a2C Adrenoceptors Inhibit Adenylyl Cyclase in Mouse Striatum: Potential Activation by Dopamine

a2C adrenoceptors occur in high density in the striatum, but the functional role of these receptors is uncertain. Mice with targeted inactivation of the a2C adrenoceptor gene (Adra2c ) and genetically related control mice expressing the wild-type a2C adrenoceptor (Adra2c ) were used to determine whether striatal a2C adrenoceptors modulate adenylyl cyclase activation. In striatal slices from Adra2c mice, the a2 adrenoceptor antagonist RX821002 facilitated forskolin-stimulated cyclic AMP accumulation in a concentration-dependent manner. In contrast, RX821002 had no effect on forskolin-stimulated cAMP accumulation in striatal slices from Adra2c mice or in striatal slices from Adra2c mice treated with reserpine and a-methyl-r-tyrosine to deplete monoamine neurotransmitters. Given the sparse innervation of the striatum by noradrenergic neurons, the possibility that dopamine can activate the mouse a2C adrenoceptor at physiologically relevant concentrations was investigated using normal rat kidney (NRK) cells transfected with the mouse a2A or a2C adrenoceptor cDNA (NRKa2A or NRK-a2C cells). Inhibition of [ H]RX821002 binding by agonists in homogenates of transfected cells revealed an affinity of dopamine for a2C adrenoceptors that was higher than the affinity of norepinephrine for its cognate receptor, the a2A adrenoceptor. Both norepinephrine and dopamine inhibited forskolin-stimulated cAMP accumulation in intact NRK-a2C cells. In NRK-a2A cells, norepinephrine facilitated forskolin-stimulated cAMP accumulation, an effect not observed for dopamine. Together, these data demonstrate that the a2C adrenoceptor is negatively coupled to adenylyl cyclase and is tonically activated in mouse striatal slices. The endogenous activator of the striatal a2C adrenoceptor may be dopamine, as well as norepinephrine. a2 adrenoceptors mediate many physiological functions and consist of three distinct subtypes: a2A, a2B, and a2C. The a2 adrenoceptor subtypes are encoded by three different genes in mouse, rat, and humans (MacDonald et al., 1997) and are expressed (mRNA) or coexpressed in many brain regions (Scheinin et al., 1994; MacDonald and Scheinin, 1995; Wang et al., 1996). Although the a2A adrenoceptor has a broad and dense distribution in the brain, the expression of the a2C adrenoceptor gene is largely limited to the hippocampus, olfactory system, and basal ganglia (Nicholas et al., 1993; Scheinin et al., 1994; MacDonald and Scheinin, 1995; Talley et al., 1996; Tavares et al., 1996). In fact, radioligand binding to the a2C adrenoceptor occurs in highest density in the striatum, relative to other brain regions (Ordway et al., 1993; Uhlen et al., 1997). a2 adrenoceptors are located on noradrenergic neurons or are located in brain regions that are innervated by noradrenergic neurons because presynaptic markers of noradrenergic innervation (e.g., dopamine b-hydroxylase and norepinephrine transporters) are in these regions (Grzanna et al. 1977; Moore and Card, 1984; Ordway et al., 1993; Jursky et al., 1994; Ordway, 1995). However, the high concentration of a2C adrenoceptor mRNA and protein in the striatum is peculiar given the paucity of noradrenergic innervation to this brain region (Moore and Card, 1984; Nicholas et al., 1993; Jursky et al., 1994; Scheinin et al., 1994; Rosin et al., 1996; Wang et al., 1996). The lack of a selective ligand for the a2C adrenoceptor has slowed the discovery of its functions in the brain. Recently, molecular biological techniques have advanced understanding of this receptor. For example, using mice lacking a functional a2C adrenoceptor and mice overexpressing this receptor, Sallinen et al. (1997) demonstrated that this receptor mediates, at least in part, hypothermia in response to s.c. administration of the a2 adrenoceptor agonist dexmedetomidine. Furthermore, the a2C adrenoceptor modulates acoustic startle reflex and its prepulse inhibition, as well as isolationinduced aggression (Sallinen et al., 1998). The functional role of the a2C adrenoceptor in the striatum is uncertain. Using antisense oligonucleotide infusions directly into the rat striatum, we demonstrated that the a2C adrenoceptor is negatively coupled to adenylyl cyclase and that this receptor appears to be tonically activated in striatal Received for publication September 24, 1998. 1 This work was supported by a gift from the Hoechst-Marion-Roussel, Inc. W.Z. was supported by a graduate student stipend from the University of Mississippi Medical Center. ABBREVIATION: NRK, normal rat kidney. 0022-3565/99/2893-1286$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 289, No. 3 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 289:1286–1292, 1999 1286 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from slices (Lu and Ordway, 1997b). Given the dense dopaminergic innervation and sparse noradrenergic innervation of the striatum, we postulated that the striatal a2C adrenoceptor may be activated promiscuously by dopamine. In the present study, we used mice with a targeted inactivation of the a2C adrenoceptor gene (Adra2c) and normal rat kidney (NRK) cells transfected with mouse a2A or a2C adrenoceptor genes to determine 1) whether the a2C adrenoceptor is linked to adenylyl cyclase in mouse striatum, 2) the affinities of dopamine and norepinephrine at mouse a2A and a2C adrenoceptors, and 3) whether dopamine could activate these receptors at physiologically relevant concentrations. Materials and Methods Animals. Male mice with a targeted inactivation of the a2C adrenoceptor gene (Adra2c) and wild-type mice (Adra2c) were generated at Stanford University (Stanford, CA) and maintained at Roche Biosciences (Palo Alto, CA). The method of generation of the two mice has been detailed by Link et al. (1995). In brief, one copy of the murine Adra2c gene was inactivated in R1 129/Sv embryonic stem cells (Nagy et al., 1993), which lacks critical structural sequences required for G protein coupling and ligand binding. These cells were injected into C57BL/6J blastocyst, and (C57BL/6J 3 DBA/ 2J)F1 mice were used to breed resulting chimeric animals. These mice were crossed back for several generations to C57BL/6J mice and intercrossed to form the colony. All experimental Adra2c mice were homozygous for the mutation. The genetic control of the wild-type strain was constituted primarily by C57BL/6J with a small contribution from 129/Sv and DBA/2J stains. The mice from both strains are viable and fertile and appear grossly normal. The mouse tail was used to monitor continuously the strain propagation of the mutation by Southern (DNA) blot. On arrival at the University of Mississippi, genotypic identification within the animal facility was maintained with the use of ear punches. Mice (weighing 26–31 g) were housed in group cages in a room maintained at constant temperature and humidity and illuminated 12 h/day. Mice were provided free access to water and food and sacrificed by decapitation. All procedures using animals were in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” and were approved by a local institutional animal care and use committee. Cell Culture. NRK cells stably transfected with mouse a2A and a2C adrenoceptors were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and gentamicin (G418; 250 mg/ml) at 37°C in 95% humidified air with 5% CO2. Transfected cell lines were obtained from Brian K. Kobilka (Stanford University, Stanford, CA). Culture medium and supplements were obtained from GIBCO BRL (Grand Island, NY). Transfections of these NRK cells have been described by Daunt et al.