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

Abstract

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.

Comparison of the ORL1 receptor‐mediated inhibition of noradrenaline release in human and rat neocortical slices

“…This assumption is supported by the observation, that the affinity (K m ) of NA for the (human) 5‐HT transporter is about 50 times lower than for the corresponding NA transporter (Tatsumi et al ., 1997). Moreover, the difference in the rate of [ 3 H]‐NA uptake by the two different uptake carriers may be even higher, since previous data suggest that also the V max of NA at the 5‐HT transporter is much lower than that at the NA carrier (James & Bryan‐Lluka, 1997; Paczkowski et al ., 1996).…”

Further characterisation of the interaction of haloperidol metabolites with neurotransmitter transporters in rat neuronal cultures and in transfected COS-7 cells

“…This has been found previously to be a sensitive method of distinguishing between substrates and inhibitors of monoamine transporters in studies in a range of model systems, including rat vas deferens (Langeloh et al , 1991, rabbit platelets (Wölfel and Graefe 1992), rat lungs Westwood et al 1996;James and Bryan-Lluka 1997) and NAT or DAT cDNA-transfected COS-7 cells (Pifl et al 1997). There is a rapid and marked increase in the rate constant for efflux of preloaded [ 3 H]amine when a substrate is added during efflux, but at most only a small and slow increase in the rate constant for efflux occurs when a nontransported inhibitor is added (see references above and results in the present study with the transporter substrates and inhibitors).…”

“…There is a rapid and marked increase in the rate constant for efflux of preloaded [ 3 H]amine when a substrate is added during efflux, but at most only a small and slow increase in the rate constant for efflux occurs when a nontransported inhibitor is added (see references above and results in the present study with the transporter substrates and inhibitors). In the present study, each test compound was added at a concentration of about five times its K i value for inhibition of amine uptake, since the effects of substrates on efflux have been shown to be dependent on their affinities for the transporter (James and Bryan-Lluka 1997).…”

Presynaptic opioid receptors on noradrenergic and serotonergic neurons in the human as compared to the rat neocortex

“…In contrast, during preincubation of neocortical tissue with 0.1 μ M [ 3 H]‐NA, false labelling of serotonergic axon terminals seems to be less probable due to significantly lower affinity ( K m ) and V max values of NA at the (human) 5‐HT transporter, as compared to the corresponding values at the NA transporter (Paczkowski et al ., 1996; James & Bryan‐Lluka, 1997; Tatsumi et al ., 1997). Moreover, also false labelling of dopaminergic nerve endings in the neocortex with [ 3 H]‐NA also seems of minor importance in view of the low density of dopaminergic axons terminals in neocortical tissue.…”