Presence Of Uptake Inhibitors Research Articles

Cationic cholesterol derivative efficiently delivers the genes: in silico and in vitro studies.

The aims of the research work were to synthesize ethyl(cholesteryl carbamoyl)-L-arginate (ECCA), an arginine-conjugated cholesterol derivative, and to evaluate its application as a gene delivery vector. The interactions of ECCA with DNA duplex were studied using molecular dynamics (MD) simulations. It was found that the guanidine group of ECCA could interact with the phosphate group of DNA through ionic interactions as well as hydrogen bonds. The structure of DNA was stable throughout the simulation time. Liposomes were formulated using ECCA and soya phosphatidylcholine (SPC) by a thin-film hydration method. They had the particle size of ~ 150nm and the zeta potential of + 51mV. To ensure the efficient binding of DNA to the liposomes, the ratio of DNA to ECCA was optimized using gel retardation assay. Further, serum stability, haemolysis and cytotoxicity studies were carried out to determine the stability and safety of the lipoplexes. Circular dichroism spectroscopy was used to determine the interaction of DNA and cationic liposomes. Cellular uptake pathway was determined by studying the uptake of coumarin-loaded lipoplexes at 4°C and in the presence of uptake inhibitors, i.e. genistein, chlorpromazine and methyl-β-cyclodextrin. Transfection studies were carried out to evaluate the transfection efficacy of the ECCA-loaded lipoplexes. The binding of DNA and lipoplexes was found to be stable in the presence of serum, and no degradation of DNA was observed. The lipoplexes showed low haemolysis and cytotoxicity. The uptake of coumarin-loaded liposomes was decreased up to ~ 20% in the presence of clathrin- and caveola-mediated uptake inhibitors, indicating a role of both the pathways in the uptake of the inhibitors. Satisfactory transfection efficiency was obtained compared to Lipofectamine®. Thus, cationic cholesterol derivative is a useful tool for gene delivery vector.

The presence of a serotonin uptake inhibitor alters pharmacological manipulations of serotonin release

The present study investigated the effects of the presence of the serotonin uptake inhibitor citalopram in the perfusion medium on pharmacological manipulations which increased and decreased striatal serotonin release using in vivo microdialysis. A high performance liquid chromatography detection system equipped with a microbore column was used which reduced the detection limit to 0.5 fmol serotonin/5 μl sample and enabled basal striatal serotonin release to be measured without the addition of a serotonin uptake inhibitor to the perfusion medium. Although serotonin uptake inhibitors have frequently been used to enhance the serotonin content of dialysate samples, the effects of the presence of serotonin uptake inhibitors on pharmacological manipulations which increased and decreased the release of serotonin have not yet been characterized. Serotonin release was reduced by the systemic administration of the 5-HT IA receptor agonist 8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT). Although 5-HT release was reduced by 8-OH-DPAT after the addition of citalopram, the 5-HT IA receptor agonist did not reduce absolute levels of extracellular serotonin below basal values of serotonin measured in the absence of citalopram. In addition, citalopram dramatically prevented the four-fold increase in the release of serotonin produced by the systemic administration of the serotonin-releasing agent fenfluramine. The blockade of fenfluramine's effects by citalopram supports the hypothesis that transport of fenfluramine into serotonergic neurons is necessary to increase serotonin release. This study demonstrates that the use of an HPLC detection system equipped with a microbore column can reliably measure basal serotonin release using in vivo microdialysis. Evaluation of the effects of the addition of citalopram to the perfusion medium suggests that the response to pharmacological manipulations of serotonin release may be substantially altered by the presence of an uptake inhibitor.

Serotonin uptake blockers influence serotonin autoreceptors by increasing the biophase concentration of serotonin and not through a ?molecular link?

The mechanism of the attenuation, by serotonin uptake blockers, of the release-inhibiting effect of exogenous serotonin autoreceptor agonists was studied in rabbit brain cortex and rat hypothalamus slices. The slices were preincubated with 3H-serotonin and then superfused and stimulated electrically. In rabbit brain slices stimulated by trains of 4 pulses at 100 Hz, 5-carboxamidotryptamine and 5-methoxytryptamine reduced the evoked overflow of tritium, and their concentration-response curves were not changed by any of three serotonin uptake inhibitors, namely citalopram, fluvoxamine and 6-nitroquipazine. In contrast, when the slices were stimulated by trains of 10 pulses at 0.033 Hz, fluvoxamine shifted the concentration-response curve of 5-methoxytryptamine to the right. Experiments with the autoreceptor antagonist metitepine indicated that little, if any, endogenous autoinhibitory tone developed in the course of trains of 4 pulses/100 Hz, irrespective of the absence or presence of uptake inhibitors, as well as during trains of 10 pulses/0.033 Hz in the absence of uptake inhibitors, whereas marked autoinhibition developed when 10 pulses/0.033 Hz were applied in the presence of fluvoxamine. In rat hypothalamic slices stimulated by trains of 4 pulses at 100 Hz, citalopram also failed to change the concentration-response curve of 5-methoxytryptamine. These results indicate that serotonin uptake blockers attenuate the effect of exogenous autoreceptor agonists by an increase in the biophase concentration of released serotonin and, hence, in endogenous autoinhibitory tone, and not by some direct "molecular link" unrelated to the biophase concentration of released serotonin.

Sodium-dependence of the potency of inhibitors of the neuronal noradrenaline carrier in the rat vas deferens.

Vasa deferentia obtained from reserpine-pretreated rats were incubated (monoamine oxidase and catechol-O-methyltransferase inhibited) in media containing various concentrations of 3H-(-)noradrenaline and Na+ and initial rates of the neuronal uptake of 3H-noradrenaline measured both in the absence and presence of uptake inhibitors after 1 min of incubation. When rates of uptake were determined at various 3H-noradrenaline (1.0-12.2 mumol/l) and two fixed Na+ concentrations (25 and 140 mmol/l), the inhibition of uptake produced by (+)amphetamine, (-)metaraminol, desipramine, nomifensine and cocaine was competitive with respect to 3H-noradrenaline at both Na+ concentrations. While the Ki for (+)amphetamine, (-)metaraminol desipramine and nomifensine increased when the Na+ concentration was lowered, that for cocaine decreased. When the Na+ concentration was varied (10-140 mmol/l) and the 3H-noradrenaline concentration held constant (1.2 mumol/l), (+)amphetamine, (-)metaraminol, nomifensine and desipramine acted as mixed-type inhibitors with respect to Na+, and the inhibition of uptake produced by these drugs was the more pronounced, the higher the Na+ concentration. On the other hand, cocaine was competitive with Na+ and the inhibition produced by this drug was the more pronounced, the lower the Na+ concentration. It is concluded that the inhibitors of neuronal uptake tested here act in dependence on the external Na+ concentration. Desipramine and nomifensine resemble alternative amine substrates in being more potent at high than at low Na+ concentrations. On the other hand, cocaine is more potent at low than at high Na+ concentrations.

Tryptamine, a substrate for the serotonin transporter in human platelets, modifies the dissociation kinetics of [3H]imipramine binding: possible allosteric interaction.

Tricyclic antidepressants and nontricyclic serotonin (5-hydroxytryptamine) uptake blockers monophasically inhibit [3H]imipramine binding in human platelets. Similarly, serotonin and tryptamine inhibit the binding of [3H]imipramine in the low micromolar range and with a pseudo-Hill coefficient near unity. Dissociation of the [3H]imipramine receptor complex in the presence of uptake inhibitors follows first-order kinetics with a half-life of approximately 60 min. Although serotonin and tryptamine do not decrease [3H]imipramine binding when added under equilibrium conditions, simultaneous addition of serotonin or tryptamine with serotonin uptake inhibitors decreases the rate of ligand-receptor dissociation in a concentration-dependent manner. These data suggest a common site of action for serotonin, which is the substrate of the transporter system, and of tryptamine, its nonhydroxylated analog. This hypothesis is supported by the identification of a high-affinity (Km = 0.55 microM), saturable, and temperature-dependent uptake of [3H]tryptamine in human platelets. Uptake of [3H]tryptamine was inhibited potently by imipramine and nontricyclic serotonin uptake inhibitors with a potency similar to that observed for [3H]serotonin uptake. These data support the hypothesis that in platelets, [3H]imipramine, tricyclic, and nontricyclic serotonin uptake inhibitors bind to a common recognition site that is associated with the serotonin transporter but that differs from the substrate recognition site of the carrier through which serotonin and tryptamine exert a heterotropic allosteric modulation on [3H]imipramine binding.

The metabolic pattern of spontaneously released [3H]norepinephrine in the presence of uptake inhibitors.

The effect of uptake inhibitors on the spontaneous release of [3H]norepinephrine was studied using rat isolated vas deferens. Cocaine, desmethylimipramine, phenoxybenzamine, and guanethidine caused an increase in the spontaneous outflow of tritium, but did not alter the metabolic pattern of released [3H]norepinephrine. Bretylium caused an increase of both total tritium outflow and norepinephrine content. It is suggested that these drugs caused release of norepinephrine from storage sites and that norepinephrine was metabolized within the nerve terminals before reaching the biophase.