Min Of Efflux Research Articles

Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.

High Affinity Transport of Taurine by the Drosophila Aspartate Transporter dEAAT2

Excitatory amino acid transporters (EAATs) are structurally related plasma membrane proteins known to mediate the Na(+)/K(+)-dependent uptake of the amino acids l-glutamate and dl-aspartate. In the nervous system, these proteins contribute to the clearance of glutamate from the synaptic cleft and maintain excitatory amino acid concentrations below excitotoxic levels. Two homologues exist in Drosophila melanogaster, dEAAT1 and dEAAT2, which are specifically expressed in the nervous tissue. We previously reported that dEAAT2 shows unique substrate discrimination as it mediates high affinity transport of aspartate but not glutamate. We now show that dEAAT2 can also transport the amino acid taurine with high affinity, a property that is not shared by two other transporters of the same family, Drosophila dEAAT1 and human hEAAT2. Taurine transport by dEAAT2 was efficiently blocked by an EAAT antagonist but not by inhibitors of the structurally unrelated mammalian taurine transporters. Taurine and aspartate are transported with similar K(m) and relative efficacy and behave as mutually competitive inhibitors. dEAAT2 can mediate either net uptake or the heteroexchange of its two substrates, both being dependent on the presence of Na(+) ions in the external medium. Interestingly, heteroexchange only occurs in one preferred substrate orientation, i.e. with taurine transported inwards and aspartate outwards, suggesting a mechanism of transinhibition of aspartate uptake by intracellular taurine. Therefore, dEAAT2 is actually an aspartate/taurine transporter. Further studies of this protein are expected to shed light on the role of taurine as a candidate neuromodulator and cell survival factor in the Drosophila nervous system.

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

The aim of the present study was to further characterise the noradrenaline and 5-hydroxytryptamine [5-HT] transporters in rat lungs by examining the efflux of noradrenaline and 5-HT, respectively. Lungs from rats were isolated and perfused via the pulmonary artery. After loading the tissue with 3H-5-HT or 3H-noradrenaline the efflux of the relevant amine from the lungs was examined for 15-25 min. The rate constant for efflux of 3H-5-HT increased by 81% when Na+ ions were removed from the perfusion solution; increased gradually when a selective 5-HT transporter inhibitor, 200 nM citalopram, was added to the perfusion solution for the final 6 min of efflux; and increased markedly and rapidly when substrates of the 5-HT transporter, tryptamine (18 microM) and 7-methyltryptamine (12 microM), were added for the final 6 min of efflux. These effects of the substrates were abolished by 1 microM citalopram, but were not significantly affected by 1 microM desipramine, a selective uptake, inhibitor. On the other hand, the previously described substrate-induced increase in the rate of efflux of noradrenaline was significantly reduced by desipramine but was unaffected by citalopram. The results show that efflux of 5-HT is mediated only by the 5-HT transporter, with no significant contribution of uptake1, and efflux of noradrenaline from rat lungs is mediated only by uptake1 and not by the 5-HT transporter. The effects of dopamine on the efflux of noradrenaline over a concentration range of 100-600 nM were investigated and the results showed that 50% of the maximal increase in the rate of efflux occurred at a concentration of 275 nM. This value did not differ from the Km 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 (0.35 and 1.5 microM). 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.

Amezinium and debrisoquine are substrates of uptake1 and potent inhibitors of monoamine oxidase in perfused lungs of rats.

Previous studies have resulted in the classification of amezinium as a selective inhibitor of neuronal monoamine oxidase (MAO), because it is a much more potent MAO inhibitor in intact tissues, in which it is accumulated in noradrenergic neurones by uptake1, than in tissue homogenates. In the present study, the effects of amezinium on the deamination of noradrenaline were investigated in intact lungs of rats, since the pulmonary endothelial cells are a site where the catecholamine transporter is non-neuronal uptake1. In addition, another drug that is both a substrate of uptake1 and a MAO inhibitor, debrisoquine, was investigated in the study. The first aim of the study was to show whether amezinium and debrisoquine are substrates of uptake1 in rat lungs. After loading of isolated perfused lungs with 3H-noradrenaline (MAO and catechol-O-methyltransferase (COMT) inhibited), the efflux of 3H-noradrenaline was measured for 30 min. When 1 mumol/l amezinium or 15 mumol/l debrisoquine was added for the last 15 min of efflux, there was a rapid and marked increase in the fractional rate of loss of 3H-noradrenaline, which was reduced by about 70% when 1 mumol/l desipramine was present throughout the efflux period. These results showed that both drugs were substrates for uptake1 in rat lungs. In lungs perfused with 1 nmol/l 3H-noradrenaline (COMT inhibited), 10, 30 and 300 nmol/l amezinium caused 58%, 76% and 74% inhibition of noradrenaline deamination, respectively, and 30, 300 and 3000 nmol/l debrisoquine caused 56%, 89% and 96% inhibition of noradrenaline deamination, respectively. When MAO-B was also inhibited, 10 nmol/l amezinium caused 84% inhibition of the deamination of noradrenaline by MAO-A in the lungs. In contrast, in hearts perfused with 10 nmol/l 3H-noradrenaline under conditions where the amine was accumulated by uptake2 (COMT, uptake1 and vesicular transport inhibited), 10 nmol/l amezinium had no effect and 300 nmol/l amezinium caused only 36% inhibition of deamination of noradrenaline. The results when considered with previous reports in the literature show that amezinium is about 1000 times more potent and debrisoquine is about 20 times more potent for MAO inhibition in rat lungs than in tissue homogenates, and the reason for their high potencies in the intact lungs is transport and accumulation of the drugs in the pulmonary endothelial cells by uptake1. Amezinium is much less potent as a MAO inhibitor in cells with the uptake2 transporter, such as the myocardial cells of the heart. The results also confirmed previous reports that amezinium is highly selective for MAO-A.

Forskolin and caffeine induce Ca2+ release from intracellular stores in rabbit aorta

We combined techniques of 45Ca efflux and computer-assisted kinetic analysis to investigate the effects of forskolin and caffeine on intracellular Ca2+ metabolism in intact rabbit aortic segments. When either 1 microM forskolin or 10 mM caffeine was present during the 45Ca load and efflux, the amount of 45Ca released from the tissue was reduced during the interval between 50 and 250 min of efflux. In contrast caffeine but not forskolin induced an acute increase in 45Ca efflux when added to the perfusion medium after 1 h of efflux in physiological salt solution. Preincubation with caffeine abolished phenylephrine-stimulated 45Ca release, but preincubation with forskolin had no effect. Kinetic analysis of these data indicated that caffeine reduced the Ca2+ content of the same intracellular compartment depleted by phenylephrine, whereas forskolin depleted a different intracellular Ca2+ store. Forskolin-induced depletion of an intracellular Ca2+ store was surprising in light of current evidence suggesting adenosine 3',5'-cyclic monophosphate stimulation of sarcoplasmic reticular Ca2+ uptake, but hypotheses that included only this mechanism are inconsistent with our findings.

Solute Accumulation by Grape Pericarp Cells III. Sugar Changes in vivo and the Effect of Shading

Summary The endogenous glucose, fructose and sucrose of the skin of the grape berry has been partitioned into diffusible and residual fractions during 1G d of the phase of rapid sugar accumulation following veraison. The data have been related to total soluble solids of the perirarp juice (°Brix) as an index of development: strong correlations were found between the partitioned hexose fractions and 0 Brix, but with the notable exception of non-diffusing glucose which was most variable during this period. Sucrose did not show good correlations with other attributes. A large proportion of both glucose and fructose diffused from the skin into an osmotic buffer during 30 min of efflux: at 5 °Brix, 40—45% of each hexose diffused, and at 16 °Brix this fraction was 70 to 75%. However, the degree of eompartmentation of glucose consistently exceeded that of fructose by a small, constant amount for any given total level. A series of berries were shaded individually, a treatment known to lessen the rate of global sugar accumulation but, as shown here, having no qualitative effect upon the relationship between glucose and fructose nor upon their degrees of “compartmentation”. A tentative calculation indicates a substantial concentration gradient between the diffusible and compartmented space (compartmented space having the lower concentration) both before veraison and during rapid sugar accumulation in the tissue. Aceumulation seems essentially an increase in diffusible sugars, in whir h the primary process is probably unloading from the phloem into the apoplast rather than eventual compartmentation within the cell.

The kinetics of the active and de-energized transport of O-methyl glucose in Ustilago maydis

The kinetics of the uptake and efflux of 3- O-methyl-glucose in sporidia of Ustilago maydis were measured, both in active cells and in cells whose metabolic activity had been inhibited by azide and iodoacetate. The de-energized transport system proved to be carrier mediated with apparent affinity constants 13 ± 2 mM outside ( K 0) and 18 ± 2 mM inside ( K 1). The apparent maximum rate constants for the same system were 0.66 ± 0.05 mmol/l cell water per min for uptake ( V + and 0.53 ± 0.04 mmol/l cell water per min for efflux ( V -. For the active system K 0 = 0.08 ± 0.01, K 1 > 40, V + = 9.7 ± 0.5 and V − 1.1 ± 0.9 (in equivalent units). These results are discussed in the context of the carrier mechanism as proposed by Regen and Morgan (Regen, D.M. and Morgan, H.E. (1964) Biochim. Biophys. Acta 79, 151–166). The antifungal compound carboxin had no effect on de-energized transport but was shown to decrease both K 0 and V + in the active system. Phloretin and phlorizin were also found to be without effect on de-energized cells but the former enhanced while the latter inhibited active uptake.