Introduction

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₂ adrenoceptors mediate many physiological functions and consist of three distinct subtypes: _{2A, 2B}, and _2C. The ₂ 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 _2A adrenoceptor has a broad and dense distribution in the brain, the expression of the _2C 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 _2C adrenoceptor occurs in highest density in the striatum, relative to other brain regions (Ordway et al., 1993; Uhlen et al., 1997). ₂ 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 -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 _2C 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 _2C 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 _2C 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 ₂ adrenoceptor agonist dexmedetomidine. Furthermore, the _2C adrenoceptor modulates acoustic startle reflex and its prepulse inhibition, as well as isolation-induced aggression (Sallinen et al., 1998).

The functional role of the _2C adrenoceptor in the striatum is uncertain. Using antisense oligonucleotide infusions directly into the rat striatum, we demonstrated that the _2C adrenoceptor is negatively coupled to adenylyl cyclase and that this receptor appears to be tonically activated in striatal slices (Lu and Ordway, 1997b). Given the dense dopaminergic innervation and sparse noradrenergic innervation of the striatum, we postulated that the striatal _2C adrenoceptor may be activated promiscuously by dopamine. In the present study, we used mice with a targeted inactivation of the _2C adrenoceptor gene (Adra2c^/) and normal rat kidney (NRK) cells transfected with mouse _2A or _2C adrenoceptor genes to determine 1) whether the _2C adrenoceptor is linked to adenylyl cyclase in mouse striatum, 2) the affinities of dopamine and norepinephrine at mouse _2A and _2C adrenoceptors, and 3) whether dopamine could activate these receptors at physiologically relevant concentrations.

	Materials and Methods

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Animals. Male mice with a targeted inactivation of the _2C 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 × 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.

Cell Culture. NRK cells stably transfected with mouse _2A and _2C adrenoceptors were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and gentamicin (G418; 250 µg/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. (1997).

Homogenate Binding. Saturation and competition binding experiments using [³H]RX821002 (methoxy idazoxan) were performed as described by Bylund et al. (1988) and Ordway (1995), with minor modifications. Frozen cells were thawed quickly at 37°C, immediately put on ice, and then centrifuged at 1000g for 8 min. Pellets were washed twice with ice-cold PBS (10 ml) and centrifuged at 1000g for 8 min. Resulting pellets were homogenized in 10 ml of ice-cold Tris buffer (50 mM Tris, pH 8.0) using a Polytron (setting 6, for 20 s) and then centrifuged at 40,000g for 30 min. Pellets were then washed once with Tris buffer, centrifuged as above (40,000g for 30 min), homogenized in 25 mM glycylglycine buffer (pH 7.4), and centrifuged again. Final pellets were resuspended in ice-cold glycylglycine buffer.

cAMP Assay. Levels of cAMP in slices of mouse striatum were measured by radioimmunoassay (Ordway et al., 1987). Mice were sacrificed by decapitation, and the brains were removed. To deplete monoamines, mice were treated with 2.5 mg/kg reserpine 24 h before and 50 mg/kg -methyl--tyrosine 1 h before decapitation. A combination of reserpine and -methyl--tyrosine pretreatment can cause up to 99% and 80% depletion of striatal dopamine and norepinephrine, respectively (Koss and Christensen, 1979; White et al., 1988). Striata were dissected and chopped into 300-µm prisms using a McIlwain tissue chopper (Brinkmann Instruments, Westbury, NY). Striatal slices prepared from two mice were dispersed in 25 ml of oxygenated (95% O₂/5% CO₂) Krebs-Ringer buffer (2.4 mM MgSO₄, 0.1 mM KH₂PO₄, 118 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl₂, and 0.01 mM disodium EDTA) at 37°C. Slices were preincubated for 1 h and washed with fresh oxygenated buffer every 15 min. After preincubation, slices were dispersed in 850-µl aliquots (about 300 µg protein/tube, in triplicate) into individual reaction tubes for 10 min. The volumes of drug and tissue aliquots were adjusted so that the final volume of the slice preparation was 1 ml. The phosphodiesterase inhibitor 3-isobutyl-methyxanthine (0.5 mM) and RX821002 (in concentrations noted) were added to each tube 15 min before the addition of forskolin. Modestly higher concentrations of RX821002 and forskolin were used in the depletion experiments (compared with the experiment with Adra2c^/ and Adra2c^+/+ mice) in an effort to increase the net change in cAMP between the forskolin-alone and forskolin-plus-RX821002 conditions. After the addition of forskolin (in concentrations noted), reactions proceeded for 10 min and were stopped by the addition of ice-cold 2.5% perchloric acid (500 µl). Samples were sonicated for 15 s and centrifuged at 30,000g for 15 min. Pellets were dissolved in 0.1 N NaOH for protein measurement. The supernates were neutralized with CaCO₃ (40 mg/tube) and centrifuged twice at 30,000g for 15 min to remove excess CaCO₂. Final supernates were assayed for cAMP by radioimmunoassay. Aliquots of samples (100 µl) were incubated, in duplicate, with an [¹²⁵I]succinyl derivative of cAMP and antiserum (New England Nuclear, Boston, MA) in an ice bath for 18 h. Antibodies were precipitated with ice-cold ethanol (95%). After centrifugation at 10,000g for 30 min, supernatants were aspirated and tubes were inverted and dried. Radioactivity was estimated using a gamma counter (Beckman Gamma 4000; Packard Instrument Company, Meriden, CT) at an efficiency of 80%. cAMP concentrations (pmol/mg protein) in each sample were determined by comparing the inhibition of [¹²⁵ I]cAMP binding to antibody caused by known concentrations of cAMP. Protein was assayed according to the method of Lowry et al. (1951).

Statistics. All saturation and competition binding data were analyzed using nonlinear regression analysis (Prism, version 1.0; GraphPad Software, San Diego, CA). Data were fit first to a model assuming binding to one site and then to a model assuming two sites of interaction. The best fit was determined statistically by a comparison of the sum of squares of residuals using the equation F = [(SS₁  SS₂)/(df₁  df₂]/(SS₂/df₂), in which SS₁ and df₁ are the sum of squares and degrees of freedom from the one binding-site model and SS₂ and df₂ are those from the two-binding site model. A two-site fit was considered a significantly better fit if the F value was larger than that reported in the F statistic table at p < .05 for the numerator of (df₁  df₂) and the denominator of df₂. Data from cAMP experiments were analyzed by one-way repeated measures ANOVA followed by a Student-Newman-Keuls test. All data are shown as mean ± S.E.M.

	Results

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Effects of RX821002 on Forskolin-Stimulated cAMP levels in Striatum. RX821002 is an ₂ adrenoceptor antagonist that is not selective with respect to the ₂ adrenoceptor subtypes. RX821002 alone has been shown to enhance forskolin-stimulated cAMP accumulation in a dose-dependent manner in rat striatal slices (Lu and Ordway, 1997b). Initial experiments were performed to verify that this effect occurs in the mouse striatum. RX821002 enhanced forskolin-stimulated cAMP accumulation in a concentration-dependent manner in slices of striata obtained from Adra2c^+/+ mice (Fig. 1). An enhancement of 60% was observed at the highest concentration of RX821002 (100 µM). To determine whether adenylyl cyclase activation in the striatum is mediated by blockade of the _2C adrenoceptor, experiments were performed to examine the ability of RX821002 (10 nM) to enhance forskolin (1 µM)-stimulated cAMP accumulation in Adra2c^+/+ and Adra2c^/ mice. As in Fig. 1, RX821002 significantly enhanced forskolin-stimulated cAMP accumulation in striatal slices of Adra2c^+/+ mice (p < .05; Fig. 2). In contrast to Adra2c^+/+ mice, no enhancement of cAMP accumulation by RX821002 was observed in striatal slices of Adra2c^/ mice. Rather, RX821002 tended to reduce forskolin-stimulated cAMP accumulation in Adra2c^/ mice, although this effect did not reach statistical significance. Both basal and forskolin-stimulated cAMP concentrations were moderately higher in Adra2c^/ striata than in Adra2c^+/+ striata.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Enhancement of forskolin (1 µM)-stimulated cAMP accumulation by RX821002 in striatal slices of Adra2c+/+ mice. Each point is the mean ± S.E.M. percentage increase in forskolin-stimulated cAMP determined in four separate experiments. Basal and forskolin-stimulated levels of cAMP were 21.5 ± 1.5 and 42.2 ± 5.3 pmol/mg protein, respectively. *p < .05 or **p < .01, significance differences from the forskolin-alone condition.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Forskolin-stimulated cAMP accumulation in the absence or presence of RX821002 (10 nM) in striatal slices from Adra2c/ and Adra2c+/+ mice. Left, each pair of connected points, representing forskolin (1 µm) alone and forskolin in the presence of RX821002 (10 nM), comes from an individual experiment in which two striata from either Adra2c/ or Adra2c+/+ mice were pooled. Right, bars illustrate the mean ± S.E.M. of the net changes in cAMP for Adra2c/ and Adra2c+/+ mice. Repeated measures ANOVA revealed a significant effect of RX821002 in Adra2c+/+ mice (P < .05). Basal values of cAMP were 39.9 ± 8.7 and 23.9 ± 5.5 pmol/mg protein for Adra2c/ and Adra2c+/+ mice, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Forskolin-stimulated cAMP accumulation in the absence or presence of RX821002 in striatal slices from control and monoamine-depleted mice. Left, each pair of connected points, representing forskolin (2 µM) alone and forskolin in the presence of RX821002 (100 nM), comes from an individual experiment in which two striata from either control or monoamine-depleted mice were pooled. Right, bars illustrate the mean ± S.E.M. of the net changes in cAMP for control and monoamine-depleted mice. Repeated measures ANOVA revealed a significant effect of RX821002 in control mice (p < .02). Basal values of cAMP were 20.6 ± 1.1 and 22.0 ± 1.7 pmol/mg protein for control and monoamine-depleted mice, respectively.

Affinities of Dopamine and Norepinephrine at Mouse _2A and _2C Adrenoceptors. Initial experiments examined the saturation binding of [³H]RX821002 in homogenates of NRK-_2A and NRK-_2C cells. The K_D and B_max values of the binding of [³H]RX821002 were 0.95 ± 0.27 nM and 8.02 ± 3.18 pmol/mg protein, respectively, in NRK-_2A cells and 0.71 ± 0.08 nM and 9.21 ± 1.62 pmol/mg protein, respectively, in NRK _2C cells. Saturation isotherms of [³H]RX821002 binding could not be fit significantly better by a model assuming two sites of interaction. Curves of the inhibition of [³H]RX821002 binding by norepinephrine and dopamine were generated to compute affinities of these agonists at the two ₂ adrenoceptor subtypes. Inhibition curves were generated in six experiments, and all data were analyzed by nonlinear regression analysis (Fig. 4). For NRK-_2A and NRK-_2C cells, inhibition curves for both dopamine and norepinephrine were fit to a model assuming two sites of interaction (F = 12.33, p < .0001 for norepinephrine, and F = 10.18, p < .002 for dopamine in _2A cells and F = 38.57, p < .0001 for norepinephrine, and F = 21.51, p < .0001 for dopamine in _2C cells; Fig. 4). High-affinity binding of dopamine was abolished by 100 µM guanosine-5'-(,-imido)triphosphate (data not shown). K_i values at high- and low-affinity states of the receptors were computed from IC₅₀ values generated from inhibition curves from individual experiments that were fit to two sites, using the Cheng-Prusoff correction (Table 1). Dopamine had an affinity for the high-affinity state of the _2C-adrenoceptor that was 18-fold higher than its affinity for the high-affinity state of the _2A adrenoceptor. Dopamine and norepinephrine had comparable affinities for the _2A adrenoceptor. Furthermore, the percentage of high-affinity states of the _2A adrenoceptor bound by dopamine and norepinephrine were similar. Similarly, norepinephrine and dopamine had comparable affinities for _2C adrenoceptors. However, the percentage of high-affinity states of the _2C adrenoceptor bound by dopamine was significantly smaller than that bound by norepinephrine (p < .05).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Inhibition of [3H]RX821002 binding (2.2 nM) to membrane homogenates of NRK-2A and NRK-2C cells by dopamine (left) and norepinephrine (right). For both cell lines, inhibition curves for both dopamine and norepinephrine were fit to a model assuming two sites of interaction. Binding parameters are noted in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Binding parameters for inhibition of [3H]RX821002 by dopamine and norepinephrine at 2 adrenoceptor subtypes

Effects of Dopamine and Norepinephrine on Forskolin-Stimulated cAMP Accumulation in NRK-_2A and NRK-_2C Cells. NRK-_2A cells were incubated with forskolin alone or in the presence of increasing concentrations of dopamine or norepinephrine. At concentrations of 50 nM to 5 mM, dopamine reduced and norepinephrine increased forskolin-stimulated cAMP levels (data not shown). The inhibition produced by dopamine (40 µM) and the enhancement produced by norepinephrine (5 µM) were reversed by RX821002 (1 µM), demonstrating that these responses were mediated by _2A adrenoceptors (Fig. 5). In NRK-_2C cells, dopamine inhibited forskolin-stimulated cAMP accumulation in a concentration-dependent manner, with maximal inhibition occurring near 40 µM (Fig. 6A). Concentrations of dopamine greater than 40 µM, up to 500 µM, had no further effect. Norepinephrine also attenuated forskolin-stimulated cAMP accumulation concentration-dependently, between concentrations of 0.1 nM and 1 µM, in NRK-_2C cells (Fig. 6B). At concentrations of 10 µM and greater, the direction of change in forskolin-stimulated cAMP levels reversed, although cAMP levels were still significantly lower than the forskolin-alone condition at 10 µM. The inhibitions produced by 40 µM dopamine and 5 µM norepinephrine were reversed by RX821002 (1 µM; Fig. 7), confirming that these effects were mediated by _2C adrenoceptors.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Inhibition and facilitation of forskolin-stimulated cAMP accumulation by dopamine (40 µM) and norepinephrine (5 µM), respectively, in intact NRK-2A cells. Where noted, the 2 adrenoceptor antagonist RX821002 (1 µM) was added 10 min before agonists. RX821002 alone (not shown) or with forskolin had no effects on cAMP levels. Each bar represents the mean value of six separate experiments. *p < .05, **p < .01, significant differences from forskolin alone and from forskolin plus agonist in the presence of RX821002.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Inhibition of forskolin (10 µM)-stimulated cAMP accumulation by dopamine (A) and norepinephrine (B) in intact NRK-2C cells. Micromolar concentrations of norepinephrine and dopamine are indicated. Each bar represent the mean value of four separate experiments. *p < .05 or **p < .01, significance differences from forskolin-alone conditions.

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Inhibition of forskolin-stimulated cAMP accumulation by dopamine (40 µM) or norepinephrine (5 µM) in intact NRK-2C cells. Where noted, the 2 adrenoceptor antagonist RX821002 (1 µM) was added 10 min before agonists. RX821002 alone (not shown) or with forskolin had no effects on cAMP levels. Each bar represents the mean value of six separate experiments. *p < .05, significant differences from forskolin alone and from forskolin plus agonist in the presence of RX821002.

	Discussion

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We reported previously that rat striatal _2C adrenoceptors are negatively coupled to adenylyl cyclase (Lu and Ordway, 1997b). In that study, brimonidine (UK14,304) induced a concentration-dependent inhibition of forskolin-stimulated cAMP accumulation in rat striatal slices, an effect that was antagonized by RX821002, an ₂ adrenoceptor antagonist that lacks selectivity for any of the subtypes of ₂ adrenoceptors. Furthermore, RX821002 alone significantly enhanced forskolin-stimulated cAMP accumulation in a concentration-dependent manner in rat striatal slices (Lu and Ordway, 1997b). Enhancement of forskolin-stimulated adenylyl cyclase by an ₂ adrenoceptor antagonist implies that striatal ₂ adrenoceptors are tonically activated by endogenous agonist. Striatal infusions of an antisense oligonucleotide targeting mRNA encoding the _2C adrenoceptor results in a selective, but incomplete, reduction in the expression of striatal _2C adrenoceptors (Lu and Ordway, 1997a) and significantly enhances forskolin-stimulated cAMP accumulation (Lu and Ordway, 1997b). The enhancement of forskolin-stimulated adenylyl cyclase produced by _2C adrenoceptor antisense infusion resembles the effect of RX821002. The present study corroborated these previous findings by demonstrating that RX821002 enhances forskolin-stimulated cAMP accumulation in striatal slices from Adra2c^+/+ mice but has no significant effect on this event in Adra2c^/ mice or in monoamine-depleted Adra2c^+/+ mice (see Figs. 2 and 3). Furthermore, RX82102 has no apparent intrinsic activity at _2A or _2C adrenoceptors, as indicated by a lack of effect of RX821002 on forskolin-stimulated cAMP accumulation in transfected cells expressing these receptors (see Figs. 5 and 7). Together, these findings demonstrate that the _2C adrenoceptor is coupled negatively to adenylyl cyclase in the striatum and imply that this receptor is tonically activated by endogenous transmitter in striatal slices.

The obvious candidate transmitter for the activation of the _2C adrenoceptor in brain is its cognate neurotransmitter, norepinephrine. In fact, many brain regions that express the _2C adrenoceptor are innervated by noradrenergic neurons. Oddly, the striatum receives little or no noradrenergic innervation. Little or no dopamine -hydroxylase immunostaining or radioligand binding to norepinephrine transporters has been found in striatum of rat brain (Grzanna et al., 1977; Jursky et al., 1994; Ordway, 1995). This occurs despite the fact that the striatum has the most dense _2C adrenoceptor gene expression (mRNA) and radioligand binding to _2C adrenoceptors relative to other brain regions (Ordway et al., 1993; Wang et al., 1996; Uhlen et al., 1997). Another possible candidate transmitter in the striatum is the related catecholamine, dopamine. There is a dense dopaminergic innervation of the striatum originating from the substantia nigra (Lindvall and Bjorklund, 1974). Concentrations of norepinephrine in striatum have been reported to be one-fifteenth to one-fortieth of the concentration of dopamine in the striatum (Laurent et al., 1975; Jacobowitz and Richardson, 1978). Versteeg et al. (1976) reported no detectable level of norepinephrine but dopamine concentrations of approximately 70 pg/µg protein in the rat striatum. Hence, there is an apparent paucity of noradrenergic innervation of striatal _2C adrenoceptors. Segawa et al. (1998) found that exogenously applied dopamine contracted femoral veins through the activation of ₂ adrenoceptors, demonstrating that dopamine can act as an agonist at peripheral ₂ adrenoceptors. Together, these observations led to the goal of the present study to examine the affinity and potency of dopamine at _2C adrenoceptors.

Here, we found that the affinities of dopamine at _2A and _2C adrenoceptors are similar to the affinities of norepinephrine at these receptors in NRK cells transfected with mouse _2Aor _2C adrenoceptors. In fact, the affinity of dopamine at the mouse _2C adrenoceptor is approximately equivalent to the affinity of norepinephrine for the _2C adrenoceptor and 3- to 8-fold higher than the affinity of norepinephrine for the _2A adrenoceptor. Furthermore, dopamine inhibited forskolin-stimulated accumulation of cAMP in both NRK-_2A and NRK-_2C cells, effects that were reversed by the ₂ adrenoceptor antagonist RX821002. These results suggest that dopamine could indeed play a physiological role in the activation of ₂ adrenoceptors in the striatum.

Little is known with regard to the function of _2C adrenoceptors in brain because highly selective agonists and antagonists for subtypes of ₂ adrenoceptors have not yet been identified. However, considerable progress has been made recently in understanding functional roles of the individual ₂ adrenoceptor subtypes through the use of molecular biological techniques. For example, a decreased hypothermic response to dexmedetomidine (₂ adrenoceptor agonist) and reduced brain concentrations of HVA have been reported in Adra2c^/ mice (Sallinen et al., 1997). Furthermore, an increased sensitivity of the hypothermic response to dexmedetomidine and higher brain concentrations of HVA and DA have been observed in transgenic mice overexpressing the _2C adrenoceptor compared with wild-type mice (Sallinen et al., 1997). Sallinen et al. (1998) also showed that Adra2c^/ mice had enhanced startle responses, shortened aggressive response latencies, and decreased prepulse inhibition in an isolation-aggression test; mice overexpressing the Adra2c^+/+ gene had opposite effects. The present demonstration of the functional coupling of the _2C adrenoceptor to adenylyl cyclase in the striatum suggests that the _2C adrenoceptor may modulate some striatal behaviors.

The classic biochemical response to ₂ adrenoceptor activation is an inhibition of adenylyl cyclase via the coupling of the receptor to the G_i protein (Jansson et al., 1994a,b). ₂ Adrenoceptors also exhibit the ability to stimulate adenylyl cyclase through coupling to G_s protein in several cell types that have been transfected with these receptors (Eason et al., 1992, 1994; Eason and Liggett, 1993; Jansson et al., 1994a, 1995). Hence, the stimulation or inhibition of cAMP production by ₂ adrenoceptor activation is both subtype and target cell specific in transfected cells. In the present study, we found that norepinephrine and the selective ₂ adrenoceptor agonists clonidine and brimonidine (data not shown for clonidine and brimonidine) increased cAMP production stimulated by forskolin in NRK-_2A cells. In contrast, dopamine only inhibited cAMP accumulation stimulated by forskolin, even at relatively high concentrations. These data indicate that the molecular mechanisms of _2A adrenoceptor activation by norepinephrine and dopamine are different. It is unlikely that this difference is related to differences in the general ability of these agonists to induce or stabilize coupling of the _2A adrenoceptor to G proteins. Curves of the inhibition of [³H]RX821002 binding revealed that there were approximately equal percentages of high-affinity binding for dopamine and norepinephrine in NRK _2A cells. Hence, differences in the effects of dopamine and norepinephrine on cAMP accumulation mediated by _2A adrenoceptors may result from induction of conformational changes in these receptors that are different for the two agonists, leading to differences in the affinity of binding of the receptor to G_s protein.

Interestingly, the percentage of high-affinity binding sites for dopamine was significantly lower than that for norepinephrine in NRK-_2C cells. The lower percentage of high-affinity binding sites for dopamine might indicate that dopamine is less capable of stabilizing or inducing the coupling of the ₂ adrenoceptor to G protein. This may account for an apparent lower potency (as could be determined by estimating the concentration to produce a 50% inhibition, or EC₅₀, in Fig. 6) of dopamine, relative to norepinephrine, for the inhibition of forskolin-stimulated cAMP accumulation, despite the near-equal affinities of these two monoamines for the _2C adrenoceptors. It should be noted that determination of potency of norepinephrine for _2C adrenoceptor-induced inhibition of forskolin-stimulated cAMP accumulation is complicated by the fact that a maximal inhibition could not be reliably obtained. This is because opposing enhancement of cAMP accumulation was initiated as the concentration of norepinephrine was increased, presumably by recruitment of G_s protein by the norepinephrine-₂ adrenoceptor complex. Hence, although maximal inhibition of cAMP accumulation was observed at 40 µM dopamine and 1 µM norepinephrine, the true maximum inhibition induced by norepinephrine was clouded by a concomitant activation.

In conclusion, RX821002 facilitated forskolin-stimulated cAMP accumulation in striatal slices in Adra2c^+/+ mice and failed to enhance forskolin-stimulated cAMP accumulation in Adra2c^/ mice. These data, along with our previous findings (Lu and Ordway, 1997b), indicate that striatal _2C adrenoceptors are negatively coupled to adenylyl cyclase and are under tonic activation by an endogenous agonist. The inhibition of forskolin-stimulated cAMP production in NRK-_2C cells by catecholamines and its antagonism by RX821002 further demonstrate that the _2C adrenoceptor is coupled to inhibition of adenylyl cyclase. The high affinity of dopamine for the _2C adrenoceptor and the ability of dopamine to activate this receptor at physiologically relevant concentrations bolster the conjecture that dopamine may act as a promiscuous activator of _2C adrenoceptors in the brain, particularly in areas such as the striatum, where this receptor occurs in conjunction with dense dopaminergic innervation and sparse or absent noradrenergic innervation.