Substrate-mediated regulation of gamma-aminobutyric acid transporter 1 in rat brain

The uptake of neurotransmitter by plasma membrane transporters is a principal method for regulating extracellular transmitter levels. Neurotransmitter-mediated signals in turn are able to regulate transporter expression and function. Thus, there is a continual interplay between transporters and the transmitters they transport. Previously we showed that extracellular gamma-aminobutyric acid (GABA) increases the expression of the GABA transporter 1 (GAT1) on a time scale of minutes by acting via the transporter to slow transporter internalization. This mechanism requires in part direct tyrosine phosphorylation of the transporter. In the present study we show that the presence of GABA on a longer time scale causes a net decrease in GAT surface expression. The decrease in expression represents the contributions of transporter-mediated up-regulation and a more substantial GABA-receptor-mediated down-regulation. This receptor-mediated down-regulation is the result of both changes in the rates of transporter trafficking and in the number of transporters available for trafficking. As with transporter-mediated regulation of GAT1, the receptor-mediated regulation is associated with changes in the direct phosphorylation of GAT1. These data suggest that multiple pathways, perhaps converging upon mechanisms involving protein phosphorylation, act to regulate GAT1 expression in neurons.     

纳米粒摄取机制的研究进展

随着纳米技术的发展,纳米材料在药物传输领域有着潜在的应用空间.研究细胞对纳米粒的细胞摄取机制,有助于从细胞层次上理解生命体的生理过程和药物的作用机制,掌握细胞治疗的机理;同时也可为构建更加安全有效的纳米药物载体提供依据.综述了纳米粒摄取机制的最新研究进展,在简要介绍纳米粒内吞途径的基础上着重讨论了影响内吞的因素,同时详细介绍了纳米粒内吞途径的常用研究方法.     

Study Protocol for the Most Effective Recall Method in a Cervical Cancer Screening Program in Klang,Malaysia

Background: Cervical cancer is the second most common cancer among Malaysian women with an ASR of17.9 and a mortality rate of 5.6 per 100,000 population in 2008 (GLOBOCAN, 2008). The 5 year prevalence wasestimated to be 14.5 per 100,000 population. As the second most common cancer affecting productive females,cervical cancer imposes an impact to the socioeconomic aspect of the country. However, the poor uptake ofcervical cancer screening is a major problem in detecting early pre-cancerous lesions and thus, delay in initiatingtreatment for cervical cancer. Realizing the urgency to increase the uptake of PAP smear, besides enhancing thepromotion of PAP smear screening for women above 35 years old, the call-recall system for pap smear screeninghad been piloted in one of the suburban districts which aimed to improve regular participation of women forcervical and breast cancer screening. This is of public health importance as identifying the best feasible optionto increase patient’s respond to participate in the screening program effectively in our setting will be helpfulin implementing an organized regular population based screening program tailored to our setting. The pilotprogram of cervical cancer screening in Klang was an opportunity to assess different options in recalling patientsfor a repeat pap smear to increase their participation and adherence to the program. Methods and Results: Thiswas a population based randomized control trial. Women aged 20-65 years in the population that matched theinclusion and exclusion criteria were re-called for a repeat smear. There are four different intervention groups;letter, registered letters, short messages services (SMS) and phone calls where 250 subjects were recruited intoeach group. Samples were generated randomly from the same population in Klang into four different groups.The first group received a recall letter for a repeat smear similar to the one that has been given during the firstinvitation. The intervention groups were either be given a registered letter, an SMS or a phone call to re-callthem. The socio-demographic data of the patients who came for uptake were collected for further analysis. Allthe groups were followed up after 8 weeks to assess their compliance to the recall. Conclusions: The study willprovide recommendations about the most effective methods for recall in a population based pap smear screeningprogram on two outcomes: i) patients response; ii) uptake for repeat pap smear.     

A rapid screening system to determine drug affinities for the intestinal dipeptide transporter 1: system characterisation

Purpose: To establish an in vitro system for the rapid assessment of the affinities of potential substrates for the di/tri/oligopeptide transport system (DTS). Methods: Monolayers of Caco-2 cells were cultured in plastic wells for 7–9 days and the uptake of Gly-[³H]L-Pro, a specific and relatively stable substrate for the DTS was used as an affinity probe. Gly-[³H]L-Pro (50 nM), together with excess L-Pro (10 mM), to suppress uptake of any [³H]L-Pro produced by degradation of the probe, was incubated with the test compound (usually 1 mM) at pH 6 for 3 min. The uptake of radiolabel was determined by liquid scintillation counting. Results: High specific-uptake (>85%) of Gly-[³H]L-Pro was obtained with cells grown for 7–9 days. Gly-[³H]L-Pro uptake had a substantial active concentration-dependent component (K_m of 0.39±0.02 mM, V_max of 0.98±0.04 nmol min⁻¹ (mg protein)⁻¹. This process was shown to be specific for the DTS as evidenced by the significant inhibition by compounds reported to be transported by this system and the lack of inhibition by amino acids. The use of low competitor concentrations (1 mM) enabled a range of inhibition values (0–89%) of a series of competitors (amino acids, dipeptides and β-lactam antibiotics) to be estimated, illustrating that structurally similar compounds can be ranked for affinity to the DTS. Conclusion: A screening system, using Caco-2 cells and the dipeptide Gly-[³H]L-Pro as a displaceable probe, was developed to assess a variety of compounds for recognition by the di/tri/oligopeptide transport system. This fully describes the first system that allows structurally related compounds to be ranked on the basis of their affinity for the DTS recognition site.     

Inhibition of uptake1 by (+)-oxaprotiline reveals a differential central regulation of noradrenaline and adrenaline release

Summary Inhibition of uptake, in the central nervous system leads to a decrease of sympathetic outflow to many tissues; central a2-adrenoceptors are involved in this decrease. The aim of the present study was to compare the effects of the selective uptake, inhibitor (+)-oxaprotiline on the plasma kinetics of noradrenaline and adrenaline in anaesthetized and in conscious rabbits. [³H]Noradrenaline and [^3H]adrenaline were infused iv. The arterial plasma concentrations of endogenous and radiolabelled noradrenaline and adrenaline were measured, and the clearance from and spillover into the plasma of noradrenaline and adrenaline were calculated.Results obtained in conscious and anaesthetized rabbits were similar. (+)-Oxaprotiline 0.2, 0.6 and 1.8 mg kg^–1 iv. dose-dependently reduced the clearance of [³H]noradrenaline from the plasma. The clearance of [³H]adrenaline was reduced less. The spillover of endogenous noradrenaline was decreased by up to 35%. In contrast, the spillover of adrenaline tended to be enhanced. Prazosin 0.1 and 1 mg kg^–1 was injected iv. in a second part of each experiment. It lowered the blood pressure and caused a marked increase in noradrenaline spillover but no increase or even a decrease in adrenaline spillover.The results are compatible with the following hypothesis. The sympathetic outflow from the central nervous system is subject to a twofold a-adrenoceptor-mediated modulation: -adrenoceptor-mediated inhibition and ₁-adrenoceptor-mediated excitation. In the control of the sympathetic outflow to many extra-adrenal tissues, the ₂-adrenergic inhibition prevails. Uptake₁ inhibitors depress sympathetic outflow to such tissues by enhancing the ₂-adrenergic inhibition. In the regulation of the sympathetic outflow to the adrenal medulla, in contrast, ₂-adrenergic inhibition and ₁-adrenergic excitation have a similar impact. Uptake, inhibitors, hence, cause little change in adrenaline release: the two opposing influences cancel out. Prazosin produces an increase in noradrenaline but not adrenaline release because the loss of the central ₁ sympathoexcitation attenuates at best slightly the baroreflex to most extra-adrenal tissues but dampens markedly the baroreflex to the adrenal medulla. Correspondence to B. Szabo at the above address     

An examination of factors influencing adrenergic transmission in the pithed rat,with special reference to noradrenaline uptake mechanisms and post-junctional α-adrenoceptors

Summary Adrenergic mechanisms were analysed in the pithed rat to determine to what extent the actions of drugs observed in vitro are relevant in situ.The drugs examined were those which are known to block the neuronal or extraneuronal uptake of noradrenaline (cocaine, desipramine and corticosterone) or to be antagonists at post and/or pre-junctional -adrenoceptors (prazosin and yohimbine) together with the antidepressant, mianserin, which has been implicated in several of these actions. These drugs were tested against the arterial diastolic pressor, cardiac chronotropic and vas deferens responses to sympathetic nerve stimulation, to indirect sympathomimetics or to direct sympathomimetics, which were chosen according to whether they were substrates for noradrenaline uptake processes or selective between adrenoceptors.Pressor and cardiac responses to sympathetic nerve stimulation or to intravenous noradrenaline were potentiated by blockade of neuronal uptake but only the pressor effect of noradrenaline was potentiated by blockade of extraneuronal uptake. The effects of the antagonists suggested that the pressor effects of noradrenaline and of sympathetic nerve stimulation result from a combination of activation of ₁- and ₂-adrenoceptors, but that the effect of noradrenaline had a relatively greater contribution from the ₂-adrenoceptors. Mianserin was found to potentiate adrenergic responses at low doses, to produce limited antagonism at post-junctional ₁-adrenoceptors in high doses but to have no detectable effect at postjunctional ₂-adrenoceptors.     

1,1′-Diisopropyl-2,4′-cyanine (disprocynium24), a potent uptake2 blocker, inhibits the renal excretion of catecholamines

1,1′-Diisopropyl-2,4′-cyanine (disprocynium24), a potent inhibitor of the extraneuronal monoamine transport system (uptake₂), was previously shown to reduce the clearance of catecholamines from plasma not only by blocking uptake₂ but presumably also by blocking organic cation transport. To provide more direct evidence for the latter conclusion, the present study was carried out in anaesthetized rabbits. It aimed at determining the effect of disprocynium24 on the renal excretion of catecholamines which is known to be, at least in part, a consequence of organic cation transport in the kidney. To this end, the plasma clearance due to renal excretion (Cl_u) of endogenous as well as infused ³H-labelled adrenaline, noradrenaline and dopamine was determined for 60-min periods of urine collection in rabbits treated either with disprocynium24 (270 nmol kg^-1 i.v followed by i.v. infusion of 80 nmol kg^-1 min^-1) or vehicle. Two groups of animals were studied: group I (monoamine oxidase and catechol-O-methyltransferase intact) and group II (monoamine oxidase and catechol-O-methyltransferase inhibited). A third group of animals with intact monoamine oxidase and catechol-O-methyltransferase was used to study the effect of disprocynium24 on the glomerular filtration rate (as determined by measuring the plasma clearance of inulin). In vehicle controls, Cl_u of endogenous adrenaline, noradrenaline and dopamine was 7.2, 5.2 and 153.6 ml kg^-1 min^-1, respectively, in group I and 10.4, 7.0 and 134.3 ml kg^-1 min^-1, respectively, in group II. Similar control values of Cl_u were obtained for infused ³H-adrenaline and ³H-noradrenaline, but not for infused ³H-dopamine; Cl_u of ³H-dopamine (4.9 ml kg^-1 min^-1 in group I and 15.4 ml kg^-1 min^-1 in group II) was considerably smaller than Cl_u of endogenous dopamine, indicating that most of the dopamine in urine (i.e., 98% in group I and 92% in group II) was derived from the kidneys rather than from the circulation. By contrast, only about one quarter of the noradrenaline in urine (32% in group I and 24% in group II) and none of the urinary adrenaline were of renal origin. In both groups, disprocynium24 markedly reduced the Cl_u of endogenous catecholamines (by 72-90%) and of infused ³H-catecholamines (by 49-69%). Moreover, it preferentially inhibited the renal excretion of those components of urinary dopamine and noradrenaline which were derived from the kidney. Therefore, disprocynium24 inhibits the tubular secretion of catecholamines and, hence, organic cation transport in the kidney. This conclusion was substantiated by the observation that disprocynium24 did not alter the glomerular filtration rate. Received: 6 February 1997 / Accepted: 12 April 1997     

Extracellular dopamine, norepinephrine, and serotonin in the ventral tegmental area and nucleus accumbens of freely moving rats during intracerebral dialysis following systemic administration of cocaine and other uptake blockers

Extracellular levels of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) were measured by microdialysis in conscious rats equipped with dual probes, one in the ventral tegmental area (VTA) and another one in the contralateral nucleus accumbens (NACC). Dialysate content of all amines in both regions was essentially abolished by local infusion of tetrodotoxin (1 μM) or Ca²⁺-free buffer. Injection of the selective DA uptake blocker GBR 12935 (15 mg/kg IP) increased DA, as well as NE and, to a lesser extent, 5-HT in the VTA; it increased DA more than 5-HT in the NACC. The selective NE uptake blocker desipramine (10 mg/kg IP) increased NE but also 5-HT in the VTA and NACC; the DA level was persistently enhanced in the VTA, whereas in the NACC it initially rose and then fell below baseline value. The selective 5-HT uptake blocker citalopram (15 mg/kg IP) was generally more effective in elevating dialysate level of 5-HT than that of other amines in both regions. Cocaine (20 mg/kg IP) was non-selective in enhancing all three amines in both regions. There is considerable crosstalk between monoamine systems occurring upon systemic administration of uptake blockers, and the VTA and NACC are notably different in the time course of the DA effect (long-lasting versus transient). Received: 25 February 1997 /Final version: 2 June 1997     

Efflux studies allow further characterisation of the noradrenaline and 5hydroxytryptamine transporters in rat lungs

The aim of the present study was to further characterise the noradrenaline and 5hydroxytryptamine [5HT] transporters in rat lungs by examining the efflux of noradrenaline and 5HT, respectively. Lungs from rats were isolated and perfused via the pulmonary artery. After loading the tissue with ³H5HT or ³Hnoradrenaline the efflux of the relevant amine from the lungs was examined for 1525min. The rate constant for efflux of ³H5HT increased by 81% when Na⁺ ions were removed from the perfusion solution; increased gradually when a selective 5HT transporter inhibitor, 200nM citalopram, was added to the perfusion solution for the final 6min of efflux; and increased markedly and rapidly when substrates of the 5HT transporter, tryptamine (18μM) and 7methyltryptamine (12μM), were added for the final 6min of efflux. These effects of the substrates were abolished by 1μM citalopram, but were not significantly affected by 1μM desipramine, a selective uptake₁ inhibitor. On the other hand, the previously described substrateinduced increase in the rate of efflux of noradrenaline was significantly reduced by desipramine but was unaffected by citalopram. The results show that efflux of 5HT is mediated only by the 5HT transporter, with no significant contribution of uptake₁, and efflux of noradrenaline from rat lungs is mediated only by uptake₁ and not by the 5HT transporter. The effects of dopamine on the efflux of noradrenaline over a concentration range of 100-600nM were investigated and the results showed that 50% of the maximal increase in the rate of efflux occurred at a concentration of 275nM. This value did not differ from the K_m for uptake of dopamine. This result implies that the only factor affecting the substrate-induced increase in noradrenaline efflux is the affinity of the substrate for uptake₁. The efflux of noradrenaline was also examined in the absence and presence of two concentrations of desipramine (0.35and 1.5μM). Analysis of these results showed that uptake₁ contributed approximately 81% and diffusion 19% to the total efflux of noradrenaline and that 90% of the total noradrenaline efflux was subject to reuptake by uptake₁ into the pulmonary endothelial cells. Received: 23 January 1997 / Accepted: 26 March 1997     

Effect of available nitrogen on phytoavailability and bioaccumulation of hexavalent and trivalent chromium in hankow willows (Salix matsudana Koidz)

The effect of available nitrogen in nutrient solution on removal of two chemical forms of chromium (Cr) by plants was investigated. Pre-rooted hankow willows (Salix matsudana Koidz) were grown in a hydroponic solution system with or without nitrogen, and amended with hexavalent chromium [Cr (VI)] or trivalent chromium [Cr (III)] at 25.0±0.5 °C for 192 h. The results revealed that higher removal of Cr by plants was achieved from the hydroponic solutions without any nitrogen than those containing nitrogen. Although faster removal of Cr (VI) than Cr (III) was observed, translocation of Cr (III) within plant materials was more efficient than Cr (VI). Substantial difference existed in the distribution of Cr in different parts of plant tissues due to the nitrogen in nutrient solutions (p<0.05): lower stems were the major sink for both Cr species in willows grown in the N-free nutrient solutions and more Cr was accumulated in the roots of plants in N-containing ones. No significant difference was found in the removal rate of Cr (VI) between willows grown in the N-free and N-containing solutions (p>0.05). Removal rates of Cr (III) decreased linearly with the strength of nutrient solutions with or without N addition (p<0.01). Translocation efficiencies of both Cr species increased proportionally with the strength of N-containing nutrient solutions and decreased with the strength of N-free nutrient solutions. Results suggest that uptake and translocation mechanisms of Cr (VI) and Cr (III) are apparently different in hankow willows. The presence of easily available nitrogen and other nutrient elements in the nutrient solutions had a more pronounced influence on the uptake of Cr (III) than Cr (VI). Nitrogen availability and quantities in the ambient environment will affect the translocation of both Cr species and their distribution in willows in phytoremediation.