**4. L-arginine transport in human fetal endothelium**

The amino acid L-arginine is taken up by endothelial cells through the transporter systems y+ , y+ L, b0,+, and y B0,+ [32–35]. Of these systems, there are two that have been described in HUVEC, that is, y+ system [36–38] and y+ L system [39]. The y+ system family is currently known to include at least five cationic amino acid transporters (CATs) called CAT-1, CAT-2A, CAT-2B, CAT-3, and CAT-4. CAT-1 is expressed ubiquitously, CAT-2A and CAT-3 are constitutively expressed in liver and brain, respectively, while CAT-2B is induced in a variety of cell types in response to bacterial endotoxins and pro-inflammatory cytokines [40, 41]. CAT-4 corresponds to a cDNA sequence with 41–42% identity with the other members of the CATs family, but its transport activity has not yet been determined [32, 34, 35]. CAT-1, CAT-2B, and CAT-3 are characterized by high affinity to the substrate (*K*m = 100–400 μM) and independency of Na+ , while CAT-2A has low affinity for cationic amino acids (*K*m = 2–5 mM). Two members of CATs have been reported to be expressed in HUVEC, that is, hCAT-1 and hCAT-2B, while hCAT-2A and hCAT-3 transporters have not been detected in this cell type [34, 36–39] (**Table 1**). Although the hCAT-1 and hCAT-2B transporters have similar kinetic characteristics, it is possible to differentiate them by their different sensitivities to L-lysine trans-stimulation. In *Xenopus laevis* oocytes injected with hCAT-1 and hCAT-2B mRNA, L-lysine increases L-arginine transport by 9.8-fold and 1.8-fold, respectively [42]. Thus, for L-lysine trans-stimulation assays in HUVEC, it has been possible to determine that

**51**

*L-Arginine/Nitric Oxide Pathway and KCa Channels in Endothelial Cells: A Mini-Review*

*SLC7A1* CAT-1 70–250 All tissues except liver and lacrimal gland *SLC7A2* CAT-2A 2.2–5.2 Liver, skeletal muscle, and pancreas *SLC7A2* CAT-2B 38–380 Endothelium, and inducible in several tissues *SLC7A3* CAT-3 40–120 Thymus, ovary, testes, and brain *SLC7A4* CAT-4 — Brain, testes, and placenta

**Gene Protein** *K***m (μM) Distribution**

the hCAT-1 transporter accounts for 60–80% of the total uptake of L-arginine in physiological conditions [36–38]. The importance of the hCAT-1 transporter in NO synthesis has been confirmed through a transgenic mouse model that overexpresses the protein exclusively in the endothelium. Aortic rings obtained from these transgenic mice have a higher sensitivity to relaxation in response to acetylcholine compared to native mice, while endothelial cell cultures obtained from these animals,

*Proteins CATs are coded in different genes (except CAT2A and 2B, same gene), have different kinetic constants for* 

Regarding the gene organization of CAT transporters, it is known that the *SLC7* family is phylogenetically composed of two subfamilies formed by cationic amino acid transporters (CATs) and glycoprotein-associated amino acid transporters (HATs). The cationic amino acid transporter family is encoded by the *SLC7A* (1–4) genes and corresponds to proteins with 14 transmembrane domains [44]. Specifically, the gene that encodes the hCAT-1 protein corresponds to *SLC7A1* whose open reading frame is formed by 11 exons and 10 introns. The gene is located on chromosome

Among the genes encoding CAT-1 in rat, mouse and human have common characteristics: the promoter region lacks TATA box, and they have multiple binding sites for the transcription factor specific protein 1 (Sp1) and they have an extensive 3′ non-translatable region (3′UTR) that could perform functions in the regulation of mRNA stability or in translation [46–49]. In rats, stress by amino acids deprivation induces an increase in the rCAT-1 mRNA expression by a mechanism related to increased mRNA stability [46]. This increased mRNA stability would be related to the presence of a regulatory region within the 3′UTR sequence of the gene [47]. Subsequent experiments have shown that the effect of amino acids deprivation on rCAT-1 expression would depend on both transcriptional [48] and posttranscrip-

In humans, it is known that insulin increases leg blood flow in healthy subjects via stimulation of endothelial NO synthase (eNOS) [51]. Insulin also increases the synthesis and release of NO and release in primary cultures of HUVEC [38, 52]. Biological effects of insulin involve activation of several transcription factors, including Sp1 in several cell types [53, 54]. Insulin increases Sp1 nuclear protein abundance and its binding to a proximal region (−177 and −105 bp from ATG) of the *SLC7A1* promoter containing four consensus sequences for Sp1 [55]. Interestingly, in patients with essential hypertension, a reduction of *SLC7A1* transcriptional activity due to reduced Sp1 activity in the promoter region has been reported [12]. So, the transcriptional regulation of *SLC7A1* is relevant for cardiovascular physiology,

that overexpress hCAT-1, exhibit a greater NO synthesis [43].

**5. Regulation of the expression of hCAT-1**

13q12-13q14 [45].

**Table 1.**

*CATs' family members.*

tional mechanisms [50].

*DOI: http://dx.doi.org/10.5772/intechopen.93400*

*the transport of L-arginine (*K*m) and distribution in tissues.*

*L-Arginine/Nitric Oxide Pathway and KCa Channels in Endothelial Cells: A Mini-Review DOI: http://dx.doi.org/10.5772/intechopen.93400*


*Proteins CATs are coded in different genes (except CAT2A and 2B, same gene), have different kinetic constants for the transport of L-arginine (*K*m) and distribution in tissues.*

#### **Table 1.** *CATs' family members.*

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

**3. Reactive oxygen-derived species (ROS) in endothelium**

Endothelial cells generate ROS, including the superoxide radical (O2

[15, 16]. In endothelial cells, the main sources of ROS are the enzymatic complex xanthine oxidoreductase (XOR) [17], the complex of membrane nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) [18], eNOS itself when it is "uncoupled" due to lack of tetrahydrobiopterin (BH4) or L-arginine [19], mito-

Among all endothelial ROS sources, NADPH oxidases are enzymes whose primary function is the generation of ROS and they play an important role in redox signaling [22]. On the other hand, the activity of NADPH oxidase can cause the uncoupling of eNOS by the oxidative degradation of BH4, leading to the eNOS-

·− is synthesized, it can act as a precursor to other ROS due to its use by superoxide dismutase (SOD) to generate H2O2 that has greater stability and capacity to cross biological membranes, and it therefore can act as a modulator of signal transduc-

a powerful oxidizing agent that causes DNA fragmentation and lipid oxidation [25].

would play a central role in the development of endothelial dysfunction that is seen in pathologies such as diabetes mellitus [26–28], preeclampsia [29, 30], and

The amino acid L-arginine is taken up by endothelial cells through the trans-

transporters (CATs) called CAT-1, CAT-2A, CAT-2B, CAT-3, and CAT-4. CAT-1 is expressed ubiquitously, CAT-2A and CAT-3 are constitutively expressed in liver and brain, respectively, while CAT-2B is induced in a variety of cell types in response to bacterial endotoxins and pro-inflammatory cytokines [40, 41]. CAT-4 corresponds to a cDNA sequence with 41–42% identity with the other members of the CATs family, but its transport activity has not yet been determined [32, 34, 35]. CAT-1, CAT-2B, and CAT-3 are characterized by high affinity to the substrate (*K*m = 100–400 μM)

(*K*m = 2–5 mM). Two members of CATs have been reported to be expressed in HUVEC, that is, hCAT-1 and hCAT-2B, while hCAT-2A and hCAT-3 transporters have not been detected in this cell type [34, 36–39] (**Table 1**). Although the hCAT-1 and hCAT-2B transporters have similar kinetic characteristics, it is possible to differentiate them by their different sensitivities to L-lysine trans-stimulation. In *Xenopus laevis* oocytes injected with hCAT-1 and hCAT-2B mRNA, L-lysine increases L-arginine transport by 9.8-fold and 1.8-fold, respectively [42]. Thus, for L-lysine trans-stimulation assays in HUVEC, it has been possible to determine that

system family is currently known to include at least five cationic amino acid

It is currently postulated that the mechanism by which O2

**4. L-arginine transport in human fetal endothelium**

derived species (ROS) [13].

dependent synthesis of O2

hypertension [31].

porter systems y+

and independency of Na+

The y+

tion pathways [24]. Furthermore, O2

, y+

have been described in HUVEC, that is, y+

O2

peroxide (H2O2), peroxynitrite (ONOO−

chondrial cytochromes [20], and hemoglobin [21].

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 oxygen-

), hydroxyl radical (.

·− and detriment of the synthesis of NO [18, 23]. Once

L, b0,+, and y B0,+ [32–35]. Of these systems, there are two that

system [36–38] and y+

, while CAT-2A has low affinity for cationic amino acids

·− reacts quickly with NO to generate ONOO−

.−), hydrogen

,

OH), among others

·− "kidnaps" NO

L system [39].

**50**

the hCAT-1 transporter accounts for 60–80% of the total uptake of L-arginine in physiological conditions [36–38]. The importance of the hCAT-1 transporter in NO synthesis has been confirmed through a transgenic mouse model that overexpresses the protein exclusively in the endothelium. Aortic rings obtained from these transgenic mice have a higher sensitivity to relaxation in response to acetylcholine compared to native mice, while endothelial cell cultures obtained from these animals, that overexpress hCAT-1, exhibit a greater NO synthesis [43].
