Presenilins are integral membrane protein involved in the production of amyloid ␤-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter ␥-secretase cleavage of the ␤-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A␤, the 4-kDa ␤-peptide. Here, we show that radiolabeled ␥-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A␤ formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N-and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of ␥-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective ␥-secretase inhibitors block A␤ formation by binding to presenilin-1 and -2.␤-Amyloid precursor protein (␤APP) 1 is a transmembrane protein that undergoes processing to A␤ by proteolytic activities known as ␤-and ␥-secretases (for review, see Refs. 1-3). The ␤-secretase cleavage occurs in the extracellular domain by a recently identified aspartyl protease variously termed BACE, memapsin, and Asp2 (4 -9), whereas the heterogeneous ␥-secretase cleavage occurs in the transmembrane domain (2, 10). Dominant mutations in either of the two human presenilin (PS-1 and PS-2) genes lead to familial Alzheimer's disease (AD). PS-1 and -2 are polytopic membrane proteins (for review, see Refs. 11-13). Presenilins are proteolytic processed. In vivo, only small amounts of the holoprotein can be detected, primarily in the nuclear envelope, whereas 30-kDa N-terminal and 20-kDa C-terminal fragments of presenilin are observed in all mammalian tissues and cell lines analyzed so far. Coimmunoprecipitation experiments revealed that presenilin fragments are assembled into a high molecular weight complex together with other proteins (for review see 11-13). The proposed mechanism through which the presenilin mutations cause AD is an alteration in the predominant ␥-secretase cleavage site which increases the amount of the longer, more amyloidogenic A␤ 1-42(43) fragments produced (11-13). A null mutation of the mouse PS-1 selectively reduces ␥-secretase activity (14), and site-directed mutagenesis of PS-1 and PS-2 at two conserved aspartyl residues, which resemble the catalytic center of aspartyl proteases, also reduces ␥-secretase activity (15, 16). These observations indicate that PS-1 and PS-2 either stimulate the activity of ␥-secretase by trafficking to appropriate cellular compartments, serve as cofactors of the ␥-secretase, or are ␥-secretase themselves.Here, we report that a series of potent and selective ␥-secretase inhibitors bind to mam...

LY248686 is an inhibitor of serotonin (S-hydroxytryptamine; 5-HT) and norepinephrine (NE) uptake in synaptosomal preparations of hypothalamus and cerebral cortex, and 5-HT uptake in human blood platelets, with inhibitor constants near nanomolar concentrations. Upon administration to rats 1 hour before sacrifi ce, LY248686 caused dose-dependent and parallel decreases of 5-HT and NE uptake in hypothalamus homogenates ex vivo. LY248686 is a positive enantiomer and was slightly more potent than its negative isomer, LY248685, as an inhibitor of 5-HT uptake. Both isomers were only weak inhibitors of dopamine (DA) uptake in striatal synaptosomes. The inhibitory effects on 5-HT and NE uptake after a single administration of LY248686 KEY WORDS: Uptake; Inhibitor; Serotonin; Norepineph rineThe extent of inhibition of serotonin (5-hydroxytrypta mine; 5-HT) and norepinephrine (NE) uptake by the tricyclic tertiary amine-containing antidepressant drugs depends on their relative degrees of metabolic N-demethylation. Amitriptyline, an inhibitor of 5-HT and NE uptake in nerve-ending preparations (synap tosomes), is converted in vivo to nortriptyline, a po tent and selective inhibitor of NE uptake, while imipra mine, a selective inhibitor of 5-HT uptake, is converted to desipramine, another potent and selective inhibitor

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In summary, fluoxetine is a highly selective serotonin uptake inhibitor in vitro and in vivo. The conformation of fluoxetine, which resembles that of sertraline and other serotonin uptake inhibitors, appears to be a key feature that enables its high affinity and selective interaction with the serotonin transporter. The para-trifluoromethyl substituent, however, is also a pivotal structural element. The molecular pharmacology of fluoxetine has been well-defined, and its in vivo pharmacological effects appear to be mediated almost exclusively by serotonin uptake inhibition. Its selectivity for the serotonin transporter, lack of affinity for neurotransmitter receptors, and retention of selectivity following metabolism to norfluoxetine make fluoxetine a useful tool to explore pharmacologically induced increases in serotonin neurotransmission. Fluoxetine has found a variety of therapeutic application. Its use in treating depression has been most extensively studied, but controlled clinical studies also suggest the drug may have a role in treating obesity and bulimia. Moreover, a variety of other psychiatric disorders may be treatable with this drug. Regardless of the outcome of these clinical trials, it is apparent that fluoxetine has found a useful niche in therapy, and can be used as a probe to determine the role of serotonin in modulating human pathophysiologies.

Background-To assess the sensitivity of biochemical, physiological, and pharmacological markers of peripheral norepinephrine (NE) transporter (NET) function, we chronically antagonized NET by a range of doses of duloxetine [(ϩ)-N-methyl-3-(1-naphthalenyloxy)-2 thiophenepropanamine], which blocks the NE reuptake process. Methods and Results-Duloxetine was administered in a randomized, placebo-controlled study in 15 healthy volunteers.Plasma from duloxetine-treated subjects (ex vivo effect) dose-dependently decreased radioligand binding to human NET (maximum inhibition was 60%) (Pϭ0.02). The dose of intravenous tyramine required to raise systolic blood pressure by 30 mm Hg (PD 30 ) increased dose-dependently with duloxetine and was significant at the end of the 120-mg/d dosage (PϽ0.001). The plasma dihydoxyphenylglycol to NE (DHPG/NE) ratio was reduced significantly at 2 weeks of treatment with 80 mg/d duloxetine (11.3 at baseline, 3.4 at 240 mg/d, PϽ0.001). Plasma NE was significantly increased starting at 120 mg/d duloxetine. Urine results (corrected for 24-hour creatinine excretion) showed a dose-dependent change from the baseline urinary excretion for NE, DHPG, and the DHPG/NE ratio. The most sensitive measure, the DHPG/NE ratio, was significant at the 80-mg dose. Urinary NE excretion was significantly raised after 2 weeks of treatment with 80 mg/d duloxetine (PϽ0.001), the lowest dose used in the study. Conclusions-These findings suggest that the degree of NET blockade can be assessed with the plasma or urine DHPG/NE ratio and the pressor effect of tyramine. Also, the DHPG/NE ratio is more sensitive at the lower end of NET inhibition, whereas tyramine exhibits a linear relation, with NET inhibition commencing at a higher dose.