Extraneuronal noradrenaline transport (uptake2) in a human cell line (Caki-1 cells)

Summary This study describes for the first time an experimental system for the extraneuronal transport mechanism of noradrenaline (uptake₂) which is based on a clonal cell line (Caki-1). Caki-1 cells were originally derived from a human renal cell carcinoma. The conclusion that these cells express uptake₂ is supported by several experimental findings. (1) The initial rate of ³H-noradrenaline uptake in Caki-1 cells is saturable, the K _m being 450 mol/l. (2) Inhibitors of uptake₂ such as corticosterone (1 mol/l) and O-methyl-isoprenaline (100 Emol/l) largely inhibit ³H-noradrenaline uptake in Caki-1 cells. Whereas inhibitors of the neuronal transport mechanism for noradrenaline (uptake₁) such as desipramine (1 mol/l) and cocaine (10 mol/l) do not reduce it. (3) Depolarization of Caki-1 cells by the elevation of extracellular potassium inhibits ³H-noradrenaline uptake. (4) There is a highly significant correlation between the IC₅₀'s of various compounds for the inhibition of ³H-noradrenaline uptake in Caki-1 cells and rabbit aorta known to possess uptake₂.Interestingly enough, uptake₂ in Caki-1 cells and rabbit aorta is inhibited by cimetidine, quinidine and procainamide which are substrates of the renal transport mechanism for organic cations. Moreover, ³H-cimetidine is shown to be a substrate of uptake₂ in the isolated perfused rat heart. These results indicate a striking similarity between uptake₂ and the renal transport mechanism for organic cations. Send offprint requests to E. Schömig at the above addressSupported by the Deutsche Forschungsgemeinschaft (SFB 176, Scho 373) and the Dr. Robert Pfleger Stiftung     

The force driving the extraneuronal transport mechanism for catecholamines (uptake2)

Summary Recently, uptake₂ was shown to exist in the clonal Caki-1 cell line. The aim of this study was two-fold: a) to determine, in Caki-1 cells, the intracellular fate of ³H-noradrenaline after its translocation by uptake₂ and b) to analyse the force driving uptake₂.Caki-1 cells have the characteristics of a metabolizing system in which the activity of catechol-O-methyl transferase (COMT) greatly exceeds that of monoamine oxidase (MAO). In all subsequent experiments these enzymes were inhibited. The determination of initial rates of uptake₂ into Caki-I cells at an extracellular pH between 6.9 and 7.9 indicated that the protonated species of ³H-noradrenaline is transported. Depolarization of Caki-1 cells (by three different procedures) inhibited the inward transport. Determination of the time course of the specific accumulation of ³H-noradrenaline in Caki-I cells and of ³H-isoprenaline in the perfused rat heart (both mediated by uptake₂) revealed that depolarization (by high K⁺) reduced the rate constant for inward transport (k_IN) and increased that for outward movement (k_OUT). Consequently, depolarization reduced the steady-state factor of accumulation.It is proposed that, as the protonated species of the substrates of uptake₂ is transported, the membrane potential is likely to provide the driving force for uptake₂. The fact that depolarization decreased k_IN and increased k_OUT agrees with this proposal, as do the magnitudes of the steady-state accumulation factors determined in Caki-I cells and perfused rat heart.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - MAO monoamine oxidase - NMN normetanephrine - OMDA O-methylated and deaminated metabolites Supported by the Deutsche Forschungsgemeinschaft (SFB 176) and the Dr. Robert Pfleger-Stiftung Send offprint requests to E. Schömig at the above address     

Expression of the extraneuronal monoamine transporter (uptake2) in human glioma cells

Tritiated methylphenylpyridinium ([³H]MPP⁺), a substrate of the neuronal and extraneuronal noradrenaline transporter (uptake₁ and uptake₂, respectively) and of the organic cation transporter (OCT1), was used to characterize the amine transport system of the established human glioma cell line SK-MG-1.Uptake of [³H]MPP⁺ (25 nM) into SK-MG-1 cells increased linearly with time for up to 15 min. Selective uptake₁ inhibitors (e.g. (+)oxaprotiline) or omission of Na⁺ or Cl^– ions did not affect [³H]MPP⁺ uptake, whereas uptake₂ inhibitors such as O-methyl-isoprenaline (OMI) or corticosterone as well as depolarizing concentrations of K⁺ or Ba²⁺ strongly reduced [³H]MPP⁺ uptake. Initial rates of OMI(100 M)-sensitive [³H]MPP⁺ uptake were saturable, with a K_m of about 17 M and a maximal rate of about 50 pmol/ (min × mg protein). IC₅₀ (or K_i) values for inhibition of [³H]MPP⁺ uptake by substrates and inhibitors of uptake₂ or OCTI were highly significantly correlated with published IC₅₀ values for inhibition of uptake₂ but not with corresponding values for inhibition of OCT1.The results presented here clearly demonstrate that human glioma cells express an uptake₂ transporter. Thus, glial cells in the human central nervous system endowed with this transporter are likely to contribute to the inactivation of neuronally released noradrenaline.     

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 mol/l amezinium or 15 mol/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 mol/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.Abbreviations COMT catechol-O-methyltransferase - DOMA 3, 4-dihydroxy-mandelic acid - DOPEG 3, 4–'dihydroxyphenylglycol - ECS extracellular space - FRL fractional rate of loss - IC ₅₀ inhibitor concentration that causes 50% inhibition - K _{m uptake} Michaelis or half-saturation constant for uptake - k _{M AO} rate constant for deamination - k _{out NA} rate constant for efflux of noradrenaline - MAO monoamine oxidase - MAO-Aa type A monoamine oxidase - MAO-B type B monoamine oxidase - T/M _NA tissue to medium ratio of noradrenaline - U-0521 3, 4-dihydroxy-2-methylpropiophenone - V _max maximal rate - v _st–st steady-state rate of metabolite formation Preliminary results of this study were presented to the 1993 Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Bryan-Lluka 1993).     

Inward transport of 3H-MPP+ in freshly isolated rat hepatocytes: evidence for interaction with catecholamines

1-Methyl-4-phenylpyridinium (MPP⁺), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is efficiently taken up and accumulated by rat hepatocytes. However, the nature of the mechanism(s) involved in the hepatic uptake of MPP⁺ remains partially unknown. The aim of the present study was to further characterize the hepatic uptake of ³H-MPP⁺, namely by investigating the interactions of catecholamines (which are also efficiently taken up by rat hepatocytes) with MPP¹ transport.The accumulation of ³H-MPP⁺ in isolated rat hepatocytes occurred through saturable and non-saturable mechanisms. The kinetics of the saturable component of ³H-MPP⁺ uptake was as follows: V_max = 181.3 ± 11.1 pmol mg protein^–1 min^–1 and K_m = 47.1 M (27.9, 66.3) (n = 5). The diffusion constant (in ml mg protein^–1 min^–1) for the non-saturable uptake of ³H-MPP⁺ was 0.00068 (0.00052, 0.00083) (n = 5). From the analysis of the time course of ³H-MPP⁺ accumulation at a substrate concentration of 100 nM ³H-MPP⁺, it was found that the rate constant of inward transport of ³H-MPP⁺ into hepatocytes (k_in) was 15.7 ± 3.8 l mg protein^–1 min^–1, the rate constant of outward transport of ³H-MPP⁺ from hepatocytes (k_out) was 0.077 ± 0.023 min^–1 and the equilibrium accumulation (A_max) of ³H-MPP⁺ was 20.2 ± 2.0 pmol mg protein^–1 (n = 36). Decynium22 (1,1-diethyl-2,2-cyanide; 1 M) significantly reduced k_in to 6.1 ± 1.8 l mg protein^–1 min^–1 (P < 0.05) and the equilibrium accumulation (A_max) of ³H-MPP⁺ to 9.6 ± 1.3 pmol mg protein^–1 (P < 0.005) (n = 36). ³H-MPP⁺ accumulation (in cells incubated with 200 nM ³H-MPP⁺) was sensitive to (–)-adrenaline, (–)-isoprenaline, (–)-dopamine, (±)-adrenaline and (–)-noradrenaline. The most potent catecholamine in inhibiting ³H-MPP⁺ uptake was (–)-adrenaline, with an IC₅₀ of 99 (22, 449) M (n = 6). (–)-Adrenaline competitively inhibited ³H-MPP⁺ uptake, as it significantly increased the K_m value of ³H-MPP⁺ uptake (to 125.4 M (63.6; 187.1); P < 0.02; n = 3) but did not change the V_max value. The cyanide-derivatives decynium22 and cyanine863 (1-ethyl-2-([1,4-dimethyl-2-phenyl-6-pyrimidinylidene]methyl)quinolinium), which inhibit uptake₂ as well as the apical type of the renal transporter for organic cations, potently inhibited ³H-MPP⁺ uptake with IC₅₀'s of 1.4 (0.4–5.3) (n = 6) and 6.5 (2.6–16) (n = 4) M, respectively. Under conditions of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) inhibition with either pargyline (500 M + Ro01-2812) (3,5-dinitropyrocatechol; 2 M) or pargyline (500 M) + U-0521(3,4-dihidroxy-2-methyl-propiophenone; l2 M)), (–)-adrenaline (up to 1 mM) had no inhibitory effect on the uptake of ³H-MPP⁺. Moreover, the uptake of ³H-MPP⁺ in the presence of pargyline + Ro 01-2812 was significantly lower (66.9 ± 30.4%; P < 0.05; n = 4) than in the absence of these compounds. Therefore, the effect of these MAO and COMT inhibitors on ³H-MPP⁺ uptake was examined. Interestingly enough, pargyline, Ro 01-2812 and U-0521 were found to inhibit the uptake of ³H-MPP⁺ (in cells incubated with 200 nM ³H-MPP⁺): 500 M pargyline, 2 M Ro 012812 and 100 M U-0521 decreased the accumulation of ³H-MPP⁺ to 38.1 ± 6.8 (n = 5), 60.5 ± 10.1(n = 7) and 71.3 ± 14.5 (n = 7) % of control, respectively.It is concluded that ³H-MPP⁺ is efficiently taken up by rat hepatocytes by a carrier-mediated mechanism sensitive to catecholamines, decynium22 and cy anine863, and to the enzyme inhibitors pargyline, Ro 01-2812 and U-0521.     

Extraneuronal uptake of noradrenaline in rabbit dental pulp: evidence of identity with uptake

Summary The extraneuronal removal and disposition of noradrenaline in rabbit dental pulp was examined in view of earlier evidence that the tissue possessed an extra-neuronal uptake process resembling neuronal uptake₁. Pulp, which had been depleted of sympathetic nerves by homolateral superior cervical ganglionectomy, was incubated in vitro with ³H-noradrenaline in low concentrations (0.025 or 0.18 mol/l). When the metabolising enzymes (monoamine oxidase, catechol-O-methyl transferase) were active, ³H retention by the denervated pulp, as indicated by the ³H content after the tissue had been washed for 30 min following incubation with ³H-noradrenaline, was less than 30% of that of the innervated pulp. When the enzymes were inhibited, retention rose to approximately 30% of that of the innervated pulp. Analysis of the time course of the ³H efflux indicated that the ³H-noradrenaline in the denervated pulp had accumulated in a single compartment characterised by a t_1/2 for efflux of several hours. Accumulation did not occur under Na⁺-free conditions, and was inhibited by desipramine (IC₅₀ < 0.03 mol/l) and by substrates of neuronal uptake₁. Mean IC₅₀, values of the latter were very similar to those for inhibition of neuronal uptake₁ and comprised (in mol/l): (+)amphetamine (0.29), dopamine (0.31), tyramine (0.39), (–)noradrenaline (0.70), (–)adrenaline (1.50), 5-hydroxytryptamine (20) and bretylium (35). Uptake₂ inhibitors were less active (O-methyl isoprenaline, IC₅₀ = 60 mol/l) than uptake₁ inhibitors, or were without inhibitory effects at the concentrations tested (hydrocortisone, 210 mol/l; 2-methoxy oestrone, 10 mol/l).The effects of Na⁺ omission, of (+)amphetamine, and of O-methylisoprenaline on ³H-normetanephrine formation (measured in the absence of catechol-O-methyl transferase inhibition) matched their effects on ³H-noradrenaline accumulation. The results provide strong support for the presence in rabbit dental pulp of extraneuronal uptake₁ which is linked with catechol-O-methyl transferase in the removal of noradrenaline. Send offprint requests to D. A. S. Parker at the above address     

Uptake of 3H-catecholamines by rat liver cells occurs mainly through a system which is distinct from uptake1 or uptake2

Isolated rat hepatocytes were incubated with 200 nmol/l ³H-(–)-noradrenaline or 50 nmol/l ³H(–)-adrenaline for 15 min, in Krebs-Henseleit solution at 37°C, gassed with 95% O₂ 5010 CO₂. Monoamine oxidase and catechol-O-methyl transferase were inhibited withpargyline (500 mol/l)and Ro 01-2812 (3,5-dinitropyrocatechol; 2 ol/l), respectively. Total radioactivity present in the cells, which corresponded mostly to intact ³H-amine, was measured.The content of ³H-noradrenaline increased with time of incubation, a plateau having been reached after 15 min of incubation. After 15 min of incubation,the cell: medium ratio for ³H-noradrenaline and ^3H-adrenaline was 0.6–0.7. Desipramine (an inhibitor of the neuronal uptake of catecholamines — uptake,; 1 mol/l) did not affect the uptake of either ³H-noradrenaline or ³H-adrenaline into hepatocytes. Corticosterone (an inhibitor of the extraneuronal uptake of catecholamines — uptake₂; 40 mol/l) slightly inhibited (by 28%) the uptake of ³H-adrenaline, and did not significantly reduce ³H-noradrenaline uptake. Probenecid (an inhibitor of the renal transport of organic anions; 100 mol/l) did not influence the amount of either ³H-noradrenaline or ³H-adrenaline in hepatocytes. Cyanine 863 (an inhibitor of the renal transport of organic cations; 10 mol/l) decreased by 62% the uptake of ³H-adrenaline into cells but did not significantly affect ³H-noradrenaline uptake. Bilirubin (a substrate of a hepatic transport for organic anions; 200 ol/l) produced a significant increase (50%) in the amount of ³H-noradrenaline and ³H-adrenaline present in the cells. When isolated hepatocytes were incubated in a sodium-free medium (sodium being replaced by choline or lithium) there was a very marked inhibition of ³H-noradrenaline and 3H-adrenaline uptake (by 85–97%). An increase in potassium content of the medium (from 6.6 to 50 mmol/l) did not affect the uptake of either ³H-amine into isolated cells.In conclusion, the uptake of catecholamines by isolated liver cells possesses characteristics that distinguish it from the classic uptake systems for catecholamines (uptake₁ and uptake₂): (1) it is sodium-dependent but not affected by desipramine; (2) it is only slightly sensitive to corticosterone and not affected by potassium-induced depolarization; (3) it is partially sensitive to cyanine 863. Moreover, the increase of ³H-amine content in the cells in the presence of bilirubin suggests the possibility of catecholamines being excreted from the hepatocytes through the bilirubin transporter.PhD student with a grant from JNICT (Programa Ciência)Abbreviations COMT catechol-O-methyl transferase - MAO monoamine oxidase - RTOC renal transport of organic cations Supported by Programa STRIDE (STRDA/P/SAU/259/92) Correspondence to: F. Martel at the above address     

Effect of thiamine on H-MPP uptake by Caco-2 cells

Recent studies on the intestinal uptake of the organic cation 1-methyl-4-phenylpyridinium (MPP⁺) showed that transport of this compound occurs through human extraneuronal monoamine transporter (hEMT). Moreover, it was recently described that alkaline phosphatase (ALP), an ecto-phosphatase anchored to the plasma membrane and able to dephosphorylate extracellular substrates or cell-surface proteins, is directly or indirectly involved in the modulation of MPP⁺ uptake by Caco-2 cells. The present study investigated a putative modulation of MPP⁺ intestinal apical uptake and ecto-ALP activity by thiamine (T⁺) and thiamine pyrophosphate (TPP, a T⁺ dietary precursor). For this purpose, we used Caco-2 cells, an enterocyte-like cell line derived from a human colonic adenocarcinoma, as an intestinal model. Ecto-ALP activity and N-[methyl-³H]-4-phenylpyridinium acetate (³H-MPP⁺) uptake were evaluated in intact Caco-2 cells. T⁺ and TPP were able to increase ecto-ALP activity, with an equal potency, and to decrease ³H-MPP⁺ apical uptake, with a similar potency. The effects of both compounds on ecto-ALP activity and ³H-MPP⁺ uptake were concentration-dependent. The results suggest that the effect of T⁺ and TPP on ecto-ALP activity may lead to inhibition of the intestinal absorption of other organic cations present in the diet. Another important conclusion is that the intestinal absorption of T⁺ may occur through hEMT, in Caco-2 cells.     

Inhibition of neuronal noradrenaline uptake (uptake1) and desipramine binding by N-ethylmaleimide (NEM)

Summary The inhibition by N-ethylmaleimide (NEM) of uptake₁ and desipramine binding was studied on clonal rat phaeochromocytoma cells (PC12 cells) in different experimental settings: (1) ³H-noradrenaline uptake into intact PC12 cells; (2) ³H-noradrenaline uptake into isolated PC12 plasma membrane vesicles; (3) ³H-desipramine binding to isolated PC12 plasma membrane vesicles.In plasma membrane vesicles, NEM inhibited ³H-desipramine binding and ³H-noradrenaline uptake with similar potency (the IC₅₀'s were 1.36 mmol/l and 1.04 mmol/l, respectively). However, in intact cells, NEM was about 75 times more potent in inhibiting ³H-noradrenaline uptake (IC₅₀ = 0.014 mmol/l). The increased potency of NEM in intact cells is probably due to an inhibition of the Na^{+/K +}-ATPase and not to a direct interaction with the noradrenaline carrier.The inactivation by NEM of ³H-desipramine binding to PC12 plasma membrane vesicles was irreversible. Both an inhibitor (cocaine, 1 mmol/l) and a substrate of uptake₁ (amezinium, 1 mmol/l) protected desipramine binding from inactivation.These results are compatible with the hypothesis of a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier.This study was supported by the Deutsche Forschungsgemeinschaft (SFB176) Send offprint requests to E. Schömig at the above address     

Uptake and metabolism of 3H-adrenaline and 3H-noradrenaline by isolated hepatocytes and liver slices of the rat

Summary Isolated rat hepatocytes were incubated with 0.05 mol/l or 0.2 mol/l ³H-(–)-noradrenaline or 0.05 mol/l ³H-(–)-adrenaline for 15 min and the content of amines as well as the formation of metabolites was measured.The removal Of both amines from the incubation medium was quantitatively similar, and mainly due to metabolism (which represented 96% of the removal of ³H-adrenaline and 98% of the removal of ³H-noradrenaline). O-methylation predominated for ³H-adrenaline: O-methylated and deaminated metabolites (³H-OMDA) and ³H-metanephrine (³H-MN) were the most abundant metabolites, accounting for 63% and 34% of total metabolite formation, respectively. Deamination predominated for ³H-noradrenaline: ³H-OMDA and ³H-dihydroxymandelic acid (³H-DOMA) were the most abundant metabolites, representing respectively 56% and 36% of total metabolite formation. The following activities of monoamine oxidase and catechol-O-methyl transferase were determined for ³H-noradrenaline: k_COMT 0.70±0.15 min^–1 and k_MAO 2.27±0.14 min^–1 In experiments with ³H-noradrenaline, inhibition of monoamine oxidase reduced the formation of ³H-OMDA and deaminated metabolites [³H-dihydroxyphenylglycol (³H-DOPEG) and ³H-DOMA] and increased the formation of ³H-normetanephrine (³H-NMN). Inhibition of catechol-O-methyl transferase, On the Other hand, decreased ³H-NMN and increased ³H-DOPEG formation. When both enzymes were inhibited, the formation of all metabolites was strongly reduced but surprisingly there was no accumulation of ³H-amines in the cells, as the cell: medium ratio for ³H-noradrenaline or ³H-adrenaline was about unity. In experiments with either ³H-noradrenaline or ³H-adrenaline, specific inhibitors of either uptake, or uptake₂ produced discrete effects, slightly decreasing the formation of ³H-OMDA and ³H-NMN or ³H-MN, and having no effect on ³H-amine content of the cells. Additional experiments were carried Out with rat liver slices incubated for 15 min with ³H-noradrenaline 0.2 mol/l. The pattern of metabolism of ³H-noradrenaline (³H-OMDA and ³H-DOMA were the most abundant metabolites) as well as the degree of metabolism of the amine removed from the incubation medium (91% of the removal) were similar to those of the isolated cells. Likewise, there was no accumulation of intact ³H-noradrenaline in the tissue. Moreover, the results obtained with enzyme inhibitors as wells as with uptake inhibitors were similar to those obtained with hepatocytes.In conclusion, isolated hepatocytes remove and metabolize catecholamines very efficiently, being one of the most active systems studied in this respect. Uptake₁ and uptake₂ are responsible for part of the removal of catecholamines by hepatocytes; the system(s) involved in the remaining removal seem(s) to be active, but possess(es) characteristics that do not allow us to characterize it (them) either as uptake₁ or uptake₂.Abbreviations COMT catechol-O-methyl transferase - DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MAO monoamine oxidase - MN metanephrine - NMN normetanephrine - OMDA O-methylated and deaminated metabolites (i.e., MOPEG = 4hydroxy-3-methoxyphenylglycol and VMA = 4-hydroxy-3-methoxymandelic acid) Supported by Programa STRIDE (STRDA/P/SAU/259/92)PhD student with a grant from JNICT (Programa Ciência) Correspondence to: F. Martel at the above address     

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-0-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 mol/l) and adrenaline (40 mol/l), both caused a rapid and marked increase in the efflux of noradrenaline, while noradrenaline (4 mol/l) had a similar effect on the efflux of dopamine. The ^uptake 1 inhibitors, imipramine (3 and 10 mol/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.Abbreviations COMT Catechol-O-methyltransferase - FRL Fractional rate of loss - K _m Michaelis or half-saturation constant - t _out rate constant for efflux - k _uptake rate constant for uptake - MAO monoamine oxidase - t _/12 half-time for efflux - U-0521 3,4-dihydroxy-2-methylpropiophenone - V _max maximal rate of uptake Preliminary results of this study were presented to the 1993 Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Scarcella et al. 1993).     

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 uptake, 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 uptake₁ in rat lungs and of uptake, 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/1 ³H-5-HT or ³H-noradrenaline for 2 min in the absence or presence of 1 mol/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 mol/l, did not affect 5-HT uptake in lungs perfused with 2 nmol/l ³H-5-HT for 2 min (uptake₁ inhibited), but 50 mol/l 5-HT inhibited noradrenaline uptake by 56% in lungs perfused with 2 nmol/l ³H-noradrenaline for 2 min (5HT 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 uptake, 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.Preliminary results of this study were presented to the December 1993 meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Paczkowski and Bryan-Lluka 1993).     

Comparison between uptake2 and rOCT1: effects of catecholamines, metanephrines and corticosterone

Active and specialized transmembrane transport systems are responsible for the functional inactivation of catecholamines. Uptake₂, the classical extraneuronal uptake system, and rOCT1, a recently cloned organic cation transporter, share a number of properties. The present study was undertaken to investigate putative differences between these two transporters that might clarify their relative physiological roles. Uptake of [³H]MPP⁺ ([³H]1-methyl-4-phenylpyridi-nium) by Caki-1 cells (to study uptake₂) and by primary cultured rat hepatocytes (to study rOCT1) was kinetically and pharmacologically characterized. In both cell types, [³H]MPP⁺ was avidly taken up and accumulated. All compounds tested (catecholamines, metanephrines and corticosterone) inhibited [³H]MPP⁺ uptake, albeit with different potencies. In Caki-1 cells, the ranking order of inhibitory potency was: (–)isoprenaline > (–)adrenaline >> (–)noradrenaline > dopamine. Metanephrine and normetanephrine were equipotent. Corticosterone had an IC₅₀ of 102 nM. In cultured hepatocytes, the ranking order of inhibitory potency was: (–)isoprenaline > dopamine > (–)adrenaline >> (–)noradrenaline. Metanephrine was about seven times more potent than normetanephrine. Corticosterone had an IC₅₀ of 72 μM, being about 700-fold less potent in inhibiting rOCT1 than uptake₂. The results showed that uptake₂ and rOCT1 can be clearly distinguished on a functional basis. On the one hand, uptake₂ prefers adrenaline among the endogenous catecholamines, whereas rOCT1 has similar affinity for adrenaline and dopamine. On the other hand, corticosterone and normetanephrine are significantly more potent in inhibiting uptake₂ than rOCT1. The results are compatible with a possible physiological role of corticosteroids in the modulation of adrenaline effects in tissues equipped with uptake₂, without significant interference with the hepatic and renal excretion of catecholamines. Received: 2 October 1998 / Accepted: 23 December 1998     

Transport of small organic cations in the rat liver

The kidneys and the liver are the principal organs for the inactivation of circulating organic cations. Recently, an organic cation transporter (OCT1) has been cloned from rat kidney. In order to answer the question whether OCT1 is involved also in hepatic uptake of organic cations, the pharmacological characteristics of organic cation transport in hepatocytes were compared to the characteristics of transiently expressed OCT1.Primary cultures of rat hepatocytes avidly accumulated the small organic cation ³H-1-methyl-4-phenylpyridinium (³H-MPP⁺). At equilibrium, the hepatocytes accumulated ³H-MPP⁺ 56-fold. Initial rates of specific ³H-MPP⁺ transport in hepatocytes were saturable. The half-saturating concentration was 13 mol/l. ³H-MPP⁺ transport was sensitive to quinine (K_i = 0.79 mol/l) and cyanine863 (K_i = 0.097 µmol/l). Quinine and cyanine863 are known inhibitors of type I hepatic transport of cationic drugs and of renal excretion of organic cations, respectively. To compare the functional characteristics of ³H-MPP⁺ transport in hepatocytes with those of OCT1, OCT1 has been heterologously expressed and characterized in a mammalian cell line (293 cells). Initial rates of ³H-MPP⁺ transport were saturable, the K_m being 13 mol/l. The rank order of inhibitory potencies of various inhibitors was almost identical in hepatocytes and 293 cells transiently transfected with OCT1. There was a positive correlation between the K_i's for the inhibition of ³H-MPP⁺ transport in isolated hepatocytes and transfected 293 cells (r = 0.85; P<0.01; n = 8).The results indicate that OCT1 is functionally expressed not only in the kidney but also in hepatocytes where it is responsible for the transport of small organic cations which, in the past, have been classified as type I substrates.     

β-Endorphin-(1-27) is a naturally occurring antagonist of the reinforcing effects of opioids

Summary The influence of inhibitors of metabolism and uptake of noradrenaline on the ³H-noradrenaline removal from the perfusion fluid by the isolated rat liver was studied. Livers were perfused with 3 nmol/l ³H-noradrenaline and ³H-noradrenaline and ³H-metabolites were determined in effluent, liver and bile. After the perfusion with 14,900 ±920 dpm · g^–1 · min^–1 during 90 min, cumulative removal of tritium was 323,574 ± 63,103 dpm/g. ³H-metabolites recovered from the liver after 90 min perfusion represented 71.1 ± 9.0% of total metabolite formation. Only the OMDA-fraction appeared in the perfusate; its approach to steady state of efflux was slow. The inhibition either of MAO or COMT changed neither the total removal of tritium nor the ³H-metabolites recovered from the liver. Cocaine (10 mol/l) reduced the accumulation of ³H-noradrenaline in the liver. The uptake₂ inhibitor corticosterone (30 mol/l) diminished total removal of tritium and the ³H-metabolites recovered from the liver without changing the accumulation of ³H-noradrenaline. The hypothesis of two different compartments, one responsible for the metabolism and the other for the accumulation of the amine is discussed.Abbreviations NA noradrenaline - NMN normetanephrine - OMDA O-methylated deaminated metabolites - MAO monoamine oxidase - COMT catechol-O-methyl transferase Send offprint requests to M. C. Rubio     

Release of noradrenaline from isolated rat tail artery induced by bafilomycin A1

The macrolide antibiotic bafilomycin A₁, a selective inhibitor of the vesicular H⁺-transporting ATPase, increased irreversibly the overflow of 3,4-dihydroxyphenylethylene glycol from isolated segments of the rat tail artery. Maximum increase in the overflow was produced by exposing the tissues to 0.5 mol/l bafilomycin As. Unless the Na^–-dependent neuronal amine carrier (uptake₁) was inhibited, overflow of noradrenaline was below the detection limit. The bafilomycin As-induced increase in overflow of noradrenaline from tissues with inhibited uptakes was accompanied by a significant decrease in the (noradrenaline overflow:glycol overflow) ratio. Unlike reserpine and tetrabenazine, the antibiotic did not alter the (noradrenaline overflow:glycol overflow) ratio in arteries incubated in Ca²⁺-free, 120 mmol/1 K⁺ medium.Bafilomycin A₁ increased overflow of noradrenaline and normetanephrine from tissues with inhibited monoamine oxidase. Inhibitors of extraneuronal catecholamine transport (uptake₂), corticosterone, 3-O-methylisoprenaline and 1,1-diethyl-2,2-cyanine, suppressed overflow of normetanephrine while increasing that of noradrenaline. Further increase in overflow of noradrenaline was produced by concomitant inhibition of uptake₁. A similar effect was observed in tissues previously exposed to phenoxybenzamine. After exposure to bafilomycin As, tyramine and (+) amphetamine (10 mol/l) were equally effective in increasing overflow of noradrenaline from tissues with inhibited monoamine oxidase into corticosterone-containing medium.Bafilomycin A₁ promotes leakage of noradrenaline from storage vesicles without affecting its conversion to 3,4-dihydroxyphenylethylene glycol. When uptake₁ is inhibited, axoplasmic noradrenaline can be translocated effectively across the axonal membrane by the diffusional efflux. When uptakes is inhibited, spontaneous quantal release contributes significantly to overflow of noradrenaline into normal media. The diffusional efflux of noradrenaline is unaffected by inhibitors of uptake₂. Even at highly elevated concentrations of axoplasmic noradrenaline, the uptake₁-mediated influx of noradrenaline exceeds the uptake₁-mediated efflux. Enhancement of noradrenaline overflow from tissues with inhibited monoamine oxidase by indirectly acting sympathomimetic amines depends primarily on their ability to induce leakage of the transmitter from storage vesicles rather than its translocation across the axonal membrane.     

The acceleration of the extraneuronal efflux of 3H-(±)-isoprenaline induced by high extracellular potassium

Summary After loading of the extraneuronal tissues of the perfused rat heart with ³H-isoprenaline the elevation of extracellular K⁺ concentration (from 2.7 to 15 mmol/1) in the perfusion solution about doubled the rate constant for efflux of the ³H-amine. As this increase was not seen in the presence of 100 mol/13-O-methyl-isoprenaline (OMI, a potent inhibitor of the uptake₂-carrier), it is concluded that the change in the concentration of K⁺ modulates OMI-sensitive outward transport of ³H-isoprenaline by uptake₂, not the diffusional efflux of the amine.Abbreviations MAO monoamine oxidase - COMT catechol-O-methyltransferase - OMI 3-O-methyl-isoprenaline - U-0521A 3,4-dihydroxy-2-methyl propiophenone     

The effect of (±)-dobutamine on adrenergic nerve endings

Summary Possible effects of (±)-dobutamine on adrenergic nerve endings were determined in experiments with ghosts of bovine chromaffin granules, with rat phaeochromocytoma (PC-12) cells and with the rat vas deferens. Dobutamine inhibited the vesicular uptake of a mixture of 70% adrenaline + 30% ³H-noradrenaline into ghosts, with an IC₅₀ of 1.7 mol/l. Dobutamine inhibited uptake, of ³H-noradrenaline in PC-12 cells (with an IC₅₀ of 0.38 mol/l) without being a substrate. However, dobutamine easily entered PC-12 cells by diffusion. After inhibition of MAO, COMT and vesicular uptake dobutamine (15 and 45 mol/l) released tritium from rat vasa deferentia preloaded with ³H-noradrenaline. Equi-inhibitory concentrations of dobutamine and desipramine (against uptake₁) were equireleasing. On the other hand, when MAO and vesicular uptake were intact, dobutamine (15 mol/l) increased the efflux of tritium from preloaded vasa deferentia much more than did an equi-inhibitory concentration of desipramine. Most of the released tritium was then ³H-DOPEG.Dobutamine is a potent inhibitor of uptake₁ as well as of vesicular uptake; moreover, it easily diffuses into adrenergic nerve endings. Hence, it blocks the neuronal and the vesicular re-uptake of noradrenaline; consequently, when MAO and vesicular uptake are intact, dobutamine increases the net leakage of noradrenaline from the storage vesicles, thereby leading to an efflux of deaminated metabolites. However, dobutamine is virtually unable to release noradrenaline into the extracellular space.Abbreviations COMT catechol-O-methyl transferase - DOPEG dihydroxyphenylglycol - DOMA dihydroxymandelic acid - MAO monoamine oxidase Supported by the Deutsche Forschungsgemeinschaft (Gr 490/5 and SFB 176) Send offprint requests to P. Fischer at the above address     

The mechanism of the 3H-noradrenaline releasing effect of various substrates of uptake1: multifactorial induction of outward transport

Summary The mechanism of action of indirectly acting sympathomimetic amines was studied in the rat vas deferens, after inhibition of vesicular uptake (by reserpine), of MAO (by pargyline) and of COMT (by U-0521). 1. K _m-values for the neuronal uptake of 12 substrates were determined as the IC₅₀ of the unlabelled substrate inhibiting the initial rate of neuronal uptake of 0.2 mol/l ³H-(–)-noradrenaline. The IC₅₀ ranged from 0.35 mol/l (for (+)-amphetamine) to 44.3 mol/l (for 5-HT). The V _max (determined for 8 substrates) was substrate-dependent. 2. Tissues were loaded with 0.2 mol/l ³H-(–)-noradrenaline and then washed out with amine-free solution. All 12 substrates of uptake₁, induced an outward transport of ³H-noradrenaline, and equieffective concentrations were positively correlated with K _m. Moreover, the EC₅₀ for release greatly exceeded K _m. It is proposed that this discrepancy between EC₅₀ and K _m is indicative of the fact that at least four factors (each one in strict dependence on K _m) contribute to the initiation of outward transport of ³H-noradrenaline: a) the appearance of the carrier on the inside of the axonal membrane (facilitated exchange diffusion), b) the co-transport of Na⁺, c) the co-transport of Cl^– (both lowering the K _m for ³H-noradrenaline at the inside carrier), and d) inhibition of the re-uptake of released ³H-noradrenaline (through competition for the outside carrier). 3. At least for amezinium, V _max. appears to limit the maximum rate of outward transport. 4. For some substrates (especially for the highly lipophilic ones) bell-shaped concentration-release curves were obtained. Apparently, inward diffusion of the substrates can lead to partial saturation of the inside carrier. Moreover, if release is expressed as a FRL (fractional rate of loss), loading with 37 mol/l ³H-(–)-noradrenaline decreased the releasing effect of various substrates. In this case the inside carrier appears to be partially saturated by the high axoplasmic concentration of ³H-noradrenaline. 5. Very high concentrations (especially of highly lipophilic substrates) were able to induce an additional intraneuronal release mechanism, presumably by increasing the pH inside storage vesicles.Abbreviations COMT catechol-O-methyl transferase - DOMAA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - FRL fractional rate of loss (rate of efflux/tissue tritium content) - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase - OM-fraction sum of all O-methylated metabolites of noradrenaline, deaminated or not This study was supported by the Deutsche Forschungsgemeinschaft (Bo 521, Tr 96 and SFB 176). Some of the results were presented to the German Pharmacological Society (Langeloh 1986)A. L. was the recipient of a fellowship of the Humboldt-Foundation Send offprint requests to: U. Trendelenburg     

Evidence for uptake2-mediated O-methylation of noradrenaline in the human amnion FL cell-line

Summary The uptake and metabolism of ³H-noradrenaline has been examined in the FL cell-line derived originally from human amnion. Cell cultures metabolised ³H-noradrenaline (1.0 mol/l) to ³H-normetanephrine and, to a lesser extent, to metabolites (not distinguished) of the O-methylated deaminated fraction; primary deaminated metabolites were not detected. ³(H-normetanephrine formation a) was not saturable in the noradrenaline concentration range 0.2–150 mol/l, b was decreased to 20%–30% of control levels by uptake₂ inhibitors (O-methylisoprenaline, 20 and 100 mol/l; cimetidine, 10 mol/l; hydrocortisone, 200 mol/l) and c, was almost insensitive to uptake₁ inhibitors (cocaine, 30 mol/l; desipramine, 3 mol/l).Uptake of noradrenaline was manifested after 30 minutes as a 6-fold increase in the cell content of the amine following inhibition of catechol-O-methyl transferase, either alone or in conjunction with inhibition of monoamine oxidase. Uptake was decreased maximally to 40% of control levels by O-methylisoprenaline. IC₅₀ values for inhibition of the O-methylisoprenaline-sensitive component of uptake were (in mol/l): corticosterone (0.3), papaverine (1.1), O-methylisoprenaline (3.0), cimetidine (6.0), (–)noradrenaline (460), and tetraethylammonium (2230). Except for the last agent, for which a comparative value is not available, the IC₅₀'s are in good agreement with those for inhibition of uptake₂ in the Caki-1 cell-line reported by other investigators.The component of uptake resistant to O-methylisoprenaline was depressed by papaverine (a 50% decrease at 50 mol/l), but was not affected by the other uptake₂ inhibitors or by cocaine (30 mol/l).It is concluded that the FL cell possesses an extraneuronal metabolising system very similar to the system in tissues such as heart and smooth muscle where transport of noradrenaline into the cell by uptake₂ is followed by rapid O-methylation via catechol-O-methyl transferase. The only difference appears to be the absence of saturation of ³H-normetanephrine formation in the FL cell at low micromolar concentrations of ³H-noradrenaline. The presence of a second uptake process is suggested by the inhibitory effect of papaverine on uptake resistant to O-methylisoprenaline; lack of effect of cocaine implies that this second process is not uptake,.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylethylene glycol - MAO monoamine oxidase - NMN normetanephrine - OMDA O-methylated and deaminated metabolite fraction - OMI 3-O-methylisoprenaline - TEA tetraethylammonium Correspondence to I. S. de la Lande at the above address