A robust GTP-induced shift in α2-adrenoceptor agonist affinity in tissue sections from rat brain
Introduction
Most of the effects of agonist-stimulation of the seven transmembrane superfamily of receptors are mediated by coupling to guanine nucleotide binding regulatory proteins (G proteins) (Gilman, 1987). This coupling is not constant, but varies with development, physiological status and pathological conditions, and can be altered by drug treatment. Thus it is important to have assays that monitor the degree of functional coupling between a receptor and its G protein(s). Several types of assays have been developed to fill this need. The activity of the enzyme adenylyl cyclase has been a frequent assay for receptors coupled to Gs (stimulation of cyclase activity) or Gi (inhibition of cyclase activity). The GTP-induced decrease in the affinity of agonists in inhibiting antagonist radioligand binding is a second type of assay. More recently, the agonist-stimulated increase in GTPγS binding has been developed for Gi/o-coupled receptors (Cerione et al., 1986, Hilf et al., 1989).
These assays were initially developed in intact cell or membrane preparations and thus lack the anatomic precision provided by receptor autoradiography, which allows the investigation of small brain regions and discrete nuclei. To date, techniques for adapting the adenylyl cyclase assay to provide information with anatomic specificity comparable to receptor autoradiography have not been developed. GTPγS binding assays using autoradiographic techniques have become available recently for a variety of Gi/o-coupled receptors (Sim et al., 1997) including the opioid, cannabinoid and GABA-B receptor systems (Sim et al., 1995), as well as for the α2-adrenoceptor (Happe et al., 2000). In this report, we present the characterization of an agonist potency GTP shift assay for the α2-adrenoceptor (Bylund et al., 1994) in rat brain sections, which is applicable to the autoradiographic study of G protein-receptor coupling. We found that GTP, but not ATP, induces a decrease in agonist affinity of greater than 100-fold.
Section snippets
Materials
[3H]RX821002 (53–67 Ci/mmol) was obtained from Amersham Pharmacia (Arlington Heights, IL). GTP, ATP and GppNHp, were purchased from Sigma (St. Louis, MO), and AppNHp was purchased from Calbiochem (La Jolla, CA). Brimonidine (UK 14,304) was a gift from Allergan (Irvine, CA). All other chemicals were research grade.
Animals
Adult Sprague-Dawley rats, 180–250 g (SASCO, Kingston, NY) were housed three or four per cage and fed ad libitum. Animals were sacrificed by decapitation under halothane anesthesia.
GTP-induced shift in agonist potency
As expected (Turner et al., 1985), 100 μM GTP reduced the affinity of norepinephrine in inhibiting the binding of the α2-adrenoceptor-selective antagonist [3H]RX821002 to tissue sections of rat brain (Fig. 1); the magnitude of the shift, however, was remarkably large. In the absence of GTP, the inhibition data fit a two-site model significantly better (P<0.05) than a one-site model. The high affinity site with a Ki of 45 nM accounted for an average of 76% of the sites, whereas the low affinity
Discussion
The GTP-induced decrease in the affinity of α2-adrenoceptor agonists in inhibiting the binding of an antagonist radioligand has been well established in studies using membrane preparations. Here we describe a more robust GTP-induced effect on agonist affinity using slide mounted tissue sections. In the absence of GTP, the agonist, receptor and G protein form a ternary complex which has high affinity for the agonist. The presence of GTP results in the disassociation of the G protein from the
Acknowledgements
This work was supported by grant NS 33197 from the National Institutes of Health.
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