAMP hydrolysis, whereas PDE1 and PDE5 are mainly responsible for cGMP-hydrolysis [25,26]. PDE1A and -1B, are expressed in differentiated VSMC. PDE1A has the particularity to be localized in different cell compartments according to the VSMC phenotype; it is predominantly cytoplasmic in medial contractile VSMC and becomes nuclear in neointimal synthetic VSMC [27]. PDE1C is specifically induced in transdifferentiated VSMC [28]. PDE3A, the main isoform expressed in arterial tissue, platelets and cardiac tissue is found is VSMCs as well as PDE3B. The largest PDE family to date, the cAMP specific PDE4 family, is expressed in numerous tissues, notably in vascular tissue. Four genes (PDE4A/B/C/D) encode over 20 distinct PDE4 isoforms as a result of mRNA splicing and the use of distinct promoters [29]. It was reported that two PDE4 "long forms", PDE4D3 and PDE4D5 are expressed in rat and human VSMC [30,31] and that the two "short forms" PDE4D1 and PDE4D2 are specifically expressed in trans-differentiated VSMC [32]. PDE5A is the major cGMP hydrolyzing PDE expressed in arterial tissues[33,34].

#### *2.2.3. Effectors of cAMP action*

#### *2.2.3.1. PKA*

**2.2. Components of the c-AMP signalling pathway**

124 Current Trends in Atherogenesis

heterotrimeric complex.

cAMP production [18].

identified in higher plants (Embryophyta).

*2.2.1. Formation of c-AMP is regulated by adenylyl cyclases*

In mammals, cAMP is synthesized from ATP by members of the Class-III AC (Adenylyl Cy‐ clase)/ADCY family (E.C 4.6.1.1)1 [6]. This class is comprised of nine trans-membrane (tm) AC enzymes and one soluble AC (sAC). tmAC are grouped into three major sub-families: group 1: AC1, AC3, AC8; group 2: AC2, AC4, AC7; and group 3: AC5, AC6. All nine tmAC can be activated by GTP-bound Gαs and, with the exception of AC9, by the plant diterpen forskolin. Nevertheless, each isoform has a specific pattern of regulation by G proteins, calci‐ um/calmodulin, and proteine kinases [7-9]. For example, differences in patterns of regula‐ tion by G proteins have been associated with isoform-specific differences in AC activation. Whereas AC1, AC5, AC6 and AC8 are inhibited by Gαi, AC2, AC4, AC7 are not. Further‐ more, whereas Gβγ subunits inhibit isoforms AC1 and AC8, they stimulate AC2, AC4 and AC7. GTP-bound Gαs, the activator of all tmAC, is the result of the exchange of GDP for GTP on the α-subunit of G protein and its subsequent dissociation from the βγ dimer. This activation can be a consequence of the binding of GPCR by several different hormones or neurotransmitters (e.g., β-adrenergic, H2-histamine, EP2-prostaglandin, α2a adrenergic and M2-muscarinic receptor), making GPCRs guanine nucleotide exchange factors (GEFs) for Gα subunits. The exchange of GDP for GTP can also be mediated independently from con‐ ventional GPCR/G protein signalling. This way involves entities called "non-GPCR GEFs", such as the recently identified cholinesterase -8a (Ric8a), a cytosolic protein reported to bind to and act as a GEF for numerous Gα in mammalian cells [10]. Signal de-activation is ach‐ ieved by Gα-mediated GTP hydrolysis (endogenous GTPase activity) allowing return of the G*α* subunit to the inactive GDP-bound and its association with G*βγ* dimer to form a G*α βγ*-

Beyond their synthase activity, ACs can function as scaffolds, and therefore contribute to the cAMP signalling compartmentalization. Indeed, several works have shown that specific AC isoforms have the capacity to interact with several proteins/enzymes on their N-terminus allowing an isoform selective coupling with specific downstream signalling cascades [11,12]. AC isoforms are themselves confined in several structural specific cellular compartments. The best characterized is their association with caveolar, lipid-rafts and the anchoring proteins AKAP [13,14]. Selective adenylyl cyclase isoform localization, regulation and coupling with specific downstream targets provide adenylyl cyclase isoform-selective patterns of signalling, that links specific AC isoforms to distinct cell processes [15,16]. For example, alteration of the AC population expressed in DDT1-MF2 cells (derived from hamster vas deferens smooth muscle) changes the processing of stimulatory and inhibitory input [17] and differential expression of AC isoforms in two VSMC models account for opposite effect of isoprenaline on

1 Adenylyl cyclases (ACs) are currently grouped in six classes based on their primary amino acid sequences. Class I ACs have been found exclusively in γ -proteobacteria. Class II ACs are toxins secreted by Bacillus anthracis, Bordetella pertussis and Pseudomonas aeruginosa. Only few members of class IV, V and VI ACs have been described to date and consists in bacterial enzymes. Class III ACs is universal. Class III ACs is found in metazoa, protozoa, fungi, eubacteria, some archaebacteria and certain green algae. Neither class III ACs nor any other type of AC has ever been conclusively

The first intracellular target of cAMP identified is the well characterized PKA holoenzyme. cAMP-PKA-mediated signalling is known to affect numerous intracellular targets in response to a wide variety of molecular signals. Numerous studies over the past 40 years have identified hundreds of PKA substrates in the plasma membrane, nucleus, and cytoplasm of cells. The PKA holoenzyme is a tetramere consisting of two catalytic subunits (C) that are maintained in an inactive conformation by a regulatory (R) subunit dimer [35]. Binding of two cAMP molecules on each R subunit leads to a conformational change and dissociation of two catalycally active C monomers, which phosphorylate serine and threonine residues on specific substrate proteins. Molecular cloning identified 4 R subunits and 4 C subunits called respec‐ tively RIα, RIβ, RIIα, RΙΙβ, Cα, Cβ, Cγ, and PRKX (the human X chromosome-encoded protein kinase X, a cAMP dependent kinase that forms a catalytically inactive holoenzyme only with the RI subunit). The R subunits exhibit different cAMP binding affinities and can form both homo and heterodimers leading to a large number of combinations. The subcellular localiza‐ tion of PKA is determined by PKA binding to A kinase ankoring proteins, AKAPs. AKAPs act as scaffolds which give PKA access to substrates localized in specific compartments within the cell and participate to cAMP signalling compartmentalization as depicted below [36,37].

found in both the endothelium and media of human arteries [43]. Functionally, CNG channels play an important role in endothelium dependent vascular dilatation to a number of cAMPelevating agents including adenosine, adrenaline and ATP [45-47]. Concerning the function of CNG in differentiated VSMC, to our knowledge, only one report demonstrates that CNG contributes to thromboxaneA2-induced contraction of rat small mesenteric arteries[48].

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**Figure 3.** The Rap1 GTPases cycle between a GTP-bound (active state) and GDP- bound (inactive state). Cycling be‐ tween the active and inactive states is facilitated by guanine nucleotide exchange factors (GEFs) that release GDP and

The idea of compartimentalized pools of cAMP originated in 1979 when Brunton et al. showed that while both the β-adrenergic receptor agonist isoprotrenol and prostaglandin E1 increased cAMP concentration in perfused rat hearts, only isoproterenol increased glycogen metabolism and phosphorylation of troponin [49]. These results illustrated the fact that different hormones may act through the same messenger to generate different pools of cAMP and mediate distinct physiological responses. An increasing number of results support now the existence of distinct cAMP microdomains that control cAMP signalling. ACs, PDEs and the scaffolding proteins AKAPs are at the foundation of this cAMP signalling compartmentalization [50,51]. As mentioned, -ACs can orchestrate their own microenvironment by recruiting a variety of signalling and scaffolding molecules, - PDEs mediate local cAMP degradation and literally sculpt gradients of cAMP surrounding specific signalling complexes and therefore regulate

allow binding of GTP, as well as GTPase activation proteins (GAPs) wich accelerate GTP hydrolysis.

**2.3. ACs, PDEs and AKAPs are essential to cAMP signaling**

**compartimentalization**

#### *2.2.3.2. Epac family*

Epac proteins are the most recent addition to the group of cAMP signalling effectors. Their discovery explains various effects of cAMP that could not be attributed to the established targets PKA and CNGs. Epac was identified in a database screen conducted to explain the independent activation of the small G protein Rap by cAMP [38]. At the same time, a screen for proteins containing cyclic-nucleotide-binding domains revealed the presence of two isoforms of Epac, Epac1 and Epac2 [39]. Epac proteins function as guanine nucleotide exchange factors (GEFs) both for Rap1 and Rap2. Rap1 and rap2 proteins belong to the Ras family of small G proteins, which cycle between an inactive GDP-bound state and an active GTP-bound state. The GTP-bound Rap mediates signalling by associating with and activating effector proteins. GEFs catalyze the exchange of GDP for GTP and thereby the activation of the small G protein (Figure 3). Herein, Epac1 and Epac2 proteins are also called cAMP-GEF I and II respectively. Their subcellular localizations are determined, like PKA, by binding to AKAPs. Epac1 and Epac2 are present in most tissues, though with different expression levels. Epac1 is highly abundant in blood vessels, kidney, adipose tissue, central nervous system, ovary and uterus, whereas Epac2 is mostly expressed in the central nervous system, adrenal gland, and pancreas. Epac proteins are implicated in many cAMP-regulated processes such as insulin secretion, cardiac contraction, vascular permeability, cell migration, neurotransmitter release and immunity [40,41].

#### *2.2.3.3. CNG famly*

Cyclic nucleotide-gated (CNG) channels are non-selective cation channels first identified in retinal photoreceptors and olfactory sensory neurons. They are opened by the direct binding of cAMP and cGMP. Although their activity shows very little voltage dependence, CNG channels belong to the super-family of voltage-gated ion channels.

CNG channels consists in heterotetrameric complexes resulting from the association of two or three subunits. Six different genes encoding CNG channels, four A subunits (A1 to A4) and two B subunits (B1 and B3), give rise to different channels. Their activity is modulated, at least in part, by Ca2+/calmodulin and by phosphorylation. The role of CNG channels has been established in retinal photoreceptors and in olfactory sensory neurons. Mutations in CNG channel genes give rise to retinal degeneration and color blindness [42].

CNG channels are widely expressed in vascular tissues across species and vascular beds [43,44]. Specifically, CNGA1 was found to be very expressed in the endothelium layer and, with a much lower extent, in VSMC [44]. In contrast, strong expression of CNGA2 has been found in both the endothelium and media of human arteries [43]. Functionally, CNG channels play an important role in endothelium dependent vascular dilatation to a number of cAMPelevating agents including adenosine, adrenaline and ATP [45-47]. Concerning the function of CNG in differentiated VSMC, to our knowledge, only one report demonstrates that CNG contributes to thromboxaneA2-induced contraction of rat small mesenteric arteries[48].

kinase X, a cAMP dependent kinase that forms a catalytically inactive holoenzyme only with the RI subunit). The R subunits exhibit different cAMP binding affinities and can form both homo and heterodimers leading to a large number of combinations. The subcellular localiza‐ tion of PKA is determined by PKA binding to A kinase ankoring proteins, AKAPs. AKAPs act as scaffolds which give PKA access to substrates localized in specific compartments within the cell and participate to cAMP signalling compartmentalization as depicted below [36,37].

Epac proteins are the most recent addition to the group of cAMP signalling effectors. Their discovery explains various effects of cAMP that could not be attributed to the established targets PKA and CNGs. Epac was identified in a database screen conducted to explain the independent activation of the small G protein Rap by cAMP [38]. At the same time, a screen for proteins containing cyclic-nucleotide-binding domains revealed the presence of two isoforms of Epac, Epac1 and Epac2 [39]. Epac proteins function as guanine nucleotide exchange factors (GEFs) both for Rap1 and Rap2. Rap1 and rap2 proteins belong to the Ras family of small G proteins, which cycle between an inactive GDP-bound state and an active GTP-bound state. The GTP-bound Rap mediates signalling by associating with and activating effector proteins. GEFs catalyze the exchange of GDP for GTP and thereby the activation of the small G protein (Figure 3). Herein, Epac1 and Epac2 proteins are also called cAMP-GEF I and II respectively. Their subcellular localizations are determined, like PKA, by binding to AKAPs. Epac1 and Epac2 are present in most tissues, though with different expression levels. Epac1 is highly abundant in blood vessels, kidney, adipose tissue, central nervous system, ovary and uterus, whereas Epac2 is mostly expressed in the central nervous system, adrenal gland, and pancreas. Epac proteins are implicated in many cAMP-regulated processes such as insulin secretion, cardiac contraction, vascular permeability, cell migration, neurotransmitter release

Cyclic nucleotide-gated (CNG) channels are non-selective cation channels first identified in retinal photoreceptors and olfactory sensory neurons. They are opened by the direct binding of cAMP and cGMP. Although their activity shows very little voltage dependence, CNG

CNG channels consists in heterotetrameric complexes resulting from the association of two or three subunits. Six different genes encoding CNG channels, four A subunits (A1 to A4) and two B subunits (B1 and B3), give rise to different channels. Their activity is modulated, at least in part, by Ca2+/calmodulin and by phosphorylation. The role of CNG channels has been established in retinal photoreceptors and in olfactory sensory neurons. Mutations in CNG

CNG channels are widely expressed in vascular tissues across species and vascular beds [43,44]. Specifically, CNGA1 was found to be very expressed in the endothelium layer and, with a much lower extent, in VSMC [44]. In contrast, strong expression of CNGA2 has been

channels belong to the super-family of voltage-gated ion channels.

channel genes give rise to retinal degeneration and color blindness [42].

*2.2.3.2. Epac family*

126 Current Trends in Atherogenesis

and immunity [40,41].

*2.2.3.3. CNG famly*

**Figure 3.** The Rap1 GTPases cycle between a GTP-bound (active state) and GDP- bound (inactive state). Cycling be‐ tween the active and inactive states is facilitated by guanine nucleotide exchange factors (GEFs) that release GDP and allow binding of GTP, as well as GTPase activation proteins (GAPs) wich accelerate GTP hydrolysis.
