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     

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     

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 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     

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     

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     

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     

Effect of progesterone on the metabolism of noradrenaline in rabbit uterine endometrium and myometrium

The metabolism of (-)-3H-noradrenaline was examined in the endometrium and the myometrium from rabbits which had received 17 beta-oestradiol, either alone (oestrogen-dominated) or with progesterone (progesterone-dominated). The progesterone treatment resulted in a 2.5-fold increase in 3H-NMN formation in the endometrium, with no change in 3H-DOPEG, 3H-DOMA or 3H-OMDA formation. In the myometrium, progesterone caused a 5-fold increase in 3H-NMN formation and a 2.5-fold increase in 3H-OMDA formation, but did not affect 3H-DOPEG or 3H-DOMA formation. In the progesterone-dominated endometrium, both 3H-NMN and 3H-OMDA formation were strongly inhibited by cocaine 30 mumol/l. When O-methylation was inhibited by a COMT inhibitor, cocaine prevented the resultant increases in deamination of noradrenaline to 3H-DOPEG and in the accumulation of 3H-noradrenaline by the tissue. The 3H-noradrenaline which accumulated in endometria, in which both MAO and COMT were inhibited, was firmly bound; desipramine 3 mumol/l and (+)-amphetamine 10 mumol/l were equieffective with cocaine 30 mumol/l in inhibiting the accumulation. Cocaine 30 mumol/l was without effect on 3H-NMN and 3H-OMDA formation in the progesterone-dominated myometrium, nor did it prevent the increase in 3H-DOPEG formation produced by COMT inhibition. Fluorescent histochemical analysis of the endometrium indicated that the epithelial cells of the endometrial glands were the site of cocaine-sensitive noradrenaline accumulation. It is concluded that progesterone stimulates O-methylation in the endometrium and myometrium in different ways.(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

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     

Release and disposition of 3H-noradrenaline in the saphenous vein of neonate and adult dogs

Summary Release of ³H-noradrenaline and formation of ³H-metabolites were studied in the saphenous vein of newborn (mean age, 18 h) and adult dogs. Vein strips were incubated with 0.23 mol/l of ³H-noradrenaline during 1 h and washed out for 110 min; thereafter, the perifusion fluid was collected in 5-min samples. Electrical stimulation was applied at 120 min (1 Hz, 2 ms, 100 V, for 5 min). In some experiments the tissues were preincubated with 1 mmol/l pargyline (to inhibit monoamine oxidase). In these experiments, 12 mol/l cocaine (to inhibit uptake₁), 41 mol/l hydrocortisone (to reduce uptake₂) and 50 mol/l U-0521 (to inhibit COMT) were present during the perifusion. ³H-noradrenaline, ³H-DOPEG, ³H-NMN, ³H-DOMA and ³H-OMDA were separated by column chromatography. The noradrenaline content of the tissue was estimated by HPLC followed by electrochemical detection. A morphological study was also carried out by light and electron microscopy.The endogenous noradrenaline content of the saphenous vein was 4.3 times higher in adults than in neonates. The number of varicosities was similar in adults and newborns but the number of vesicles per varicosity profile was 5 times higher in adults. Hence, the endogenous noradrenaline content per vesicle was about the same in adults and newborns. The accumulation of ³H-noradrenaline per vesicle was about 5 times higher in newborns than in adults. On the other hand, the vein wall media of neonates was about 3 times thinner than that of adults. The evoked fractional release of tritium was about 10 times higher in neonates than in adults, whether the inactivation pathways were blocked or not. This difference in the evoked fractional release therefore cannot be ascribed to any difference in the efficacy of the inactivation pathways between neonates and adults. On the other hand, the difference also cannot be ascribed to a different alpha₂-adrenoceptor-mediated inhibition of ³H-noradrenaline release since, as previously shown, this mechanism is as effective in newborns as in adults.It is concluded that the evoked fractional release of ³H-noradrenaline is higher in neonates than in adults because in neonates the majority of varicosities (and vesicles) is situated closer to the surface of the tissue such that both uptake from the surrounding media into the stores and release from the stores into the surrounding media become easier in the neonates.Abbreviations COMT catechol-O-methyl transferase - DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - HPLC high pressure liquid chromatography - MAO monoamine oxidase - NMN normetanephrine - OMDA 3-methoxy-4-hydroxyphenylglycol (MOPEG) plus 3-methoxy-4-hydroxymandelic acid (VMA) - U-0521 3,4-dihydroxy-2-methyl propiophenone Correspondence to D. Moura at the above address     

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     

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

Summary In a recent study, we have shown that hyperpolarization of cells by -adrenoceptor agonists results in stimulation of the uptake₂ process for catecholamines. The aim of the present study was to further explore the hypothesis that uptake₂ is dependent on membrane potential by examining the effects of the K⁺-channel opening drug, (–)-cromakalim, and the K⁺-channel blocking drug, glibenclamide, on uptake₂ of isoprenaline. The effects of these drugs were examined in guinea-pig trachealis muscle, in which isoprenaline and cromakalim cause hyperpolarization, and in rat heart, in which isoprenaline and cromakalim have little effect on membrane potential.In guinea-pig trachealis muscle segments, 1 mol/l glibenclamide reduced uptake₂ (as measured by the steady-state rate of corticosterone-sensitive formation of ³H-3-O-methylisoprenaline normalized for the isoprenaline concentration) in tissues incubated in concentrations of ³H-(±)-isoprenaline that hyperpolarize the muscle (25 and 250 nmol/l) but not at an isoprenaline concentration that did not hyperpolarize the muscle (1 nmol/l). (–)-Cromakalim (10 mol/l), which hyperpolarizes the trachealis muscle, increased uptake₂ of isoprenaline (1 or 25 nmol/l) and this effect of (–)-cromakalim was inhibited by glibenclamide. In rat hearts perfused with 1 or 25 nmol/l ³H-(±)-isoprenaline and 10 mol/l U-0521 to inhibit catechol-O-methyltransferase, the rate of uptake₂ of isoprenaline was unaffected by cromakalim or glibenclamide.The results show that hyperpolarization of cells by various mechanisms can result in stimulation of uptake₂ of catecholamines and provide further evidence to support the hypothesis that the uptake₂ transport process is driven by the membrane potential of cells.Preliminary results of part of this study were presented to the German Society for Pharmacology and Toxicology (Bryan-Lluka and Vuocolo 1991)Correspondence to L. J. Bryan-Lluka at the above address     

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).     

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     

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     

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.