Abnormal Regulation of the Sympathetic Nervous System in a2A-Adrenergic Receptor Knockout Mice

a2-Adrenergic receptors (ARs) play a key role in regulating neurotransmitter release in the central and peripheral sympathetic nervous systems. To date, three subtypes of a2-ARs have been cloned (a2A, a2B, and a2C). Here we describe the physiological consequences of disrupting the gene for the a2AAR. Mice lacking functional a2A subtypes were compared with wild-type (WT) mice, with animals lacking the a2B or a2C subtypes, and with mice carrying a point mutation in the a2A-AR gene (a2AD79N). Deletion of the a2A subtype led to an increase in sympathetic activity with resting tachycardia (knockout, 581 6 21 min; WT, 395 6 21 min), depletion of cardiac tissue norepinephrine concentration (knockout, 676 6 31 pg/mg protein; WT, 1178 6 98 pg/mg protein), and downregulation of cardiac b-ARs (Bmax: knockout, 23 6 1 fmol/mg protein; WT, 31 6 2 fmol/mg protein). The hypotensive effect of a2 agonists was completely absent in a2A-deficient mice. Presynaptic a2-AR function was tested in two isolated vas deferens preparations. The nonsubtype-selective a2 agonist dexmedetomidine completely blocked the contractile response to electrical stimulation in vas deferens from a2B-AR knockout, a2C-AR knockout, a2AD79N mutant, and WT mice. The maximal inhibition of vas deferens contraction by the a2 agonist in a2A-AR knockout mice was only 42 6 9%. [ H]Norepinephrine release studies performed in vas deferens confirmed these findings. The results indicate that the a2A-AR is a major presynaptic receptor subtype regulating norepinephrine release from sympathetic nerves; however, the residual a2-mediated effect in the a2A-AR knockout mice suggests that a second a2 subtype (a2B or a2C) also functions as a presynaptic autoreceptor to inhibit transmitter release. a2-Adrenergic receptors (ARs) play a prominent role in the regulation of the sympathetic nervous system (Ruffolo et al., 1991). Activation of a2-ARs in the brainstem leads to a reduction in sympathetic tone, with a resultant decrease in heart rate and blood pressure. This effect is augmented by stimulation of a2-ARs on sympathetic nerve terminals. These presynaptic a2-ARs serve as autoreceptors regulating catecholamine release. There are three a2-AR subtypes (a2A, a2B, and a2C), and studies using gene-targeting strategies indicate independent functions for each (Link et al., 1996; MacMillan et al., 1996; Sallinen et al., 1997). Resting blood pressure and heart rate were not significantly altered by disruption of either the a2B or a2C gene, indicating that neither receptor is necessary for normal sympathetic regulation (Link et al., 1996). Intra-arterial administration of clonidine-like a2 agonists produced a biphasic blood pressure response in wild-type (WT) mice so an initial brief pressor effect was followed by a sustained fall in arterial blood pressure (Link et al., 1996; MacMillan et al., 1996). This is a characteristic cardiovascular response pattern of clonidinelike drugs in other mammals (Hoefke and Kobinger, 1966). The vasopressor response to a2 agonists was absent in the a2B knockout (KO) mice, indicating that the a2B-AR mediates vasoconstriction in some vascular beds. The response to a2 agonist was not altered in the a2C-deficient mice. Recent studies indicate that the a2C-AR plays a role in several aspects of behavior (Sallinen et al., 1998). Much has been learned about the function of the a2A-AR from mice with a targeted mutation of the a2A-AR in the second transmembrane at position 79 (a2AD79N) (MacMillan et al., 1996). In cultured cell lines, the a2AD79N mutant receptor failed to activate K currents but exhibited normal inhibition of voltage-gated calcium channels and cAMP production (Surprenant et al., 1992). The a2AD79N mice were developed to study the physiological importance of K current regulation by the a2A-AR. Surprisingly, targeted mutation of the a2A-AR gene reduced expression of the a2AD79N mutant receptor by 80% as determined by radioligand binding assays in brain (MacMillan et al., 1996). a2AD79N muThis work was supported by Deutsche Forschungsgemeinschaft Grants SFB 355 (to M.J.L. in support of L.H.) and SFB 1523 (to A.U.T. and K.S.) and the Howard Hughes Medical Institute (to B.K.). ABBREVIATIONS: AR, adrenergic receptor; KO, knockout; ES, embryonic stem; KAC, potassium acetate; CYP, iodocyanopindolol. 0026-895X/99/010154-08$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 56:154–161 (1999).