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     

Characterization of the [3H]-desipramine binding site of the bovine adrenomedullary plasma membrane

Summary The specific (i.e. nisoxetine-sensitive) binding of [³H]desipramine was studied in membranes prepared from bovine adrenal medullae. (1) [³H]desipramine bound reversibly and with high affinity (K _D = 2.8 nmol/l) to a single class of non-interacting binding sites (Hill coefficient = 0.96); the maximal number of binding sites (B_max) was 2.1 pmol/mg protein. (2) Binding of [³H]desipramine was dependent on [Na⁺] and [Cl^–]. Increasing the concentrations of these ions increased binding. (3) Substrates and inhibitors of the neuronal noradrenaline transport system (uptake,) inhibited binding of [³H]desipramine with a rank order of potency typical for an interaction with the uptake, carrier.The characteristics of [³H]desipramine binding remained essentially unchanged after solubilization of adrenomedullary membranes with the non-ionic detergent digitonin.The results indicate that the plasma membrane of bovine adreno-medulary cells is endowed with the neuronal uptake₁ transporter. Correspondence to: H. Bönisch     

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).     

EFFECT OF BILE DUCT OBSTRUCTION ON HEPATIC UPTAKE OF 1-METHYL-4-PHENYLPYRIDINIUM IN THE RAT

Previous studies have demonstrated that the organic cation 1-methyl-4-phenylpyridinium (MPP⁺) is avidly taken up by rat freshly isolated hepatocytes through at least two distinct transport mechanisms: the type I hepatic transporter of organic cations and P-glycoprotein. In this study, the effects of extrahepatic cholestasis induced by bile duct ligation for 4 days on the uptake of [³H]MPP⁺by rat freshly isolated hepatocytes and liver slices were determined. Bile duct ligation produced no significant alterations in the characteristics of [³H]MPP⁺uptake by freshly isolated hepatocytes. The strong correlation found between the effect of various drugs on [³H]MPP⁺uptake by hepatocytes from control and treated rats (r=0.958;P<0.0001;n=15) suggests that neither the type I hepatic transporter of organic cations nor P-glycoprotein were affected by bile duct ligation. On the contrary, uptake of [³H]MPP⁺by liver slices was markedly changed after bile duct ligation: (1) there was a significant increase (≅40%) in the amount of [³H]MPP⁺taken up by liver slices from bile duct-ligated rats; (2) there was no correlation between the effect of various drugs on [³H]MPP⁺uptake by liver slices from control and treated rats (r=0.772;P=0.072;n=6). On the basis of (1) the lack of effect of bile duct ligation on [³H]MPP⁺uptake by isolated hepatocytes; and (2) the profound morphological alterations of liver tissue observed 4 days after bile duct ligation (increase in volume density of bile ductules, ductular cells and infiltration of inflammatory cells), we suggest that non-parenchymal liver cells have an important participation in the hepatic uptake of [³H]MPP⁺after bile duct ligation in the rat.     

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

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

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

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

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     

Pertussis toxin inhibits noradrenaline accumulation by bovine adrenal medullary chromaffin cells

Summary Bovine adrenal medullary chromaffin cells maintained in tissue culture accumulated [³H]-noradrenaline by a high affinity, Na⁺-dependent, desipramine-sensitive process. The accumulation was linear with time (1–90min) and had an apparent K_m of 0.52±0.24 mol/l and V_max of 1.70±0.48 pmol/(10⁵ cells · 15 min). Pretreatment of the cells with the ADP-ribosylating agent pertussis toxin resulted in a reduction in the V_max, [0.81±0.39 pmol/(10⁵cells · 15 min)] but no significant change in the apparent affinity (K_m = 0.42±0.07 mol/l). This inhibition of [³H]noradrenaline accumulation was distinct from that produced by the vesicular transport inhibitor reserpine. Pertussis toxin inhibition probably did not arise through an indirect action on the Na⁺-gradient because while, as expected, Na⁺, K⁺-ATPase inhibition reduced [³H]noradrenaline accumulation, pertussis toxin pretreatment always caused a further significant reduction even in the presence of maximally effective concentrations of ouabain. Stimulation of the CAMP-protein kinase A system by forskolin or 8-bromocyclic AMP also caused a reduction in [³H] noradrenaline accumulation but again pertussis toxin pretreatment always resulted in a further reduction. Thus, the data provide evidence for a pertussis toxin-sensitive element in the catecholamine accumulation process and are consistent with an action at a site directly associated with the transporter itself rather than with an indirect action via secondary processes.Correspondence to S. J. Bunn at the above address     

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     

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)     

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     

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

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

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

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

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 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     

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     

Characterization of the K+-channel-coupled adenosine receptor in guinea pig atria

Summary In the present work we studied the pharmacological profile of adenosine receptors in guinea pig atria by investigating the effect of different adenosine analogues on⁸⁶Rb⁺-efflux from isolated left atria and on binding of the antagonist radioligand 8-cyclopentyl-1,3-[³H]dipropylxanthine ([³H]DPCPX) to atrial membrane preparations. The rate of⁸⁶Rb⁺-efflux was increased twofold by the maximally effective concentrations of adenosine receptor agonists. The EC₅₀-values for 2-chloro-N⁶-cyclopentyladenosine (CCPA), R-N⁶-phenylisopropyladenosine (R-PIA), 5-N-ethylcarboxamidoadenosine (NECA), and S-N⁶-phenylisopropyladenosine (S-PIA) were 0.10, 0.14, 0.24 and 12.9 M, respectively. DPCPX shifted the R-PIA concentration-response curve to the right in a concentration-dependent manner with a K_B-value of 8.1 nM, indicating competitive antagonism. [³H]DPCPX showed a saturable binding to atrial membranes with a B_max-value of 227 fmol/mg protein and a K_D-value of 1.3 nM. Competition experiments showed a similar potency for the three agonists CCPA, R-PIA and NECA. S-PIA is 200 times less potent than R-PIA. Our results suggest that the K⁺ channel-coupled adenosine receptor in guinea pig atria is of an A₁ subtype.Abbreviations CCPA 2-chloro-N⁶-cyclopentyladenosine - DPCPX 8-cyclopentyl-1,3-dipropylxanthine - NECA 5-N-ethylcarboxami-doadenosine - PIA N⁶-phenylisopropyladenosine Send offprint requests to H. Tawfik-Schlieper 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     

Interaction of two sulfhydryl reagents with a cation recognition site on the neuronal dopamine carrier evidences small differences between [3H]GBR 12783 and [3H]cocaine binding sites

We have compared the effect of treating rat striatal cell membranes with ionic hydrophilic sulfhydryl reagents on the specific bindings of [³H]cocaine and of [³H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-[1-³H]propenyl)-piperazine) to the neuronal transporter of dopamine. Treatment with 1 mmol/1 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in similar time-and concentration-dependent reductions of the specific binding of both radioligands. None of the uptake blockers tested afforded any protection against 1 mmol/1 DTNB. Addition of (sub)millimolar concentrations of CaCl₂ or MgCl₂, or 250 mmol/1 KCl to a treatment medium containing 10 mmol/l Na + significantly increased the DTNB-induced reduction of the specific binding of both radioligands. Cations were likely to be responsible for this effect since ions in combination with DTNB induced similar reductions in binding when either 1 mmol/l CaCl₂ or 50–250 mmol/l NaCl were added. Effects of cations on the DTNB-induced inhibition of binding were generally more marked on [³H]GBR 12783 than on [³H]cocaine binding. When added to a medium containing 10 mmol/1 Na⁺ 1 mmol/1 DTNB induced a reduction in the B_max of the specific binding of both radioligands. Addition of 1 mmol/l Ca²⁺ maintained or increased this B_max reduction and elicited a decrease in affinity which was significant for [³H]GBR 12783 binding.Treatment of membranes with the sodium salt of p-hydroxymercurybenzenesulfonate (pHMBS) induced time-and concentration-dependent decreases in [³H]GBR 12783 binding which were significantly greater than decreases in [³H]cocaine binding. However, 50mol/lpHMBS produced a similar decrease in the B_max of the specific binding of both radioligands. The pHMBS-induced reduction of [³H]GBR 12783 binding was not reversed by drugs whose action is purely that of uptake inhibition or by substrates of the dopamine carrier. Some of these drugs (100 mol/l dopamine, 1 mol/l mazindol or 100 mol/l cocaine) protected the specific binding of [³H]cocaine against the effects of pHMBS, whereas 1 mmol/1 p-tyramine, 10 mol/l nomifensine and 10 nmol/l GBR 12783 were ineffective. Addition of 120 mmol/l Na⁺, 1 mmol/l Ca²⁺ or 10 mmol/l Mg²⁺ to a treatment medium containing 10 mmol/l Na⁺ significantly reduced the effects of pHMBS on the specific binding of both radioligands. When striatal cell membranes were treated in a medium containing 130 mmol/1 Na⁺, there was a general decrease in the effects of ions on the reductions of specific binding produced by DTNB or pHMBS. Cation concentrations which interfered with the actions of DTNB and pHMBS were approximately those which blocked the specific binding of [³H]GBR 12783 when they were present during association of the radioligand (K⁺, Ca ²⁺, Mg²⁺), or, in the case of Na⁺, which are effective in reducing this blockade (Bonnet et al. 1988).The present data are consistent with the existence of mutually exclusive binding sites for [³H]GBR and [³H]cocaine on the neuronal dopamine transporter. The hypothesis of a cation recognition site which could gate admission of uptake inhibitors or carrier substrates to their binding domain on the transporter is discussed.