No abstract

The radioligand-binding assay is a relatively simple but powerful tool for studying G-protein-coupled receptors. There are three basic types of radioligand-binding experiments: (1) saturation experiments from which the affinity of the radioligand for the receptor and the binding site density can be determined; (2) inhibition experiments from which the affinity of a competing, unlabeled compound for the receptor can be determined: and (3) kinetic experiments from which the forward and reverse rate constants for radioligand binding can be determined. Detailed methods for typical radioligand-binding assays for G-protein-coupled receptors in membranes and intact cells are presented for these types of experiments. Detailed procedures for analysis of the data obtained from these experiments are also given.

Neuropharmacological and genetic association studies have implicated norepinephrine and adrenergic receptors in the pathogenesis of ADHD. The purpose of this study was to compare genetic association studies of three polymorphisms of the alpha-2A adrenergic receptor gene (ADRA2A) with radioligand binding studies of the alpha-2A adrenergic receptor protein in platelets from a sample of children without or with ADHD. The pediatric subjects ranged from 6 to 18 years of age. A thorough clinical assessment of each child resulted in one of the following DSM-IV ADHD diagnoses: inattentive, hyperactive/impulsive, combined, or no ADHD. No significant linkage was found between the ADRA2A polymorphisms (MspI, HhaI, and DraI) and any of the phenotypes tested. Association analysis, however, did detect significant linkage disequilibrium for the DraI polymorphism. Association was also evaluated considering the three ADRA2A single nucleotide polymorphisms as haplotypes. The HhaI-DraI and the MspI-HhaI-DraI haplotypes were significantly associated with ADHD. The platelet alpha-2 adrenergic receptor density did not differ between children without or with ADHD. The affinity of the receptor for the radioligand however, differed significantly between those without and with ADHD. In addition, there were some significant correlations between binding parameters and severity of ADHD in this well-characterized clinical population, and significant association was found between these measures of receptor function and MspI and DraI polymorphisms. Thus, both the genetic and the binding studies indicate that the alpha-2 adrenergic receptor may play a role in ADHD.

Although the tricyclic antidepressants, such as desipramine (DMI), are among the most efficacious treatments for adult depression, they are not effective in treating childhood and adolescent depression. Because the adrenergic nervous system is not fully developed until late adolescence, we hypothesized that the mechanisms regulating receptor density may not yet be mature in young mammals. To test this hypothesis, the effects of DMI treatment on cortical ␣-1-, ␣-2-, and ␤-adrenergic receptors were compared in juvenile and adult rats. DMI was delivered either by 4 days of twice daily injections to postnatal day 9 to 13 (4 and 7 mg/kg/day) and adult (20 mg/ kg/day) rats, or by 2 weeks of continual drug infusion (osmotic minipumps) to postnatal day 21-35 (15 mg/kg/day) and adult (10 mg/kg/day) rats. These delivery paradigms gave juvenile brain concentrations of DMI similar to those in adult rats. The ␤-adrenergic receptor was down-regulated with both treatment paradigms in both juvenile and adult rats. By contrast, in the postnatal day 9 to 13 rats, there was a dose-dependent upregulation of the ␣-1 in the cortex and ␣-2-adrenergic receptor in the prefrontal cortex, whereas there was no change in density in adult rats. These differences in the ␣-adrenergic receptor regulation after DMI treatment suggest that the lack of efficacy of tricyclic antidepressants in treating childhood depression may be related to immature regulatory mechanisms for these receptors.

The tricyclic antidepressants, including desipramine (DMI), are no better than placebo in treating childhood and adolescent depression, but are effective in adult depression. Animal studies comparing the effects of DMI in juveniles and adults are complicated by age-related variations in elimination rates. Thus, different dosing regiments are needed to achieve similar brain drug levels in juvenile and adult rats. We compared the half-life of DMI as well as the brain and serum concentrations of DMI and its active metabolite desmethyldesipramine in juvenile and adult rats after various drug administration paradigms. After acute i.p. administration DMI is eliminated from the brain more slowly in postnatal day (PND) 21 and 28 rats as compared to adults. After chronic i.p. administration (for 4-5 days between PND 9 and 28), lower doses of DMI are needed with juvenile rats to obtain the same brain DMI concentrations as adults. By contrast, two weeks of continuous drug delivery (minipump) to PND 21-35 and adult rats result in similar brain DMI concentrations. Thus, the pharmacokinetic properties of DMI varies with the age of the animal and dosing of DMI and needs to be carefully adjusted in order to have appropriate brain levels of the drug.