alpha2-Adrenergic receptors (alpha2ARs) present in the brainstem decrease blood pressure and are targets for clinically effective antihypertensive drugs. The existence of three alpha2AR subtypes, the lack of subtype-specific ligands, and the cross-reactivity of alpha2AR agonists with imidazoline receptors has precluded an understanding of the role of individual alpha2AR subtypes in the hypotensive response. Gene targeting was used to introduce a point mutation into the alpha2aAR subtype in the mouse genome. The hypotensive response to alpha2AR agonists was lost in the mutant mice, demonstrating that the alpha2aAR subtype plays a principal role in this response.

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...

alpha2-Adrenergic receptors (ARs) play a key role in regulating neurotransmitter release in the central and peripheral sympathetic nervous systems. To date, three subtypes of alpha2-ARs have been cloned (alpha2A, alpha2B, and alpha2C). Here we describe the physiological consequences of disrupting the gene for the alpha2A-AR. Mice lacking functional alpha2A subtypes were compared with wild-type (WT) mice, with animals lacking the alpha2B or alpha2C subtypes, and with mice carrying a point mutation in the alpha2A-AR gene (alpha2AD79N). Deletion of the alpha2A subtype led to an increase in sympathetic activity with resting tachycardia (knockout, 581 +/- 21 min-1; WT, 395 +/- 21 min-1), depletion of cardiac tissue norepinephrine concentration (knockout, 676 +/- 31 pg/mg protein; WT, 1178 +/- 98 pg/mg protein), and down-regulation of cardiac beta-ARs (Bmax: knockout, 23 +/- 1 fmol/mg protein; WT, 31 +/- 2 fmol/mg protein). The hypotensive effect of alpha2 agonists was completely absent in alpha2A-deficient mice. Presynaptic alpha2-AR function was tested in two isolated vas deferens preparations. The nonsubtype-selective alpha2 agonist dexmedetomidine completely blocked the contractile response to electrical stimulation in vas deferens from alpha2B-AR knockout, alpha2C-AR knockout, alpha2AD79N mutant, and WT mice. The maximal inhibition of vas deferens contraction by the alpha2 agonist in alpha2A-AR knockout mice was only 42 +/- 9%. [3H]Norepinephrine release studies performed in vas deferens confirmed these findings. The results indicate that the alpha2A-AR is a major presynaptic receptor subtype regulating norepinephrine release from sympathetic nerves; however, the residual alpha2-mediated effect in the alpha2A-AR knockout mice suggests that a second alpha2 subtype (alpha2B or alpha2C) also functions as a presynaptic autoreceptor to inhibit transmitter release.