Muscarinic activation of Airway smooth muscle (ASM) is of major importance to the physiological and patho-physiological actions of acetylcholine, which induces bronchoconstriction, airway smooth muscle thickening, and the modulation of cytokine and chemokine production by these cells as described in . The parasympathetic nervous system is the dominant neuronal pathway in the control of airway smooth muscle tone. Stimulation of cholinergic nerves causes bronchoconstriction, and mucus secretion as in . The human airways are innervated via efferent and afferent autonomic nerves, which regulate many aspects of airway function. It has been suggested that neural control of the airways may be abnormal in asthmatic patients, and that neurogenic mechanisms may contribute to the pathogenesis and pathophysiology of asthma. Although abnormalities of the cholinergic innervation have been suggested in asthma, thus far the evidence for cholinergic dysfunction in asthmatic subjects is not convincing as mentioned in references [1,2].
Acetylcholine (Ach) is the predominant parasympathetic neurotransmitter, acting as an autocrine or paracrine hormone in the airways and its role in the regulation of bronchomotor tone and mucus secretion from airway submucosal glands in the respiratory tract is well established as described in . More recent findings suggest that acetylcholine regulates additional functions in the respiratory tract, may promoting airway inflammation and remodelling, including airway smooth muscle thickening in inflammatory lung diseases as reported in references [3-5]. Another source of ACh is the non-neuronal cells and tissues, particularly inflammatory cells and the airway epithelium as described in references [6-8].
Collectively, these findings indicate that acetylcholine, derived from the vagal nerve and from non-neuronal origins such as the airway epithelium, may induce cell responses associated with airway wall remodelling and trigger proinflammatory Cytokines as IL-6 and IL-8 release in  by structural cells of the airways, including the airway smooth muscle itself. In addition, muscarinic receptors regulate proliferative and proinflammatory functions of the airway smooth muscle as mentioned in references [9,10].
Acetylcholine acting on muscarinic receptors (mAChRs) anchored at the airway smooth muscle sarcolemma are involved in the generation of a number of signal transducing cascades allowing the activation of the smooth muscle machinery as described in . Airway smooth muscle muscarinic activation is mediated through mAChRs, which are members of the so called G protein-coupled receptors (GPCR) family, which are cell surface receptors, that activate intracellular responses by coupling G proteins as described in  to specific effectors in . Molecular cloning studies have revealed the existence of five mammalian subtypes of muscarinic receptors (m1-m5) as described in . In trachea, smooth muscle expresses mRNAs coding for both m2 and m3 receptors as reported in . Airways smooth muscarinic receptors have been identified as a mixed population of M2 and M3 subtypes roughly in a 4:1 ratio using pharmacological ligand binding studies in [16,17] being the M2 subtype, the most abundant muscarinic receptor in tracheal plasma membranes as reported in references [16,18,19]. In addition, a muscarinic antagonist heterogeneity associated with the M3AchR subtype present in plasma membrane fractions from tracheal smooth muscle has been described in .
It has been claimed that M3AChR represents a primary target of acetylcholine in the airways, involved in the regulation of bronchoconstriction as stated in references [17,18,21,22]. Classically, M3AChRs in ASM are coupled to phospholipase C (PLC)/protein kinase C (PKC) pathway via pertussis toxin (PTX)-insensitive G proteins of the Gq/11 family. The contractile response evoked by M3AChRs stimulation is attributed to the formation of inositol trisphosphate (IP3), the subsequent release of Ca2+ from intracellular stores, the additional influx of extracellular calcium, and the Ca2+-sensitizing effect of PKC as mentioned in [21,23,24].
On the other hand, the stimulation of M2 muscarinic receptors (M2AChRs) in ASM inhibits adenylyl cyclase via activation of PTX-sensitive G proteins of the Gi/o family in [25,26] and therefore M2AChRs are thought to counteract relaxation as reported in . Experimental evidence has been provided that M2AChRs participate directly in ASM contraction but the molecular mechanisms by which the M2AChRs in ASM induce contraction is, still unknown.
Recently, it has been shown that M2AChRs stimulate Gi/o proteins to released βγ dimer, which inhibit the Large Conductance Ca2+-activated K+ Channel Activity (BK channels) as described in . The inhibition of BK channel activity favors contraction of ASM and these BK channels are opposed to the M2AChR-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges as mentioned in .
In addition, the influence of M2AChRs to modulate the relaxant effects of atrial natriuretic peptide (ANP) has been reported. Thus, the stimulation of M2AChRs suppresses ANP-induced activation of particulate guanylyl cyclase via a PTX-sensitive G protein as reported in .
More recently, we showed that muscarinic agonists, via M2AChR induced a massive and selective α1β1-NOsGC migration from cytoplasm to plasma membranes in a dose-dependent manner. Such migration was blocked by PTX, suggesting the involvement of Go/Gi proteins in .
Some of the signal cascades activated by mAChRs at TSM are linked to the generation of second messengers such as cyclic nucleotides: cAMP and cGMP as described in reference . In this review, we address the cGMP generation as a product of the muscarinic activation of ASM. It is well known that in mammalian cells, cGMP is produced by the action of two distinctive guanylyl cyclases, named the NO-sensitive soluble guanylylcyclases (NO-sGC) as reported in  and the single membrane-spanning guanylyl cyclases as published in references [33-35].
2. Body: Cyclic GMP signals during muscarinic activation
The muscarinic activation of tracheal smooth muscle (TSM) fragments associated with smooth muscle contraction, involves the generation of two cGMP signals, at 20-s and 60-s as described in reference  and a kinetic behavior is shown in Figure 1.
Interestingly, the 20-s signal is linked to the onset and the 60s signal is related to the plateau of the smooth muscle contraction as described in . The 20-s signal is associated with the activity of a Soluble guanylyl cyclases, which are nitric oxide stimulated guanylyl cyclase (NO-sGC), which are described in  and the second, the 60-s signal, is linked to membrane-bound natriuretic peptide receptor guanylyl cyclase (NPR-GC) in references [33-35], which has been previously characterized at TSM in [37,38].
The details of the activation of an NO-sGC, generating the early 20-s cGMP signal, presents in the plasma membrane of BTSM, has been previously established in reference .
Soluble guanylyl cyclases are nitric oxide stimulated guanylyl cyclase (NO-sGC) due to the fact that the primary and best-studied endogenous activator is nitric oxide (NO). NO-sGC is a heterodimeric hemoprotein formed by two different subunits, α- and β-subunits, which exist in four types (α1, α2, β1, and β2), each the product of a separate gene as described in [39,40]. Structurally, each subunit consists of N-terminal H-NOX domain, a central domain related to the dimerization, and a C-terminal consensus nucleotide catalytic cyclase domain as described in [41,42]. For the formation of a catalytically active enzyme, both α- and β-subunits are required as reported in . Although the α1β1 isoform is ubiquitous, the α2β1 isoform is less broadly distributed described in [39-43].
The best-characterized heterodimers are the α1/β1 and the α2/β1 isoforms as mentioned in  being the first ones, relevant in our studies as in . At molecular level, His-105 at the amino terminus of the β1 subunit of NO-sGC is the axial ligand of the pentacoordinated reduced iron center of heme, which is required for NO activation of the enzyme. Thus, NO activates sGC by binding to the sixth position of the heme ring, which breaks the bond between the axial histidine and iron to form a 5-coordinated ring with NO in the fifth position as reported in .
By using several experimental approaches as biochemical, pharmacological and molecular biology methods, we established that the first 20-s signal is a product of NO-sGC being sensitive to ODQ, as shown in Figure 2, which is translocated from cytoplasm to the inner face of the airway smooth muscle sarcolemma under muscarinic activation.
In addition, there is a coupling mechanism between M2AChRs and NO-sGC involving a Go/Gi heterotrimeric proteins as previously demonstrated in . Thus, in intact smooth muscle fragments and isolated plasma membranes from bovine tracheal smooth muscle, we showed that the heterodimer of NO-sGC involved in such translocation is the α1β1sGC. The experimental evidence using Western blotting with specific antibodies against all subunits of NO-sGC demonstrate that under muscarinic activation, the α1β1NO-sGC-heterodimer isoform is translocated from cytoplasm to plasma membranes of BTSM. These experimental data are shown in Figure 3.
Since the capability of this α1β1-sGC to migrate to plasma membranes under muscarinic activation, a purification procedure and further identification of this α1β1-sGC heterodimer was also performed. Such NO-sGC translocation involves a M2AChR subtype as previously described in .
Identification of α1/β1 sGC heterodimer in membrane fractions isolated from BTSM strips under muscarinic agonist exposure. Isolated BTSM were incubated in KRB at 37°C in the presence and absence of muscarinic agonist CC (1 x10−5 M) during 0, 20 s and 60 s. The samples were immediately frozen and pulverized with a mortar in liquid nitrogen, following the protocols described to isolate crude plasma membranes fraction as described in [31,54]. The final membranes sediments were electrophoresed in 12% PAGE-SDS, transferred to nitrocellulose membranes, and probed with specific antibodies against the α1, α2, (A) β1 and β2 (B) of NO-sGC subunits. The specific antibodies against α2 and β2 produced negative results. This experiment was performed three times with similar results.
Thus, we demonstrate that the first 20-s cGMP signal is a product of a novel signaling cascade involving M2AChR coupled to Go/i proteins, which facilitates the α1β1-sGC isoform migration from cytoplasm to the BTSM sarcolemma. These experimental evidences support a model for the M2AChR, Gi/o, and heterodimer of α1β1 of NO-sGC novel signal transducing cascade as illustrated in Figure 4.
Proposed model for a novel signal transducing cascade in mammalian cells involving three distinct molecular entities: M2AChR, Gi/o protein, and heterodimer of α1β1 of NO-sGC. (A) Basal condition: The hetero-dimer (α1β1NO-sGC) is located in the cytoplasm and the M2AChR and Gi/o proteins are macromolecules spanning and associated respectively with the plasma membrane bilayer. (B) Under muscarinic exposure, the agonist (Cch) binds at the extracellular domains of M2AChR causing the activation of M2AChR. This induces conformational changes at the M2AChR cytoplasm domains stimulating a PTX-sensitive G protein (Gi/o), which may induce a migration and further activation of the α1β1 heterodimer NO-sGC from cytoplasm to the plasma membrane resulting in a fast rise in cGMP production, which is related to the 20-s cGMP signal generated during the muscarinic activation of tracheal smooth muscle cell.
In the other hand, the 60-s cGMP signal is a product of a Natriuretic Peptide Receptor Guanylylcyclase-B (NPR-GC-B), which was previously identified at TSM, using biochemical as reported in  and molecular biology approaches as described in . The NPR-GC-B (GC-B) is a membrane-spanning homodimer form, which contains an extracellular ligand-binding, trans-membrane, kinase homology, dimerization and carboxyl-terminal catalytic domains that was published in references [46,47]. NPR-GC-B, which is also called NPR-B or NPR2, is activated by CNP, which exists in 22 and 53 amino acid forms that are structurally similar to ANP and BNP as reported in references [48,49].
Furthermore, this GC-B from TSM is a novel G-protein coupled guanylyl cyclase presents in isolated plasma membranes fractions from this smooth muscle subtype as described in . This G-protein-coupled NPR-GG-B showed complex kinetics and regulation, which is summarized in a proposed model as shown in Figure 5 as illustrated in reference . This NPR-B was activated by Natriuretic Peptides (CNP-53>CNP-22>ANP-28) at the Ligand Extracellular Domain, stimulated by Gq-protein activators such as mastoparan, and inhibited by a chloride sensitive-Gi/o, interacting at the Juxtamembrane Domain. The Kinase Homology Domain was evaluated by the ATP inhibition of Mn2+-activated-NPR-B, which was partially reversed by mastoparan. The Catalytic Domain was studied by its kinetics of Mn2+/Mg2+ and GTP, and the catalytic effect with GTP analogs with modifications of the βγ phosphates and ribose moieties. Most NPR-B biochemical properties remained after detergent-solubilization but the mastoparan-activation and chloride-inhibition of NPR-B disappeared. This NPR-GC-B is a highly regulated nano-machinery with domains acting at crosstalk points with other signal transducing cascades initiated by G-Protein Coupled Receptors (GPCR) and affected by intracellular ligands such as chloride, Mn2+, Mg2+, ATP and GTP. In addition, this model contains a novel GPRM domain, a G-protein-regulatory site, which was identified and characterized as the binding domain for the G proteins subunits as described in .
GC-B is abundantly expressed in brain, lung, bone, heart and ovary tissue as reported in . GC-B contains three intramolecular disulfide bonds and is highly glycosylated on asparagine residues. Moreover, GC-B is highly phosphorylated and dephosphorylation is associated with receptor inhibition as described in . ATP increases the enzymatic activity of GC-B by reducing the Michaelis-Menten constant for GTP, an order of magnitude and this nucleotide seems to be essential for maximal activity as demonstrated in reference .
On the other hand, this Natriuretic Peptide Receptor Guanylylcyclase–B (NPR-GC-B), which has been previously identified as the predominant functional TSM membrane-bound GC subtype by using molecular biology in  and biochemical approaches as described in . Nucleotide sequence of membrane-bound GC transcripts retrieved by RT-PCR correspond to the bovine GC-B isoform, indicating the predominance of this NPR-sensitive GC-B over GC-A and GC-C, which are also expressed in TSM in reference . Both ANP and CNP stimulates the TSM membrane-bound GC activity in a concentration-dependent manner, but the CNP effect (10-8 to 10-5 M) was significantly higher than that exerted by ANP, whereas guanylin (a GC-C activator) showed no effect. Given that CNP specifically activates GC-B receptors and that it activates GC-A receptors only marginally, the significant CNP effect on TSM cGMP production indicates that NPR-GC-B is the most abundantly expressed tracheal membrane-spanning GC isoform, further confirming the RT-PCR results in .
This NPR-GC-B is regulated in an opposite way by two MAChRs signaling cascades, acting the M2AChR as an inhibitor and the M3AChRs as an activator of this NPR-GC-B as mentioned in .
This novel signal transducing system is illustrated as a model in Figure 6. The relevant feature of these two opposite signal cascades that regulate this NPR-GC-B is the coupling of two MAChRs and distinctive G proteins. Thus, this activation system coupled a M3AChR to a Gq16 being stimulated by mastoparans (tetradecapeptides from wasp venoms), whereas, on the inhibitor cascade of NPR-GC-B, via M2AChR coupled to a Go/Gi protein has been previously described in references [35,38,56].
A model for coupling M3AChR, via Gqα16ßγ to activate NPR-GC-B in plasma membranes from BTSM. This model is composed of three separate and different molecular entities, M3AChR, a GPCR seven transmembrane receptor, a heterotrimeric G protein and homodimeric NPR-GC (cGMP producing enzyme) as the effector. The drawings do not take into account the actual structural biology (molecular mass) of these entities; it is a scheme to suggest the ﬂow of information in this novel signal transducing cascade, which is indicated by the dashed lines. Thus, a muscarinic agonist (ACh) binds at extracellullar domains of M3AChR inducing conformational changes at the cytoplasmic i3M3AChR domain, which stimulates the Gqα16ßγ, to release its active subunits that interact with NPR-GC. Mastoparan and its active analogues may act at the interactions between i3M3AChR domain and the Gqα16ßγ protein as indicated in the scheme.
Mastoparans are tetradecapeptides isolated from wasp venom as reported in [57,58] and they are well known as heterotrimeric G-protein activators as described in [59,60]. Moreover, mastoparan is able to activate the NPR-GC-B associated with plasma membranes fractions from TSM in . Following these results, we evaluate the effect of these peptides on TSM contraction. It was found that mastoparan and analogs were able to inhibit in a selective manner the BTSM contraction induced by muscarinic agonist as carbachol (CC) as shown in Figure 7. Thus, our original findings indicated that mastoparans in the nM range decreased the contractile maximal responses induced by CC without changing its EC50. One explanation for the decrement on the contractile maximal responses by mastoparans may be related to the ability of these tetradecapeptides to disturb the function and the contractile machinery of the BTSM type through cytotoxic mechanisms, which have been described in other biological models, specifically in the μM concentration range as described in [61,62].
This assumption is not supported by our results on the effects of classic TSM spasmogens as 5-HT in as described in reference , which produced potent contractions even in the presence of mastoparan (nM) as shown in Figure 8. These findings can be explained since this bioactive amine has been claimed to exert their physiological effects on TSM through specific GPCRs. These receptors are the 5-HT2A, which induced activation of the Gq/11 protein and its downstream effector phospholipase C (PLC) leading to intracellular phosphatidylinositol turnover and Ca2+mobilization as reported in . These Gq/11 proteins are mastoparan-insensitive ones. The latter facts can explain the mastoparan-insensitivity of the serotoninergic transducing cascades at TSM. Furthermore, our results demonstrated that mastoparan inhibits selectively the muscarinic activation without altering other spasmogens transducing cascades at TSM.
The disappearance of the second signal of cGMP (60-s) correlates well with a significant reduction on the contractile maximal responses as here described in Figure 7. This 60-s cGMP signal is a product of NPR-GC-B as above mentioned in references [35,54,55]. Recently, we further recognized that muscarinic agonist and mastoparan activations of NPR-GC-B in isolated BTSM plasma membranes fraction involve this mastoparan-sensitive-Gq16 protein in . Thus, the pre-exposure of TSM strips to mastoparan stimulated the Gq16 protein, disrupting this signal cascade inducing the failure of this muscarinic-dependent TSM contraction. However, the serotoninergic-dependent TSM contraction remained functionally active indicating that the smooth muscle machinery remains fully active.
Until now, the BTSM muscarinic activation is unique biological system that involves two cGMP signals as second messengers in references [35,36]. In addition, this activation is a highly regulated biological process, which starts with M2/M3AChRs coupled to two different heterotrimeric G proteins, leading to a fine time regulation and stimulation of two distinctive guanylyl cyclases that accomplish the generation of these two (20-s and 60-s) cGMP signal peaks.
Based in our results, it can be postulated that the first cGMP signal (20-s) is a product of the activation of one M2AChR subtype coupled to a PTX-sensitive-Gi/o protein that leads to the translocation and further activation of the heterodimer α1β1 NO-sGC isoform, anchored to plasma membrane. Recently, it has been suggested that a plasma membrane-bound GC, may provide a localized pool of cGMP in , which seems to be in a similar trend suggested by our work. The second signal peak is a product of a the activation of M3AChR coupled to the stimulation of mastoparan-sensitive Gq16 as reported in reference  to turn on a transmembrane-homodimer as NPR-GC-B as described in [35,55].
Thus, a dysfunction of these M2/M3AChR signal transducing cascades has been implied in the pathophysiological mechanisms of bronchial asthma as mentioned in references [1,3,4] and Chronic Obstructive Pulmonary Disease (COPD) in . In this sense, we attempted to evaluate these novel signal transducing cascades involving guanylyl cyclase activities above discussed, in an experimental asthma model in rats as described in reference . In addition, it has been claimed that excessive NO production that occurs in asthma induces a down-regulation of NO-sGC as reported in .
Following these rationale, we used cultured Airway Smooth Muscle Cells (ASMC) from Control and an experimental asthma model (Ovoalbumin exposed rats or OVA-ASMC), which were sensitized to Ovoalbumin using a procedure described in [70,71]. In these ASMC, we evaluate the cGMP production by NPR-GC-B stimulated by muscarinic agonists, mastoparans, natriuretic peptides (ANP, CNP) and for NO-sGC, a classic NO donor as Sodiun NitroPrusside (SNP) and a selective inhibitor for the NO-sGC as 1H-[1, 2,4] Oxadiazolo[ 4,3-a]quinoxalin-1-one (ODQ) were used.
All ASMC exposed to a NO-donor compound as SNP and muscarinic agonist as CC increased cGMP intracellular levels, which were inhibited by ODQ, suggesting that NO-sGC is present and this activity is partially responsible for cGMP production in these ASMC. However, OVA-ASMC showed low basal cGMP production compared to CONTROL ASMC in reference  as shown in Figure 10 possibly due to substantial reduced NO-sGC expression reflected in decrease the steady state levels of NO-sGC subunit mRNAs and protein level expression, as described in intact lung tissue from OVA-sensitized mice described elsewhere in reference .
Trying to understand the role of the NPR-GC-B in the ASMC, we studied the effect of several activators of this NPR-GC such as natriuretic peptides (ANP and CNP) and mastoparans as previously described in BTSM in references [37,38,71]. Thus, ASMC from Control and OVA-exposed rats were cultured and exposed to these NPR-GC-B activators. Thus, in Figure 11, the total GC activity at OVA-ASMC was stimulated by CNP, ANP and muscarinic agonist (Cch) indicating that NPR-GC-B was present in Control ASMC, which are similar results described in intact BTSM strips and isolated plasma membranes from BTSM in references [37,38,53,55]. In addition, the OVA-ASMC showed a more significant stimulation by these NPR-GC activators. Interestinly, the combination of CC plus ANP and CNP increased in more than 6 times the GC activity. As expected, mastoparan, an activator of NPR-GC-B, via Gq16 dramatically increased in 5 times, the GC activity as reported in . These data showed for the first time that there is an hyperstimulation of M3AChR/Gq16/NPR-GC-B cascade in ASMC from a experimental asthma model.
Taking together all these results indicate that the ASMC from OVA-sensitized rats express a reduced NO-sGC activity and an increased in the NPR-GC-B activity. This imbalance between these two guanylyl cyclases can contribute to airway hyperreactivity and might be implicated in the hyperplastic smooth muscle responses and remodeling present in asthma. All these previous experimental data unravels some of complex molecular mechanisms associated with the muscarinic activation of ASM. This muscarinic activation is the most physiological, pathophysiological and pharmacological relevant mechanisms because Ach is the neurotransmitter-linked stimulation of ASM in asthma and COPD. Thus, this works opens new trends for the pathophysiological and pharmacological mechanisms, which may lead to new therapeutic approaches for the treatment of these chronic respiratory diseases, such as asthma and COPD, in which, the ASM is involved.
In this review, we exposed the recent experimental evidences, in relation to the generation of cGMP, on the muscarinic activation of ASM, which is the essential element in the bronchoconstriction presents in asthma.
Moreover, this muscarinic activation seems to be involved in the remodelling and functional changes of ASM described in asthma and COPD as described in references [1-6,73]. We discussed the existence of two cGMP signals, at 20-s and 60-s in , which are products of two distinctive guanylyl cyclases, the NO-sensitive soluble guanylyl cyclases (NO-sGC) and Natriuretic Peptide Receptor Guanylyl Cyclase-B (NPR-GC-B). The 20-s cGMP signal is linked to the activation of a M2AChR coupled to Go/Gi proteins inducing a massive and transient α1β1-NO-sGC translocation from cytoplasm to plasma membranes of ASM as reported in .
The 60-s cGMP signal is associated with a NPR-GC-B in , a novel G-protein coupled NPR-GC-B as described in references [38,53,55], which is nano-machine regulated by GPCR and also modulated, in an opposite way, by an activator M3AChRs coupled to Gq16 to activate NPR-GC-B (M3AChR/Gq16/NPR-GC-B cascade) that is stimulated by mastoparan as reported in  and an inhibitor M2AChR signal cascade that was partially characterized.
Mastoparan inhibited in a selective manner the muscarinic-dependent ASM contraction and affected the two cGMP signals. The ASM muscarinic activation is unique biological system involving two cGMP signals and a dysfunction of these M2/M3AChR cascades has been implied in asthma and COPD as published in references [1-6].
We used another mammalian model as ASM from rats, which are more prone to develop experimental asthma using Ovoalbumin leading to the OVA-sensitized rats model as described in [70,71]. In isolated and cultured Airway Smooth Muscle Cells (ASMC) from Control and OVA, we evaluated the cGMP production in these ASM cultured cells. All ASMC showed cGMP increments by a classic NO donor as SNP being ODQ-sensitive, indicating that NO-sGC is present, but OVA-ASMC showed low basal cGMP production compared to Control ASMC described in , which confirmed the molecular biology results reported elsewhere in reference . Moreover, NPR-GC-B is present in Control ASMC but OVA ASMC displayed an hyperstimulation of M3AChR/Gq16/NPR-GC-B cascade, which is an original experimental findings as reported in .
These results indicate that the OVA-ASMC express a reduced NO-sGC and increased NPR-GC-B activities. This imbalance between these two GCs can contribute to airway hyperreactivity and might be implicated in the abnormal ASM responses present in asthma and COPD.Acknowledgement
This work was supported by grants from CDCH-UCV # PG -09-7401-2008/2 (RGA) and CDCH-UCV # PI -09-7726.2009/2 (ILB) and financial support for the publication of this book chapter. WHS is a Graduate student at Ph-D program of Curso de Postgrado en Ciencias Fisiológicas. Facultad de Medicina, Universidad Central de Venezuela (UCV). The authors thank to Dr. Marcelo Alfonzo-González for the editing process of this manuscript.
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