Examples of progress made during the prior year are summarized below. 1) We demonstrated in rats that PET can measure the activation status of an enzyme that may be involved in depression (Itoh et al., 2010. The 3',5'-cyclic adenosine monophosphate (cAMP) cascade is a major signal transduction system in brain and may be involved in psychiatric illnesses such as mood disorders and drug addiction. cAMP is synthesized by adenylyl cyclase and is metabolized by phosphodiesterases (PDEs). PDE type 4 (PDE4) is the major isozyme that hydrolyzes cAMP in brain. By 11C-labeling rolipram, a selective inhibitor of PDE4, the cAMP cascade has been imaged with PET in rat, monkey, and man. cAMP-dependent protein kinase (PKA) phosphorylates PDE4 and increases both enzyme activity and affinity for rolipram. In the present PET study, we examined effects of PKA modulators in conscious rats on the binding of 11C(R)-rolipram in comparison to the much less active enantiomer 11C(S)-rolipram. We found that the intrastriatal injection in conscious rats of PKA activator and inhibitor significantly increased and decreased, respectively, the in vivo binding of 11C(R)-rolipram measured with PET. These alterations reflected specific binding of 11C(R)-rolipram to PDE4, since there were no effects on 11C(S)-rolipram uptake. These results provide strong evidence that 11C(R)-rolipram can monitor the in vivo activity and phosphorylation state of PDE4, an important regulatory enzyme in the cAMP second messenger cascade. We will use this radioligand to measure PDE4 in brains of patients with major depressive disorder. Based upon this work in animals, we now know that our imaging will be sensitive not only to the amount of enzyme but also its activation status. 2) We found that our PET radioligand, 11C-desmethyl-loperamide (a metabolite of Imodium), is selective at the blood-brain barrier for one drug transporter that may cause resistance to treatment in several disorders, including cancer, epilepsy, and HIV infection of brain (Kannan et al., 2010). Efflux transporters of the ATP binding cassette (ABC) family block the entry into cells of subsets of foreign compounds and thereby protect the body from potential toxins. At the blood-brain barrier, the three most prevalent ABC transporters (ABCB1, ABCC1, and ABCG2) are expressed in the walls of the blood vessels and not only protect the brain from toxic substances but also impede delivery of therapeutic drugs. Among these three transporters, ABCB1 (commonly known as P-gp) has been most studied, in part because it likely causes pathology in both brain and periphery. At the blood-brain barrier, over-expression of ABCB1 may contribute to drug-resistance in epilepsy and in HIV infection of the brain. In the periphery as well as in the brain, over-expression of ABCB1 in cancer cells is one cause of multidrug resistance to chemotherapy. The goal of this study was to determine the selectivity of dLop for ABCB1 in human tissues using three human cell lines that over-express ABCB1, ABCC1, or ABCG2 as well as in vivo using three strains of mice that were selectively knocked out for the gene that encodes ABCB1, ABCC1, or ABCG2. Both in vitro and in vivo results showed that our radioligand is selective for ABCB1. We are now using this radioligand 11Cdlop, to study the function of ABCB1 (P-gp) at the blood brain barrier in healthy subjects. (See Dr. Inniss companion report on clinical studies MH002852-06). The importance of the above studies is that we know we are selectively measuring the function of ABCB1 and not two other transporters prevalent at the blood-brain barrier. 3) We used genetically modified mice to demonstrate that PET can measure the in vivo internalization of receptors that mediate the actions of stimulants like amphetamine (Skinbjerg et al. 2010). Dopamine released by amphetamine decreases the in vivo binding of PET radioligands to the dopamine D2 receptor. Although concentrations of extracellular dopamine largely return to baseline within one to two hours after amphetamine treatment, radioligand binding remains decreased for several hours. The purpose of this study was to determine whether the prolonged decrease of radioligand binding after amphetamine administration is caused by receptor internalization. To distinguish dopamine displacement from receptor internalization, we used wild-type and arrestin3 (arr3) knockout mice, which are incapable of internalizing D2 receptors. In addition, we used both the D2 selective agonist 11CMNPA (which is thought to bind to the high affinity state of the receptor) and the D2 selective antagonist 18Ffallypride (which does not differentiate between high and low affinity state). We found that the prolonged decrease of radioligand binding after amphetamine is mainly due to internalization of the D2 receptor rather than dopamine displacement. In addition, this study demonstrates the utility of small animal PET to study receptor trafficking in vivo in genetically modified mice.
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