Evidence for uptake1-mediated efflux of catecholamines from pulmonary endothelial cells of perfused lungs of rats

Abstract

Previous pharmacological studies have demonstrated that pulmonary endothelial cells and noradrenergic neurones possess the same transporter for inward transport of catecholamines, uptake1. In noradrenergic neurones, it has been shown that uptake1 is also involved in the carrier-mediated outward transport, or efflux, of noradrenaline and dopamine. The aim of the present study was to examine the efflux of noradrenaline and dopamine from perfused lungs of rats to determine whether uptake1, in addition to diffusion, mediates efflux of catecholamines from pulmonary vascular endothelial cells. The effects of reducing the cellular sodium gradient and of substrates and inhibitors of uptake1 on the efflux of 3H-noradrenaline and 3H-dopamine from rat lungs were measured. Isolated perfused lungs of rats (monoamine oxidase and catechol-O-methyltransferase inhibited) were loaded with 3H-(-)-noradrenaline or 3H-dopamine for 10 min followed by perfusion with either (1) a low sodium, amine-free Krebs solution, in which NaCl was replaced by either Tris.HCl or LiCl, for 15 or 10 min, respectively or (2) amine-free Krebs solution for 30 min in the absence or presence of a substrate or inhibitor of uptake1 for the last 15 min. The rate constants for spontaneous efflux of noradrenaline and dopamine from the lungs were 0.0163 min-1 and 0.0466 min-1, respectively. When NaCl was replaced by Tris.HCl during efflux, the rate constants for efflux of noradrenaline and dopamine were increased 2.5-fold and 3-fold, respectively, whereas, when NaCl was replaced by LiCl, the rate constants were increased 8-fold and 4-fold, respectively. The uptake1 substrates, dopamine (1 and 3 mumol/l) and adrenaline (40 mumol/l), both caused a rapid and marked increase in the efflux of noradrenaline, while noradrenaline (4 mumol/l) had a similar effect on the efflux of dopamine. The uptake1 inhibitors, imipramine (3 and 10 mumol/l) and nisoxetine (50 nmol/l), caused small and gradual increases in the efflux of noradrenaline and dopamine from rat lungs. These results demonstrate that efflux of noradrenaline and dopamine from rat lungs is affected by alterations in the normal sodium gradient across the cell and by drugs that interact with the uptake1 transporter. Thus, it can be concluded that the spontaneous efflux of catecholamines from pulmonary vascular endothelial cells is mediated predominantly by uptake1. In addition, efflux of catecholamines from the lungs has a diffusional component, which, combined with inhibition of reuptake, accounts for the small increase in amine efflux by inhibitors of uptake1.

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

“…In efflux experiments, the rate of efflux of 3H-noradrenaline from the lungs for each venous effluent sample and the 3H-noradrenaline content of the lungs at the end of the experiment were calculated as described previously (Westwood et al 1996). Values for the fractional rate of loss (FRL) of 3H-noradrenaline, which is an instantaneous measure of ko~t NA, the rate constant for efflux of noradrenaline from the lungs (Graefe and B6nisch 1988), were then calculated over the entire efflux period as described previously (Westwood et al 1996).…”

“…Values for the fractional rate of loss (FRL) of 3H-noradrenaline, which is an instantaneous measure of ko~t NA, the rate constant for efflux of noradrenaline from the lungs (Graefe and B6nisch 1988), were then calculated over the entire efflux period as described previously (Westwood et al 1996).…”

“…Adult, male, specific pathogen-free, Wistar rats (200-260 g) were pretreated with 75 mg/kg pargyline intraperitoneally 18 h and 2 h prior to the experiment to inhibit MAO and were then anaesthetized with 60 mg/kg pentobarbitone sodium intraperitoneally. The lung preparations were dissected as described by Westwood et al (1996). All experiments were carried out according to the 'Principles of laboratory animal care' and the guidelines of the National Health and Medical Research Council of Australia.…”

“…The Krebs solution contained (in retool/l) : NaC1 118, KC1 4.7, CaC12 25, KHzPO4 1.2, MgSO~ 1.2, NaHCO3 25, glucose 11.7, ascorbic acid 0.57 and Na2EDTA 0.040. 3H-Noradrenaline (1 nmol/1) was added by infusing the 3H-amine into the perfusion solution at 0.1 ml/min for the last 10 rain of the preperfusion to load the lungs with 3H-amine (Westwood et al 1996). The lungs were then perfused at 37°C for 30rain with aerated (95% Oz and 5% CO2) Krebs solution containing 10 gmol/1 U-0521, but no bovine serum albumin or 3H-amine and, in some experiments (see Results), 1 ~tmol/1 desipramine was present to inhibit uptake1.…”

“…Aduit, male, specific pathogen-free, Wistar rats (185-250 g) were anaesthetized with 60 mg/kg pentobarbitone sodium intraperitoneally and the lung preparations were dissected as described previously (Westwood et al 1996). The lungs were initially perfused via the puimonary artery at 37 ° C and a flow rate of 10 ml/min for 20 rain with Krebs soiution containing 5% bovine serum albumin, 10 ~mol/1 U-0521 and, where appropriate, 10, 30 or 300 nmol/1 amezinium or 30, 300 or 3000 nmol/l debrisoquine.…”