Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells. Relationship with the mitogenic action of serotonin.
-The increased delivery of serotonin (5-hydroxytryptamine, 5-HT) to the lung aggravates the development of hypoxia-induced pulmonary hypertension in rats, possibly through stimulation of the proliferation of pulmonary artery smooth muscle cells (PA-SMCs). In cultured rat PA-SMCs, 5-HT (10(-8) to 10(-6) mol/L) induced DNA synthesis and potentiated the mitogenic effect of platelet-derived growth factor-BB (10 ng/mL). This effect was dependent on the 5-HT transporter (5-HTT), since it was prevented by the 5-HTT inhibitors fluoxetine (10(-6) mol/L) and paroxetine (10(-7) mol/L), but it was unaltered by ketanserin (10(-6) mol/L), a 5-HT2A receptor antagonist. In PA-SMCs exposed to hypoxia, the levels of 5-HTT mRNA (measured by competitive reverse transcriptase-polymerase chain reaction) increased by 240% within 2 hours, followed by a 3-fold increase in the uptake of [3H]5-HT at 24 hours. Cotransfection of the cells with a construct of human 5-HTT promoter-luciferase gene reporter and of pCMV-beta-galactosidase gene allowed the demonstration that exposure of cells to hypoxia produced a 5.5-fold increase in luciferase activity, with no change in beta-galactosidase activity. The increased expression of 5-HTT in hypoxic cells was associated with a greater mitogenic response to 5-HT (10(-8) to 10(-6) mol/L) in the absence as well as in the presence of platelet-derived growth factor-BB. 5-HTT expression assessed by quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization in the lungs was found to predominate in the media of pulmonary artery, in which a marked increase was noted in rats that had been exposed to hypoxia for 15 days. These data show that in vitro and in vivo exposure to hypoxia induces, via a transcriptional mechanism, 5-HTT expression in PA-SMCs, and that this effect contributes to the stimulatory action of 5-HT on PA-SMC proliferation. In vivo expression of 5-HTT by PA-SMC may play a key role in serotonin-mediated pulmonary vascular remodeling. (+info)
Nongenomic steroidal modulation of high-affinity serotonin transport.
The ability of steroids to modulate high-affinity 5-HT transport was investigated using cell-based models which stably manifest all known properties of this transport system. beta-Estradiol (E2) exhibited noncompetitive, and possibly allosteric, inhibition of both radiolabeled serotonin ([3H]5-HT) transport by, and radiolabeled cocaine congener ([3H]CFT) binding to, this system. Such inhibitory effects were observed within short time courses and unlikely to result from genomic effects normally ascribed to estrogen action. Rather, such nongenomic effects on 5-HT uptake were more akin to modulatory effects of select steroid metabolites on other plasma membrane systems such as neurotransmitter receptors and ionic channels. Beyond E2, preliminary examination of other steroid metabolites and synthetic steroid receptor agonists/antagonists revealed that inhibition of 5-HT transport is additionally attributable only to estriol (E3, an E2 metabolite) and tamoxifen (a nonsteroidal, E2 receptor antagonist). These findings indicate that the present form of transport modulation is only rendered by select compounds and not a general property of steroidal and related agents. Assessments of covalent conjugates of E2 suggested that E2 interacts with the transporter protein at allosteric site(s) inaccessible from the extracellular domain. These findings collectively suggest that steroid-mediated regulation of 5-HT transport may be a physiologically relevant mechanism, and that antidepressant as well as psychostimulant effects in vivo may contain a steroidal component. (+info)
Modeling geriatric depression in animals: biochemical and behavioral effects of olfactory bulbectomy in young versus aged rats.
Geriatric depression exhibits biological and therapeutic differences relative to early-onset depression. We studied olfactory bulbectomy (OBX), a paradigm that shares major features of human depression, in young versus aged rats to determine mechanisms underlying these differences. Young OBX rats showed locomotor hyperactivity and a loss of passive avoidance and tactile startle. In contrast, aged OBX animals maintained avoidance and startle responses but showed greater locomotor stimulation; the aged group also exhibited decreased grooming and suppressed feeding with novel presentation of chocolate milk, effects which were not seen in young OBX. These behavioral contrasts were accompanied by greater atrophy of the frontal/parietal cortex and midbrain in aged OBX. Serotonin transporter sites were increased in the cortex and hippocampus of young OBX rats, but were decreased in the aged OBX group. Cell signaling cascades also showed age-dependent effects, with increased adenylyl cyclase responses to monoaminergic stimulation in young OBX but no change or a decrease in aged OBX. These data indicate that there are biological distinctions in effects of OBX in young and aged animals, which, if present in geriatric depression, provide a mechanistic basis for differences in biological markers and drug responses. OBX may provide a useful animal model with which to test therapeutic interventions for geriatric depression. (+info)
Characteristics of drug interactions with recombinant biogenic amine transporters expressed in the same cell type.
We characterized the effects of drugs on the uptake of [3H]neurotransmitter by and the binding of [125I](3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester ([125I]RTI-55) to the recombinant human dopamine (hDAT), serotonin (hSERT), or norepinephrine (hNET) transporters stably expressed in human embryonic kidney 293 cells. RTI-55 had similar affinity for the hDAT and hSERT and lower affinity for hNET (Kd = 1. 83, 0.98, and 12.1 nM, respectively). Kinetic analysis of [125I]RTI-55 binding indicated that the dissociation rate (k-1) was significantly lower for hSERT and the association rate (k+1) was significantly lower for hNET compared with the hDAT. The potency of drugs at blocking [3H]neurotransmitter uptake was highly correlated with potency at blocking radioligand binding for hDAT and hSERT. Substrates were more potent at the inhibition of [3H]neurotransmitter uptake than radioligand binding. The potency of drugs was highly correlated between displacement of [3H]nisoxetine (Kd = 6.0 nM) and [125I]RTI-55 from the hNET, suggesting that these radioligands recognize similar sites on the transporter protein. The correlation observed between inhibitory potency for uptake and binding of either ligand at the hNET was lower than correlations between uptake and binding for hDAT and hSERT. The present results indicate that the cocaine analog [125I]RTI-55 has unique binding properties at each of the transporters and that the use of recombinant transporters expressed by a single cell type can provide a powerful screening tool for drugs interacting with biogenic amine transporters, such as possible cocaine antagonists. (+info)
Molecular chaperones stimulate the functional expression of the cocaine-sensitive serotonin transporter.
The serotonin transporter (SERT) is an N-glycosylated integral membrane protein that is predicted to contain 12 transmembrane regions. SERT is the major binding site in the brain for antidepressant drugs, and it also binds amphetamines and cocaine. The ability of various molecular chaperones to interact with a tagged version of SERT (Myc-SERT) was investigated using the baculovirus expression system. Overexpression of Myc-SERT using the baculovirus system led to substantial quantities of inactive transporter, together with small amounts of fully active and, therefore, correctly folded molecules. The high levels of inactive Myc-SERT probably arose because folding was rate-limiting due, perhaps, to insufficient molecular chaperones. Therefore, Myc-SERT was co-expressed with the endoplasmic reticulum (ER) molecular chaperones calnexin, calreticulin and immunoglobulin heavy chain binding protein (BiP), and the foldase, ERp57. The expression of functional Myc-SERT, as determined by an inhibitor binding assay, was enhanced nearly 3-fold by co-expressing calnexin, and to a lesser degree on co-expression of calreticulin and BiP. Co-expression of ERp57 did not increase the functional expression of Myc-SERT. A physical interaction between Myc-SERT-calnexin and Myc-SERT-calreticulin was demonstrated by co-immunoprecipitation. These associations were inhibited in vivo by deoxynojirimycin, an inhibitor of N-glycan precusor trimming that is known to prevent the calnexin/calreticulin-N-glycan interaction. Functional expression of the unglycosylated SERT mutant, SERT-QQ, was also increased on co-expression of calnexin, suggesting that the interaction between calnexin and SERT is not entirely dictated by the N-glycan. SERT is the first member of the neurotransmitter transporter family whose folding has been shown to be assisted by the molecular chaperones calnexin, calreticulin, and BiP. (+info)
Transmembrane domain I contributes to the permeation pathway for serotonin and ions in the serotonin transporter.
Mutation of a conserved Asp (D98) in the rat serotonin (5HT) transporter (rSERT) to Glu (D98E) led to decreased 5HT transport capacity, diminished coupling to extracellular Na+ and Cl-, and a selective loss of antagonist potencies (cocaine, imipramine, and citalopram but not paroxetine or mazindol) with no change in 5HT Km value. D98E, which extends the acidic side chain by one carbon, affected the rank-order potency of substrate analogs for inhibition of 5HT transport, selectively increasing the potency of two analogs with shorter alkylamine side chains, gramine, and dihydroxybenzylamine. D98E also increased the efficacy of gramine relative to 5HT for inducing substrate-activated currents in Xenopus laevis oocytes, but these currents were noticeably dependent on extracellular medium acidification. I-V profiles for substrate-independent and -dependent currents indicated that the mutation selectively impacts ion permeation coupled to 5HT occupancy. The ability of the D98E mutant to modulate selective aspects of substrate recognition, to perturb ion dependence as well as modify substrate-induced currents, suggests that transmembrane domain I plays a critical role in defining the permeation pathway of biogenic amine transporters. (+info)
Phosphorylation and sequestration of serotonin transporters differentially modulated by psychostimulants.
Many psychotropic drugs interfere with the reuptake of dopamine, norepinephrine, and serotonin. Transport capacity is regulated by kinase-linked pathways, particularly those involving protein kinase C (PKC), resulting in transporter phosphorylation and sequestration. Phosphorylation and sequestration of the serotonin transporter (SERT) were substantially impacted by ligand occupancy. Ligands that can permeate the transporter, such as serotonin or the amphetamines, prevented PKC-dependent SERT phosphorylation. Nontransported SERT antagonists such as cocaine and antidepressants were permissive for SERT phosphorylation but blocked serotonin effects. PKC-dependent SERT sequestration was also blocked by serotonin. These findings reveal activity-dependent modulation of neurotransmitter reuptake and identify previously unknown consequences of amphetamine, cocaine, and antidepressant action. (+info)
Excess of serotonin (5-HT) alters the segregation of ispilateral and contralateral retinal projections in monoamine oxidase A knock-out mice: possible role of 5-HT uptake in retinal ganglion cells during development.
Retinal ganglion cell (RGCs) project to the ipsilateral and contralateral sides of the brain in the dorsal lateral geniculate nucleus (dLGN) and the superior colliculus (SC). Projections from both eyes are initially intermingled until postnatal day 3 (P3) but segregate into eye-specific layers by P8. We report that this segregation does not occur in monoamine oxidase A knock-out mice (MAOA-KO) that have elevated brain levels of serotonin (5-HT) and noradrenaline. The abnormal development of retinal projections can be reversed by inhibiting 5-HT synthesis from P0 to P15. We found that in MAOA-KO mice, 5-HT accumulates in a subpopulation of RGCs and axons during embryonic and early postnatal development. The RGCs do not synthesize 5-HT but reuptake the amine from the extracellular space. In both MAOA-KO and normal mice, high-affinity uptake of 5-HT and serotonin transporter (SERT) immunoreactivity are observed in retinal axons from the optic cup to retinal terminal fields in the SC and dLGN. In the dLGN, transient SERT labeling corresponds predominantly to the ipsilateral retinal projection fields. We show that, in addition to SERT, developing RGCs also transiently express the vesicular monoamine transporter gene VMAT2: thus, retinal axons could store 5-HT in synaptic vesicles and possibly use it as a borrowed neurotransmitter. Finally we show that the 5-HT-1B receptor gene is expressed by RGCs throughout the retina from E15 until adult life. Activation of this receptor is known, from previous studies, to reduce retinotectal activity; thus 5-HT in excess could inhibit activity-dependent segregation mechanisms. A hypothesis is proposed whereby, during normal development, localized SERT expression could confer specific neurotransmission properties on a subset of RGCs and could be important in the fine-tuning of retinal projections. (+info)