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  • 1
    Electronic Resource
    Electronic Resource
    Effects of Monovalent Ions on the Transport of Noradrenaline Across the Plasma Membrane of Neuronal Cells (PC-12 Cells) (1985)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 45 (1985), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The dependence on Na+, K+, and C1− of uptake and accumulation of [3H]noradrenaline was studied in plasma membrane vesicles isolated from PC-12 pheochromocytoma cells. Plasma membrane vesicles accumulated [3H]noradrenaline when an inward-directed gradient for Na+ and an outward-directed gradient for K+ were imposed across the vesicle membrane. Under these conditions, initial rates of uptake of [3H]noradrenaline were saturable (Km= 0.14 μM) and inhibited by a series of substrates and inhibitors of “uptake1.’ The IC50 values were positively correlated with those for inhibition of uptake into intact PC-12 cells. Uptake and accumulation [3H]noradrenaline in plasma membrane vesicles were absolutely dependent on external Na+ and C1−; they were dependent on an inwardly directed gradient for Na+ but less dependent on an inwardly directed gradient for C1−. Internal K+ strongly enhanced uptake and accumulation of [3H]noradrenaline. Rb+, but not Li+, had the capacity to replace internal K+. Two explanations are proposed for this effect of internal K+: (a) creation of a K+ diffusion potential (inside negative) provides a driving force for inward transport, and/or (b) K+ increases the turnover rate by formation of a highly mobile potassium–carrier complex. A hypothetical scheme for the transport of noradrenaline is presented.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1985.tb05536.x
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  • 2
    Electronic Resource
    Electronic Resource
    Sodium-dependence of the saturability of carrier-mediated noradrenaline efflux from noradrenergic neurones in the rat vas deferens (1986)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 332 (1986), S. 131-134 
    Details
    ISSN: 1432-1912
    Keywords: Neuronal efflux ; Noradrenaline carrier ; Veratridine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. The carrier-mediated transport of 3H-noradrenaline out of noradrenergic neurones was studied in vasa deferentia obtained from rats after pretreatment with reserpine and pargyline (to inhibit vesicular storage and monoamine oxidase, respectively). The tissue was first preincubated with various concentrations of 3H-noradrenaline (0.3–100 μmol/l; 30 min) and then washed out for 110 min with amine-free medium. During the last 10 min of washout, carrier-mediated neuronal efflux of 3H-noradrenaline was elicited by exposure to either Na+-free medium or 100 μmol/l veratridine; it was measured at 1-min intervals. 2. While the peak rates of carrier-mediated 3H-noradrenaline efflux elicited by Na+-free medium were linearly related to the 3H-noradrenaline content of the tissue (which cannot be raised beyond a certain maximal value, since uptake is saturable), those evoked in response to veratridine approached saturation as the 3H-noradrenaline level in the tissue was raised. Hence, saturation of 3H-noradrenaline outward transport was demonstrated at high (exposure to veratridine), but not at low (exposure to Na+-free medium) intraneuronal Na+ concentrations. 3. The results indicate that the K m for the mediated outward transport of noradrenaline across the plasma membrane of noradrenergic neurones is inversely related to the internal Na+ concentration, just as the K m for the mediated inward transport of noradrenaline (i.e., the neuronal noradrenaline uptake) is inversely related to the external Na+ concentration.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00511402
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  • 3
    Electronic Resource
    Electronic Resource
    Binding of3H-desipramine to the neuronal noradrenaline carrier of rat phaeochromocytoma cells (PC-12 cells) (1986)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 334 (1986), S. 403-411 
    Details
    ISSN: 1432-1912
    Keywords: 3H-desipramine ; Neuronal noradrenaline carrier ; PC-12 cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The specific (i.e., nisoxetine-sensitive) binding of3H-desipramine was studied in purified plasma membranes of PC-12 cells (rat phaeochromocytoma cells).3H-desipramine bound reversibly and with high affinity (K D=4.5 nmol/l) to a single, non-interacting site (Hill coefficient=1.04); the maximal number of binding sites (B max) was 19.6 pmol/mg protein. Like the uptake of noradrenaline (by uptake1), the binding of3H-desipramine was dependent on both sodium and chloride. The stimulation of binding by chloride and sodium was characterized by a Hill coefficient of about 1 and 2, respectively. Both, chloride and sodium, slowed the rate of dissociation of bound3H-desipramine. Increasing concentrations of sodium decreased theK D of3H-desipramine binding without altering theB max. The binding of3H-desipramine was inhibited by tricyclic antidepressants and other noradrenaline uptake blockers. There was a highly significant correlation between the potencies of a series of drugs for the inhibition of3H-desipramine binding and for the inhibition of3H-noradrenanne uptake into intact PC-12 cells. Both, binding of3H-desipramine and uptake of3H-noradrenaline, were stereoselectively inhibited by the enantiomers of cocaine and oxaprotiline. However, for most of the substrates of uptake1 the IC50 for inhibition of3H-desipramine binding was much higher than that for inhibition of3H-noradrenaline uptake. Nevertheless, noradrenaline competitively inhibited3H-desipramine binding and unmasked dissociation of bound3H-desipramine. Thus,3H-desipramine probably binds to the substrate recognition site. From theB max of3H-desipramine binding to PC-12 membranes and from theV max of3H-noradrenaline uptake into PC-12 cells, a duration of about 400 ms for the transport cycle for a single noradrenaline molecule was calculated. In addition, from theB max the maximum number of3H-desipramine binding sites (carriers) for a single PC-12 cell was calculated to be 55,000.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00569378
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  • 4
    Electronic Resource
    Electronic Resource
    Solubilization and characterization of the3H-desipramine binding site of rat phaeochromocytoma cells (PC12-cells) (1986)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 334 (1986), S. 412-417 
    Details
    ISSN: 1432-1912
    Keywords: Neuronal uptake ; Desipramine binding sites ; Membrane solubilization ; Clonal rat phaeochromocytoma cells (PC12)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 3H-Desipramine binding sites of the plasma membranes of rat phaeochromocytoma cells (PC12-cells) were solubilized with the nonionic detergent digitonin (0.5%). With the method described here, the binding characteristics of the desipramine binding site were essentially unaltered by solubilization. Binding of3H-desipramine to the solubilized binding site showed the following characteristics: (1)3H-desipramine bound with high affinity (K D=16.6 nmol/l) to a single class of noninteracting (Hill-coefficient=1.01) binding sites; (2) binding was reversible; (3) binding of unlabelled desipramine had the same dissociation constant as had3H-desipramine; (4) increasing concentrations of sodium- and chloride-ions stimulated the binding of3H-desipramine; (5) binding was inhibited by various inhibitors and substrates of neuronal uptake of noradrenaline; and (6) inhibition of binding by the optical isomers of cocaine, oxaprotiline, and amphetamine showed marked stereoselectivity (with preference for (−)cocaine, (+)oxaprotiline, and (+)amphetamine). The finding that the binding of3H-desipramine to the solubilized binding site was dependent on sodium and chloride, as the neuronal uptake of noradrenaline is, and the finding that all substrates of uptake1 inhibited the binding of3H-desipramine, is consistant with the view that desipramine binds to the substrate recognition site of the neuronal carrier for noradrenaline.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00569379
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  • 5
    Electronic Resource
    Electronic Resource
    Veratridine-induced outward transport of 3H-noradrenaline from adrenergic nerves of the rat vas deferens (1987)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 336 (1987), S. 621-630 
    Details
    ISSN: 1432-1912
    Keywords: Veratridine ; Ouabain ; Rat vas deferens ; Adrenergic nerve endings ; Neuronal outward transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. The neuronal release by 100 μmol/l veratridine of preloaded 3H-noradrenaline was studied in the rat vas deferens, the MAO, COMT and vesicular uptake of which were inhibited. To prevent any exocytotic release of the 3-Hamine, all solutions were calcium-free. Veratridine induced an early and a late peak of tritium efflux. The early peak was abolished by the presence of 1 μmol/l desipramine, the late peak was abolished by 1 μmol/l tetrodotoxin (administered subsequently to the first peak). The administration of veratridine plus 1 mmol/l ouabain resulted in only the early peak of efflux. 2. The peak response to veratridine plus ouabain was increased by a very early administration of veratridine plus ouabain (after 40 min of wash-out instead of the usual 130 min) (i. e., when the relative size of the axoplasmic distribution compartment was increased). However, very high axoplasmic 3H-noradrenaline levels (after loading with 37 instead of the usual 0.2 μmol/l) reduced the height of the peak (when expressed as a FRL). 3. Substantially similar responses to vcratridine plus ouabain were obtained after loading with 3H-noradrenaline, 3H-adrenaline or 3H-dopamine. 4. As the second peak of veratridine-induced release is ouabain-sensitive, it appears to be caused by exhaustion of neuronal ATP stores; this, in turn, raises the intravesicular pH and induces efflux of 3H-noradrenaline from the vesicles into the axoplasm. The first peak, on the other hand, represents outward transport of 3H-noradrenaline from the axoplasmic compartment. Evidently, a pronounced vesicular distribution of 3H-noradrenaline takes place even after inhibition by reserpine of the vesicular uptake. 5. In preparations with intact vesicular uptake (MAO and COMT inhibited) a plateauresponse was obtained; in the presence of 10 μmol/l Ro 4-2184 (a reserpine-like compound) a peak response was restored after loading with 0.2 μmol/l3H-noradrenaline, less so after loading with 37 μmol/l. 6. It is confirmed that veratridine (plus ouabain) exerts a reserpine-like effect when applied to tissues with intact vesicular uptake and intact MAO.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00165752
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  • 6
    Electronic Resource
    Electronic Resource
    Neuronal efflux of noradrenaline induced by tris or lithium as substitutes for extracellular sodium (1986)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 333 (1986), S. 13-16 
    Details
    ISSN: 1432-1912
    Keywords: Neuronal efflux ; Noradrenaline ; Low sodium ; Tris ; Lithium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Vasa deferentia of either untreated or reserpine (R) and/or pargyline (P) pretreated rats were incubated with3H-noradrenaline and then washed with amine- and Ca2+-free solution until (after 100 min) the efflux of radioactivity largely originated from adrenergic nerve endings; COMT was inhibited by U-0521 (U). After 110 min of wash out, the sodium chloride in the wash-out solution was replaced by an equimolar concentration of either Tris-HCl or LiCl. This caused a despramine-sensitive (i.e., carrier-mediated) efflux of tritiated noradrenaline. The initial increase of the “low Na+”-induced efflux dependent on the experimental conditions: it was most pronounced when the axoplasmic concentration of noradrenaline was high (RPU) and relatively small when MAO and vesicular storage were intact (U). The effects of Li+ and Tris+ differed with regard to the time course of the efflux of tritium: under all three experimental conditions (RPU, PU, U), Tris+ caused the rate of efflux of tritium to increase gradually within the 30 min period of observation, while Li++ either had a “peak-effect” (RPU, PU) or a “plateau-effect” (U). Under “U-conditions” Tris+ caused a slowly increasing, pronounced increase with time of the efflux of both,3H-noradrenaline and3H-DOPEG; whereas Li+ caused only a small and sustained increase of the efflux of3H-noradrenaline and a decrease in the efflux of3H-DOPEG. Conclusions: 1) The results are compatible with the view that the buffering agent Tris can diffuse into nerve endings and then also into storage vesicles, and, thus, increases the intravesicular pH; as a consequence of the elevated pH, the leakage of noradrenaline from the vesicles increases and, thus, more noradrenaline becomes available for deamination in and outward transport from the axoplasm. 2) A decrease of the sodium-gradient (brought about by e.g. low extracellular sodium) increases the availability of carrier sites on the internal face of the axonal membrane. This results in outward transport only when the axoplasmic concentration of noradrenaline is elevated (either due to inhibition of MAO or to increased vesicular efflux of noradrenaline).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00569653
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  • 7
    Electronic Resource
    Electronic Resource
    The neuronal noradrenaline transport system of PC-12 cells: Kinetic analysis of the interaction between noradrenaline, Na+ and Cl− in transport (1986)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 333 (1986), S. 246-252 
    Details
    ISSN: 1432-1912
    Keywords: Neuronal uptake ; Noradrenaline ; PC-12 cells ; Sodium ; Chloride
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The uptake of 3H-noradrenaline into reserpine-pretreated PC-12 cells (a clonal cell line which possesses “uptake1”) was abolished when at high extracellular Cl−1 all the extracellular Na+ was replaced by Tris+ and when at high extracellular Na+ all the extracellular Cl− was replaced by isethionate. Increases in the external Cl− concentration (at a fixed high Na+ concentration) progressively increased the uptake of 3H-noradrenaline. The same was found with increase in the external Na+ concentration (at a fixed high Cl− concentration). From the anions tested only Br− and SCN− were able to partially mimic the transport-stimulating effect of Cl− (with about 40% and 20% effectiveness, respectively). When chloride was replaced by nitrate or larger anions such as sulphate, methylsulphate or isethionate, virtually no transport of 3H-noradrenaline was observed. The initial rate of uptake of 3H-noradrenaline showed saturation with increasing concentrations of noradrenaline when determined at several fixed concentrations of either Na+ or Cl−. The apparent K m for noradrenaline transport ( $$K_{m_{NA} } $$ ) progressively decreased and the $$V_{max_{NA} } $$ increased with increases in the concentration of Na+ (at a high concentration of Cl−) or Cl− (at a high concentration of Na+). The stimulation of the initial rate of uptake of 3H-noradrenaline by increasing concentrations of either Na+ or Cl− obeyed saturation kinetics when determined at several concentrations of noradrenaline. The concentration of Na+ (or Cl−) which caused half-maximal stimulation of uptake (i.e., the apparent $$K_{m_{Na + } } $$ and the apparent $$K_{m_{Cl - } } $$ ) decreased with increases in the concentration of noradrenaline. These results strongly suggest that Na+ and Cl− are co-transported with noradrenaline.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00512937
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  • 8
    Electronic Resource
    Electronic Resource
    The mechanism of the 3H-noradrenaline releasing effect of various substrates of uptake1: multifactorial induction of outward transport (1987)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 336 (1987), S. 602-610 
    Details
    ISSN: 1432-1912
    Keywords: Adrenergic nerve endings ; Outward transport of noradrenaline ; Uptake1 ; Indirectly acting sympathomimetic amines ; Release of noradrenaline
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: 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 μmol/l 3H-(−)-noradrenaline. The IC50 ranged from 0.35 μmol/l (for (+)-amphetamine) to 44.3 μmol/l (for 5-HT). The V max (determined for 8 substrates) was substrate-dependent. 2. Tissues were loaded with 0.2 μmol/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 μmol/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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00165750
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  • 9
    Electronic Resource
    Electronic Resource
    Inhibition of neuronal noradrenaline uptake (uptake1) and desipramine binding by N-ethylmaleimide (NEM) (1988)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 337 (1988), S. 633-636 
    Details
    ISSN: 1432-1912
    Keywords: Uptake1 ; Desipramine binding ; PC12 Cells ; N-Ethylmaleimide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00175788
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  • 10
    Electronic Resource
    Electronic Resource
    Kinetic evidence for a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier (1988)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 337 (1988), S. 626-632 
    Details
    ISSN: 1432-1912
    Keywords: Uptake1 ; PC12 Cells ; Desipramine binding ; K m/K D Ratio for substrates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The neuronal noradrenaline uptake mechanism (uptake1) has been further characterized. For a number of substrates of uptake, the half-saturating concentration (K m) and the maximal initial transport rate (V max) were determined. Furthermore, the dissociation constants (K D) for binding of these substrates to the desipramine binding site of the neuronal noradrenaline carrier were measured. The uptake experiments were done on rat phaeochromocytoma cells (PC12 cells), the binding experiments on purified plasma membranes of PC12 cells. The substrates differed markedly in respect of V max, K m, and K D. Neither K m and V max nor K D and V max were found to be correlated. However, the discrepancy between K m and K D expressed as the ratio, Km/KD, was negatively correlated with V max (r = − 0.9315, n = 7, p 〈 0.01). For the interpretation of these results a model on the basis of the steady-state assumption has been proposed for uptake1. From the mathematics of that model the following conclusions can be drawn. (1) The half-saturating substrate concentration (K m) is not identical with the dissociation constant for the binding of a substrate to the substrate recognition site (K D). (2) The discrepancy between K m and K D is expected to be negatively correlated with the maximal initial transport rate of the substrate (V max). The experimental results are in good agreement with the proposed model for uptake,. Especially the negative correlation between K m/K D and Vmax supports the hypothesis that desipramine inhibits uptake, via binding to the substrate recognition site of the neuronal noradrenaline carrier.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00175787
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