Substitution of a mutant α _2a -adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo

Abstract

Norepinephrine contributes to antinociceptive, sedative, and sympatholytic responses in vivo, and ␣ 2 adrenergic receptor (␣ 2 AR) agonists are used clinically to mimic these effects. Lack of subtype-specific agonists has prevented elucidation of the role that each ␣ 2 AR subtype (␣ 2A , ␣ 2B , and ␣ 2C ) plays in these central effects. ␣ 2 -adrenergic receptors (␣ 2 ARs) present in the central nervous system (CNS) respond to norepinephrine (NE) and epinephrine and mediate sympatholytic, sedative-hypnotic, analgesic, anesthetic-sparing, hypotensive, and anxiolytic responses (1). Many of these responses are therapeutically useful and are exploited clinically, for example, during anesthesia and to attenuate the symptoms of opioid withdrawal (2). Three ␣ 2 AR subtypes have been revealed by pharmacological (␣ 2A AR, ␣ 2B AR, and ␣ 2C AR) and molecular cloning (␣ 2a AR, ␣ 2b AR, and ␣ 2c AR) strategies (3), and all couple, via pertussis toxin-sensitive G i ͞G o proteins, to attenuation of adenylyl cyclase, suppression of voltage-gated Ca 2ϩ channels, and activation of inwardly rectifying K ϩ channels (4).Multiple experimental limitations have precluded clarifying the involvement of each ␣ 2 AR subtype in catecholaminemediated physiological responses in the CNS. Subtype-specific ␣ 2 AR agonists and antagonists are not available (5); even when subtype selectivity has been noted in vitro, varying and unknown in vivo bioavailability precludes confident correlation of the administered dose with the amount of drug at the receptor site. Previous studies to explore ␣ 2 AR involvement in various responses have used prazosin to block catecholamine responses mediated by ␣ 1 adrenergic receptors (␣ 1 AR); however, it is now known that the ␣ 2B AR and ␣ 2C AR subtypes also are blocked by prazosin (5), thus confounding the interpretations of these earlier studies. In addition, because ␣ 1 AR can functionally antagonize ␣ 2 AR-mediated responses in some settings, ␣ 2 AR responses in the presence of prazosin (added to block ␣ 1 AR, ␣ 2B AR, and ␣ 2C AR) may reflect the disturbance of the balance between the functionally antagonistic ␣ 2 AR and ␣ 1 AR systems rather than provide insights concerning the role of the ␣ 2A AR subtype. Consequently, we manipulated the mouse genome to provide definitive evidence regarding the role of the ␣ 2A AR subtype in CNS responses.We used the ''hit and run'' targeting variant of homologous recombination (6, 7) to substitute a subtle mutation of the ␣ 2a AR, D79N into the mouse genome as a tool to explore the role of the ␣ 2a AR in vivo (8). The aspartate residue at position 79 (D79) is highly conserved in a topologically identical position in the second transmembrane span in a large subset of G protein-coupled receptors (9). Mutation of this residue has been shown to eliminate allosteric regulation of receptor binding by monovalent cations (10-13) and to perturb receptor-G protein-effector coupling (14-17) in heterologous expression systems. Thus, the animals expressing the D79N ␣ 2a AR provi...

Alpha2A-Adrenoceptors are Important Modulators of the Effects of D-Amphetamine on Startle Reactivity and Brain Monoamines

“…The almost totally abolished startle reflex of WT mice after Dex 10 and especially 30 mg/kg was likely due to its sedative effect. The sedation elicited by a 2 -AR agonists is mainly mediated through activation of a 2A -AR, and has previously been shown to be attenuated, but not totally absent in mice lacking functional a 2A -AR (Hunter et al, 1997;Lakhlani et al, 1997;Lähdesmäki et al, 2003). The smallest Dex dose, 3 mg/ kg, is not, however, sedative in mice (Hunter et al, 1997), but still effectively reduced startle in WT mice.…”

“…The a 2A -AR subtype mainly contributes to the inhibition of both neuronal excitability and NE release; the a 2C -AR has a similar, but lesser role (Lakhlani et al, 1997;Starke, 2001). Moreover, the a 2A -AR and to a minor extent the a 2C -AR modulate the neurotransmitter release of brain dopaminergic and serotonergic neurons (Yavich et al, 1997;Scheibner et al, 2001).…”

Activation of α2 Adrenergic Receptors Suppresses Fear Conditioning: Expression of c-Fos and Phosphorylated CREB in Mouse Amygdala

“…As many of the central effects of a 2 agonists are mediated by the a 2A AR (Lakhlani et al, 1997), we sought to determine whether this AR subtype was also the major mediator of the fear amnestic effect by which dexmedetomidine acted. To test this, dexmedetomidine (20 mg/kg) was administered to a 2A AR KO mice prior to training.…”

Regulation of α2AR trafficking and signaling by interacting proteins

“…3 suggest that our in vitro findings indicating reciprocal regulation of α 2 AR functions by spinophilin and arrestin have in vivo relevance. UK 14,304, an α 2 -agonist, evokes sedation in WT mice via activation of the α 2 AAR subtype [5]. In Sp −/− mice, α 2 -agonists become more efficacious, manifest by a leftward shift in the UK 14,304 dose response curve for sedation (Fig.…”

“…In vivo, activation of the α 2 AR by endogenous ligand, epinephrine and norepinephrine, leads to decrease in epileptogenesis [1] and anxiety [2]. In response to α 2 -agonists, the α 2 AR activation can lower blood pressure by central mechanisms [3;4], evoke sedation [5], reduce pain perception [5;6], and improve working memory [7][8][9]. There are three subtypes of α 2 ARs, α 2A -, α 2B -and α 2A AR, which are encoded by three different genes but all couple to the G i/o subfamily of G proteins to inhibit adenylyl cyclase and voltage-gated Ca 2+…”