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The cytokines IL-1α and IL-1β exert powerful pro-inflammatory actions throughout the body, mediated primarily by the intracellular signaling capacity of the interleukin-1 receptor (IL-1R1). Although Il1r1 knockout mice have been informative with respect to a requirement for IL-1R1 signaling in inflammatory events, the constitutive nature of gene elimination has limited their utility in the assessment of temporal and spatial patterns of cytokine action. To pursue such questions, we have generated C57Bl/6J mice containing a floxed Il1r1 gene (Il1r1loxP/loxP), with loxP sites positioned to flank exons 3 and 4 and thereby the ability to spatially and temporally eliminate Il1r1 expression and signaling. We found that Il1r1loxP/loxP mice breed normally and exhibit no gross physical or behavioral phenotypes. Moreover, Il1r1loxP/loxP mice exhibit normal IL-1R1 receptor expression in brain and spleen, as well as normal IL-1R1-dependent increases in serum IL-6 following IL-1α injections. Breeding of Il1r1loxP/loxP mice to animals expressing a cytomegalovirus (CMV)-driven Cre recombinase afforded efficient excision at the Il1r1 locus. The Il1r1loxP/loxP line should be a valuable tool for the assessment of contributions made by IL-1R1 signaling in diverse cell types across development.

Alterations in central serotonin (5-hydroxytryptamine, 5-HT) neurotransmission and peripheral immune activation have been linked to multiple neuropsychiatric disorders, including depression, schizophrenia and autism. The antidepressant-sensitive 5-HT transporter (SERT, SLC6A4), a critical determinant of synaptic 5-HT inactivation, can be regulated by pro-inflammatory cytokine signaling. Systemic innate immune system activation via intraperitoneal lipopolysaccharide (LPS) injection rapidly elevates brain SERT activity and 5-HT clearance. Moreover, the pro-inflammatory cytokine interleukin (IL)-1β rapidly stimulates SERT activity in raphe nerve terminal preparations ex vivo, effects that are attenuated by pharmacological p38 MAPK inhibition. To establish a role of serotonergic p38α MAPK signaling in LPS/IL-1β-induced SERT regulation and attendant behavioral responses, we pursued studies in mice that afford conditional elimination of p38α MAPK in 5-HT neurons (p38α(5HT-)). We found p38α(5HT-) and control (p38α(5HT+)) littermates to be indistinguishable in viability and growth and to express equivalent levels of SERT protein and synaptosomal 5-HT transport activity. Consistent with pharmacological studies, however, IL-1β fails to increase SERT activity in midbrain synaptosomes prepared from p38α(5HT-) animals. Moreover, although LPS elevated plasma corticosterone and central/peripheral pro-inflammatory cytokines in p38α(5HT-) animals, elevations in midbrain SERT activity were absent nor were changes in depressive and anxiety-like behaviors observed. Our studies support an obligate role of p38α MAPK signaling in 5-HT neurons for the translation of immune activation to SERT regulation and 5-HT-modulated behaviors.

Background: Rare genetic variation is an important class of autism spectrum disorder (ASD) risk factors and can implicate biological networks for investigation. Altered serotonin (5-HT) signaling has been implicated in ASD, and we and others have discovered multiple, rare, ASD-associated variants in the 5-HT transporter (SERT) gene leading to elevated 5-HT re-uptake and perturbed regulation. We hypothesized that loci encoding SERT regulators harbor variants that impact SERT function and/or regulation and therefore could contribute to ASD risk. The adenosine A3 receptor (A3AR) regulates SERT via protein kinase G (PKG) and other signaling pathways leading to enhanced SERT surface expression and catalytic activity., Methods: To test our hypothesis, we asked whether rare variants in the A3AR gene (ADORA3) were increased in ASD cases vs. controls. Discovery sequencing in a case-control sample and subsequent analysis of comparison exome sequence data were conducted. We evaluated the functional impact of two variants from the discovery sample on A3AR signaling and SERT activity., Results: Sequencing discovery showed an increase of rare coding variants in cases vs. controls (P=0.013). While comparison exome sequence data did not show a significant enrichment (P=0.071), combined analysis strengthened evidence for association (P=0.0025). Two variants discovered in ASD cases (Leu90Val and Val171Ile) lie in or near the ligand-binding pocket, and Leu90Val was enriched individually in cases (P=0.040). In vitro analysis of cells expressing Val90-A3AR revealed elevated basal cGMP levels compared with the wildtype receptor. Additionally, a specific A3AR agonist increased cGMP levels across the full time course studied in Val90-A3AR cells, compared to wildtype receptor. In Val90-A3AR/SERT co-transfections, agonist stimulation elevated SERT activity over the wildtype receptor with delayed 5-HT uptake activity recovery. In contrast, Ile171-A3AR was unable to support agonist stimulation of SERT. Although both Val90 and Ile171 were present in greater numbers in these ASD cases, segregation analysis in families showed incomplete penetrance, consistent with other rare ASD risk alleles., Conclusions: Our results validate the hypothesis that the SERT regulatory network harbors rare, functional variants that impact SERT activity and regulation in ASD, and encourages further investigation of this network for other variation that may impact ASD risk.

Activation of A₃ adenosine receptors (A₃ARs) rapidly enhances the activity of antidepressant-sensitive serotonin (5-HT) transporters (SERTs) in vitro, ex vivo, and in vivo. A₃AR agonist stimulation of SERT activity is lost in A₃AR knockout mice. A₃AR-stimulated SERT activity is mediated by protein kinase G1 (PKGI)- and p38 mitogen-activated protein kinase (MAPK)-linked pathways that support, respectively, enhanced SERT surface expression and catalytic activation. The mechanisms by which A₃ARs target SERTs among other potential effectors is unknown. Here we present evidence that A₃ARs are coexpressed with SERT in midbrain serotonergic neurons and form a physical complex in A₃AR/hSERT cotransfected cells. Treatment of A₃AR/SERT-cotransfected Chinese hamster ovary cells with the A₃AR agonist N⁶-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (1 μM, 10 min), conditions previously reported to increase SERT surface expression and 5-HT uptake activity, enhanced the abundance of A₃AR/SERT complexes in a PKGI-dependent manner. Cotransfection of SERT with L90V-A₃AR, a hyperfunctional coding variant identified in subjects with autism spectrum disorder, resulted in a prolonged recovery of receptor/transporter complexes after A₃AR activation. Because PKGI and nitric-oxide synthetase are required for A₃AR stimulation of SERT activity, and proteins PKGI and NOS both form complexes with SERT, our findings suggest a mechanism by which signaling pathways coordinating A₃AR signaling to SERT can be spatially restricted and regulated, as well as compromised by neuropsychiatric disorders.

Serotonin (5-hydroxytryptamine, 5-HT) has long been implicated in regulation of mood. Medications that block the neuronal 5-HT transporter (SERT) are used as major pharmacological treatment for mood disorders. Conversely, stimuli that enhance SERT activity might be predicted to diminish synaptic 5-HT availability and increase the risk for 5-HT-related CNS disorders. We have shown that the inflammatory cytokines enhance brain SERT activity in cultured serotonergic cells and nerve terminal preparations in vitro. In this study, we establish that intraperitoneal injection of the cytokine-inducer lipopolysaccharide (LPS) stimulates brain SERT activity, acting at doses below those required to induce overt motor suppression. SERT stimulation by LPS is paralleled by increased immobility in both the tail suspension test (TST) and the forced swim test (FST); antidepressant-sensitive alterations are thought to model aspects of behavioral despair. Both the stimulation of SERT activity and induced immobility are absent when LPS is administered to interleukin-1 receptor (IL-1R)-deficient mice and in the presence of SB203580, an inhibitor of IL-1R-stimulated p38 MAPK. Moreover, the ability of LPS to enhance immobility in TST is lost in SERT knockout mice. These findings reveal an ability of peripheral inflammatory stimuli to enhance brain SERT activity through IL-1R and p38 MAPK pathways in vivo and identify a requirement for SERT expression in immune-system-modulated despair behaviors. Our studies identify IL-1R- and p38 MAPK-dependent regulation of SERT as one of the mechanisms by which environmentally driven immune system activation can trigger despair-like behavior in an animal model, encouraging future analysis of the pathway for risk factors in neuropsychiatric disorders.

The inactivation of synaptic serotonin (5-hydroxytryptamine, 5-HT) is largely established through the actions of the presynaptic, antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). Recent studies have demonstrated post-translational regulation of SERT mediated by multiple Ser/Thr kinases, including protein kinases C and G (PKC and PKG) and p38 mitogen-activated protein kinase (MAPK), as well as the Ser/Thr phosphatase PP2A. Less well studied are specific surface receptors that target these signaling pathways to control SERT surface expression and/or catalytic rates. Using rat basophilic leukemia 2H3 cell line (RBL-2H3), we previously established that activation of A(3) adenosine receptors (A(3)AR) stimulates SERT activity via both PKG and p38 MAPK (Zhu et al., 2004a). Whether A(3)ARs regulate SERT in the central nervous system (CNS) is unknown. Here we report that the A(3)AR agonist N(6)-(3-iodobenzyl)-N-methyl-5'carbamoyladenosine (IB-MECA) rapidly (10 min) and selectively stimulates 5-HT transport in mouse midbrain, hippocampal, and cortical synaptosomes. IB-MECA-induced stimulation of 5-HT uptake is blocked by the selective A(3)AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(+/-)dihydropyridine-3,5-dicarboxylate (MRS1191) and is absent from synaptosomes prepared from A(3)AR knockout mice. Kinetic analyses demonstrate that IB-MECA induces an increase of 5-HT transport V(max) with no significant change in K(m). As in RBL-2H3 cells, IB-MECA stimulation of synaptosomal 5-HT uptake can be blocked by preincubation with PKG antagonists N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) and DT-2 (YGRKKRRQRRRPPLRK(5)H), as well as by the p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Chronoamperometry studies in the anesthetized rat hippocampus support a role for A(3)ARs in SERT regulation in vivo. Together, these results identify a novel, region-specific action of CNS A(3)ARs in the modulation of SERT-mediated 5-HT transport that may be relevant for the etiology and/or therapy of 5-HT-linked brain disorders.

Proinflammatory cytokines and serotonergic homeostasis have both been implicated in the pathophysiology of major psychiatric disorders. We have demonstrated that activation of p38 mitogen-activated protein kinase (MAPK) induces a catalytic activation of the serotonin transporter (SERT) arising from a reduction in the SERT Km for 5-hydroxytryptamine (5-HT). As inflammatory cytokines can activate p38 MAPK, we hypothesized that they might also activate neuronal SERT. Indeed, Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) stimulated serotonin uptake in both the rat embryonic raphe cell line, RN46A, and in mouse midbrain and striatal synaptosomes. In RN46A cells, IL-1beta stimulated 5-HT uptake in a dose- and time-dependent manner, peaking in 20 min at 100 ng/ml. This was abolished by IL-1ra (20 ng/ml), an antagonist of the IL-1 receptor, and by SB203580 (5 microM), a p38 MAPK inhibitor. TNF-alpha also dose- and time-dependently stimulated 5-HT uptake that was only partially blocked by SB203580. Western blots showed that IL-1beta and TNF-alpha activated p38 MAPK, in an SB203580-sensitive manner. IL-1beta induced an SB203580-sensitive decrease in 5-HT Km with no significant change in Vmax. In contrast, TNF-alpha stimulation decreased 5-HT Km and increased SERT Vmax. SB203580 selectively blocked the TNF-alpha-induced change in SERT Km. In mouse midbrain and striatal synaptosomes, maximal stimulatory effects on 5-HT uptake occurred at lower concentrations (IL-1beta, 10 ng/ml; TNF-alpha, 20 ng/ml), and over shorter incubation times (10 min). As with RN46A cells, the effects of IL-1beta and TNF-alpha were completely (IL-1beta) or partially (TNF-alpha) blocked by SB203580. These results provide the first evidence that proinflammatory cytokines can acutely regulate neuronal SERT activity via p38 MAPK-linked pathways.

Human serotonin [5-hydroxytryptamine (5-HT)] transporters (hSERT, 5HTT, and SLC6A4) inactivate 5-HT after release and are prominent targets for therapeutic intervention in mood, anxiety, and obsessive-compulsive disorders. Multiple hSERT coding variants have been identified, although to date no comprehensive functional analysis of these variants has been reported. We transfected hSERT or 10 hSERT coding variants and examined total and surface protein expression, antagonist recognition, and transporter modulation by posttranslational, regulatory pathways. Two variants, Pro339Leu and Ile425Val, demonstrated significant changes in surface expression supporting alterations in 5-HT transport capacity (V(max)). Regardless of basal transport activity, all SERT variants displayed a capacity for rapid, phorbol ester-triggered down-regulation. Remarkably, five variants (Thr4Ala, Gly56Ala, Glu215Lys, Lys605Asn, and Pro612Ser) demonstrated no capacity for 5-HT uptake stimulation after acute protein kinase G (PKG)/p38 mitogen-activated protein kinase (MAPK) activation. Epstein-Barr virus (EBV)-transformed lymphocytes natively expressing the most common of these variants (Gly56Ala) exhibited a similar loss of 5-HT uptake stimulation by PKG/p38 MAPK activators. HeLa cells transfected with the Gly56Ala variant demonstrated elevated basal phosphorylation and, unlike hSERT, could not be further phosphorylated after 8-bromo cGMP (8BrcGMP) treatments. These studies reveal cellular phenotypes associated with naturally occurring human SERT coding variants and suggest that altered transporter regulation by means of PKG/p38 MAPK-linked pathways may influence risk for disorders attributed to compromised 5-HT signaling.

Presynaptic, plasma membrane serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) clear 5-HT following vesicular release and are regulated through trafficking-dependent pathways. Recently, we provided evidence for a trafficking-independent mode of SERT regulation downstream of adenosine receptor (AR) activation that is sensitive to p38 MAPK inhibitors. Here, we probe this pathway in greater detail, demonstrating elevation of 5-HT transport by multiple p38 MAPK activators (anisomycin, H(2)O(2), and UV radiation), in parallel with p38 MAPK phosphorylation, as well as suppression of anisomycin stimulation by p38 MAPK siRNA treatments. Studies with transporter-transfected Chinese hamster ovary cells reveal that SERT stimulation is shared with the human norepinephrine transporter but not the human dopamine transporter. Saturation kinetic analyses of anisomycin-SERT activity reveal a selective reduction in 5-HT K(m) supported by a commensurate increase in 5-HT potency (K(i)) for displacing surface antagonist binding. Anisomycin treatments that stimulate SERT activity do not elevate surface SERT surface density whereas stimulation is lost with preexposure of cells to the surface-SERT inactivating reagent, 2-(trimethylammonium)ethyl methane thiosulfonate. Guanylyl cyclase (1H-(1,2,4)-oxadiazolo[4,3-a]-quinoxalin-1-one) and protein kinase G inhibitors (H8, DT-2) block AR stimulation of SERT yet fail to antagonize SERT stimulation by anisomycin. We thus place p38 MAPK activation downstream of protein kinase G in a SERT-catalytic regulatory pathway, distinct from events controlling SERT surface density. In contrast, the activity of protein phosphatase 2A inhibitors (fostriecin and calyculin A) to attenuate anisomycin stimulation of 5-HT transport suggests that protein phosphatase 2A is a critical component of the pathway responsible for p38 MAPK up-regulation of SERT catalytic activity.

The serotonin (5-hydroxtryptamine, 5-HT) transporter (SERT) plays a critical role in the inactivation of synaptic 5-HT and has been implicated in multiple psychiatric and peripheral disorders. SERT regulation studies demonstrate that activation of cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG)-linked pathways can increase SERT activity. As cGMP actions are limited by cGMP-specific phosphodiesterase (PDEs), we investigated whether the cGMP-specific PDE5 inhibitor sildenafil (Viagra) can stimulate 5-HT uptake and potentiate cGMP-mediated regulation. In RBL-2H3 cells, SERT activity was stimulated by sildenafil in a concentration- and time-dependent manner. Sildenafil also enhanced the stimulation of SERT triggered by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), effects blocked by the PKG inhibitor N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8). Sildenafil stimulation of 5-HT uptake arises from an increase in 5-HT transport Vmax and is paralleled by elevated SERT surface antagonist binding, also H8-sensitive. These findings implicate cGMP-targeted PDEs in limiting the regulation of antidepressant-sensitive 5-HT transport.

Serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) are critical determinants of synaptic 5-HT inactivation and the targets for multiple drugs used to treat psychiatric disorders. In support of prior studies, we found that short-term (5-30 min) application of the adenosine receptor (AR) agonist 5'-N-ethylcarboxamidoadenosine (NECA) induces an increase in 5-HT uptake Vmax in rat basophilic leukemia 2H3 cells that is enhanced by pretreatment with the cGMP phosphodiesterase inhibitor sildenafil. NECA stimulation is blocked by the A3 AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(+/-)dihydropyridine-3,5-dicarboxylate (MRS1191), by the phospholipase C inhibitor 1-(6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl)-1H-pyrrole-2,5-dione (U73122), by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, and by the guanyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Hydroxylamine, a nitric-oxide donor, and 8-bromo-cGMP, a membrane-permeant analog of cGMP, mimic the effects of NECA on 5-HT uptake, whereas the protein kinase G (PKG) inhibitor N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) blocks NECA, hydroxylamine, and 8-bromo-cGMP effects. NECA stimulation activates p38 mitogen-activated protein kinase (MAPK), whereas p38 MAPK inhibitors block NECA stimulation of SERT activity, as does the protein phosphatase 2A (PP2A) inhibitor calyculin A. 5-HT-displaceable [125I]3beta-(4-iodophenyl)-tropane-2beta-carboxylic acid methylester tartrate (RTI-55) whole-cell binding is increased by NECA or sildenafil, and both surface binding and cell surface SERT protein are elevated after NECA or sildenafil stimulation of AR/SERT-cotransfected Chinese hamster ovary cells. Whereas p38 MAPK inhibition blocks NECA stimulation of 5-HT activity, it fails to blunt stimulation of SERT surface density. Moreover, inactivation of existing surface SERTs fails to eliminate NECA stimulation of SERT. Together, these results reveal two PKG-dependent pathways supporting rapid SERT regulation by A3 ARs, one leading to enhanced SERT surface trafficking, and a separate, p38 MAPK-dependent process augmenting SERT intrinsic activity.

Background: Serotonin (5-HT) neuronal systems have been implicated in the modulation of obsessive-compulsive disorder (OCD) symptoms. 5-HT3 receptor antagonists have been found to act as anxiolytics in selected animal models of anxiety; in particular, those involving an element of risk assessment. Since the compulsions of OCD are frequently triggered by an abnormal perception of risk, a pilot study was initiated to determine whether the 5-HT3 receptor antagonist ondansetron might have efficacy in the treatment of OCD., Method: Eight medication-free subjects with a DSM-IV diagnosis of OCD and a Yale-Brown Obsessive Compulsive Scale (YBOCS) score > or = 16 entered an 8-week open-label trial of ondansetron at a fixed dose of 1 mg 3 times daily in a study conducted between February and October 1998., Results: Six subjects completed the trial. Three subjects (37%) achieved a clinically significant response (> or = 35% reduction in YBOCS score). For these subjects, the average reduction in YBOCS-rated symptoms was 55%. In aggregate, the 8 patients exhibited a 28% reduction in YBOCS-rated symptoms over the course of the trial. The medication was well tolerated., Conclusion: These results suggest that low-dose ondansetron may have promise as a monotherapy for some patients suffering from OCD.

One-hundred ninety-one ligands were screened at 5-HT(3) receptors in membranes from rat brain and NCB20 cells for their ability to displace the selective, high-affinity 5-HT(3) receptor antagonist, [(125)I]DAIZAC ([(125)I]( S)-5-chloro-3-iodo-2-methoxy- N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide). Thirty-seven compounds having structures related to benzamide, dibenzepine, serotonin, phenylbiguanide, or arylpiperzine were selected for more extensive displacement studies in membranes from rat and mouse brains, from two cultured cell preparations expressing heteromeric mouse-derived 5-HT(3) receptor proteins (NCB20 and NG108-15 cell lines), and from recombinant Sf9 cells expressing homomeric 5-HT(3A) receptors. [(125)I]DAIZAC bound specifically to a single site in each of the five tissue preparations with high affinity ( K(D) 0.12-0.19 nM). The densities of [(125)I]DAIZAC-labeled 5-HT(3) receptors were 7.4-7.5 fmol/mg protein in membranes from murine brain, and 38, 99, and 1588 fmol/mg protein in membranes from cultured NCB20, NG108-15, and recombinant Sf9 cells, respectively. The affinity of substituted benzamides ( n=10) was similar in all five tissue preparations. The affinity of dibenzepines ( n=17) was significantly higher in membranes from cultured cells as compared to membranes from rat and mouse brain, but similar in the two brain membrane preparations, and in each of the cultured cell membrane preparations. Serotonin-, phenylbiguanide-, and quipazine-analogs ( n=10), which typically function as 5-HT (5-hydroxytryptamine) agonists, exhibited significantly higher apparent p K(i) values in membranes from rat brain and Sf9 recombinant cells than in membranes from the three preparations expressing heteromeric mouse-derived 5-HT(3) receptor proteins ( F=7.52, P<0.001). These findings confirm that there are both species and cell-type dependent differences in binding to 5-HT(3) receptors, and that care must be taken when comparing results between experimental paradigms that utilize different sources of 5-HT(3) receptors.

Behavioral effects of desamino-3-iodozacopride (DAIZAC) [(S)-5-chloro-3-iodo-2-methoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide], a selective high-affinity 5-HT(3) receptor antagonist (K(D) 0.14 nM), were evaluated in the mouse elevated plus-maze using the anxiolytic benzodiazepine, diazepam, as a positive control. DAIZAC treatment produced a significant dose-related increase in the time spent in the open arm. The increased total time in the open arm resulted from a significant dose-dependent increase in the number of entries into that arm. The minimum dose of DAIZAC associated with a statistically significant increase in entries and time spent in the open arm was 0.05 mg/kg ip, consistent with its high affinity for the 5-HT(3) receptor. DAIZAC did not affect the amount of time spent in the open arm after each entry. Thus, DAIZAC reduced apparent avoidance of the open arm when the animal was in the central compartment, without affecting active avoidance of that arm when the animal was in the exposed condition. The increase in the open-arm entries was accompanied by a corresponding reduction in the number of entries into the closed arm with a consequent reduction in the time spent in the closed arm. The time spent in the closed arm after each entry was not altered by DAIZAC administration. As such, the sole apparent effect of DAIZAC was to alter the choice of arm to enter when the animal was in the central compartment. Diazepam also significantly increased total time in the open arm; however, the increase was not attributable to a single behavioral factor. The anxiolytic-like effects of DAIZAC reached maximum by 20-30 min and returned to baseline levels by 90 min. Ex vivo binding studies found that levels of DAIZAC-like activity assayed in brains of mice 25 min after DAIZAC injection were significantly correlated with the behavioral parameters associated with anxiolysis. These results indicate that DAIZAC produces dose-dependent anxiolytic-like behavioral changes in the mouse elevated plus-maze that are correlated with brain DAIZAC-like activity.

The 5-hydroxytryptamine(HT)3 receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2. 2]oct-3-yl)benzamide] is a compound with high affinity and selectivity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB-20 cell membranes gave a Bmax of 340 +/- 58 fmol/mg protein and a KD of 0.14 +/- 0.03 nM, which is in agreement with the value previously reported in rat brain (KD = 0.15 nM). Nonspecific binding of [125I]DAIZAC in NCB-20 cells was <1% of total binding at the KD for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 microM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT3 receptor over the M1 muscarinic binding site, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3-30 microM) were ineffective in displacing [125I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3-30 microM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [125I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation current in NCB-20 cells with an IC50 of 0.24 nM. [125I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT3 receptor.

In an effort to develop selective, high-affinity radioligands for the 5-HT-3 receptor, a series of homologues of 5-chloro-2,3-dimethoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide (2b) was prepared in which individual methoxy groups were replaced by ethoxyl, (2-fluoroethoxyl), allyloxyl, propargyloxyl, or (3-iodoallyl)oxyl groups. Affinities for the 5-HT-3 receptor were determined by displacement of the binding of [125I]MIZAC (2a), a selective 5-HT-3 receptor antagonist radioligand, in rat brain homogenates. The 3-substituted homologues were more potent than the lead compound, 2b. The homologue having the largest 3-substituent, i.e., E-(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chloro-3-(3-iodo-2-propenyl)oxy- 2-methoxybenzamide (3b, THIZAC), had one of the highest affinities, Ki 0.08 nM. The 2-substituted homologues were equipotent with 2b, having Ki 0.2-0.3 nM, regardless of the size of the substituent. The corresponding iodoallyl derivative, E-(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chloro-2-(3-iodo-2-propenyl)oxy- 3-methoxybenzamide (4, LIZAC), displayed a Ki of 0.29 nM. Saturation binding of [125I]-4 gave a KD of 0.31 +/- 0.04 nM and a Bmax of 2.36 +/- 0.10 fmol/mg of entorhinal cortex. In vivo biodistribution of [125I]-4 in the rat brain showed increased accumulation in hippocampus relative to that in cerebellum. Both the high-affinity ligands [125I]-3b and [125I]-4 are potentially useful radioligands for studying the 5-HT-3 receptor.

1. Antagonists at 5-HT3 receptors have shown activity in animal models of mental illness, however, few radiolabeled 5-HT3 ligands are available for preclinical studies. MIZAC, an analogue of the selective 5-HT3 antagonist, zacopride, binds with high affinity (1.3-1.5 nM) to CNS 5-HT3 sites. The authors report here the selectivity of MIZAC for these sites in rat brain homogenates. 2. Ninety-seven percent of total specific binding of [125I]MIZAC (0.1 nM) of was displaced by bemesetron (3 microM), a selective 5-HT3 antagonist. Competition studies using ligands with known affinities for 5-HT3 sites give a high correlation with reported pKi values (r2 0.98). Bemesetron displaceable binding has a regional distribution consistent with that of the 5-HT3 receptor, i.e. highest in cortex and hippocampus, and lowest in striatum and cerebellum. 3. Potent antagonists present at concentrations sufficient to occupy 95% of other 5-HT receptor populations (1A, 1B, 1D, 2A, 2B, 2C, 5A, 5B, 6, and 7) showed minimal ability to displace [125I]MIZAC binding (3 nM). Specificity studies using radioligand binding assays selective for 5-HT4, 5-HT6, and 5-HT7 receptors, and for binding sites of other neurotransmitters indicate a high degree of selectivity of [125I]MIZAC for the 5-HT3 receptor. 4. [125I]MIZAC binds to an apparent low affinity (benzac) site having a unique pharmacology. Low affinity binding was displaceable by benztropine, but not by other muscarinic agents nor inhibitors of dopamine uptake. The regional distribution of the low affinity site differed markedly from that of the high affinity site. The apparent affinity of [125I]MIZAC for the benzac site is two orders of magnitude lower than for the 5-HT3 receptor. Given its high selectivity for 5-HT3 binding sites, [125I]MIZAC appears to be a promising ligand for labeling 5-HT3 receptors in vitro and in vivo.

We report an improved synthesis of unlabeled (S)-iodozacopride, the radiolabeling of (S)-[125I]iodozacopride via deschloro-(S)-zacopride, and a re-evaluation of its affinity for the 5-HT3 receptor. Unlabeled (S)-iodozacopride was prepared in seven steps from 4-aminosalicylic acid via alkaline hydrolysis of its 4-acetamide derivative. Catalytic hydrogenation of (S)-iodozacopride gave deschloro-(S)-zacopride, identical to that obtained from (S)-3-amino-quinuclidine and 4-amino-2-methoxybenzoic acid via its corresponding 1-imidazole derivative. Radioiodination to produce (S)-[125I]iodozacopride was accomplished by treatment of deschloro-(S)-zacopride with 5 mCi sodium 125iodide and chloramine-T in hydrochloric acid. Purification of the reaction products using an HPLC system capable of detecting chlorinated side-products revealed a mixture of 2.1 mCi (1.3 nmol) (S)-[125I]iodozacopride and (S)-zacopride (1.5 nmol). Saturation analysis of the binding of the purified (S)-[125I]iodozacopride to whole rat brain homogenates gave an estimated KD of 1.10 +/- 0.07 nM. As anticipated, this is approximately half the KD reported for binding of racemic [125I]iodozacopride, and differs from the previously reported value by an order of magnitude. Analysis of the apparent binding affinity of a 1:1 mixture of (S)-[125I]iodozacopride and (S)-zacopride suggests that the previous result may have been confounded by contamination of the product with unlabeled (S)-zacopride. Competition analysis of the displacement of (S)-[125I]iodozacopride binding by unlabeled (S)-iodozacopride and (S)-zacopride gave Ki values of 0.95 and 0.21 nM, respectively.

Serotonergic reuptake inhibitors have been the primary medications for treatment of obsessive-compulsive disorder (OCD); however, other serotonergic and alpha 2-adrenergic medications also have been reported to reduce obsessive-compulsive symptoms. In this study, we compare three medications with reported efficacy in OCD to a control medication, diphenhydramine, a medication without theoretical or demonstrated treatment benefit. The three active medications were clomipramine, a serotonergic reuptake inhibitor; clonazepam, a benezodiazepine with putative serotonergic properties; and clonidine, an alpha 2-adrenergic agonist. Twenty-eight subjects with DSM-III-R diagnosis of OCD rotated through 6-week trials of each of the four medications in a randomized, double-blind, multiple crossover protocol. Clomipramine and clonazepam were both effective relative to the control medication in reducing OCD symptoms. There was a significant cross-response between these two medications; however 40% of subjects failing clomipramine trials had a clinically significant response to clonazepam treatment. The control medication, diphenhydramine, itself produced a significant decrement in symptoms, whereas clonidine was ineffective in reducing OCD symptoms. Clonazepam improvement was unrelated to changes in anxiety and occurred early in treatment. Clonazepam was significantly more effective than the other medications during the first 3 weeks of treatment. The results confirm the efficacy of clomipramine in the treatment of OCD and suggest that clonazepam might be a useful alternative treatment for patients with this disorder.

The success of serotonergic reuptake inhibitors in the treatment of obsessive-compulsive disorder (OCD) has suggested that serotonergic neurotransmission may play a role in the pathogenisis of this disorder. Prolactin responses to a 60-mg oral dose of fenfluramine in 26 medication-free patients with a DSM-III-R diagnosis of OCD were compared with those of 20 controls subjects. Fenfluramine produced a significant elevation of prolactin levels in both OCD patients and controls. Prolactin responses were significantly blunted in OCD patients compared with responses in control subjects. Female subjects in both groups showed greater prolactin responses to fenfluramine than did their male counterparts. There was a significant interaction between sex and the presence of OCD such that female patients had lower prolactin responses than their controls, while the difference between male patients and controls was not significant. Prolactin responses were not correlated with age, weight, drug level, depression, anxiety, or degree of OCD symptoms. The results are consistent with a relative reduction in serotonergic efficacy in the setting of OCD.

Obsessive compulsive disorder has recently been successfully treated with antidepressants that selectively inhibit serotonin reuptake, and a serotonergic hypothesis related to the etiology and treatment of obsessive compulsive disorder has been proposed. Clonazepam, a novel benzodiazepine, uniquely affects serotonergic neurotransmission. It has been employed in the treatment of other neuropsychiatric syndromes that respond to serotonergic medications. Three patients with obsessive compulsive disorder who were treated with clonazepam for periods of up to 1 year experienced substantial improvement in their symptoms. Clonazepam had a rapid onset of antiobsessive action with accompanying decreases in both depression and anxiety. One patient showed reductions in obsessions and compulsions that were equivalent to or greater than those seen during previous treatment with clomipramine: Clonazepam may be a useful alternative to serotonergic antidepressants in patients who cannot tolerate the toxic effects of these medications.

Opiate binding sites in five brain regions were labeled with the mu and delta markers, 3H-morphine and 3H-[D-Ala2,D-leu5]enkephalin, respectively. The highest densities of both 3H-morphine and 3H-DADLE labeled sites are found in striatum and frontal cortex. Hypothalamus and midbrain contain predominantly 3H-morphine labeled sites. The selectivity of the opioid peptides [D-Ala2,D-leu5]enkephalin, beta-endorphin and dynorphin(1-13) for the two opiate sites was investigated by comparing the potency of these unlabeled compounds against the mu and delta markers in different brain regions. This determination has the effect of controlling for the breakdown of peptides within each region. While the enkephalin analogue shows a preference for the delta binding site and beta-endorphin is more nearly equipotent towards the two binding sites, dynorphin(1-13) shows a high affinity and selective preference for the mu binding site over the delta site. The potency of the opioid peptides in displacing the mu and delta markers varies from region to region according to the relative densities of the two opiate binding site populations.

The biological activity of many proteins and peptides can be profoundly affected by enzyme-catalyzed covalent modifications such as acetylation, sulfation, glycosylation, or amidation. This article describes the cloning of such an enzyme, a peptide acetyltransferase from rat brain that catalyzes the amino-terminal acetylation of endorphins and perhaps other substrates in vivo. Blot-hybridization analysis suggests that the mRNA encoding the acetyltransferase is approximately 2.0 kilobases, is present in whole rat brain and rat hypothalamus, and is slightly larger in mouse AtT20 tumor cells. The acetyltransferase was cloned by using a strategy whereby a cDNA expression library was screened with a solid-phase enzyme-activity assay; this technique combines the use of the substrate coupled to a solid support and subsequent recognition of the product by using a specific antiserum. We have called this method the enzyme immunodetection assay (EIDA). The EIDA should prove useful in the isolation of other clones for proteins that possess enzymatic activity upon expression in bacterial hosts.

The binding of tritiated beta-endorphin (3H-beta-EP) to brain homogenates is described. This has been difficult to achieve due to the lack of availability of 3H-beta-EP and to technical difficulties associated with high non-specific binding of beta-EP. We now report that 3H-beta-EP binding is saturable, stereospecific, has high affinity and is inhibited by sodium. Its dissociation rate is ten-fold longer than that of naloxone. Its regional distribution exhibits interesting differences from naloxone and enkephalin binding. ACTH1-24 appears to displace it more effectively than it displaces 3H-naloxone. The results are discussed in terms of multiple transmitter systems and the multiple opiate receptor hypothesis.