, such as activation of the extracellular signal-regulated kinases ERK1/2 and PI3K

[5155]. Gi-coupled receptors have been shown to regulate nonreceptor tyrosine

kinases, such as Src, which acts as an intermediate between Gi and other
molecules like Ras, PI3K [54, 56], Akt, and thus eNOS. 3-AR is mainly located on
ECs and acts in conjunction with 1-AR and 2-AR to mediate relaxation through
the activation of NO synthase pathways and subsequent increase in tissue cGMP
content and is reduced by endothelium removal or in the presence of
monomethyl-L-arginine, monoacetate (L-NMMA) [57]. This 3-AR-mediated aortic
relaxation seems to be independent of Gi protein stimulation, since the blockade
of Gi proteins by pertussis toxin does not modify 3-AR agonist-induced
relaxation. On the contrary, selective blockers of KCa, KATP, and Kv channels
decreased 3-AR agonist-induced relaxation. Thus, it appears that this effect
results from the activation of several potassium channels (KCa, KATP, Kv) [58].
Another intriguing mechanism in eNOS regulation upon adrenergic stimulation
involves the G-protein-coupled receptor kinases (GRKs). In order to control their
overstimulation, -ARs are phosphorylated by several kinases to induce receptor
desensitization [59, 60]. Both 1-AR and 2-AR can be phosphorylated by PKA
and by certain GRKs. Because PKA can phosphorylate -ARs in the absence of
agonist, PKA is thought to mediate heterologous (agonist-independent)
desensitization without affecting homologous (agonist-dependent)
desensitization. In contrast to PKA, GRKs selectively phosphorylate agonist-
occupied receptors, primarily on Ser/Thr residues located in their carboxyl-
terminal tails. Among the different GRKs, GRK2 has been involved at the level of
eNOS regulation. When the receptor is activated by an agonist, GRK2
translocates to the plasma membrane by means of its interaction with free G
subunits and induces receptor desensitization [61, 62]. Indeed, following GRK2-
mediated phosphorylation of -ARs, -arrestins are recruited to these receptors
and these adaptor molecules block further G protein activation [63] while
promoting internalization of the receptors leading to their degradation or
resensitization (recycling to the membrane) [64]. In terms of eNOS activation and
regulation, GRK2 can interact with eNOS directly or through Akt [65, 66]. In both
mechanisms, GRK2 appears to reduce eNOS activity and this is particularly
evident in pathophysiological conditions like hypertension [25]. Of note, GRK2
appears also to modulate Akt/eNOS activation by non-GPCR receptors such as
the insulin receptor (IR). Indeed, IR is thought to promote eNOS activation by a
complex interaction involving -arrestin2, Akt, and eNOS. Increased levels of
GRK2 at the plasma membrane upon insulin stimulation, as observed in ob/ob
mice, interfere with this signaling, preventing -arrestin 2 binding to Akt [67].