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  • 1
    Electronic Resource
    Electronic Resource
    Relationship between the uptake of 3H-(±) metaraminol and the density of adrenergic innervation in isolated rat tissues (1977)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 296 (1977), S. 99-110 
    Details
    ISSN: 1432-1912
    Keywords: Metaraminol ; Neuronal uptake ; Extraneuronal uptake ; Density of adrenergic innervation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. The uptake of 3H-(±)metaraminol (MA) by tissue slices or pieces was studied in vitro in several peripheral rat organs of varying density of sympathetic innervation (the tissue level of endogenous noradrenaline ranging from 1.7–99.1 nmoles/g). In each individual tissue preparation amine uptake was corrected for entry into the 14C-d-sorbitol space. 2. When the tissues were incubated with 1.4 μM MA, the rate of total amine uptake (i.e., neuronal plus extraneuronal uptake of MA) remained virtually constant for up to 7 min. Therefore, rates of uptake were determined after 2 min of incubation with substrate concentrations ranging from 0.25–12.2 μM. In all tissues the total uptake of MA was saturable. 3. Under the condition of inhibition of neuronal uptake by the presence of 100 μM cocaine, the uptake of MA (considered as extraneuronal amine uptake) was no longer saturable. When tissues were exposed to 1.4 μM MA, the relative contribution by extraneuronal (measured in the presence of cocaine) to total amine uptake (measured in the absence of cocaine) was inversely correlated with the log endogenous noradrenaline content. 4. After correction of the rates of total MA uptake for the cocaine-resistant distribution of the amine, a saturable component of uptake was obtained for each tissue. This uptake was considered to be neuronal; it was subjected to kinetic analysis. 5. Apparent K m values for the neuronal uptake of MA were very similar in all tissues and did not show any dependence on the tissue level of endogenous noradrenaline (average K m=1.2μM). 6. V max values for the neuronal uptake of MA were linearly correlated with the endogenous noradrenaline content of the tissues (r=0.976; P〈0.001), the V max for the vas deferens being excluded. When related to the content of endogenous noradrenaline, the V max obtained in the vas deferens was lower than that for all other tissues. 7. The results presented here strongly suggest that the membrane site involved in neuronal amine uptake (operationally characterized by the K m of MA) is likely to be identical in all rat tissues and that the number of uptake sites available per nerve terminal does not vary greatly between tissues.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00508460
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  • 2
    Electronic Resource
    Electronic Resource
    Stereoselectivity in the metabolism of 3H-noradrenaline during uptake into and efflux from the isolated rat vas deferens (1977)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 299 (1977), S. 225-238 
    Details
    ISSN: 1432-1912
    Keywords: Stereoselective metabolism of noradrenaline ; Neuronal efflux ; Cocaine ; Phenoxybenzamine ; Rat vas deferens
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. The metabolism of 3H-(-)- and 3H-(±)-noradrenaline (NA) was studied in the isolated rat vas deferens either under conditions of uptake or of efflux of the amine. Any differences obtained between 3H-(-)-and 3H-(±)NA as substrate were interpreted as being a reflection of differences between the two isomers of the amine. 2. Uptake experiments (0.13 μM; 7.5 min) showed that neuronal mechanisms of amine disposition prevail over extraneuronal ones. Thus, most of the metabolites of 3H-NA formed during incubation with the amine (including the O-methylated products) were of neuronal origin. The acid deaminated metabolite 3,4-dihydroxymandelic acid (DOMA), tended to be much better retained by the tissue than the neutral deaminated metabolite, 3,4-dihydroxyphenylethyleneglycol (DOPEG). While neuronal uptake exhibited no stereoselectivity, a pronounced stereoselectivity was found for monoamine oxidase (MAO) [(-)NA〉 (+)NA] as well as for the enzymes which are in series with MAO, namely, aldehyde reductase and aldehyde dehydrogenase [(-)DOPEG〉 (+)DOPEG; (-)DOMA 〈(+)DOMA]. 3. After about 2 h of washout, the efflux of radioactivity from the tissue [which was previously incubated for 30 min with 1.2 μM of either 3H-(-)- or 3H-(±)NA] originated from one neuronal compartment with no stereoselectivity of the rate constant for the efflux of total tritium. The rate-limiting step for the neuronal efflux of tritium resided either in the net efflux of amine from the storage vesicles (normal tissues) or in the net efflux across the axonal membrane (tissues with the amine metabolizing enzymes inhibited). The effects of cocaine and phenoxybenzamine on the neuronal efflux of tritiated compounds strongly depended on the intraneuronal distribution of the 3H-amine. The results indicate that cocaine has only one site of action (neuronal uptake), while phenoxybenzamine exerts reserpine-like as well as cocaine-like effects. 4. The neuronal efflux of tritium from normal tissues preloaded with 3H-(-)- or 3H-(±)NA consisted mainly of amine metabolites (90% of the total; most of this was DOPEG). Since after 2 h of washout the tissue contained hardly any metabolites, these metabolites did not represent pre-formed metabolites (formed during the period of preloading) but newly formed metabolites resulting from the catabolism of the neuronally stored amine. This catabolism was brought about through the activity of presynaptic enzymes and was stereoselective in that more DOPEG, less DOMA and less O-methylated metabolites were formed from (-)-than from (+)NA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00500315
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  • 3
    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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  • 4
    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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  • 5
    Electronic Resource
    Electronic Resource
    The rate constants for the efflux of the metabolites of noradrenaline from rabbit aortic strips (1978)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 302 (1978), S. 207-215 
    Details
    ISSN: 1432-1912
    Keywords: Rate constants for efflux ; Efflux of noradrenaline metabolites ; Rabbit aorta ; Metabolism of noradrenaline
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. Rabbit aortic strips were incubated with 1.18 μM labelled noradrenaline for 30 min and then washed with amine-free solution for at least 110 min. From the last efflux sample and from the metabolite content of the strip analysed at the end of the experiment, rate constants for the efflux of the metabolites were calculated in two ways: a) as k = rate of efflux/metabolite content of strip, or b) as the slope of the regression line relating rate of efflux to metabolite content of the strip. Both determinations yielded essentially the same ranking order, and the results of b) indicated that there was no tight binding of metabolites in the tissue. 2. The rate constants for the efflux of glycols (DOPEG and MOPEG) and normetanephrine were much higher than those of the acid metabolites (DOMA and VMA). Although this ranking order agrees with results obtained with perfused hearts, k-values obtained from experiments with incubated strips tended to be lower (by a factor of 1.6 to 14.1) than k-values derived from experiments with perfused hearts. Since this difference was smallest for the acid metabolites and highest for the glycols, it is likely that considerable redistribution of the metabolites with high rate constants takes place in incubated, but less so in perfused tissues. 3. The rate constants for the efflux of metabolites also influence the rate of the approach of the metabolite content of the strip to steady state (during the incubation with noradrenaline): the rate of approach to steady state increases with increasing rate constant for efflux. 4. The apparent half time for the efflux of a metabolite (obtained from the slope of the efflux curve) equals the half time calculated from the rate constant for efflux $$\left( {t/2 = \frac{{1{\text{n 2}}}}{k}} \right)$$ , provided there is no formation of the metabolite during the relevant period of wash out. However, a discrepancy between the two parameters becomes the more noticeable, the higher the rate of formation of the metabolite during the period of observation. 5. In conjunction with earlier observations, the present results show that the efflux of metabolites observed during wash out of tissues previously loaded with noradrenaline is determined by a) the rate constant for efflux (k) and b) the formation (or lack of formation) of the metabolites during the period of observation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00517987
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  • 6
    Electronic Resource
    Electronic Resource
    The determination of the rate constant for the efflux of an amine from efflux curves for amine and metabolite (1978)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 304 (1978), S. 147-155 
    Details
    ISSN: 1432-1912
    Keywords: Rate constant for efflux of amine ; Isoprenaline ; Simulated efflux curves ; Extraneuronal mechanism ; Mathematical model
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. Rat hearts were perfused with 0.1 μM 3H-isoprenaline for 10 min in the presence of 10 μM U-0521 to inhibit catechol-O-methyl transferase (COMT) and then washed out with amine-free solution. Analysis of efflux curves revealed a preferential filling of one (compartment III) of the two extraneuronal compartments described by Bönisch et al. (1974). U-0521 inhibited the efflux of isoprenaline from compartment III. Omission of U-0521 from the wash out solution quickly restored COMT activity. It was then possible to determine the rate constant for the efflux (k s) of isoprenaline from rate of efflux and amine content of tissue. 2. A procedure is developed which permits the calculation of k s from efflux curves for amine and metabolite without any need for determining the amine content of the tissue. With this procedure, k s can be determined even when there is a “bound fraction” (i.e., a second compartment, the amine content of which does not contribute to the experimentally determined efflux). The procedure is based on the fact that, for a single compartment in which the amine is metabolized and from which there is efflux of amine and metabolite, parallel efflux curves (i.e., plots of log rate of efflux against time) are obtained, if the rate constant for the efflux of the metabolite (k p) is higher than the rate constant for the loss of amine from the compartment (k system). The activity of the metabolizing enzyme determines k system and the ratio “initial rate of efflux of metabolite/initial rate of efflux of amine” (F 0). 3. A mathematical model (simulating metabolism in, and efflux of amine and metabolite from a single compartment) was used to determine the distortion of F 0 by “k system/k P” (when k P limits the efflux of the metabolite). An estimate of k s obtained from F 0 and from k system agrees well with the estimate of k s obtained directly (see 1, above).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00495551
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  • 7
    Electronic Resource
    Electronic Resource
    Kinetics of the uptake and metabolism of 3H-(±) isoprenaline in the rat submaxillary gland (1978)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 305 (1978), S. 51-63 
    Details
    ISSN: 1432-1912
    Keywords: Isoprenaline ; Submaxillary gland ; Extraneuronal catecholamine uptake ; “O-methylating systems” ; Corticosteroids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. The uptake and O-methylation of 3H-(±)isoprenaline was studied in slices of the rat submaxillary gland. 2. The initial uptake of 3H-isoprenaline after inhibition of catechol-O-methyl transferase (COMT) was described by a single saturable process with relatively high K m (311 μM) and V max (101 nmoles·g−1·min−1). Both corticosterone and normetanephrine were competitive inhibitors of uptake. 3. When examined at substrate concentrations lower than the K m for uptake (and after block of COMT), 3H-isoprenaline distributed into two compartments in the tissue which approached equilibrium with half times of 2.4 and 15.8 min. The filling of both compartments was inhibited by corticosterone or phenoxybenzamine and also by high-K+ medium (in which 118 mM NaCl of the incubation medium had been replaced by KCl), but remained unaffected on substituting 118 mM NaCl with Tris-HCl. 4. In tissues in which COMT was not inhibited, the metabolism of 3H-isoprenaline to 3H-O-methylisoprenaline proceeded at a constant rate from the beginning of the incubation with the amine. When the substrate concentration was very low, little unchanged 3H-isoprenaline was found in the tissue. On the other hand, at high substrate concentrations the parent amine accumulated in the tissue, and at a time when 0-methylation had reached a steady state, the accumulation of 3H-isoprenaline was continuing. 5. The formation of 3H-O-methylisoprenaline was impaired by the presence of corticosterone, normetanephrine, phenoxybenzamine or 17-β-oestradiol with no indication of inhibition of COMT. While lowering the external Na+ concentration (on replacing 118 mM NaCl by 236 mM sucrose) did not affect the formation of 3H-O-methylisoprenaline, replacement of 118 mM NaCl by KCl reduced it. 6. The dependence of the steady-state rate of formation of 3H-O-methylisoprenaline on the substrate concentration in the incubation medium showed that two saturable components participated in the O-methylation of 3H-isoprenaline (low K m system: K m =7.2 μM and V max=1.2 nmoles·g−1·min−1; high-K m system: K m =339 μM and V max=4.6 nmoles·g−1·min−1). Corticosterone and normetanephrine competitively inhibited both the low-K m and the high-K m O-methylation. 7. The results indicate that the submaxillary gland of the rat resembles other tissues in having a low-K m (high-affinity) “O-methylating system” as well as a high-K m (low-affinity) extraneuronal uptake mechanism for catecholamines.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00497006
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  • 8
    Electronic Resource
    Electronic Resource
    Kinetic analysis of the interaction between noradrenaline and Na+ in neuronal uptake: Kinetic evidence for CO-transport (1979)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 309 (1979), S. 99-107 
    Details
    ISSN: 1432-1912
    Keywords: Neuronal uptake ; Noradrenaline ; Effects of Na+ ; Na+-coupled membrane transport ; Rat vas deferens
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. Vasa deferentia obtained from reserpine-pretreated rats were incubated (under conditions of inhibition of both monoamine oxidase and catechol O-methyltransferase) in medium containing various concentrations of 3H-(−)noradrenaline (1.25–30.25 μmol·l−1) and Na+ (0–143 mmol·l−1; isosmolality maintained by Tris+). Initial rates of neuronal uptake (v i ) were determined in each single vas from the difference between the uptake of noradrenaline occurring in the absence and that occurring in the presence of 100 μmol·l−1 cocaine. 2. The uptake of noradrenaline observed after exposure to cocaine was virtually identical with that observed after incubation in Na+-free medium (containing or not containing cocaine). Under these experimental conditions, 70% of the uptake was due to extracellular distribution of the amine, and not only this part of uptake, but also the remaineder was linearly related to the noradrenaline concentration in the medium. 3. The neuronal uptake of noradrenaline showed saturation with increasing concentrations of noradrenaline or Na+. When determined at several fixed concentrations of Na+ (or noradrenaline), the plots of 1/v i vs. 1/[noradrenaline] (or 1/[Na+]) were all linear and intersected at a common point to the left of the ordinate and above the abscissa. Increases in the fixed concentration of Na+ (or noradrenaline) progressively increased the apparent V max and progressively decreased the apparent K m of the system for noradrenaline (or Na+). Moreover, the vertical intercept (1/apparent V max) and the slope (apparent ratio of K m /V max) of the Lineweaver-Burk plots were linearly related to the reciprocal of the concentration of the “fixed” substrate. 4. Thus, the neuronal uptake mechanism exhibits the kinetic properties of a two-substrate sequential reaction in which both noradrenaline and Na+ (1:1) must bind to the carrier for transport of noradrenaline to occur and in which noradrenaline and Na+ act as mutually cooperative co-substrates.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00501216
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  • 9
    Electronic Resource
    Electronic Resource
    The inhibitory effect of some monovalent cations on the stimulation by Na+ of the neuronal uptake of noradrenaline (1979)
    Springer
    Naunyn-Schmiedeberg's archives of pharmacology 309 (1979), S. 89-97 
    Details
    ISSN: 1432-1912
    Keywords: Neuronal noradrenaline uptake ; Na+-dependent noradrenaline transport ; Effect of monovalent cations ; Rat vas deferens
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. Vasa deferentia obtained from reserpine-pretreated rats were incubated (after inhibition of both monoamine oxidase and catechol O-methyltransferase) in media containing 1.1 μmol·l−1 3H-(−)noradrenaline and various concentrations of Na+ (0–140 mmol·l−1; isosmolality maintained by sucrose or by several monovalent cations). Initial rates of neuronal uptake were determined in each single vas from the difference between “total” and “cocaine-resistant” uptake of 3H-noradrenaline. 2. The “cocaine-resistant” uptake (i.e., the distribution of 3H-noradrenaline observed in the presence of 100 μmol·l−1 cocaine) was considered to be nonneuronal. It was entirely independent of both the external Na+ concentration and the substance used to replace Na+ (or NaCl) in the medium. 3. The neuronal uptake of 3H-noradrenaline was virtually absent in Na+-free medium and was progressively stimulated by increasing Na+ concentrations. The stimulation of uptake by low Na+ concentrations was most pronounced when Tris+ was used to replace Na+; i.e., all other substitutes tested here (including sucrose, Li+, choline+ and K+) inhibited neuronal uptake when compared with Tris+. 4. While the Na+-dependent stimulation of neuronal uptake followed Michaelis-Menten kinetics in Tris+- or Li+-containing media, the kinetics of uptake stimulation by Na+ were rather complex in media containing choline+ or K+ as the substitute cation. 5. Li+ and K+ acted as competitive inhibitors with respect to Na+, whereas the inhibition of neuronal uptake by choline+ was the more pronounced, the higher the concentration of external Na+. 6. At concentrations higher than 25 mmol·l−1, the impairment of neuronal uptake by K+ exceeded that predictable from competitive inhibition of the action of Na+. This was due to the fact that high external K+ concentrations decelerated net uptake very early in the time course of amine accumulation, so that initial rates of uptake are likely to be underestimated under these conditions. 7. Thus, apart from maintaining isosmolality, several substances used to replace Na+ in the medium have inhibitory effects which must be considered in experiments designed to examine the role of Na+ in membrane transport of noradrenaline.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00501215
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