**2. Synthesis of nitric oxide in the endothelium**

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

E-selectin, and NFκB (**Figure 1** [8]).

a central mechanism [10–13].

produced through a factor that was released by the endothelium in response to agonists [4]. This factor was called the endothelial-derived relaxing factor (EDRF) [5]. Between 1986 and 1990, it was concluded that this factor corresponded to nitric oxide (NO) [6, 7]. The endothelium responds to mechanical stimuli such as pressure and flow stress ("shear stress"), hormonal stimuli, and vasoactive substances that regulate the vascular tone. The endothelial cells release molecules that regulate vasomotor function, inflammation, and hemostasis. Vasodilators agents include NO, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). Vasoconstrictors agents include endothelin 1, angiotensin II, thromboxane A2, and reactive oxygen-derived species (ROS). Inflammatory mediators include NO, intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1),

Since the discovery of NO, the mechanisms of endothelial cell activation and endothelial dysfunction have been studied. In this way, the quiescent endothelial cells express a vasodilator, anticoagulant, and anti-adhesive phenotype, while the activated endothelial cell expresses procoagulant, pro-adhesive, and vasoconstrictive properties [9]. It has been considered that the decrease in the capacity of the vascular endothelium to stimulate vasodilation generates endothelial dysfunction, a phenomenon that is observed in several pathological conditions such as hypertension, hypercholesterolemia, diabetes mellitus, hyperhomocysteinemia, chronic kidney failure, chronic heart failure, etc. Although the molecular basis for endothelial dysfunction is not fully understood, numerous studies point to decreased biosynthesis and/or NO activity as

*Vascular tone regulation. The vascular tone is partially regulated by the local factors secreted by endothelial cells (ECs) in response to physical factors like shear stress and humoral and chemical factors like hormones and oxygen levels. The changes in blood flow are detected by membrane proteins, mainly receptors (Rs), transporters (Ts), and ion channels (ICs). There is a network connecting the activities of these proteins through signaling pathways that induce the release of different mediators like thromboxane A2 (TxA2), endothelin 1, reactive oxygen species (ROS), nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), or prostacyclin (PGI2), among others. The equilibrium between the vasoconstrictors and vasodilators factors maintains the* 

**48**

**Figure 1.**

*endothelial function and vascular health.*

NO is synthesized from the semi-essential cationic amino acid L-arginine, which must be transported from the extracellular space into the endothelial cell by a family of cationic amino acid transporters (CATs) [14]. This amino acid is the substrate in a reaction where the metabolic product corresponds to L-citrulline in an equimolar proportion with the coproduct NO [15, 16]. This reaction is catalyzed by the enzyme NO synthase (NOS), which can be classified into their constitutive forms (cNOS) and their inducible form (iNOS) [17]. The cNOS includes the endothelial isoform (eNOS) and the neuronal isoform (nNOS), both producing NO in short bursts at low concentrations (nM) and in a calcium-dependent manner to fulfill the physiological functions of NO. The physiological activity of eNOS is dependent on several cofactors and is regulated by signaling pathways that induce phosphorylation in different sites for activation (serine 1177) or inhibition (threonine 495) [17]. NO diffuses from endothelial cells to smooth muscle cells (SMCs) and activates the soluble guanylate cyclase (sGC) pathway, to reduce the intracellular calcium and induce vasodilation (**Figure 2**). iNOS is mainly expressed in cells that participate in the inflammatory response after induction by cytokines and other inflammatory mediators, producing NO in high concentrations (μM) and independently of calcium [18–20].

The availability of NO in vivo is regulated by a combination of NO synthesis and inactivation. The decrease in the availability of NO may be due to a lower expression

#### **Figure 2.**

*L-arginine transport and nitric oxide synthesis in endothelial cells. hCAT-1 is a protein expressed in plasma membrane of endothelial cells, mainly in plasma membrane invagination called caveolae. The L-arginine enters to the cell from blood and is used by eNOS to synthesize L-citrulline and nitric oxide (NO). The eNOS needs different cofactors to maintain its function, which include tetrahydrobiopterin (BH4), nicotidamine adenine dinuclotide phosphate (NADPH), and heat shock protein 90 (Hsp90). Nitric oxide diffuses through the cell membranes and enters the smooth muscle cells to activate the soluble guanlylate cyclase (sGC). The sGC synthesizes cyclic GMP (cGMP), which activates protein kinase G and, after subsequent steps, the intracellular calcium decreases to induce the vasodilation.*

or activity of eNOS, as a result of the action of endogenous and exogenous inhibitors or due to the lower availability of the substrate L-arginine [8, 14]. The availability of NO can also be diminished by the rapid reaction between NO and reactive oxygenderived species (ROS) [13].
