Vascular Sympathetic Neurotransmission and Endothelial Dysfunction Vascular Sympathetic Neurotransmission and Endothelial Dysfunction

Endothelium is an important regulator of vascular tone via release of various endothelium- derived substances. Several studies have reported that endothelium may decrease the release of noradrenaline from vascular postganglionic sympathetic nerves and thus neuro- genic vasoconstriction. Endothelium derived-mediators (adenosine and NO) can modify vascular sympathetic neurotransmission and are relevant for vascular homeostasis. This is a relevant issue in terms of vascular homeostasis and, any modification, may lead to a deregulation process and to pathologies. Focus on NO-mediated effects on vascular sym- pathetic transmission will be done, discriminating the effects ascribed to NO generated by NO synthases located in the different vascular layers. A comparison between mesenteric/ tail arteries will also be explored, particularly the relevance of the transsynaptic modulation on noradrenaline release mediated by endothelial NO and adenosine in normotensive/ hypertensive vascular tissues. Adenosinergic system, namely adenosine, nucleoside transporters and adenosine receptors, can be influenced by endothelium mediators, namely by NO, causing alterations on the way these players interact with each other. In conditions where endothelium is compromised, a deregulation occurs with an increase in vascular sympathetic neurotransmission (as a consequence of adenosinergic system dynamic alter-ation). In summary, the impact of endothelial dysfunction on vascular neurotransmission is debated with particular focus on adenosinergic and nitroxidergic system dynamics.


Introduction
Endothelium has been described to present key roles in the vascular physiology: various endothelium-derived endogenous substances [1], namely contracting (endothelin, prostaglandin F2a and thromboxane A2) and/or relaxing (prostaglandin I2 and nitric oxide, NO) factors [2,3] can modulate blood vessel tone. These substances, known as endothelium-derived contracting factors (EDCF) or endothelium-derived relaxing factor (EDRF), can modify the vascular smooth muscle tone directly, acting on smooth muscle cells, or indirectly, by altering sympathetic transmission [4]. Nevertheless, when endothelium integrity and/or function is compromised, such regulation can be impaired. Indeed, evidence suggests that endothelial dysfunction (present an altered NO production and oxidative stress) may contribute to the pathogenesis of hypertension. As a consequence, an increase in peripheral vascular resistance occurs in conditions where endothelium is somehow injured. For example, endothelium dysfunction leads to the enhancement of contractile responses to vasoconstrictor agents [2,[5][6][7][8]. Nevertheless, in the literature, there are also innumerous other factors that can also influence endothelium function and, therefore, vascular responsiveness, such as tetrahydrobiopterin (BH4), sex hormones and gender, angiotensin, insulin, vascular endothelial growth factor, vitamin D, adiponectin, uric acid, lipids, oxygen-derived free radicals, aldosterone and epithelial sodium channels.
In this chapter, the impact of endothelial dysfunction on vascular neurotransmission is debated with particular focus on adenosinergic and nitroxidergic system dynamics.

Endothelium and vasodilation
The vascular wall is composed of layers that can be identified by their respective morphology and by the different functions exhibited by respective cells which, ultimately, are responsible for the vascular tone, influencing blood pressure. Arteries and veins have a similar structure presenting three layers: intima or endothelium, media or smooth muscle and adventitia.
The tunica intima is the inner and thinnest layer and surrounds the lumen. It is made up of endothelial cells lining the entire vasculature and includes circular elastic bands, the internal elastic lamina. The tunica media, also called muscle layer, is composed of vascular smooth muscle, which helps regulate the size of the lumen and externally present circular elastic bands, the external elastic lamina. This tunica differs between arteries and veins: arteries contain more smooth muscle than the tunica media of their counterpart, the veins, and this allows arteries to constrict and dilate to adjust the volume of blood needed by the tissues that they support. Additionally, the structure of arteries differs between large arteries and resistant arteries: in the first type, arteries present a media with large amount of elastic fibers disposed between smooth muscle cells and the thickness of the vascular wall is thinner than that exhibited by resistant arteries that often have multiple strands of smooth muscle layers. The external layer, adventitia layer is composed of connective tissue allowing the blood vessel to withstand forces acting on the vessel wall and of collagen fibers that anchor the vessel to surrounding tissues.
The endothelium can evoke effects, dilation or contraction of the underlying vascular smooth muscle, by releasing endothelium-derived relaxing factors (EDRF) such as NO or endotheliumderived contracting factors (EDCF) such as endothelin or prostanoids.

NO effects on vasodilation and endothelial dysfunction
NO is a well-known EDRF that induces vasodilation through the activation of soluble guanylyl cyclase in the vascular smooth muscle cells producing cyclic guanosine monophosphate (therefore, through the signaling pathway that can be represented as NO-cGMP/cGMP-dependent kinases).
It is well accepted that the benefits of NO released from endothelium are compromised in vascular diseases and aging since there is a reduced amount of NO. However, evidence also show that the production of NO can be upregulated, for example, by estrogens, exercise and dietary factors and downregulated by oxidative stress, smoking, pollution and oxidized lowdensity lipoproteins.
Moreover, when endothelium is dysfunctional, the vasodilation induced by endothelial mediators is impaired and it can even lead to vascular smooth muscle cells contraction. For instance, in aged subjects and in vascular diseases (essential hypertension and diabetes) when the production of NO is compromised, endothelium-dependent contractions are intensified.
NO is produced by three isoforms of NO synthase, presenting a more general distribution in the human body than that initially predicted: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). nNOS is constitutively expressed in central and peripheral nervous system contributing to regulation of blood pressure, smooth muscle dilation and vasodilation via peripheral nitrergic nerves. iNOS is expressed in several cell types and generates large amounts of NO, which is involved in the pathophysiology of inflammatory diseases, as regulatory effector molecule of the innate immune response and septic shock. eNOS is expressed mainly in endothelial cells and has several vasoprotective and anti-atherosclerotic effects as well as an important role in vascular tone and thus blood pressure regulation.
Many cardiovascular risk factors lead to oxidative stress, eNOS uncoupling and endothelial dysfunction in the vasculature. eNOS generates NO which results from the activity of two domains, the oxygenase domain that convert L-arginine to L-citrulline plus NO and the reductase domain that convert nitrites to NO [9].
As mentioned above, NO production from endothelium can be upregulated or downregulated by a number of factors of which vascular endothelial growth factor (VEGF) can upregulate eNOS. Interestingly, a chronic side effect of VEGF inhibitors is the occurrence of hypertension, suggesting a physiological role for VEGF in maintaining endothelial control of vasomotor tone [10][11][12]. In humans, in hypertension, VEGF inhibitors may cause increased production of endothelin-1 [13,14] and reduced vascular response to acetylcholine [15,16].
Acute and chronic increases in flow as well as the resulting augmentation in shear stress of the blood on the endothelial cells can be altered through Ca 2+ -dependent and Ca 2+ -independent pathways. It has been described that Ca 2+ -independent pathway can increase both the expression and activity of eNOS and thus the release of NO [17]. The role played by the endothelial cells to protect against thrombin and other platelet products by increasing the activity of eNOS has been demonstrated both in vitro [18][19][20][21][22][23][24][25][26] and in vivo [27]. Serotonin and adenosine diphosphate are mediators released by aggregating platelets, which may activate eNOS and increase NO production. When endothelium is absent/dysfunctional, vasodilation is no longer observed, and aggregating platelets induce contractions, because they release vasoconstrictors (thromboxane A2 and serotonin). When platelet aggregation occurs in a healthy artery (i.e, with an intact and physiologically active endothelium), serotonin (and ADP) release by the platelets as well as production of thrombin will increase NO release from endothelial cells. Thus, NO will be increased in the vicinity of smooth muscle cells inducing dilation, and consequently, increasing blood flow.
Another important factor influencing NO production relies on the presence of reactive oxygen/ nitrogen species (ROS/RNS). Indeed, several enzymes from endothelium can produce superoxide anions such as nicotinamide adenine dinucleotide phosphate oxidase (NOX), xanthine oxidase (XO), cyclooxygenases (COX) and also eNOS but only when there is a deficient supply of substrate or of the cofactor BH4. Under pathophysiological conditions, superoxide anions scavenge NO resulting in the formation of peroxynitrite, reducing considerably the bioavailability of NO. Moreover, ROS can inactivate eNOS through S-glutathionylation. Taken together, these may explain why oxidative stress is often associated with endothelial dysfunction.
Moreover, intake of a number of natural products, such as flavonoids and other polyphenols, favors endothelium-dependent dilations and protects endothelium from dysfunction through increased production of NO. The protective effects of polyphenols against endothelial dysfunction involve increased production of NO in response to endothelium-derived vasodilators resulting from: facilitation of the effects of NO on the vascular smooth muscle cells, increased levels of BH4, calcium-independent phosphorylation of eNOS, antioxidant properties preventing the uncoupling of eNOS, activation of estrogen receptors and upregulation of AMP-activated protein kinase (AMPK) and of NAD(+)-dependent deacetylase (SIRT1) [28][29][30][31].

Influence of NO on another EDRF
Besides its direct role as a vasodilator, NO also modulates the release of other endotheliumderived mediators. Thus, in a number of larger arteries, endothelium-derived hyperpolarization (EDH)-mediated dilations become prominent only when the synthesis of NO is inhibited [32,33]. Hence, EDH is able to take over, at least temporarily, in the case of 'classical' endothelial dysfunction associated with a loss of NO synthesis, demonstrating strong compensatory efficiency of EDH-mediated responses. Intriguingly, exogenous NO attenuates EDH-mediated responses in coronary arteries in vitro [34] and in coronary circulation in vivo [35,36]. Moreover, NO has been shown to exert a negative feedback effect on endothelium-dependent dilation through cGMP-mediated desensitization in isolated coronary arteries [32]. Indeed, clinical studies show that chronic therapy with nitrate, used as a NO donor, in patients with ischemic heart disease does not yield a benefit on mortality [37,38], confirming the importance of the physiological balance between NO and EDH. Moreover, the amount of NO formed in the endothelial cells controls the release of vasoconstrictor prostanoids [39,40].

Endothelium and sympathetic neurotransmission
The sympathetic nervous system (SNS) is known to play a fundamental role in the short-and long-term regulation of different vascular functions. Vessels contain sympathetic nerves distributed between smooth muscle and adventitia layers [41]. Sympathetic nerve fibers are enveloped in Schwann cells: most nerve fibers travel through individual channels in the Schwann cell, but small fibers are sometimes bundled together within a single channel [42]. The SNS signals to dilate or constrict the vessel, changing the lumen size, i.e., regulating vascular tone and, therefore, affecting blood pressure.
Nowadays, it is well established that SNS contributes to the modulation of vascular function and that this relationship is a key factor in the development of cardiovascular diseases. Several factors, such as the renin-angiotensin system, NO, ROS and endothelin, influence this modulation at central and peripheral level [43][44][45]. Moreover, endothelial function also seems to be regulated by SNS, mainly in the control of vascular tone. Additionally, endothelial dysfunction as well as increase in sympathetic activity has been associated to cardiovascular risk factors and disease. For example, in studies carried out in healthy subjects, an increase in sympathetic activity was associated with a decrease in endothelial function [46] Moreover, in humans, stiffness of large artery was also associated with an increased activity of SNS [47]. On the other hand, large artery stiffness can interfere with autonomic regulation by impairing carotid baroreflex sensitivity [48].
The influence of endothelium in noradrenaline release has also been previously demonstrated [49,50]. This conclusion was obtained not only in arteries without endothelium but also in a model of endothelial dysfunction (i.e, essential hypertensive arteries), which is shown in Table 1. This type of information can be obtained from experiments where synapse events are mimicked allowing the evaluation of putative players able to alter neurotransmitter release from the nerves. Indeed, in such experiments, the use of selective pharmacological tools, such as agonists/antagonists of receptors or of activators/inhibitors of proteins or enzymes, can reveal their respective role in the neurotransmission dynamic. For instance, in experiments where rat vascular tissues, preincubated with [ 3 H]-noradrenaline, are electrically stimulated (5 Hz, 100 pulses, 1 ms, 50 mA), the release of 3 H is induced (which mimics a physiological depolarization) and can be measured by liquid scintillation spectrometry. In addition, by altering the receptors or proteins activated (with pharmacological tools), it is possible to evaluate the activity/role of a specific player in neurotransmitter release (please see previous articles from our group where the methodology is described in detail [49,51,52]). For example, in Table 1, data refer to tissues that were stimulated twice at 30-min interval: outflow (b n ) refers to the 5-min period immediately before each stimulation period. The electrically evoked tritium overflow (S n ) was calculated by subtracting the estimated basal outflow from total outflow observed during and in the 25-min period subsequent to S 1 and expressed as a percentage of the tissue 3 H content at the onset of stimulation. Two animal models have been used: spontaneously hypertensive rats (SHR), a well-established model of essential hypertension [53,54], and the respective controls, the Wistar Kyoto (WKY) rats. Moreover, in WKY animals, some arteries were endothelium denuded. The influence of these conditions on the release of S 1 was evaluated, and the results are presented in Table 1.
The results in this table show that the outflow observed in the endothelium-denuded vascular tissue is lower than that obtained in intact tissue. Also, the S 2 values obtained in the endothelium-denuded arteries are altered, with values higher than those observed in intact tissues. These data reveal the importance of a healthy endothelium to the sympathetic neurotransmission homeostasis, once it seems to present a transsynaptic influence mediated by endothelium. In pathological conditions, this influence can be impaired augmenting the amount of noradrenaline release and causing vasoconstriction.
Several substances produced in endothelial cells, such as NO, adenosine, ROS and/or RNS (e.g. peroxides, superoxide, hydroxyl radical, and singlet oxygen) and prostaglandins can influence sympathetic transmission [55,56]. Also, the activity of some enzymes, such as adenosine kinase, adenosine deaminase, NOX, XO and COX, can be altered leading to changes in the bioavailability of their respective products, influencing, indirectly, sympathetic neurotransmission.

NO and vascular neurotransmission
There is evidence demonstrating that NO can modulate sympathetic neurotransmission modifying vascular smooth muscle tone, in various vascular beds, such as in coronary [57,58], mesenteric [50,59,60] and pulmonary arteries [61][62][63]. Indeed, and as illustrated in Figure 1, a NO donor, DEA-NONOATE (10 μM) altered noradrenaline release (measured as explained above, i.e., by determining the amount of 3 H overflow using liquid scintillation spectrometry) in differential mode depending on the vascular territory: an increase of noradrenaline release occurs in tail artery contrasting to mesenteric territory where noradrenaline release is reduced.
Another relevant data are related with NO source, i.e., the type of NOS that generates NO ( Figure 1): in tail arteries, NO production is ascribed mainly to eNOS isoform, particularly to eNOS oxygenase domain with residual activity of the eNOS reductase domain [50], while in mesenteric arteries, nNOS, with both reductase and oxygenase domains being equally active, seems to be the most relevant isoform producing NO.
These differences in vascular neurotransmission elicited by NO can be explained by the activation of different pathways, leading to opposite outcomes. In resistant arteries, such as tail artery, the well-established NO-cGMP/cGMP-dependent kinases activating voltagedependent-Ca 2+ seem to be the predominant pathway [64], leading to vasoconstriction. However, in other vascular territories, such as the mesenteric artery, NO actions, in addition to the classically accepted activation of intracellular cGMP-dependent pathway [65], can also activate cGMP-independent pathways, namely by eliciting an energy decrease in mitochondria (i.e., ATP), particularly with an increase in ATP catabolism, with subsequent adenosine accumulation. Adenosine will then act on presynaptic A 1 receptors causing a reduction in cAMP formation and, consequently, of PKA. Therefore, a reduction of Ca 2+ channels phosphorylation (by PKA) will occur reducing the intracellular amount of Ca 2+ . Presynaptically, the amount of intracellular Ca 2+ is critical for neurotransmission; therefore, lower amounts of calcium will cause a reduction of noradrenaline release and of the postsynaptic signal events triggered by noradrenaline, leading to vasodilation [66].
The location of enzyme isoforms is also relevant: nNOS in mesenteric arteries are located mostly in Schwann cells contrasting to tail arteries where their presence is very scarce (Figure 2).

Adenosine, endothelium and vascular neurotransmission
It is well established that adenosine can act as a physiological neuromodulator through activation of four types of adenosine receptors, A 1 , A 2A , A 2B and A 3 in the vasculature [67]. These receptors present differential affinities for adenosine, with adenosine A 1 receptor requiring lower concentrations to get activated (KdA 1 , 0.3-3 nM), followed by A 2A receptors with a Kd  1-20 nM. Pharmacological studies have also revealed that A 2B and A 3 receptors are low affinity receptors for adenosine requiring concentrations higher than 1 μM, although these adenosine receptor subtypes present different Kd (A 3 subtype requires higher concentrations than A 2B receptors) [68].
Adenosine, a well-known nucleoside, results from the sequential catabolism of ATP, forming ADP, AMP and adenosine with this later step being mediated by 5 0 -nucleotidase. Adenosine can then be further converted in inosine by adenosine deaminase or, instead, can be reconverted to AMP by adenosine kinase. In addition to adenosine receptors and adenosine, adenosinergic system is also composed by nucleoside transporters (NTs), which are responsible for nucleoside transport into the cells and vice versa. Some of the NTs in particular some equilibrative nucleoside transporters (ENT)s have already been identified as capable of promoting adenosine transport in vasculature, namely the subtype ENT1 and ENT 4 [69,70].
In vascular tissues as well as in some diseased states, such as hypertension, the bioavailability of adenosine varies [71], as presented in Table 2.
The amount of adenosine present in the vicinity of adenosine receptors depends on the adenosinergic system dynamics which, in turn, can be influenced by innumerous factors, such as NO, ROS, lipid peroxidation, endothelium dysfunction, etc., that can be altered in several pathological conditions, namely in hypertension, diabetes, aging and inflammation.
Another factor related with the relevance of vascular adenosine-mediated effects relies on adenosine receptor subtype distribution in the vasculature. All adenosine receptor subtypes have been identified not only in arteries, such as pulmonary [72], mesenteric [73][74][75][76][77], ear [73], aorta [78] and tail [51,52,[79][80][81], but also in veins [75,82]. In renal vessels, a role of adenosine receptors in sympathetic regulation was also demonstrated [83], conditioning the blood efflux Tissue preparations of mesenteric and tail arteries from WKY and SHR animals were superfused with Krebs-Henseleit. Tissues were stimulated twice at 30-min interval (S 1 -S 2 ; 100 pulses, 5 Hz, 1 ms, 50 mA): b 1 refers to the 5-min period immediately before S 1 . The superfusate was collected in 5-min period before and after stimulation, and each sample was heated at 80 C and derivatized using chloroacetaldehyde for 50 min at 70 C in a dry bath incubator. Identification of the ε-adenosine formed in this collected samples was confirmed by a gradient HPLC using a fluorescent detector at 230 nm excitation and 420 nm emission wavelengths. Values presented are means AE SEM of adenosine per mg of tissue, and n denotes the number of tissue preparations. Significant differences from WKY vessels: * P < 0.05. Table 2. Basal (b 1 ) and electrically evoked (S 1 ) adenosine release from sympathetic nerve terminals from normotensive (WKY) and hypertensive (SHR) vessels of the rat. in the afferent arteriole and, consequently, of renal filtration. In hypertensive arteries and veins, an impairment of the neuromodulation exerted by adenosine A 1 receptors [75][76][77]82] was described, contrasting with a preserved adenosine A 2A receptor-mediated facilitation of noradrenaline release [75][76][77]. Note that a redistribution of adenosine A 1 receptors from sympathetic nerves to Schwann cells was reported in hypertensive state while adenosine A 2A receptors, in sympathetic nerves, were preserved [77]. Particular relevant information relies on the location of adenosine receptors on the vascular wall layers contributing to the understanding of the functional role ascribed to adenosine receptors.
In endothelium, the four adenosine receptor subtypes have been identified by functional and immunohistochemical assays, for instance in tail artery [80,84] and aorta [84,85]. The influence of endothelium in adenosine-mediated responses has been demonstrated, with endogenous adenosine inducing an inhibition on noradrenaline release, through activation of adenosine A 1 receptors, (Figure 3, effect of DPCPX, a selective A 1 receptor antagonist). Adenosine availability is a crucial factor (effect demonstrated by pentostatin and α,β-methylene ADP, which inhibit adenosine deaminase and ecto-5'nucleotidase, respectively), conditioning the type of adenosine receptor that is activated. In resistant arteries, this effect is impaired when endothelium is compromised (arteries denuded of endothelium or in essential hypertensive arteries) and, instead, a facilitatory effect mediated by adenosine A 2A receptors, revealed by a selective A 2A receptor antagonist, the SCH 58261, and by inhibition of adenosine kinase, revealed by an adenosine kinase inhibitor, 5 0 -iodotubercidin (ITU), and by ecto-5'nucleotidase inhibitor, α,β-methylene ADP, demonstrating the relevance of adenosinergic dynamics both in physiological and pathophysiological contexts, such as in hypertension [49]. The adenosinergic system dynamic is adjusted to the unfavorable conditions created by endothelium injury, with enzymes involved in adenosine formation, such as adenosine deaminase and 5 0 -nucleotidase operating to promote an increase in the adenosine amount available and favoring the activation of A 2A receptors. This occurs despite the efforts of nucleoside transporters to equilibrate the concentration of adenosine between the inner and outer space of cells. In mesenteric arteries, A 2A receptor effect is enough to counteract the existing inhibitory tonus mediated by adenosine A 1 , but in resistant arteries, the facilitatory effect mediated by A 2A receptors (upon noradrenaline release) predominates.

Interplay between nitroxidergic and other pathways in neurotransmission
NO signaling events, in mesenteric arteries, cause an accumulation of adenosine (as previously described in Section 3.1). This condition may favor adenosine neuromodulation, namely by activation of adenosine A 1 receptors (which is revealed when blockade of A 1 receptors occurs in the presence of the NO donor, DEA-NONOATE; Figure 4) in the mesenteric artery. Activation of A 1 receptors leads to a reduction of noradrenaline release, and subsequently, the activation of α 1adrenoceptors in vascular smooth muscle cells is reduced, leading to vasodilation. In the tail artery, such interplay does not occur, at least in the experimental conditions tested.
Furthermore, in the cardiovascular system, NO can also interplay with the adrenergic pathway. NO source is, most probably, endothelial since noradrenaline release in the presence of a β-adrenoceptor agonist, isoprenaline (300 nM), caused an increase of noradrenaline release (175.10 AE 13.8%, n = 11), but the increase observed was lower in endothelium-denuded arteries (129.92 AE 13.1%, n = 7). Therefore, these data support the possibility, previously raised by Balligand et al. [86] and by Conti et al. [87], that NO production can lead to an increase in noradrenaline release, as a consequence of adrenergic receptors activation, namely of facilitatory β-adrenoceptors.

Current and future developments
In addition to the direct effects exerted by several substances on smooth muscle cells, which can cause vasodilation or vasoconstriction, the evidence that endothelium-derived factors can also influence sympathetic neurotransmission that reinforces the importance of endothelium and of its putative role in pathologies. Indeed, vascular sympathetic neurotransmission and the interplay exerted by endothelium-derived substances are, therefore, relevant in the homeostasis of vascular tone. In pathophysiological conditions, especially when endothelium is injured, their impact on neurotransmission account, at least in part, for the occurring vasoconstriction. NO has been viewed as a vasodilator substance since its direct effect on vascular smooth muscle cells causes dilation. However, NO can influence neurotransmission, and the interplay with adenosinergic and adrenergic pathways altering neurotransmission can, in some cases, cause an increase in noradrenaline release, which consequently will promote vasoconstriction. Therefore, the importance of NO is renewed as well as its ability to interplay with other signaling pathways involving sympathetic regulation such as the adrenergic and adenosinergic ones. Additional information and research on this field are, therefore, required to extend the knowledge on the insights of transsynaptic modulation of vascular neurotransmission. This is particularly important and can be useful to develop new therapeutic strategies, particularly in pathologies or clinical conditions, where the sympathetic system is hyperactivated.