Role of proline residues in the expression and function of the human noradrenaline transporter.

The aim was to investigate the roles of proline residues in extracellular loop 2 (P172, P183, P188 and P209) and transmembrane domains 2, 5, 11 and 12 (P108, P270, P526, P551, P552 and P570) in determining noradrenaline transporter (NET) expression and function. Mutants of human NET with these residues mutated to alanine were pharmacologically characterized. Mutation of P108, P270 and P526 disrupted cell surface expression, from [3H]nisoxetine binding and confocal microscopy data. Mutations of P526, P551 and P570 reduced transporter turnover (Vmax of [3H]noradrenaline uptake/Bmax of [3H]nisoxetine binding) by 1.5-1.7-fold compared with wild-type NET, so these residues might be involved in conformational changes associated with substrate translocation. Conversely, mutations of P172, P183, P188 and P209 increased Vmax/Bmax by 2-3-fold compared with wild-type, indicating that the presence of these proline residues limits turnover of the NET. The mutations had few effects on apparent affinities of substrates or affinities of inhibitors, except decreases in inhibitor affinities after mutations of the P270 and P570 residues, and increases after mutation of the P526 residue. Hence, proline residues in extracellular loop 2 and in transmembrane domains have a range of roles in determining expression and function of the NET.

Tyrosine residue 271 of the norepinephrine transporter is an important determinant of its pharmacology.

The aim was to examine the functional importance in the norepinephrine transporter (NET) of (i) the phenylalanine residue at position 531 in transmembrane domain (TMD) 11 by mutating it to tyrosine in the rat (rF531Y) and human (hF531Y) NETs and (ii) the highly conserved tyrosine residues at positions 249 in TMD 4 of human NET (hNET) (mutated to alanine: hY249A) and 271 in TMD 5, by mutating to alanine (hY271A), phenylalanine (hY271F) and histidine (hY271H). The effects of the mutations on NET function were examined by expressing the mutant and wildtype NETs in COS-7 cells and measuring the K(m) and V(max) for uptake of the substrates, [3H]norepinephrine, [3H]MPP(+) and [3H]dopamine, the K(D) and B(max) for [3H]nisoxetine binding and the K(i) of the inhibitors, nisoxetine, desipramine and cocaine, for inhibition of [3H]norepinephrine uptake. The K(m) values of the substrates were lower for the mutants at amino acid 271 than hNET and unaffected for the other mutants, and each mutant had a significantly lower V(max) than NET for substrate uptake. The mutations at position 271 caused an increase in the K(i) or K(D) values of nisoxetine, desipramine and cocaine, but there were no effects for the other mutations. Hence, the 271 tyrosine residue in TMD 5 is an important determinant of NET function, with the mutants showing an increase in the apparent affinities of substrates and a decrease in the apparent affinities of inhibitors, but the 249 tyrosine and 531 phenylalanine residues do not have a major role in determining NET function.

Effects of short- and long-term exposure to c-AMP and c-GMP on the noradrenaline transporter.

The effects of short- and long-term exposure of cells to elevated cyclic adenosine monophosphate (c-AMP), using dibutyryl-c-AMP, 8-bromo-c-AMP, cholera toxin or forskolin, or cyclic guanosine monophosphate (c-GMP), using dibutyryl-c-GMP or 8-bromo-c-GMP, on the activity and expression of the noradrenaline transporter (NAT) were examined. Short- or long-term c-GMP elevation had no effects on (3)H-noradrenaline uptake by rat PC12 phaeochromocytoma cells or human SK-N-SH-SY5Y neuroblastoma cells. Short-term c-AMP elevation (for 17 min experiment duration) caused a decrease in (3)H-noradrenaline uptake by PC12 cells, but had no effects on SK-N-SH-SY5Y cells or COS-7 cells transfected with human or rat NAT cDNA. c-AMP did not affect (3)H-nisoxetine binding to PC12 cells. Long-term (24 h) exposure to elevated c-AMP levels caused a decrease in (3)H-noradrenaline uptake and NAT mRNA in PC12 cells, but had no effects on SK-N-SH-SY5Y cells and caused a small increase in (3)H-noradrenaline uptake in COS-7 cells heterologously expressing rat or human NAT. Hence, c-AMP, but not c-GMP, causes a cell type-dependent reduction in NAT activity after short-term exposure and a reduction in NAT expression after long-term exposure.

Specific uptake of 5-hydroxytryptamine is reduced in lungs from hypoxic pulmonary hypertensive rats.

In this study, the aim was to determine whether 5-hydroxytryptamine (5-HT) removal by the pulmonary endothelium is reduced in 1-week hypoxic, pulmonary hypertensive rats by directly measuring [3H]5-HT uptake in isolated lungs. In lungs from hypoxic rats, specific 5-HT uptake was reduced. This was due to a 50% decrease in the maximal initial rate of uptake rather than a decrease in affinity of 5-HT for its transporter. It is possible that reduced removal of 5-HT may contribute to the elevation in plasma levels of this vasoactive amine in pulmonary hypertension.

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

It has been proposed that the extrapyramidal symptoms such as tardive dyskinesia developed by patients on long-term haloperidol treatment may be the result of uptake of haloperidol metabolites into neurons via the monoamine neurotransmitter transporters followed by neurotoxic events, as occurs for MPP+, the pyridinium metabolite of MPTP. We recently showed that haloperidol and its metabolites are inhibitors of the human noradrenaline transporter (NAT), dopamine transporter (DAT) and serotonin transporter (SERT), and determined their Ki values for inhibition of the three transporters expressed in transfected COS-7 cells. In this study, we extended the investigation of these compounds to their inhibitory effects on DAT, SERT and the high affinity choline uptake (HACU) in neuronal cultures from embryonic rat brain, and investigated whether the compounds are substrates or non-transported inhibitors of the NAT, DAT and SERT in transfected COS-7 cells and DAT and SERT in the neuronal cultures. Haloperidol and its metabolites inhibited DAT, SERT and HACU in the neuronal cultures, indicating that they are not specific inhibitors of the monoamine neurotransmitter transporters. The ratio of the Ki values of the least and most potent inhibitors were found to be 2.8 for DAT, 24 for SERT and 7.6 for HACU. The compounds were more potent inhibitors of DAT and SERT in neuronal cultures than we found previously in transfected COS-7 cells. The question of whether the compounds are substrates or non-transported inhibitors of the monoamine transporters was investigated by determining whether they caused an increase in efflux of [3H]amine in transfected COS-7 cells or neuronal cultures preloaded with [3H]amine. Haloperidol metabolites were weak substrates for SERT, but not for NAT or DAT, in transporter-transfected COS-7 cells. In neuronal cultures, the metabolites appeared to be non-transported inhibitors or very weak substrates of DAT and SERT. Despite inhibition of the monoamine transporters by haloperidol and its metabolites, there is little evidence to support the proposal that these compounds are likely to cause neurotoxic effects via neuronal uptake using the monoamine transporters. The mechanisms of the side effects of haloperidol therapy, such as tardive dyskinesia, are still unclear, but are unlikely to depend on interactions of the drug or its metabolites with NAT, DAT or SERT.

Potencies of haloperidol metabolites as inhibitors of the human noradrenaline, dopamine and serotonin transporters in transfected COS-7 cells.

Extrapyramidal symptoms, such as tardive dyskinesia, often develop in patients on long-term treatment with haloperidol. It has been proposed that these symptoms could be caused by neurotoxic effects of haloperidol metabolites following uptake by monoamine transporters, in an analogous mechanism to the neurotoxic effect of MPP+ (1-methyl-4-phenylpyridinium) metabolised from MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). In this study, the hypothesis was partially investigated by determining the potencies of haloperidol and reduced haloperidol and the corresponding pyridinium and tetrahydropyridine metabolites, compared with MPP+ and MPTP, as inhibitors of the noradrenaline transporter (NAT), dopamine transporter (DAT) and 5-HT transporter (SERT). Two days after COS-7 cells were transiently transfected with the cDNA for the human NAT, DAT or SERT (Lipofectamine method), the cells were incubated with 10 nM [3H]noradrenaline, dopamine or 5-HT, respectively, for 2 min at 37 C, in the absence or presence of various concentrations of the eight compounds or a specific uptake inhibitor (NAT: nisoxetine 1 microM; DAT: GBR 12909 1 microM; SERT: citalopram 10 microM). Specific amine uptake (fmol/ mg protein) was calculated as the difference in uptake in the absence and presence of the specific uptake inhibitor. Ki values were calculated for the eight compounds for inhibition of NAT, DAT and SERT. Haloperidol, its five metabolites and MPP+ and MPTP all inhibited NAT, DAT and SERT. For the pyridinium and tetrahydropyridine metabolites of haloperidol, there were not marked differences between their potencies as inhibitors between each other for NAT or DAT or between NAT and DAT, with all of the Ki values in the range of 5.8-16 microM. However, there were more marked differences for SERT, with all but one of the metabolites showing selectivity for inhibition of SERT relative to NAT and DAT. Haloperidol and reduced haloperidol had similar inhibitory potencies for all three transporters, and were clearly less potent than the other haloperidol metabolites only for inhibition of SERT. The lack of correlation between the inhibitory potencies of the haloperidol metabolites and their structural analogues, MPTP and MPP+, suggests that they are not likely to cause neurotoxicity by a mechanism analogous to that of the latter neurotoxin.

Comparison of the pharmacological properties of cloned rat, human, and bovine norepinephrine transporters.

The aims of this study were to characterize the recently cloned rat norepinephrine transporter (NET) in more detail and in particular to study possible species differences in its pharmacological properties compared with the human and bovine NETs. The study was carried out by measuring the uptake of [(3)H]norepinephrine in COS-7 cells expressing the NET after transient transfection with rat, human, or bovine NET cDNA. There were small but significant differences between the rat NET and the human or bovine NETs with respect to the affinities of sodium ions (greater for rat than for bovine) of the substrates norepinephrine, epinephrine, and 1-methyl-4-phenylpyridinium (greater for human than for rat), and of the inhibitor cocaine (greater for human and bovine than for rat), whereas the affinities of dopamine and of most inhibitors, including tricyclic antidepressants, showed no species differences. The fact that the affinities for some substrates, cocaine and sodium ions exhibited small but significant interspecies differences among the rat, human, and bovine NETs suggests that ligand recognition, the translocation process, and sodium ion dependence are influenced differentially by just a few amino acid exchanges in the primary sequences of the transporters. On the other hand, the lack of any major differences in the pharmacological properties of the rat, human, and bovine NETs in this study suggests that data obtained in previous studies on rat tissues and bovine cells can be extrapolated, in all except the most quantitative analyses, to the properties of the human NET.

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.

Lanthanides inhibit the human noradrenaline, 5-hydroxytryptamine and dopamine transporters.

Transporters for the monoamine neurotransmitters, including noradrenaline, 5-hydroxytryptamine [5-HT] and dopamine, have twelve transmembrane spanning regions and cotransport Na+ and Cl- ions. Another family of Na(+)-dependent transporters is that containing the Na+/glucose and Na+/proline cotransporters that are found in the epithelial cells of renal and intestinal brush border membranes. It has been shown that various trivalent lanthanides can substitute for Na+ for transport of glucose and proline. The aim of this study was to determine the effects of lanthanides on the activities of the human noradrenaline, 5-HT and dopamine transporters. Cultured cells were incubated for 2 min with 10 nM 3H-noradrenaline (SK-N-SH-SY5Y human neuroblastoma cells), 3H-5-HT (JAR human placental choriocarcinoma cells) or 3H-dopamine (COS-7 cells transfected with the cDNA of the human dopamine transporter). Specific amine uptake was determined as the difference between accumulation of the amine in the cells in the absence and presence of a corresponding uptake inhibitor. Under both isotonic (150 mM NaCl or LiCl or 90 mM lanthanide salt) and hypertonic (150 mM NaCl +100 mM LiCl, 250 mM LiCl or 150 mM lanthanide salt) conditions, replacement of Na+ by Li+, La3+, Eu3+ or Sm3+ abolished the specific uptake of noradrenaline in SK-N-SH-SY5Y cells and replacement of Na+ by Li+ or Eu3+ decreased the specific uptake of 5-HT in JAR cells by 94-100% and that of dopamine in transfected COS-7 cells by 95-99%. The direct effects of Eu3+ (with Na+ present) on the human noradrenaline transporter in SK-N-SH-SY5Y cells were also examined. Eu3+ inhibited noradrenaline uptake into the cells (IC50 2.6 mM) and nisoxetine binding to crude membranes of SK-N-SH-SY5Y cells (IC50 4.7 mM) with similar potencies. Further experiments showed that 4.5 mM EuCl3 in the presence of 150 mM Na+ caused a 3.5-fold increase in the K(m) of noradrenaline and no change in the maximal rate of noradrenaline uptake. EuCl3 (4.5 mM) also caused a pronounced inhibition of the Na(+)-dependent stimulation of noradrenaline uptake by SK-N-SH-SY5Y cells. It can be concluded from these data that, in contrast with the Na+/glucose and Na+/proline cotransporters, the lanthanides cannot substitute for Na+ in the transport of substrates by the monoamine neurotransmitter transporters and that the lanthanides inhibit the latter transporters by interacting with sites of the transporters involved in amine and Na+ binding.

The rat norepinephrine transporter: molecular cloning from PC12 cells and functional expression.

The rat norepinephrine transporter (rNET) cDNA from the PC12 pheochromocytoma cell line has been cloned by RT-PCR and characterized. The cDNA encodes an integral membrane protein consisting of 617 amino acids which contains twelve putative transmembrane domains, two potential N-glycosylation sites, two potential phosphorylation sites for protein kinase C and one phosphorylation site for casein kinase II. The nucleotide and deduced amino acid sequence shows a high level of homology to the human and the bovine norepinephrine transporter and less homology to the rat dopamine transporter (rDAT). Heterologous expression of rNET in HEK293 cells revealed that uptake of [3H]norepinephrine is sodium- and chloride-dependent and highly sensitive to the selective norepinephrine transporter inhibitors desipramine and nisoxetine. The cloned rNET cDNA provides the opportunity to investigate this transporter in heterologous expression systems and adds a new member to the family of sodium- and chloride-dependent neurotransmitter transporters.

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

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.

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.

Conclusive evidence for distinct transporters for 5-hydroxytryptamine and noradrenaline in pulmonary endothelial cells of the rat.

The aims of this study were to obtain conclusive evidence about the roles of a 5-hydroxytryptamine [5-HT] transporter and uptake1 in the dissipation of 5-HT in the lungs of the rat and to compare the properties of the 5-HT transporter in rat lungs with that in other tissues, including brain and platelets. In the first part of the study, the IC50 values of a range of selective inhibitors and substrates of the 5-HT transporter or uptake1 were determined for inhibition of uptake of 5-HT or noradrenaline in intact perfused lungs of rats. Monoamine oxidase was inhibited and, in experiments with noradrenaline, catechol-O-methyltransferase was also inhibited. Initial rates of uptake of 5-HT or noradrenaline were measured in lungs perfused with 2 nmol/l 3H-5-HT or 3H-noradrenaline for 2 min, in the absence or presence of at least three concentrations of paroxetine, citalopram, fluoxetine, 7-methyltryptamine, tryptamine, nisoxetine, imipramine, 5-HT, desipramine, (+)-oxaprotiline, cocaine or tyramine. The results showed that pharmacologically distinct transporters are involved in the uptake of 5-HT and noradrenaline in rat lungs, since there was no significant correlation between the IC50 values for inhibition of 5-HT and noradrenaline uptake in the lungs. However, there were significant correlations between the IC50 values for (a) inhibition of 5-HT uptake in rat lungs and of uptake by the 5-HT transporter in rat brain and (b) inhibition of noradrenaline uptake in rat lungs and of uptake1 in rat phaeochromocytoma PC-12 cells. The results support the conclusion that 5-HT uptake in rat lungs occurs, at least predominantly, by a 5-HT transporter which is very similar to or the same as that in other tissues, such as the brain, and provide further evidence for transport of noradrenaline by uptake1. Further experiments were carried out to determine whether there is any transport of 5-HT by uptake1 or of noradrenaline by the 5-HT transporter in rat lungs. Lungs were perfused with 2 nmol/l 3H-5-HT or 3H-noradrenaline for 2 min in the absence or presence of 1 mumol/l citalopram, desipramine, or citalopram and desipramine. The results showed that there was no evidence of any transport of 5-HT in the lungs by uptake1 or of noradrenaline by the 5-HT transporter, in that desipramine had no effect on 5-HT uptake (in the absence or presence of citalopram) and citalopram had no effect on noradrenaline uptake (in the absence or presence of desipramine). The final series of experiments was carried out to determine whether, at high concentrations of the amine, there is any interaction of 5-HT with uptake1 or of noradrenaline with the 5-HT transporter. Noradrenaline, at a concentration of 10 mumol/l, did not affect 5-HT uptake in lungs perfused with 2 nmol/l 3H-5-HT for 2 min (uptake1 inhibited), but 50 mumol/l 5-HT inhibited noradrenaline uptake by 56% in lungs perfused with 2 nmol/l 3H-noradrenaline for 2 min (5-HT transporter inhibited). These and the above results show that the 5-HT transporter appears to be exclusively responsible for 5-HT uptake in rat lungs, despite the possible interaction of 5-HT at high concentrations with the uptake1 transporter in the cells. On the other hand, noradrenaline is transported exclusively by uptake1 in the lungs, and there is no evidence that it interacts with the 5-HT transporter, even at high concentrations.

A kinetic investigation of the pulmonary metabolism of dopamine in rats shows marked differences compared with noradrenaline.

The aim of this study was to investigate the deamination of dopamine in the intact pulmonary circulation of isolated lungs of the rat. The first part of the study showed that dopamine is not converted to noradrenaline by dopamine-beta-hydroxylase (DBH) when dopamine is perfused through isolated lung preparations with monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) inhibited. Hence, it was not necessary to inhibit DBH in subsequent experiments. The metabolite profile for deamination of dopamine in the lungs was examined by determining whether MAO and semicarbazide-sensitive amine oxidases (SSAO) contribute to the deamination of dopamine (and noradrenaline), and by determining the activity of MAO (kMAO) for the metabolism of dopamine. Lungs were perfused with 1 nmol/l 3H-dopamine or 3H-noradrenaline with COMT inhibited and, in experiments to determine the contribution of SSAO to deamination, with MAO inhibited. Inhibition of MAO reduced the deamination of dopamine and noradrenaline by 99.8% and 98.6%, respectively, indicating that MAO, and not SSAO, was responsible for deamination of the catecholamines in the lungs. The kMAO value for deamination of dopamine was 3.89 min-1. Further experiments were carried out to determine the contributions of MAO-A and MAO-B to the deamination of dopamine in lungs perfused with 1 nmol/l 3H-dopamine and 100 nmol/l lazabemide or 300 nmol/l Ro41-1049, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for saturation of catechol-O-methyltransferase by low concentrations of noradrenaline in perfused lungs of rats.

Previous studies on the pulmonary removal and metabolism of catecholamines in rat lungs have shown that, when the lungs are perfused with a low concentration (1 nmol/l) of noradrenaline, the amine is metabolized by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), but is predominantly O-methylated, and the activities of COMT and MAO are 0.357 min-1 and 0.186 min-1, respectively. The aim of the present study was to examine the changes in the metabolic profile of noradrenaline in rat lungs over a range of concentrations, and to examine the kinetics of the pulmonary O-methylation of noradrenaline and adrenaline. In isolated lungs perfused with 3H-noradrenaline, there was a progressive decrease in the proportion of O-methylated metabolites and a corresponding increase in the proportion of deaminated metabolites, as the noradrenaline concentration in the perfusion solution was increased from 1 to 10 to 100 to 1000 nmol/l. Experiments designed to determine the rate of uptake of noradrenaline in lungs perfused with 1 nmol/l 3H-noradrenaline, under conditions of MAO inhibited, COMT inhibited and COMT and MAO inhibited, showed that the results were compatible with co-existence of COMT and MAO in the pulmonary endothelial cells. Hence, it appeared that the changing metabolic profile with amine concentration in the previous series of experiments was not due to saturation of noradrenaline uptake into cells that contained COMT but not MAO.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of albumin sensitization and challenge of guinea pigs on uptake2 of isoprenaline in trachealis muscle.

There is evidence that hyperpolarization of cells by various mechanisms results in stimulation of uptake2 of catecholamines and, conversely, that depolarization causes inhibition of uptake2. The aim of this study was to examine whether changes that have been shown to occur in the resting membrane potential of the trachealis muscle of guinea pigs that have been sensitized to albumin (hyperpolarization) or albumin-sensitized followed by inhalational albumin challenge (depolarization) are reflected in changes in uptake2 in the smooth muscle. Uptake2 of isoprenaline (as measured by the steady-state rate of specific 3H-O-methylisoprenaline formation normalized for the isoprenaline concentration) was determined in isolated segments of trachealis muscle that were incubated in 3H-(+/-)-isoprenaline and were from guinea pigs from three treatment groups: (i) controls, (ii) albumin-sensitized and (iii) albumin-sensitized and challenged. At an isoprenaline concentration that does not hyperpolarize the trachealis muscle (1 nmol/l), uptake2 was significantly greater in the muscle from sensitized guinea pigs than that from control or sensitized and challenged guinea pigs. When a drug that hyperpolarized the trachealis muscle was present (25 nmol/l isoprenaline or 10 mumol/l (-)-cromakalim), there were no differences in uptake2 between the three groups. Propranolol prevented the stimulation of uptake2 by isoprenaline and glibenclamide prevented stimulation of uptake2 by (-)-cromakalim. In the presence of propranolol or glibenclamide, there were no differences in uptake2 between the three treatment groups of guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Types A and B monoamine oxidase contribute to the metabolism of norepinephrine in perfused lungs of rats.

The aim of the study was to determine the activity of monoamine oxidase (MAO) and the contributions of the A and B forms of MAO to the metabolism of norepinephrine (NE) in isolated perfused lungs of the rat. Preliminary experiments to investigate the MAO-A and MAO-B selective inhibitors, Ro41-1049 and Ro19-6327, respectively, were carried out in isolated preparations of rat vas deferens incubated in 10 nM [3H]NE (Uptake2 and catechol-O-methyltransferase inhibited) to measure rates of deamination of NE and the rate constant for deamination (kMAO) by MAO-A. These experiments showed that 1) the IC50 of Ro41-1049 for inhibition of MAO-A was 9.62 nM with 97% inhibition by 300 nM and 2) 100 or 300 nM Ro19-6327 did not affect deamination of NE by MAO-A. Hence, 300 nM Ro41-1049 and 100 nM Ro19-6327 were used to inhibit selectively MAO-A and MAO-B, respectively, in rat lungs perfused in vitro with 10 nM [3H]NE. Catechol-O-methyltransferase was inhibited and kMAO values for metabolism of NE were determined. The kMAO value for deamination of NE in the absence of Ro41-1049 or Ro19-6327 was 0.186 min-1. Ro41-1049 and Ro19-6327 decreased this kMAO value by 79 and 33%, respectively. Thus, it was concluded that both MAO-A and MAO-B contribute to the deamination of NE in the pulmonary circulation of the rat, with MAO-A responsible for 67-79% of the total deamination and MAO-B for 21-33%.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of (-)-cromakalim and glibenclamide on uptake2 in guinea-pig trachealis muscle.

In a recent study, we have shown that hyperpolarization of cells by beta-adrenoceptor agonists results in stimulation of the uptake2 process for catecholamines. The aim of the present study was to further explore the hypothesis that uptake2 is dependent on membrane potential by examining the effects of the K(+)-channel opening drug, (-)-cromakalim, and the K(+)-channel blocking drug, glibenclamide, on uptake2 of isoprenaline. The effects of these drugs were examined in guinea-pig trachealis muscle, in which isoprenaline and cromakalim cause hyperpolarization, and in rat heart, in which isoprenaline and cromakalim have little effect on membrane potential. In guinea-pig trachealis muscle segments, 1 mumol/l glibenclamide reduced uptake2 (as measured by the steady-state rate of corticosterone-sensitive formation of 3H-3-O-methylisoprenaline normalized for the isoprenaline concentration) in tissues incubated in concentrations of 3H-(+/-)-isoprenaline that hyperpolarize the muscle (25 and 250 nmol/l) but not at an isoprenaline concentration that did not hyperpolarize the muscle (1 nmol/l). (-)-Cromakalim (10 mumol/l), which hyperpolarizes the trachealis muscle, increased uptake2 of isoprenaline (1 or 25 nmol/l) and this effect of (-)-cromakalim was inhibited by glibenclamide. In rat hearts perfused with 1 or 25 nmol/l 3H-(+/-)-isoprenaline and 10 mumol/l U-0521 to inhibit catechol-O-methyltransferase, the rate of uptake2 of isoprenaline was unaffected by cromakalim or glibenclamide. The results show that hyperpolarization of cells by various mechanisms can result in stimulation of uptake2 of catecholamines and provide further evidence to support the hypothesis that the uptake2 transport process is driven by the membrane potential of cells.

Evidence from guinea-pig trachealis that Uptake2 of isoprenaline is enhanced by hyperpolarization of the smooth muscle.

Previous studies (Bönisch et al. 1985; Trendelenburg 1986, 1987) have provided evidence that Uptake2 of catecholamines is inhibited by depolarization of cells. The aim of this study was to further examine the relationship between Uptake2 and membrane potential by testing the hypothesis that Uptake2 is, conversely, stimulated by hyperpolarization of cells. The effects of beta-adrenoceptor agonists (isoprenaline and salbutamol) and beta-adrenoceptor antagonists (propranolol and ICI 118,551) on Uptake2 of isoprenaline were examined in guinea-pig trachealis muscle, in which stimulation of beta-adrenoceptors mediates hyperpolarization of the smooth muscle cells (Allen et al. 1985), and in rat heart, in which beta-adrenoceptor agonists do not cause hyperpolarization. In guinea-pig trachealis muscle segments, propranolol and ICI 118,551 reduced Uptake2 (as measured by the steady-state rate of corticosterone-sensitive formation of 3-O-methylisoprenaline normalized for the isoprenaline concentration) in tissues incubated in 2.5-250 nmol/l 3H-isoprenaline (in the range over which isoprenaline causes hyperpolarization of the muscle), but not in 1 nmol/l 3H-isoprenaline (which does not hyperpolarize the muscle). The normalized rates were greater in tissues incubated in 25 nmol/l than 1 nmol/l isoprenaline, and were enhanced by 2.5 mumol/l salbutamol in tissues incubated in 1 nmol/l isoprenaline. In rat hearts perfused with 1 or 25 nmol/l 3H-isoprenaline and U-0521 to inhibit catechol-O-methyltransferase, the rate of Uptake2 of isoprenaline, normalized for the isoprenaline concentration, was unaffected by the isoprenaline concentration or the presence of propranolol, ICI 118,551 or salbutamol.(ABSTRACT TRUNCATED AT 250 WORDS)