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  • 1
    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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  • 2
    Electronic Resource
    Electronic Resource
    The rate constants for the efflux of deaminated metabolites of 3H-dopamine from the perfused rat heart (1980)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 314 (1980), S. 231-235 
    Details
    ISSN: 1432-1912
    Keywords: Rat heart ; 3H-Dopamine ; Neuronal uptake ; Extraneuronal uptake ; Rate constants for efflux ; Dopamine metabolites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Hearts of rats pretreated with reserpine and FLA 63 were perfused for 30 min with 1 μmol/l 3H-dopamine and in the presence of an inhibitor of either neuronal (30 μmol/l cocaine) or extraneuronal uptake (87 μmol/l corticosterone). From the rate at which the deaminated metabolites appeared in the venous perfusate and from the tissue content of the metabolites at the end of the perfusion rate constants for efflux (k-values) were determined. The k-values for the deaminated metabolites of dopamine did not differe when the deamination of dopamine was restricted to either extraneuronal or neuronal sites. However, marked differences existed between the rate constant for efflux of the deaminated acid DOPAC (dihydroxyphenylacetic acid) and the glycol DOPET (dihydroxyphenylethanol). The relationship between the apparent lipophilicity and the rate constant for efflux of DOPAC fitted very well with that reported for other metabolites of catecholamines.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00498544
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  • 3
    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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  • 4
    Electronic Resource
    Electronic Resource
    Inhibition of 5-HT3 receptor function by imidazolines in mouse neuroblastoma cells: potential involvement of σ2 binding sites (1996)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 354 (1996), S. 245-252 
    Details
    ISSN: 1432-1912
    Keywords: Key words N1E-115 cells ; Imidazoline receptor ; σ-Binding site ; 5-HT3 receptor ; Ligand-gated ion channels ; [3H]DTG binding sites ; [3H]GR65630 binding sites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The influence of several imidazolines and σ-site ligands on cation influx through the 5-HT3 receptor channel in N1E-115 mouse neuroblastoma cells was studied by measuring the 2-min influx of the organic cation [14C] guanidinium induced by 1 μM 5-HT (in the presence of 10 μM substance P in all experiments). In addition, we determined specific binding of [3H]DTG (1,3-di(2-tolyl)- guanidine), a selective σ-site radioligand, and [3H] GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone), a selective 5-HT3 receptor antagonist, to membranes prepared from N1E-115 cells. The 5-HT-induced [14C]guanidinium influx was inhibited by the imidazolines, ondansetron, antazoline, idazoxan, BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), cirazoline, naphazoline, clonidine and by the guanidine agmatine, but not by the catecholamine adrenaline. The inhibitory effect of the imidazolines on cation influx through the 5-HT3 receptor channel was mimicked by the σ-site ligands, (±)-ifenprodil, (+)-3-PPP ((R)-3-(3-hydroxy-phenyl)-N-propylpiperidine), DTG (1,3-di-tolyl-guanidine), haloperidol, dizocilpine, and ketamine as well as by the polyamines, arcaine and spermidine. – Ondansetron inhibited [3H]GR65630 binding with high affinity, whereas inhibition of binding of this radioligand to the 5-HT3 receptor by antazoline, BDF 6143, idazoxan, cirazoline, (±)-ifenprodil, (+)-3-PPP, DTG and haloperidol occurred in the high micromolar range. In the competition experiments with [3H]DTG, (±)-ifenprodil, haloperidol, unlabelled DTG, BDF 6143 and (+)-3-PPP inhibited binding of the radioligand at moderate affinity (Ki values in the range of 1 μM or lower), whereas ondansetron, antazoline, idazoxan, cirazoline, naphazoline, clonidine, tolazoline, efaroxan, RX821002 (2-[2-(2-methoxy-1,4-benzodioxanyl)]imidazo- line), ketamine and spermidine exhibited affinity in the high micromolar or millimolar range only. Comparison of the potencies of the ligands (pIC50% values) in inhibiting 5-HT-induced [14C]guanidinium influx with their affinities (pKi values) at the 5-HT recognition sites of the 5-HT3 receptor and at the σ2-sites of the N1E-115 cells by means of multiple regression analysis revealed a significant correlation with the affinities at both sites. In conclusion, our data suggest that imidazolines and σ-ligands, which as a rule possess low affinity for the 5-HT recognition site of the 5-HT3 receptor, may be assumed to exert their inhibitory effect on cation influx through the 5-HT3 receptor channels, at least in part, by interacting with σ2-binding sites.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00171054
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  • 5
    Electronic Resource
    Electronic Resource
    Inhibition of 5-HT3 receptor function by imidazolines in mouse neuroblastoma cells: potential involvement of σ2 binding sites (1996)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 354 (1996), S. 245-252 
    Details
    ISSN: 1432-1912
    Keywords: N1E-115 cells ; Imidazoline receptor ; σ-Binding site ; 5-HT3 receptor ; Ligand-gated ion channels ; [3H]DTG binding sites ; [3H]GR65630 binding sites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The influence of several imidazolines and σ-site ligands on cation influx through the 5-HT3 receptor channel in NIE-115 mouse neuroblastoma cells was studied by measuring the 2-min influx of the organic cation [14C] guanidinium induced by 1 μM 5-HT (in the presence of 10 μM substance P in all experiments). In addition, we determined specific binding of [3H]DTG (1,3-di(2-tolyl)-guanidine), a selective σ-site radioligand, and [3H] GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone), a selective 5-HT3 receptor antagonist, to membranes prepared from NIE-115 cells. The 5-HT-induced [14C]guanidinium influx was inhibited by the imidazolines, ondansetron, antazoline, idazoxan, BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), cirazoline, naphazoline, clonidine and by the guanidine agmatine, but not by the catecholamine adrenaline. The inhibitory effect of the imidazolines on cation influx through the 5-HT3 receptor channel was mimicked by the σ-site ligands, (±)-ifenprodil, (+)-3-PPP ((R)-3-(3-hydroxyphenyl)-N-propylpiperidine), DTG (1,3-di-tolyl-guanidine), haloperidol, dizocilpine, and ketamine as well as by the polyamines, arcane and spermidine. — Ondansetron inhibited [3H]GR65630 binding with high affinity, whereas inhibition of binding of this radioligand to the 5-HT3 receptor by antazoline, BDF 6143, idazoxan, cirazoline, (±)-ifenprodil, (+)-3-PPP, DTG and haloperidol occurred in the high micromolar range. In the competition experiments with [3H]DTG, (±)-ifenprodil, haloperidol, unlabelled DTG, BDF 6143 and (+)-3-PPP inhibited binding of the radioligand at moderate affinity (Ki values in the range of 1 μM or lower), whereas ondansetron, amazoline, idazoxan, cirazoline, naphazoline, clonidine, tolazoline, efaroxan, RX821002 (2-[2-(2-methoxy-1,4-benzodioxanyl)]imidazoline), ketamine and spermidine exhibited affinity in the high micromolar or millimolar range only. Comparison of the potencies of the ligands (pIC50% values) in inhibiting 5-HT-induced [14C]guanidinium influx with their affinities (pKi values) at the 5-HT recognition sites of the 5-HT3 receptor and at the σ2-sites of the N1E-115 cells by means of multiple regression analysis revealed a significant correlation with the affinities at both sites. In conclusion, our data suggest that imidazolines and σ-ligands, which as a rule possess low affinity for the 5-HT recognition site of the 5-HT3 receptor, may be assumed to exert their inhibitory effect on cation influx through the 5-HT3 receptor channels, at least in part, by interacting with σ2-binding sites.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00171054
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  • 6
    Electronic Resource
    Electronic Resource
    The influence of the rate of perfusion on the kinetics of neuronal uptake in the rabbit isolated heart (1978)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 302 (1978), S. 275-283 
    Details
    ISSN: 1432-1912
    Keywords: Rate of perfusion ; Neuronal uptake ; Accessibility of neuronal uptake sites ; Perfusion pressure ; Rabbit heart
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. Rabbit hearts (with monoamine oxidase and catechol-O-methyl transferase inhibited) were obtained from reserpine-pretreated animals. They were perfused at rates ranging from 1.3–11.3 ml·g−1·min−1 with 0.1 mM 14C-sorbitol and various concentrations of 3H-(−)noradrenaline (NA). From measurements of the arterio-venous concentration difference of 3H and 14C activity the removal of NA and sorbitol from the perfusion fluid was followed for 2–3 min at intervals of 5 s. The uptake of NA into intracellular spaces of the heart (known to be over-whelmingly into sympathetic nerve terminals) was obtained by subtracting the removal of sorbitol from that of NA. If was cumulated and plotted against time. 2. The progress curves of NA uptake were sigmoid in shape: following a lag period, uptake proceeded at first at a constant initial rate and from then on at gradually decreasing rates. Irrespective of the NA concentration used, the lag period became shorter and the initial rate of uptake increased whenever the rate of perfusion was increased. Furthermore, at high rates of perfusion the initial rate was maintained for a shorter time than at low ones. 3. At any given perfusion rate, the initial rates of NA uptake obeyed Michaelis-Menten kinetics. While changes of the rate of flow did not alter the apparent K m (range: 2.2–2.4 μM), a rectangular hyperbolic relationship was found between V max and the perfusion rate. The V max was half-maximal at a rate of flow of 2.7 ml·g−1·min−1 and approached a maximum value of 9.0 nmoles·g−1·min−1. 4. From the lack of change in the K m it can be concluded that the uptake sites of the perfused heart are functionally arranged in parallel. The change in V max, on the other hand, indicate that the accessibility of the sites is limited by the rate of perfusion.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00508296
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  • 7
    Electronic Resource
    Electronic Resource
    Saturation kinetics of the adrenergic neurone uptake system in the perfused rabbit heart. A new method for determination of initial rates of amine uptake (1978)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 302 (1978), S. 263-273 
    Details
    ISSN: 1432-1912
    Keywords: Neuronal uptake ; Initial rates of amine uptake ; Lag period for amine uptake ; Cocaine ; Rabbit heart
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. Hearts were obtained from normal or reserpine-pretreated rabbits and perfused at a constant rate (3.6 ml·g−1·min−1) with Tyrode's solution containing 14C- or 3H-sorbitol and various concentrations of 3H-(−)noradrenaline (NA), 14C-(+)NA or 3H-(±)metaraminol; when NA was used, monoamine oxidase and catechol-O-methyl transferase were inhibited. During perfusion for 2 min the arterio-venous difference for 3H and 14C activity (and in this way the removal of amine and sorbitol from the perfusion fluid) was determined at intervals of 5 s. The uptake of amine into intracellular spaces of the heart was obtained by subtraction of the removal of sorbitol from that of amine; it was cumulatively added and plotted against time (uptake curve). Uptake was overwhelmingly neuronal. 2. The uptake curves were sigmoidal: after a brief initial lag period, uptake curves became linear; there-after, the slope of the curves decreased. The last phase of divergence from linearity occurred the earlier and was the more pronounced, the higher the amine concentration. It was interpreted to indicate that neuronal efflux of amine then began to reduce net uptake. 3. From the slope of the linear phase of the uptake curves initial rates of amine transport were obtained. They were saturable with increasing amine concentrations and obeyed Michaelis-Menten kinetics. The apparent K m values of the three amines were similar in magnitude and ranged from 2.9 to 5.9 μM. Uptake was stereoselective in that the V max of (+)NA was significantly lower than that of (−)NA. Pretreatment with reserpine affected neither the K m nor the V max for uptake. Cocaine was a potent competitive inhibitor of amine transport (K i=0.5–1.0 μM). 4. The intercept of the linear phase of the uptake curves on the time axis (t lag) (corrected for the time necessary for transit through the dead space) was taken as a measure of the lag period. It declined when uptake was progressively saturated (or inhibited) by increasing substrate (or cocaine) concentrations. Moreover, t lag was always linearly correlated with the fraction of amine removed from the perfusion fluid. These findings indicate that the equilibration of the uptake sites with the substrate concentration in the perfusion fluid is delayed by the uptake process itself, especially under low saturation conditions (i.e., when S〈K m).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00508295
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  • 8
    Electronic Resource
    Electronic Resource
    The transport of (+)-amphetamine by the neuronal noradrenaline carrier (1984)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 327 (1984), S. 267-272 
    Details
    ISSN: 1432-1912
    Keywords: (+)-amphetamine ; Neuronal uptake ; Sympathomimetic amines
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary PC-12 cells (a clonal line of rat phaeochromocytoma cells) take up noradrenaline by a transport system which is identical with the neuronal amine transport system (“uptake1”). The uptake of 3H-noradrenaline into reserpine-pretreated PC-12 cells (monoamine oxidase inhibited) was saturable (Km=0.6±0.1 μmol/l), dependent on sodium and chloride, and competitively inhibited by (+)-amphetamine (Ki=0.18±0.04 μmol/l), cocaine (Ki=0.55±0.15 μmol/l) and desipramine (Ki=4.3±0.6 nmol/l). The uptake and accumulation of 3H (+)-amphetamine showed characteristics comparable to those of 3H-noradrenaline, since the uptake of 3H (+)-amphetamine (0.1 μmol/l) was reduced by omission of sodium or chloride from the incubation medium. The sodium-sensitive component of uptake and accumulation of 3H (+)-amphetamine was fully inhibited by cocaine and desipramine. The IC50 of desipramine for inhibition of the sodium-sensitive component of the 1-min uptake of 3H (+)-amphetamine (20 nmol/l) was about 2 nmol/l, i.e., identical with the Ki for inhibition of uptake of 3H-noradrenaline. At concentrations above 1 μmol/l, desipramine additionally caused an inhibition of the sodium-independent permeation of 3H (+)-amphetamine into PC-12 cells. Hence, by using a homogeneous population of cells endowed with “uptake1”, it is possible to demonstrate — besides a pronounced lipophilic entry — a carrier-mediated uptake of 3H (+)-amphetamine.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00506235
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  • 9
    Electronic Resource
    Electronic Resource
    The role of co-transported sodium in the effect of indirectly acting sympathomimetic amines (1986)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 332 (1986), S. 135-141 
    Details
    ISSN: 1432-1912
    Keywords: Sympathomimetic amines ; Efflux of noradrenaline ; Neuronal uptake ; Amphetamine ; Tyramine ; Na+ ; K+-ATPase inhibition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The adrenergic nerve endings of vasa deferentia of either untreated or reserpine (R) and/or pargyline (P) pretreated rats were loaded with 3H-noradrenaline; COMT was inhibited by U-0521 (U). After 100 min of wash-out with Ca2+-free solution, the efflux of tritium (and of 3H-noradrenaline) from the tissue was largely of neuronal origin and remained constant with time (when expressed as fractional rate of loss; FRL). After 110 min of wash-out the effect of inhibition of the Na+,K+-ATPase (by low K+ or ouabain) on basal and on sympathomimetic amine-induced efflux of tritium (or 3H-noradrenaline, under the condition U) was studied in paired experiments. Inhibition of the Na+,K+-ATPase caused a time-dependent increase in the efflux of tritium (or 3H-noradrenaline) which was inhibited by desipramine. Inhibition of the Na+,K+-ATPase also caused a time-dependent reduction of the initial rate of neuronal uptake of 3H-noradrenaline. The effectiveness of the sympathomimetic amines tyramine and amphetamine in inducing “release” (i.e., outward-transport) of noradrenaline depended on the experimental condition: it was most pronounced under the condition RPU, followed by the condition PU and lowest under the condition U (i.e., in tissue of untreated rats). Inhibition of the Na+,K+-ATPase caused an early and transient enhancement of the “release” of noradrenaline induced by tyramine or amphetamine. This enhancement was seen already within the first min after inhibition of the ATPase, i.e., before a pronounced inhibition of uptake (of noradrenaline) and before a pronounced increase of the basal efflux was observed. It also depended on the experimental condition: RPU 〉 PU 〉 U; i.e., it was the more pronounced, the higher the free axoplasmic concentration of noradrenaline. In tissues of untreated rats, tyramine increased the rate of efflux of DOPEG, whereas amphetamine decreased it. 1) Both, tyramine and amphetamine are transported by the Na+-dependent neuronal transport system; 2) the co-transported Na+ causes a local increase in the Na+ concentration at the inside of the neuronal plasma membrane and thereby contributes to the outward-transport of axoplasmic noradrenaline induced by indirectly acting sympathomimetic amines; however, this contribution is only of importance when the axoplasmic concentration of noradrenaline is high (RPU, PU).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00511403
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