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).     

The extraneuronal transport mechanism for noradrenaline (uptake2) avidly transports 1-methyl-4-phenylpyridinium (MPP+)

Summary The corticosterone-sensitive extraneuronal transport mechanism for noradrenaline (uptake₂) removes the neurotransmitter from the extracellular space. Recently, an experimental model for uptake₂ has been introduced which is based on tissue culture techniques (human Caki-1 cells). The present study describes some properties of uptake₂ in Caki-1 cells and introduces a new substrate, i.e., 1-methyl-4-phenylpyridinium (MPP⁺).Experiments on Caki-1 cells disclosed disadvantages of tritiated noradrenaline as substrate for the investigation of uptake₂. The initial rate of ³H-noradrenaline transport [k_in = 0.58 l/(mg protein · min)] was low compared with other cellular transport systems and intracellular noradrenaline was subject to rapid metabolism (k_{O-methylation} = 0.54 min^–1). The neurotoxin MPP⁺ was found to be a good substrate of uptake₂. Initial rates of specific ³H-MPP⁺ transport into Caki-1 cells were saturable, the K_m being 24 mol/l and the V_max being 420 pmol/(mg protein · min). The rate constant of specific inward transport was 34 times higher [19.6 mol/l (mg protein · min)] than that of ³H-noradrenaline. The ratio specific over non-specific transport was considerably higher for ³H-MPP⁺ (12.6) than for ³H-noradrenaline (3.0). ³H-MPP⁺ transport into Caki-1 cells was inhibited by various inhibitors of uptake₂. The highly significant positive correlation (p < 0.001, r = 0.986, n = 7) between the IC₅₀'s for the inhibition of the transport of ³H-noradrenaline and ³H-MPP⁺, respectively, proves the hypothesis that MPP⁺ enters Caki-1 cells via uptake₂. ³H-MPP⁺ is taken up via uptake₂ not only by Caki-1 cells but also by the isolated perfused rat heart which is another established model of uptake₂.Tritiated MPP⁺ is a new and convenient tool for the investigation of uptake₂. The rate constant for inward transport, the factor of accumulation and the ratio specific over non-specific transport are considerably higher for ³H-MPP⁺ than for ³H-noradrenaline. In uptake studies with ³H-MPP⁺ inhibition of intracellular noradrenaline-metabolizing enzymes is not necessary. In tissues and tissue cultures which possess fewer uptake₂ carriers than Caki-1 cells or the rat heart, the identification and characterization of uptake₂ can be expected to be greatly facilitated by the use of ³H-MPP⁺.Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to H. Russ at the above address     

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     

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     

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     

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     

The release of3H-noradrenaline by p- and m-tyramines and -octopamines,and the effect of deuterium substitution in α-position

Summary The³H-noradrenaline-releasing effects of p- and m-tyramines and -octopamines, either deuterated or not, were studied in isolated vasa deferentia of the rat (COMT inhibited and calcium-free solution in all experiments). K _m, for uptake₁ was higher for octopamines than for tyramines, but not increased by the introduction of deuterium in -position, except for (probably contaminated) deuterated p-octopamine. Other tissues were preloaded with³H-noradrenaline. After inhibition of vesicular uptake and MAO equi-releasing concentrations of the eight amines were strictly correlated withK _m, they were 6 to 7 times higher for unsubstituted octopamines than for corresponding tyramines. When only MAO (but not vesicular uptake) was inhibited, this difference decreased to about 4-fold, but the releasing potency of the deuterated amines (relative to their parent amines) remained unchanged (except for p-octopamine). When vesicular uptake and MAO were intact, unsubstituted octopamines were only 1.5 to 2.2 times less potent than the corresponding tyramines. Analysis of the efflux of³H-DOPEG confirmed that this gain in the relative potencies of octopamines is due to their increased ability to mobilize vesicular 3H-noradrenaline; moreover, deuterated amines as well were then better mobilizers than were their parent amines.It is concluded that, provided vesicular uptake is intact, the introduction of a \-OH-group enhances the ability of indirectly acting sympathomimetic amines to mobilize vesicular noradrenaline; the introduction of deuterium in -position, on the other hand, enhances this mobilizing effect exclusively when MAO is intact.Abbreviations used here COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - MAO monoamine oxidase - OM-fraction column chromatographic fraction containingall O-methylated metabolites Supported by the Deutsche Forschungsgemeinschaft (SFB 176)     

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     

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     

The mechanism of the 3H-noradrenaline releasing effect of various substrates of uptake1: role of monoamine oxidase and of vesicularly stored 3H-noradrenaline

Summary The mechanism of action of indirectly acting sympathomimetic amines was studied in vasa deferentia of unpretreated rats (COMT inhibited), preloaded with ³H-noradrenaline. 1. Concentration-release curves were obtained for 12 unlabelled indirectly acting amines. From differences between these results and those in an accompanying report (involving tissues from rats pretreated with reserpine and pargyline), it is concluded that a mobilisation of vesicular ³H-noradrenaline is required for high and sustained rates of outward transport of ³H-noradrenaline from intact adrenergic varicosities. 2. Experiments with a reserpine-like compound (Ro 4-1284) supported the view that a mobilisation of vesicular ³H-noradrenaline is required for substantial release. 3. An atypical time course of release and abnormally high rates of release were observed in the presence of excessive concentrations of (+)-amphetamine. Such atypical effects are ascribed to the ability of basic amines to increase the intravesicular pH. 4. Analysis of the ratio NA/DOPEGG (rate of efflux of ³H-noradrenaline/rate of efflux of ³H-DOPEGG) indicated that the inward transport (by uptake,) of substrates of MAO fails to achieve axoplasmic concentrations which saturate MAO. Inhibition (or saturation) of MAO is not a prerequisite for the initiation of outward transport. 5. A larger fraction of vesicular ³H-noradrenaline is accessible to equireleasing concentrations of (+)-amphetamine (an inhibitor of MAO) than of tyramine (a substrate of MAO). 6. From the present and the accompanying report it is concluded that substantial and sustained indirect sympathomimetic effects are to be expected for substrates of uptake₁ which additionally mobilise vesicular noradrenaline. However, this mobilisation does not seem to involve a change in intravesicular pH, except at excessive concentrations.Abbreviations: COMT catechol-O-methyl transferase - DOMAA dihydroxymandelic acid - DOPEGG 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 (Tr 96 and SFB 176). Some of the results were presented to the German Pharmacological Society (Langeloh 1986)The author was the recipient of a fellowship of the Humboldt-Foundation Send offprint requests to U. Trendelenburg     

Mechanisms of the release of 3H-noradrenaline by dimethylphenylpiperazinium (DMPP) in the rat vas deferens

Summary In the rat vas deferens, DMPP is a substrate of uptake, (K_rn = 11.5 mol/I). After block of vesicular uptake, monoamine oxidase and catechol-O-methyl transferase, after loading of the tissue with ³H-noradrenaline, and in calcium-free solution (i. e., when axoplasmic ³H-noradrenaline levels were high and when depolarization-induced exocytotic release was impossible), DMPP induced a pronounced outward transport of ³H-noradrenaline. On the other hand, when, in similar experiments, vesicular uptake and monoamine oxidase were intact (i.e., when axoplasmic ³H-noradrenaline levels were low), DMPP induced very little outward transport of ³H-noradrenaline. This discrepancy indicates that DMPP has little ability to mobilize vesicularly stored ³H-amine.When the medium contained calcium (catechol-O-methyl transferase inhibited, all other mechanisms intact), 100 (but not 10) mol/l DMPP induced a hexamethonium-sensitive release of ³H-noradrenaline of short duration. Hence, in the presence of extracellular calcium, 100 mol/l DMPP elicits exocytotic release via activation of hexamethonium-sensitive nicotinic acetylcholine receptors.DMPP inhibits the monoamine oxidase of rat heart homogenate with an IC₅₀ of about 100 mol/l.Abbreviations COMT catechol-O-methyl transferase - DMPP dimethylphenylpiperazinium - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - MAO monoamine oxidase - NMN normetanephrine - OM-fraction column chromatographic fraction containing all O-methylated ³H-metabolites - OMDA fraction containing O-methylated and deaminated metabolites Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to U. Trendelenburg at the above address     

Evidence for saturation of catechol-0-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/1) 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.Further experiments to examine the kinetics of O-methylation of noradrenaline and adrenaline (MAO inhibited) showed that the O-methylation of these amines in the lungs was predominantly saturable, with half-saturation occurring at concentrations (9.8 nmol/I and 19.4 nmol/l, respectively) that were two orders of magnitude lower than those required to half-saturate uptake₁ of the amines. Saturation of O-methylation by these low concentrations of noradrenaline (1) provides the explanation for the change in the metabolic profile of noradrenaline described above and (ii) appears to occur because V_{max uptake} V_{max COMT} for the metabolizing system consisting of non-neuronal uptake₁ + COMT in the lungs, as has been described previously for the system consisting of uptake₂ + COMT in extraneuronal sites in rat heart. The results show that the metabolic profile of catecholamines in the pulmonary circulation will reflect that occurring at physiological levels only if studies are carried out with very low amine concentrations.Abbreviations COMT Catechol-O-methyltransferase - DOMA 3,4-dihydroxymandelic acid - DOPEG 3 4-dihydroxyphenylglycol - ECS Extracellular space - HSOC Half-saturating outside concentration - K_{m uptake} Half-saturation constant for uptake - k_COMT Rate constant for O-methylation - k_MAO Rate constant for deamination - k_{out NA} Rate constant for efflux of noradrenaline - MAO Monoamine oxidase - MB-COMT Membrane-bound - COMT NMN Normetanephrine - OMDA O-methylated deaminated metabolites - S-COMT Soluble COMT - T/M_NA Tissue to medium ratio of noradrenaline - U-0521 3,4-dihydroxy-2-methylpropiophenone - V_max Maximal rate of uptake or O-methylation - V_st-st Steady-state rate of metabolite formation - V_uptake Rate of uptake Preliminary results of part of this study were presented to the Seventh Meeting on Adrenergic Mechanisms, Porto, Portugal (Bryan 1990)     

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     

The uptake and metabolism of 3H-catecholamines in rat cerebral cortex slices

Summary The accumulation and metabolism of ³H-catecholamines were studied in cerebral cortex slices obtained from rats pretreated with reserpine, during 30 min of incubation with 50 nmol/l of the ³H-amines. In some experiments neuronal uptake (uptake,) was inhibited by the presence of 0.3 mol/l desipramine, in others COMT was inhibited by 30 mol/l U-0521. When both MAO and COMT were intact, most of the metabolism of ³H-noradrenaline was neuronal (i. e., desipramine-sensitive). For ³H-adrenaline rates of neuronal metabolism were much lower than for ³H-noradrenaline, non-neuronal O-methylation accounting for about 50% of total metabolism. Rates of metabolism of ³H-dopamine were similar to those of ³H-noradrenaline, but with a predominance of non-neuronal metabolism, which involved O-methylation and deamination. Under these conditions, very little ³H-catecholamine was recovered from the tissues; moreover, desipramine tended to increase tissue levels. Hence, tissue content then appears to partly reflect extracellularly distributed ³H-amines. After block of MAO rates of metabolism of ³H-noradrenaline and ³H-dopamine were greatly reduced, and tissue levels were increased. Desipramine now antagonized the accumulation of ³H-amines in the tissue, while U-0521 increased it. Rates of O-methylation (in the presence of desipramine) increased in the order ³H-noradrenaline < ³H-dopamine. It is concluded that neuronal uptake is associated with MAO only, and rates of neuronal deamination increased in the order: ³H-adrenaline < ³H-dopamine « ³H-noradrenaline. Non-neuronal uptake is associated with both, COMT and MAO, and rates of non-neuronal metabolism increased in the order: ³H-adrenaline < ³H-noradrenaline « ³H-dopamine.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPAC dihydroxyphenylacetic acid - DOPEG dihydroxyphenylglycol - DOPET dihydroxyphenylethanol - HVA homovanillic acid - MAO monoamine oxidase - MN metanephrine - MOPEG methoxyhydroxyphenylglycol - MOPET methoxyhydroxyphenylethanol - NMN normetanephrine - 3-OMT 3-O-methyl-tyramine - VMA vanillylmandelic acid Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to U. Trendelenburg at the above addresswith the technical assistance of M. Babl     

Extraneuronal uptake and O-methylation of 3H-adrenaline in the rabbit aorta

Summary The influence of uptake₂ inhibitors on the Omethylation and accumulation of ³H-adrenaline by the isolated rabbit aorta was studied. Strips were incubated with 0.05 mol/l ³H-(–)-adrenaline during 15 min. Monoamine oxidase and uptake, were inhibited and the ³H-adrenaline present in the tissue was measured as well as the metabolites found in the tissue and in the incubation fluid. In another series of experiments, monoamine oxidase, uptake₁ and catechol-O-methyl transferase (COMT) were inhibited, and tritium accumulation was measured in the tissue.When COMT was inhibited, inhibitors of uptake₂ produced a maximal reduction of ³H-adrenaline accumulation that did not exceed 50%. When COMT was in tact, inhibitors of uptake₂ diminished total ³H-removal and, more markedly, O-methylation and concomitantly increased the tissue content of ³H-adrenaline.Mineralocorticoids (corticosterone and deoxycorticosterone acetate) inhibited ³H-adrenaline uptake (when COMT was inhibited) and ³H-metanephrine formation (when COMT was functional) as effectively as did sexual steroids (17--oestradiol, progesterone and testosterone); hydrocortisone (hemisuccinate or phosphate) had no effect (for concentrations up to 120 mol/l).At the end of the incubation some strips were washed out with amine-free solution. Compartmental analysis of the efflux showed that the amine had distributed into three extraneuronal compartments (compartment I, II and III, with half times of 0.4, 4 and 15 min, respectively). Corticosterone (120 mol/l). decreased the amount of ³H-adrenaline in compartment III and simultaneously increased the amount of the amine in compartment I (extracellular space).The extraneuronal accumulation of ³H-adrenaline in rabbit aorta can be only partially ascribed to uptake₂, as already stated by other authors; our results clearly show that inhibition of uptake₂ increases the amount of ³H-adrenaline in the extracellular space, i. e., uptake₂ creates a concentration gradient for ³H-adrenaline in the extracellular space; elastin and collagen represent very probably the site of binding of ³H-adrenaline which is independent of uptake₂.Abbreviations COMT Catechol-O-methyltransferase - DOCA deoxycorticosterone acetate - DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - MAO monoamine oxidase - OMDA O-methylated and deaminated metabolites - MOPEG methoxyhydroxyphenylglycol - VMA methoxyhydroxymandelic acid A preliminary account of some of the results was presented to the 7th International Catecholamine Symposium in Amsterdam. Supported by Instituto Nacional de Investigação Cientffica (INIC, FmP1)PhD student with a grant from JNICT (Programa Ciência)Correspondence to F. Martel 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     

The sensitivity of adrenergic varicosities to the 3H-noradrenaline-releasing effect of potassium

Summary After the loading of incubated, homogeneously innervated tissues with ³H-noradrenaline (monoamine oxidase and catechol-O-methyl transferase inhibited, calcium-containing solution) high K⁺ released the ³H-amine from adrenergic varicosities. In paired experiments the sensitivity of rat atria to high K⁺ exceeded that of vasa deferentia.In the rat vas deferens the releasing effect of high K⁺ was enhanced by drugs or procedures which induce a carrier-mediated outward transport of ³H-noradrenaline, i.e., by ouabain, by glucose deprivation and by hypoxia. — In the presence of extracellular calcium desipramine failed to affect the releasing effect of high K⁺ (except in the absence of glucose or during hypox1a), but in the absence of calcium desipramine reduced it. Apparently, whenever the axoplasmic levels of ³H-noradrenaline are increased, high K⁺ is able to induce some carrier-mediated outward transport of the 3H-amine.It is suggested that organ differences with respect to the sensitivity to high K⁺ may well be due to hypoxia (plus some lack of glucose) of those varicosities that had been loaded with ³H-noradrenaline. The risk of storage of ³H-noradrenaline in hypoxic varicosities appears to be greater in incubated than in perfused organs, and in the former it is greater in sparsely than in densely innervated tissues.Abbreviations COMT catechol-O-methyl transferase - FRL fractional rate of loss - MAO monoamine oxidase Supported by the Dr. Robert Pfleger-Stiftung and the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to U. Trendelenburg 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).     

The uptake and O-methylation of 3H-(±)-isoprenaline in rat cerebral cortex slices

Summary The O-methylation and accumulation of ³H-isoprenaline in slices of the rat cerebral cortex were studied before and after inhibition of COMT. 1. Inhibition of COMT by mol/l U-0521 virtually abolished the O-methylation and increased the accumulation of ³H-isoprenaline; hence, there is evidence for the existence of a central O-methylating system (with a transport mechanism and intracellular COMT). 2. Experiments were carried out with selective uptake inhibitors for uptake, (cocaine and desipramine) or uptake2 (corticosterone and OMI), with phenoxybenzamine (known to inhibit both carriers) and with changes in the ionic composition of the incubation medium. They revealed that the central carrier differed from both, uptake, and uptake2, although exhibiting some resemblance with uptake₂ (lack of dependence on Na⁺ and Cl^–, sensitivity to K⁺ and phenoxybenzamine, ability to transport ³H-isoprenaline). 3. Although the central carrier was rather sensitive to inhibition by beta-adrenoceptor antagonists (propranolol, carteolol), the effect of propranolol was not stereoselective; hence, beta-adrenoceptors do not seem to be involved. 4. Virtually identical IC₃₀-values were obtained for inhibitors, when determined with or without inhibition of COMT. Only OMI was found to inhibit COMT as well as the central transport system; hence it was more potent in inhibiting the O-methylation than the accumulation of ³H-isoprenaline. 5. IC₅₀-values (against initial rates of accumulation of ³H-isoprenaline; COMT inhibited) were determined for various substrates and inhibitors of peripheral uptake₂. There was no correlation with the IC₅₀-values determined earlier for uptake2 in rat heart (Grohmann and Trendelenburg 1984). 6. Unlabelled catecholamines half saturated the intracellular COMT when slices were incubated with 0.22 mol/l [(±)-dobutamine] to 4.9 mol/l [(–)-noradrenaline]. As the presence of unlabelled catecholamines increased tissue levels of ³H-isoprenaline, catecholamines are substrates of the central carrier. 7. The carrier of the central O-methylating system differs from uptake₂ of peripheral organs, although it resembles the peripheral carrier in some respects.Abbreviations COMT catechol-O-methyl transferase - DOPEG dihydroxyphenylglycol - MAO monoamine oxidase - OMI 3-Omethyl-isoprenaline Supported by the Deutsche Forschungsgemeinschaft (Tr 96 and SFB 176) and by a scholarship of the Royal Society for V. G. Wilson. Some of the results were presented to the British Pharmacological Society (Trendelenburg and Wilson 1986)     

The handling of five amines by the extraneuronal deaminating system of the rat heart

Summary The handling of five amines by the extraneuronal deaminating system was studied in perfused hearts of rats (pretreated with reserpine; COMT and neuronal uptake inhibited). Hearts were perfused with 50 nmol/l ³H-noradrenaline for 30 min, in the presence of increasing concentrations of unlabelled (–)-adrenaline, (–)-noradrenaline, dopamine, tyramine and 5-HT. IC₅₀'s were determined as those concentrations of unlabelled amines which halved the steady-state rate of deamination of ³H-noradrenaline. After correction for changes in the tissue/medium ratio for ³H-noradrenaline, half-saturating outside concentrations were obtained. They increased in the order (–)-adrenaline (15 mol/l) — tyramine — dopamine — noradrenaline —5-HT (53 mol/l). The V _max for extraneuronal deamination was determined for ³H-(–)-adrenaline, ³H-(–)-noradrenaline and ³H-dopamine, as well as (by HPLC and electrochemical detection) for tyramine and 5-HT. It was low for (–)-adrenaline, intermediate for (–)-noradrenaline, dopamine and 5-HT, high for tyramine. For the three catecholamines the half-saturating outside concentrations of the extraneuronal deaminating system clearly exceeded those for the extraneuronal O-methylating system of the same organ (see Grohmann and Trendelenburg 1985), although the two enzymes appear to co-exist in the same cells, so that the same transport system is involved.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to U. Trendelenburg