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Uptake of (−) 3H-norepinephrine by storage vesicles prepared from whole rat brain: Properties of the uptake system and its inhibition by drugs
Pergamon Press
Life Sciences, Vol . 21, pp . 1075-1086 Printed in the U .S .A .
UPTAKE OF (-) 3H-NOREPINEPHRINE BY STORAGE VESICLES PREPARED FROM WHOLE RAT BRAIN : PROPERTIES OF THE UPTAKE SYSTEM AND ITS INHIBITION BY DRUGS ,Frederic J . Seidler, D . F . Kirksey, Christopher Lau, William L . Whitmore and Theodore A . Slotkin Department of Physiology and Pharmacology Duke University Medical Center 27710 Durham, North Carolina (Received in final form September 1, 1977)
Neurotransmitter storage vesicles were isolated from rat brain by differential centrifugation and the uptake of C-) 3 H-ngrepinephrine was determined in vitro . Uptake showed a marked temperature dependence, an absolute requirement for ATP-Mg 2 , and was inhibited in vitro by reserpine . Uptake was linear for 5 min at 30 0 , but not at 37 0 . The uptake was saturable and displayed a single Km value of 4 x 10 -7 M . Other phenylamines and indoleamines displayed competitive inhibition pf norepinephrine uptake ; the affinities followed the rank order : reserpine>harmaline>serotonin>epinephrine>dopamine>norepinephrine>metaraminol . Uptake was reduced in vesicles isolated from rats treated intracisternally with 6-hydroxydopamine but not from rats treated With 5,6-dihydroxytryptamine, suggesting that most of the uptake occurs in catecholaminergic, and not serotpnergic, vesicles . This method provides a ready characterization of pharmacologic effects on rat brain storage vesicle properties, as demonstrated by the prompt and complete inhibition of uptake in vitro after administration of reserpine in vivo . The uptake of catecholamines by neurons is characterized by two distinct and separable processes . One system transports amines from the synaptic cleft to the neuronal cytoplasm, utiliz ing a sodlum-potassium-activated ATPase, and uptake is inhibited by ouabain, cocaine and tricyclic antidepressants (1) . In the second process, catecholamines are translocated from the cytoplasm to the interior of the storage vesicle, a process inhibited by reserpine and tetrabenazine and dependent upon a magnesiumactiVated ATPase (2) . These two systems play different roles in neuronal function . Uptake from the synaptic, cleft is responsible primarily for termination of the actions of released neurotransmitter (3) and only secondarily for maintenance of catecholamine stores ; Indeed, during neuron stimulation, most of the norepinephrine taken back up from the synaptic cleft apperas to be destroyed by intraneuronal monamine oxidase, rather than undergoing reutilization (4), and furthermore, neither acute nor 1075
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chronic treatments of animals with drugs which block synaptic uptake cause significant transmitter depletion (5,6) . On the other hand, drugs which inhibit uptake into storage vesicles have as their primary action a profound depletion of amines (7-y) emphasizing the role of the vesicular uptake system in the maintenance of transmitter stores . The synaptic and vesicular uptake mechanisms differ also in the degree to which they have been studied in various tissues . In the peripheral nervous system, synaptic uptake into a wide variety of tissues, either in slices or perfused whole organs, has been examined extensively, as has synaptic uptake in the brain using slices or synaptosomal preparations (1, 10, 11) . The relatively (although not absolutely) specific nature of the synaptic uptake mechanisms of various biogenic amine neurons, coupled with the high yield and stability of synaptosomes has aided in the study of the properties of central synaptic uptake mechanisms for norepinephrine, dopamine and serotonin (12-14) . These also have enabled the evaluation of in vivo drug effects on synaptic uptake in preparations from smâll animals . The properties which favor ready study of synaptic uptake mechanisms are generally lacking in storage vesicle uptake systems . From most neuronal tissues, yields of isolated vesicles are low and the uptake mechanism unstable ; indeed, even short post-mortem delays in preparing vesicles from bovine splenic nerve results in substantial loss of vesicular amine stores and alterations in uptake and release capabilities of the vesicles (15) . Storage vesicles also exhibit thermolability, thus limiting the period of time that they can be incubated (16, 17) . In preparations from the central nervous system, these factors are further complicated by the apparent lack of specificity of vesicular uptake for a given amine (2) . Consequently, most vesicle uptake systems have been done with preparations from large animals (cow, sheep, pig), primarily in the adrenal medulla and splenic nerve, and to a lesser extent in heart and sympathetic ganglia (2) . An early study with storage vesicle fractions prepared from whole rat brain suggested that uptake of norepinephrine can occur in vitro (16) ; however, in this study, ATP and magnesium were not supplied in the incubation medium, and consequently high concentrations of norepinephrine and long incubation times were necessary to demonstrate a net uptake . In this preparation, reserpine reduced uptake somewhat, but not as effectively as in vesicle preparations from other tissues (10) . More recently, Philippu and coworkers (17-20) have examined uptake of biogenic amines into crude storage vesicle fractions prepared from various regions of pig brain, and have found properties similar to those of vesicles from sympathetic neurons and adrenal medulla . The quantity of tissue required for these measurements (2 g . of brain for each uptake sample), however, rendered the reported method unsuitable for use in brain regions from small animals . However, it seemed likely that, with modification, this method could be utilized with less tissue, thus enabling evaluation of drug effects on storage vesicles from rat brain . The current study describes the properties of storage vesicle
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uptake systems in rat brain in terms of requirements for uptake, relative specificity, saturation kinetics and kinetics of inhibition as well as demonstrating the utility of the method for evaluation of effects on storage vesicle uptake of drugs administered in vivo . These are measured in a system using vesicles from as little as 130 mg of brain per assay . Methods A crude fraction containing storage vesicles from whole rat brain was prepared by differential centrifugation by a modification of the method of Philippu and Beyer (17) . Sprague-Dawley rats (Zivic-Miller) were decapitated and brains were homogenized with a Dual ground-glass homogenizer in 4 volumes of 300 mM sucrose buffered with 25 mM Tris (pH 7 .4) containing 10 VM iprpniazid . The homogenate was centrifuged at 1000 x g for 15 min and the pellet discarded . The supernatant was recentrifuged at 20,000 x g for 30 min and the supernatant of this latter centrifugation was sedimented at 100,000 x g for 30 min . Using a Teflon-to-glass homogenizer, the crude vesicle pellet was resuspended gently with 2 up-down strokes in 130 mM potassium phosphate buffer, pH 7 .4, in a volume equal to that of the original homogenate . This suspension was used for subsequent determinations . Each sample contained 0 .67 ml of vesicle preparation (representing 133 mg . of brain tissue), 0 .83 ml of 2 mm ATP + MgS04 in phosphate buffer, 17 ul of 1 mM ascorbic acid, 8 .3 pl of 1 mM iproniazid ; 1 ul of 83 .3 PM (-) 3H-norepinephrine (New England Nuclear, 5 .85 Ci/mmol), and phosphate buffer to In some experiments, concentramake a final volume of 1 .7 ml . tions of ATP-Mg 2+ or of norepinephrine were varied or drugs added to the incubation medium . Unless otherwise noted, samples were incubated for 4 min at 30 ° with duplicate samples kept on ice to serve as blanks . Incubations were stopped by addition of 1 .7 ml of ice-cold phosphate buffer and the labeled vesicles either sedimented by centrifugation at 100,000 x g for 30 min or filtered on cellulose acetate paper (see below) . The 100,000 x g supernatant was discarded and the vesicle pellet washed by addition of 3 ml of fresh phosphate buffer without resuspension of the pellet . After centrifugation for 15 min at 100,000 x g the wash was repeated and the sample recentrifuged . The final pellet was resuspended in 1 ml of water using glass-to-glass homogenization, added to 10 ml of Aquafluor (New England Nuclear) and the sample counted by liquid scintillation spectrometry at an efficiency of 40% (determined by external standardization) . Equivalent results were obtained by rapid vacuum filtration of the incubation mixture on cellulose acetate filter paper (Millipore type EG, pore size 0 .2 um) immediately after stopping the uptake by addition of ice-cold phosphate buffer ; the paper was washed three times with cold buffer and counted in 10 ml of Aquafluor at an efficiency of 40% (determined by channels ratio method) . The filtration apparatus contained 12 2 .5 cm ports connected in parallel, with vacuum adjusted such that filtration took approximately 20 sec . The filtration method took far less time and was as reliable as the centrifugation method . Since the pore size is considerably larger than the diameter of storage vesicles, it is perhaps surprising that results were
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equivalent to those obtained with centrifugation, implying nearly total trapping of vesicles on the paper ; an identical phenomenon is seen with splenic nerve vesicles, and it is due probably to either vesicle aggregation or to the tortuous nature of the interspaces among the fibers in the disc (21) . For either centrifugation or filter method, uptake was determined by subtracting the 0° tissue blank from the 30° sample and expressed as pmoles of norepinephrine taken up in 4 min per original gram of brain . Typical 0° tissue blanks contained 500-700 CPM and 30 ° samples 3000-4000 CPM . In the filtration method, binding of (-) 3H-norepinephrine to the paper in the absence of tissue was approximately 350 CPM and showed no dependence on ATP -Mg2+ concentration . Results are reported as means ± standard errors, and the levels of significance calculated by Students? t-test . Results The uptake of (-) 3 H-norepinephrine into storage vesicles prepared from whole rat brain exhibited a marked dependence on temperature (Fig . 1) . In the presence of ATP and magnesium, Figure 1
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Effects of time and temperature on (-) 3 H-norepinephrine uptake into rat brain storage vesicles expressed per gram of brain . Each point is the average of duplicate ortrip2 licate determinations . ATP-Mg concentration was 1 mM .
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there was little or no uptake at 0° , but substantial uptake was observed at 20°, 30 ° or 37 ° . While uptake at the highest two temperatures was greater than at 20 ° , there was little difference between 30° and 37° . Uptake appeared to be linear with time for approximately 5 min at 20 ° and 30°, but leveled off thereafter . At 37°, an actual decline in uptake was observed between 5 and 7 min . Consequently, 4 min at 30 ° was used thereafter as the best combination of time and temperature for studying uptake . To determine the energy requirement for uptake, vesicles were incubated with varying concentrations of ATP and magnesium (Fig . 2) . In the absence of ATP and magnesium, uptake was only 0 .7 pmole/g brain; addition of ATP and magnesium at a concentration of 0 .5 mM increased the uptake 4-fold, and 5 mM increased uptake as much as 5-fold . However, since concentrations above 2 mM tended to cause substantial increases in 0° blanks, 1mM was chosen as the concentration for the subsequent studies .
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Figure 2
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Effects of ATP -Mg2+ concentration on (-) 9H-norepinephrine uptake into rat brain storage vesicles expressed per gram of brain . Each point is the average of duplicate or triplicate determinations . Incubations lasted 4 min at 30° .
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7.'o test whether the ATP -Mg t+ -activated uptake could be 2+ inhibited by reserpine, vesicles were incubated with ATP-Mg and (-) 3h-norepinephrine and reserpine added after 3 min (Fig . Reserpine completely stopped the uptake of amine . This ~) . Figure 3 Effects of reserpine and Triton X-100 on (-) 3H-norepinephrine uptake into rat brain storage vesicles expressed per gram of brain . Each point is the average of duplicate or triplicate determinations . Reserpine or Triton X-100 were added at 3 min (arrow) .
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effect was on uptake and not on amine storage stability nor on vesicle fragility as demonstrated by the different effect produced by addition of a detergent instead of reserpine . - Triton X-100 released all of the amine that had been taken up . Thus, the effect of reserpine is specifically inhibition of uptake and not non-specific release of amines or lysis of vesicles . fhe abilities of other amines to inhibit (-) 3H-norepinephrine uptake are shown in Fig . 4 . Addition of 5 x 10 -7 M unlabeled norepinephrine reduced the uptake of (-) 3H-norepineph rine by 38%, indicating that the uptake is saturable . Dopamine was more effective (56% inhibition) than norepinephrine, and serotonin inhibited uptake 78% . Reserpine was the most effective inhibitor, reducing uptake to zero even at a drug concentration of 1 x 10 -7 M. Harmaline was less effective than reserpine, but more effective than serotonin . To further characterize saturation and inhibition of uptake, studies were done in which the concentration of norepinephrine in the labeling medium was varied over a range from 5 x 10 - M to 8 x 10 -7 M, and presented as Lineweaver-Burk plots (Fig . 5) . Norepinephrine uptake exhibited a single iua value of 3 .9 x 10- 7 M . Dopamine, epinephrine,
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Figure 4
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Effects of other amines on (-) 3H-norepinephrine uptake into rat brain storage vesicles expressed per gram of brain . Bars and lines represent means and standard errors of 4-6 determinations . NE = norepinephrine, DA = dopamine, 5HT = serotonin, HAR = harmaline, RES = reserpine . metaraminol and serotonin all demonstrated purely competitive inhibition of norepinephrine u~take, with Km values of 2 .0 x 10 -7 M for dopamine, 1 .3 x 10 - M for epinephrine, 6 .3 x 10 -7 M for metaraminol and 0 .8 x 10 -7 M for serotonin . Since these studies suggested a relative lack of specificity for norepinephrine vs other amines, a study was undertaken in which rats were lightly anesthetized with ether and given either 6-hydroxydopamine (340 ug intracisternally) or 5,6-dihydroxytryptamine (120 pg intracisternally) ; each drug was dissolved in a mixture of 0 .9% NaCl and 0 .1% ascorbic acid, and 25 pl were injected into each animal . Controls received vehicle alone . These treatments cause substantial destruction of catecholaminergic (6-hydroxydopamine) and serotonergic (5,6-dihydroxytryptamine) nerve endings (22, 23) . One week after administration of these arugs, brain preparations were analyzed for storage vesicle uptake of (-) 3 H-norepinephrine as well as for synaptosomal uptake of (-) 3 h-norepinephrine as described previously (24) ; the concentration of norepinephrine in the synaptosomal uptake medium was the same as for vesicle uptake (5 x 10 -8 M) . Rats treated with 6-hydroxydopamine showed a 50% decline in storage vesicle uptake and an 80% decline in synaptosomal uptake (Table 1) . Administration of 5,6-dihydroxytryptamine did not alter norepinephrine uptake into either vesicles or synaptosomes . To test whether the isolated brain vesicles display inhibition of uptake after administration of reserpine in vivo, rats were given saline or reserpine (0 .25 mg/kg or 5 mg kg s .c .) and sacrificeu 4 hr later . While uptake of (-) 3H-norepinephrine
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Table 1 Effects of intracisternally-administered 6-hydroxydopamine or 5,6-dihydroxytryptamine on (-) 3 H-norepinephrine uptake into storage vesicles and synaptosomes isolated from rat brain . Treatment
Control (7) 6-hydroxydopamine (4) (340 ug) 5,6-dihydroxytryptamine (5) (120 ug)
Synaptosomal Uptake pmols/g . brain 15 .5 t 0 .6 3 .3 ± 0 .6*
14 .7 ± 1 .6
Vesicular Uptake pmols/g . brain 6 .7 ± 0 .5 3 .4 ± 0 .2*
6 .0 ± 0 .3
Rats were killed 7 days after drug administration . Number of animals in each group is given in parentheses . *p < 0 .001 vs . control . into isolated storage vesicles of control rats averaged 4 .38 ± 0 .63 pmols/g (6 animals), vesicles from rats given 0 .25 mg/kg of reserpine took up only 1 .70 ± 0 .14 pmols/g (6 animals), and vesicles from those given 5 mg/kg, only 0 .11 ± 0 .03 pmols/g (6 animals) . Discussion The uptake of (-) 3 H-norepinephrine into storage vesicles prepared from rat brain fulfills the requirements which typify catecholamine uptake into vesicles from a wide variety of central and peripheral neural tissues (2) . These include marked temperature dependence, saturation kinetics, an absolute requirement for ATP and magnesium, complete inhibition of uptake by reserpine in vitro and in vivo , and thermolability of the preparation . It is therefore extremely likely that a viable preparation of rat brain storage vesicles has been isolated by this procedure, and that, although this is a crude microsomal preparation, (-) 3 H-norepinephrine is being taken up almost solely by the storage vesicles present in the preparation . Similarly, the rank order of inhibitory potency of other amines is the same as in vesicles prepared from various regions of pig brain or from rat adrenal medulla (2, 17, 18, 19, 20, 25), viz . reserpine> hariaaline>serotonin>epinephrine>dopamine>norepinephrine>metaraminol . Furthermore, the kinetics of inhibition for serotonin, epinephrine, dopamine and metaraminol are competitive, as they are in other vesicle systems . Considering the relative lack of specificity of the vesicle system responsible for uptake of (-) 3 H-norepinephrine, and since the preparation was made from whole brain, the question arises whether the (-) 3H-norepinephrine is being taken up solely into noradrenergic vesicles, or whether other populations (dopaminer-
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gic, serotonergic, etc .) are also involved . The identification of a single Km and the extremely low value (4 x 10 -7 M) of the Km suggest that either a single vesicle population is involved or else that the different populations all have approximately the same Km for norepinephrine . Intracisternal administration of 5,6-dihydroxytryptamine did not produce a reduction in vesicular uptake of (-) 3H-norepinephrine ; since this drug is toxic to the serotonergic system to a much greater extent than to the catecholaminergic system, it is unlikely that uptake of (-) 3Hnorepinephrine into serotonergic vesicles represents a significant portion of the total uptake . In contrast, 6-hydroxydopamine, which affects predominantly dopaminergic and noradrenergic neurons, markedly reduced vesicular uptake of (-) 3H-norepinephrine, suggesting that most of the uptake is occurring in catecholaminergic vesicles . However, synaptosomal (-) 3H-norepinephrine uptake, which is thought to occur preferentially in norepinephrine neurons (12-14), was reduced to a greater extent by 6-hydroxydopamine treatment than was vesicular uptake ; this suggests that significant contributions may be made by vesicles containing other transmitters (dopamine?), even though a large portion of the (-) 3 H-norepinephrine is being taken up by noradrenergic vesicles . Clear identification of the vesicle population or populations responsible for (-) 3H-norepinephrine uptake must await further studies using rat brain regions . While the properties of rat brain storage vesicles qualitatively resemble those of vesicles from pig hypothalamus and striatum (17, 18), there are quantitative differences between Z+ the preparations . The degree of uptake stimulation by ATP-Mg is much lower (2-fold) in pig hypothalamic vesicles than found here for rat brain vesicles ; even high concentrations (6 x 10 -5 M) of reserpine were unable to inhibit totally the uptake in pig hypothalamic vesicles, whereas in rat brain vesicles, inhibition was .complete at 10 -7 M reserpine . Additionally, the Km and Ki values for norepinephrine, dopamine and reserpine are an order of magnitude lower in rat brain vesicles than those reported for pig striatum . These differences may reflect species variations in vesicle properties or, alternatively, potential deterioration of the vesicle preparation in the pig, perhaps due to the time lags between killing the pig, brain removal and transportation from abbatoir to laboratory . However, it must be noted that differences in amine concentrations in the medium, temperature and time requirements and stereoisomer used in the two types of preparations can influence the Km values as well as ATP-Mg Z+ requirements and reserpine sensitivities, and therefore caution must be exercised in attributing the different values to species variation alone . A similar 10-fold difference noted in the Km for catecholamine uptake in vesicles from bovine adrenal glands vs rabbit or rat adrenals, (26-29), also may be attributable to species, post-mortem delays or incubation conditions . The extension of vesicle uptake techniques to rat brain should enable future evaluation of short- and long-term effects on vesicle systems of drugs which act on neurotransmitters . As an example, rata were administered reserpine, a drug which is known to inhibit vesicular uptake mechanisms in vitro and in vivo . There was a prompt and complete inhibition of uptakéin rat brain vesicles isolated subsequent to drug administration .
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The persistence of action of this drug in brain storage vesicles can now be examined by direct measurement . Acknowledg ements Supported by USPHS HD-09713 and DA-00465 . Dr . Theodore A . Slotkin is the recipient of Research Scientist Development Award DA-00006 from the National Institute on Drug Abuse . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 " 16 . 17 . 18 . 19 . 20 . 21 . 22 .
D .M . PATON, in "The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines" (Ed . D .M . Paton) pp . 49-66, Raven Press, New York (1976) . A . PHILIPPU, in "The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines" (Ed . D .M . Paton) pp . 215-246, Raven Press, New York (1976) . G .B . KOELLE, in "The Pharmacological Basis of Therapeutics," 5th edition (Ed . L .S . Goodman and A . Gilman) pp . 404-444, MacMillan, New York (1975) " M .L . DUBOCOVICH and S .Z . LANGER, J . Pharmacol . Exp . Ther . 198 : 83-101 (1976) . .E . LEONARD and W .F . KAFOE, Biochem . Pharmacol . 25 : 1939B 1942 (1976) W .F . KAFOE and B .E . LEONARD, Biochem . Pharmacol . 26 : 10831084 (1977) . A . CARLSSON, Handb . Exp . Pharmacol . 19 : 529-592 (1965) . T .A . SLOTKIN, in Neuropoisons : Their Pathophysiological Actions" vol . 2 (Ed . L . L . Simpson and D .R . Curtis) pp . 1-60, Plenum, New York (1974) . R .E . STITZEL, Pharmacol . Rev . 28 : 179-205 (1976) . U . S . v . EULER, Handb . Exp . Pharmacol . 33 : 186-230 (1972) . S .B . ROSS, in "The Mechanism of Neuronaland Extraneuronal Transport of Catepholamines" (Ed . D .M . Paton) pp . 67-93, Raven Press, New York (1976) . K . FUXE and U . UNGERSTEDT, Histochemie 13 : 16-28 (1968) . L .L . IVERSEN, in "Advances in Biochemical Psychopharmacology" vol . 2 (Ed . E . Costa and E . Giacobini) pp . 109-132, Raven Press, New York (1969) . M .J . KUHAR, E .G . SHASKAN and S .H . SNYDER, J . Neurochem . 18 : 333-343 (1971) . TT THURESON-KLEIN, R .L . KLEIN and H . LAGERCRANTZ, J_ . Neuroc to~l . 2 : 13-27 (1973) B .L . MIRK N, N .J . GIARMAN and D .X . FREEDMAN, Biochem . Pharmacol . 13 : 1027-1035 (1964) . A . IFH=PUand J . Naun n-Schmiedeber 's Arch . Pharmacol . 278 : 387-402 BEYER4973) . A.-=L-ITPU,H. BECKE and A . BURGER . Eur . J . Pharmacol . 6 : 96-101 (1969) 1. PHILIPPU, H . MATTHAEI and H . LENTZEN, Naun nSchmiedeberg's Arch . Pharmacol . 287 : 181-190 1975) " A . PHILIPPU and H . MATTHAEI, Naun n-Schmiedeber 's Arch . Pharmacol . 287 : 191-204 (1975) . R .L . KLEIN and H . LAGERCRANTZ, Acta Physiol . Scand . 83 : 179-190 (1971) . R .M . KOSTRZEWA and D .M . JACOBOWITZ, Pharmacol . Rev . _26 : 199288 (1974) .
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23 . 24 . 25 . 26 . 27 . 28 . 29 .
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H .G . BAUMGARTEN, A . BJÖRKLUND, A . NOBIN, E . ROSENGREN and H .G . SCHLOSSBERGER, Acta Physiol . Scand . Suppl . 429, pp . 1-27 (1975) . P .V . THADANI, C . LAU, T .A . SLOTKIN and S .M . SCHANBERG, J . Pharmacol . Exp . Ther . 200 : 292-297 (1977) " T .A . SLOTKIN, T .R . ANDERSON, F .J . SEIDLER and C . LAU, Biochem . Pharmacol . 24 : 1413-1419 (1975) " P . LUNDBORG, Acta Physiol . Scand . 423-429 (1966) . J . JONASSON, E . ROSENGREN and B . WALDECK, Acta Physiol . Scand . 60 : 136-140 (1964) . O .H . VIVEROS, L . ARQUEROS, R .J . CONNETT and N . KIRSHNER, Mol . Pharmacol . 5 : 69-82 (1969) . H . 0 . GREEN and T.A . SLOTKIN, Mol . Pharmacol . 9 : 748-755 (1973) .
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Life Sciences, Vol . 21, pp . 1075-1086 Printed in the U .S .A .
UPTAKE OF (-) 3H-NOREPINEPHRINE BY STORAGE VESICLES PREPARED FROM WHOLE RAT BRAIN : PROPERTIES OF THE UPTAKE SYSTEM AND ITS INHIBITION BY DRUGS ,Frederic J . Seidler, D . F . Kirksey, Christopher Lau, William L . Whitmore and Theodore A . Slotkin Department of Physiology and Pharmacology Duke University Medical Center 27710 Durham, North Carolina (Received in final form September 1, 1977)
Neurotransmitter storage vesicles were isolated from rat brain by differential centrifugation and the uptake of C-) 3 H-ngrepinephrine was determined in vitro . Uptake showed a marked temperature dependence, an absolute requirement for ATP-Mg 2 , and was inhibited in vitro by reserpine . Uptake was linear for 5 min at 30 0 , but not at 37 0 . The uptake was saturable and displayed a single Km value of 4 x 10 -7 M . Other phenylamines and indoleamines displayed competitive inhibition pf norepinephrine uptake ; the affinities followed the rank order : reserpine>harmaline>serotonin>epinephrine>dopamine>norepinephrine>metaraminol . Uptake was reduced in vesicles isolated from rats treated intracisternally with 6-hydroxydopamine but not from rats treated With 5,6-dihydroxytryptamine, suggesting that most of the uptake occurs in catecholaminergic, and not serotpnergic, vesicles . This method provides a ready characterization of pharmacologic effects on rat brain storage vesicle properties, as demonstrated by the prompt and complete inhibition of uptake in vitro after administration of reserpine in vivo . The uptake of catecholamines by neurons is characterized by two distinct and separable processes . One system transports amines from the synaptic cleft to the neuronal cytoplasm, utiliz ing a sodlum-potassium-activated ATPase, and uptake is inhibited by ouabain, cocaine and tricyclic antidepressants (1) . In the second process, catecholamines are translocated from the cytoplasm to the interior of the storage vesicle, a process inhibited by reserpine and tetrabenazine and dependent upon a magnesiumactiVated ATPase (2) . These two systems play different roles in neuronal function . Uptake from the synaptic, cleft is responsible primarily for termination of the actions of released neurotransmitter (3) and only secondarily for maintenance of catecholamine stores ; Indeed, during neuron stimulation, most of the norepinephrine taken back up from the synaptic cleft apperas to be destroyed by intraneuronal monamine oxidase, rather than undergoing reutilization (4), and furthermore, neither acute nor 1075
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chronic treatments of animals with drugs which block synaptic uptake cause significant transmitter depletion (5,6) . On the other hand, drugs which inhibit uptake into storage vesicles have as their primary action a profound depletion of amines (7-y) emphasizing the role of the vesicular uptake system in the maintenance of transmitter stores . The synaptic and vesicular uptake mechanisms differ also in the degree to which they have been studied in various tissues . In the peripheral nervous system, synaptic uptake into a wide variety of tissues, either in slices or perfused whole organs, has been examined extensively, as has synaptic uptake in the brain using slices or synaptosomal preparations (1, 10, 11) . The relatively (although not absolutely) specific nature of the synaptic uptake mechanisms of various biogenic amine neurons, coupled with the high yield and stability of synaptosomes has aided in the study of the properties of central synaptic uptake mechanisms for norepinephrine, dopamine and serotonin (12-14) . These also have enabled the evaluation of in vivo drug effects on synaptic uptake in preparations from smâll animals . The properties which favor ready study of synaptic uptake mechanisms are generally lacking in storage vesicle uptake systems . From most neuronal tissues, yields of isolated vesicles are low and the uptake mechanism unstable ; indeed, even short post-mortem delays in preparing vesicles from bovine splenic nerve results in substantial loss of vesicular amine stores and alterations in uptake and release capabilities of the vesicles (15) . Storage vesicles also exhibit thermolability, thus limiting the period of time that they can be incubated (16, 17) . In preparations from the central nervous system, these factors are further complicated by the apparent lack of specificity of vesicular uptake for a given amine (2) . Consequently, most vesicle uptake systems have been done with preparations from large animals (cow, sheep, pig), primarily in the adrenal medulla and splenic nerve, and to a lesser extent in heart and sympathetic ganglia (2) . An early study with storage vesicle fractions prepared from whole rat brain suggested that uptake of norepinephrine can occur in vitro (16) ; however, in this study, ATP and magnesium were not supplied in the incubation medium, and consequently high concentrations of norepinephrine and long incubation times were necessary to demonstrate a net uptake . In this preparation, reserpine reduced uptake somewhat, but not as effectively as in vesicle preparations from other tissues (10) . More recently, Philippu and coworkers (17-20) have examined uptake of biogenic amines into crude storage vesicle fractions prepared from various regions of pig brain, and have found properties similar to those of vesicles from sympathetic neurons and adrenal medulla . The quantity of tissue required for these measurements (2 g . of brain for each uptake sample), however, rendered the reported method unsuitable for use in brain regions from small animals . However, it seemed likely that, with modification, this method could be utilized with less tissue, thus enabling evaluation of drug effects on storage vesicles from rat brain . The current study describes the properties of storage vesicle
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uptake systems in rat brain in terms of requirements for uptake, relative specificity, saturation kinetics and kinetics of inhibition as well as demonstrating the utility of the method for evaluation of effects on storage vesicle uptake of drugs administered in vivo . These are measured in a system using vesicles from as little as 130 mg of brain per assay . Methods A crude fraction containing storage vesicles from whole rat brain was prepared by differential centrifugation by a modification of the method of Philippu and Beyer (17) . Sprague-Dawley rats (Zivic-Miller) were decapitated and brains were homogenized with a Dual ground-glass homogenizer in 4 volumes of 300 mM sucrose buffered with 25 mM Tris (pH 7 .4) containing 10 VM iprpniazid . The homogenate was centrifuged at 1000 x g for 15 min and the pellet discarded . The supernatant was recentrifuged at 20,000 x g for 30 min and the supernatant of this latter centrifugation was sedimented at 100,000 x g for 30 min . Using a Teflon-to-glass homogenizer, the crude vesicle pellet was resuspended gently with 2 up-down strokes in 130 mM potassium phosphate buffer, pH 7 .4, in a volume equal to that of the original homogenate . This suspension was used for subsequent determinations . Each sample contained 0 .67 ml of vesicle preparation (representing 133 mg . of brain tissue), 0 .83 ml of 2 mm ATP + MgS04 in phosphate buffer, 17 ul of 1 mM ascorbic acid, 8 .3 pl of 1 mM iproniazid ; 1 ul of 83 .3 PM (-) 3H-norepinephrine (New England Nuclear, 5 .85 Ci/mmol), and phosphate buffer to In some experiments, concentramake a final volume of 1 .7 ml . tions of ATP-Mg 2+ or of norepinephrine were varied or drugs added to the incubation medium . Unless otherwise noted, samples were incubated for 4 min at 30 ° with duplicate samples kept on ice to serve as blanks . Incubations were stopped by addition of 1 .7 ml of ice-cold phosphate buffer and the labeled vesicles either sedimented by centrifugation at 100,000 x g for 30 min or filtered on cellulose acetate paper (see below) . The 100,000 x g supernatant was discarded and the vesicle pellet washed by addition of 3 ml of fresh phosphate buffer without resuspension of the pellet . After centrifugation for 15 min at 100,000 x g the wash was repeated and the sample recentrifuged . The final pellet was resuspended in 1 ml of water using glass-to-glass homogenization, added to 10 ml of Aquafluor (New England Nuclear) and the sample counted by liquid scintillation spectrometry at an efficiency of 40% (determined by external standardization) . Equivalent results were obtained by rapid vacuum filtration of the incubation mixture on cellulose acetate filter paper (Millipore type EG, pore size 0 .2 um) immediately after stopping the uptake by addition of ice-cold phosphate buffer ; the paper was washed three times with cold buffer and counted in 10 ml of Aquafluor at an efficiency of 40% (determined by channels ratio method) . The filtration apparatus contained 12 2 .5 cm ports connected in parallel, with vacuum adjusted such that filtration took approximately 20 sec . The filtration method took far less time and was as reliable as the centrifugation method . Since the pore size is considerably larger than the diameter of storage vesicles, it is perhaps surprising that results were
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equivalent to those obtained with centrifugation, implying nearly total trapping of vesicles on the paper ; an identical phenomenon is seen with splenic nerve vesicles, and it is due probably to either vesicle aggregation or to the tortuous nature of the interspaces among the fibers in the disc (21) . For either centrifugation or filter method, uptake was determined by subtracting the 0° tissue blank from the 30° sample and expressed as pmoles of norepinephrine taken up in 4 min per original gram of brain . Typical 0° tissue blanks contained 500-700 CPM and 30 ° samples 3000-4000 CPM . In the filtration method, binding of (-) 3H-norepinephrine to the paper in the absence of tissue was approximately 350 CPM and showed no dependence on ATP -Mg2+ concentration . Results are reported as means ± standard errors, and the levels of significance calculated by Students? t-test . Results The uptake of (-) 3 H-norepinephrine into storage vesicles prepared from whole rat brain exhibited a marked dependence on temperature (Fig . 1) . In the presence of ATP and magnesium, Figure 1
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Effects of time and temperature on (-) 3 H-norepinephrine uptake into rat brain storage vesicles expressed per gram of brain . Each point is the average of duplicate ortrip2 licate determinations . ATP-Mg concentration was 1 mM .
4
Y i 2 w Z
TIME (min .)
there was little or no uptake at 0° , but substantial uptake was observed at 20°, 30 ° or 37 ° . While uptake at the highest two temperatures was greater than at 20 ° , there was little difference between 30° and 37° . Uptake appeared to be linear with time for approximately 5 min at 20 ° and 30°, but leveled off thereafter . At 37°, an actual decline in uptake was observed between 5 and 7 min . Consequently, 4 min at 30 ° was used thereafter as the best combination of time and temperature for studying uptake . To determine the energy requirement for uptake, vesicles were incubated with varying concentrations of ATP and magnesium (Fig . 2) . In the absence of ATP and magnesium, uptake was only 0 .7 pmole/g brain; addition of ATP and magnesium at a concentration of 0 .5 mM increased the uptake 4-fold, and 5 mM increased uptake as much as 5-fold . However, since concentrations above 2 mM tended to cause substantial increases in 0° blanks, 1mM was chosen as the concentration for the subsequent studies .
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Figure 2
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Effects of ATP -Mg2+ concentration on (-) 9H-norepinephrine uptake into rat brain storage vesicles expressed per gram of brain . Each point is the average of duplicate or triplicate determinations . Incubations lasted 4 min at 30° .
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7.'o test whether the ATP -Mg t+ -activated uptake could be 2+ inhibited by reserpine, vesicles were incubated with ATP-Mg and (-) 3h-norepinephrine and reserpine added after 3 min (Fig . Reserpine completely stopped the uptake of amine . This ~) . Figure 3 Effects of reserpine and Triton X-100 on (-) 3H-norepinephrine uptake into rat brain storage vesicles expressed per gram of brain . Each point is the average of duplicate or triplicate determinations . Reserpine or Triton X-100 were added at 3 min (arrow) .
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effect was on uptake and not on amine storage stability nor on vesicle fragility as demonstrated by the different effect produced by addition of a detergent instead of reserpine . - Triton X-100 released all of the amine that had been taken up . Thus, the effect of reserpine is specifically inhibition of uptake and not non-specific release of amines or lysis of vesicles . fhe abilities of other amines to inhibit (-) 3H-norepinephrine uptake are shown in Fig . 4 . Addition of 5 x 10 -7 M unlabeled norepinephrine reduced the uptake of (-) 3H-norepineph rine by 38%, indicating that the uptake is saturable . Dopamine was more effective (56% inhibition) than norepinephrine, and serotonin inhibited uptake 78% . Reserpine was the most effective inhibitor, reducing uptake to zero even at a drug concentration of 1 x 10 -7 M. Harmaline was less effective than reserpine, but more effective than serotonin . To further characterize saturation and inhibition of uptake, studies were done in which the concentration of norepinephrine in the labeling medium was varied over a range from 5 x 10 - M to 8 x 10 -7 M, and presented as Lineweaver-Burk plots (Fig . 5) . Norepinephrine uptake exhibited a single iua value of 3 .9 x 10- 7 M . Dopamine, epinephrine,
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Effects of other amines on (-) 3H-norepinephrine uptake into rat brain storage vesicles expressed per gram of brain . Bars and lines represent means and standard errors of 4-6 determinations . NE = norepinephrine, DA = dopamine, 5HT = serotonin, HAR = harmaline, RES = reserpine . metaraminol and serotonin all demonstrated purely competitive inhibition of norepinephrine u~take, with Km values of 2 .0 x 10 -7 M for dopamine, 1 .3 x 10 - M for epinephrine, 6 .3 x 10 -7 M for metaraminol and 0 .8 x 10 -7 M for serotonin . Since these studies suggested a relative lack of specificity for norepinephrine vs other amines, a study was undertaken in which rats were lightly anesthetized with ether and given either 6-hydroxydopamine (340 ug intracisternally) or 5,6-dihydroxytryptamine (120 pg intracisternally) ; each drug was dissolved in a mixture of 0 .9% NaCl and 0 .1% ascorbic acid, and 25 pl were injected into each animal . Controls received vehicle alone . These treatments cause substantial destruction of catecholaminergic (6-hydroxydopamine) and serotonergic (5,6-dihydroxytryptamine) nerve endings (22, 23) . One week after administration of these arugs, brain preparations were analyzed for storage vesicle uptake of (-) 3 H-norepinephrine as well as for synaptosomal uptake of (-) 3 h-norepinephrine as described previously (24) ; the concentration of norepinephrine in the synaptosomal uptake medium was the same as for vesicle uptake (5 x 10 -8 M) . Rats treated with 6-hydroxydopamine showed a 50% decline in storage vesicle uptake and an 80% decline in synaptosomal uptake (Table 1) . Administration of 5,6-dihydroxytryptamine did not alter norepinephrine uptake into either vesicles or synaptosomes . To test whether the isolated brain vesicles display inhibition of uptake after administration of reserpine in vivo, rats were given saline or reserpine (0 .25 mg/kg or 5 mg kg s .c .) and sacrificeu 4 hr later . While uptake of (-) 3H-norepinephrine
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Table 1 Effects of intracisternally-administered 6-hydroxydopamine or 5,6-dihydroxytryptamine on (-) 3 H-norepinephrine uptake into storage vesicles and synaptosomes isolated from rat brain . Treatment
Control (7) 6-hydroxydopamine (4) (340 ug) 5,6-dihydroxytryptamine (5) (120 ug)
Synaptosomal Uptake pmols/g . brain 15 .5 t 0 .6 3 .3 ± 0 .6*
14 .7 ± 1 .6
Vesicular Uptake pmols/g . brain 6 .7 ± 0 .5 3 .4 ± 0 .2*
6 .0 ± 0 .3
Rats were killed 7 days after drug administration . Number of animals in each group is given in parentheses . *p < 0 .001 vs . control . into isolated storage vesicles of control rats averaged 4 .38 ± 0 .63 pmols/g (6 animals), vesicles from rats given 0 .25 mg/kg of reserpine took up only 1 .70 ± 0 .14 pmols/g (6 animals), and vesicles from those given 5 mg/kg, only 0 .11 ± 0 .03 pmols/g (6 animals) . Discussion The uptake of (-) 3 H-norepinephrine into storage vesicles prepared from rat brain fulfills the requirements which typify catecholamine uptake into vesicles from a wide variety of central and peripheral neural tissues (2) . These include marked temperature dependence, saturation kinetics, an absolute requirement for ATP and magnesium, complete inhibition of uptake by reserpine in vitro and in vivo , and thermolability of the preparation . It is therefore extremely likely that a viable preparation of rat brain storage vesicles has been isolated by this procedure, and that, although this is a crude microsomal preparation, (-) 3 H-norepinephrine is being taken up almost solely by the storage vesicles present in the preparation . Similarly, the rank order of inhibitory potency of other amines is the same as in vesicles prepared from various regions of pig brain or from rat adrenal medulla (2, 17, 18, 19, 20, 25), viz . reserpine> hariaaline>serotonin>epinephrine>dopamine>norepinephrine>metaraminol . Furthermore, the kinetics of inhibition for serotonin, epinephrine, dopamine and metaraminol are competitive, as they are in other vesicle systems . Considering the relative lack of specificity of the vesicle system responsible for uptake of (-) 3 H-norepinephrine, and since the preparation was made from whole brain, the question arises whether the (-) 3H-norepinephrine is being taken up solely into noradrenergic vesicles, or whether other populations (dopaminer-
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gic, serotonergic, etc .) are also involved . The identification of a single Km and the extremely low value (4 x 10 -7 M) of the Km suggest that either a single vesicle population is involved or else that the different populations all have approximately the same Km for norepinephrine . Intracisternal administration of 5,6-dihydroxytryptamine did not produce a reduction in vesicular uptake of (-) 3H-norepinephrine ; since this drug is toxic to the serotonergic system to a much greater extent than to the catecholaminergic system, it is unlikely that uptake of (-) 3Hnorepinephrine into serotonergic vesicles represents a significant portion of the total uptake . In contrast, 6-hydroxydopamine, which affects predominantly dopaminergic and noradrenergic neurons, markedly reduced vesicular uptake of (-) 3H-norepinephrine, suggesting that most of the uptake is occurring in catecholaminergic vesicles . However, synaptosomal (-) 3H-norepinephrine uptake, which is thought to occur preferentially in norepinephrine neurons (12-14), was reduced to a greater extent by 6-hydroxydopamine treatment than was vesicular uptake ; this suggests that significant contributions may be made by vesicles containing other transmitters (dopamine?), even though a large portion of the (-) 3 H-norepinephrine is being taken up by noradrenergic vesicles . Clear identification of the vesicle population or populations responsible for (-) 3H-norepinephrine uptake must await further studies using rat brain regions . While the properties of rat brain storage vesicles qualitatively resemble those of vesicles from pig hypothalamus and striatum (17, 18), there are quantitative differences between Z+ the preparations . The degree of uptake stimulation by ATP-Mg is much lower (2-fold) in pig hypothalamic vesicles than found here for rat brain vesicles ; even high concentrations (6 x 10 -5 M) of reserpine were unable to inhibit totally the uptake in pig hypothalamic vesicles, whereas in rat brain vesicles, inhibition was .complete at 10 -7 M reserpine . Additionally, the Km and Ki values for norepinephrine, dopamine and reserpine are an order of magnitude lower in rat brain vesicles than those reported for pig striatum . These differences may reflect species variations in vesicle properties or, alternatively, potential deterioration of the vesicle preparation in the pig, perhaps due to the time lags between killing the pig, brain removal and transportation from abbatoir to laboratory . However, it must be noted that differences in amine concentrations in the medium, temperature and time requirements and stereoisomer used in the two types of preparations can influence the Km values as well as ATP-Mg Z+ requirements and reserpine sensitivities, and therefore caution must be exercised in attributing the different values to species variation alone . A similar 10-fold difference noted in the Km for catecholamine uptake in vesicles from bovine adrenal glands vs rabbit or rat adrenals, (26-29), also may be attributable to species, post-mortem delays or incubation conditions . The extension of vesicle uptake techniques to rat brain should enable future evaluation of short- and long-term effects on vesicle systems of drugs which act on neurotransmitters . As an example, rata were administered reserpine, a drug which is known to inhibit vesicular uptake mechanisms in vitro and in vivo . There was a prompt and complete inhibition of uptakéin rat brain vesicles isolated subsequent to drug administration .
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The persistence of action of this drug in brain storage vesicles can now be examined by direct measurement . Acknowledg ements Supported by USPHS HD-09713 and DA-00465 . Dr . Theodore A . Slotkin is the recipient of Research Scientist Development Award DA-00006 from the National Institute on Drug Abuse . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 " 16 . 17 . 18 . 19 . 20 . 21 . 22 .
D .M . PATON, in "The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines" (Ed . D .M . Paton) pp . 49-66, Raven Press, New York (1976) . A . PHILIPPU, in "The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines" (Ed . D .M . Paton) pp . 215-246, Raven Press, New York (1976) . G .B . KOELLE, in "The Pharmacological Basis of Therapeutics," 5th edition (Ed . L .S . Goodman and A . Gilman) pp . 404-444, MacMillan, New York (1975) " M .L . DUBOCOVICH and S .Z . LANGER, J . Pharmacol . Exp . Ther . 198 : 83-101 (1976) . .E . LEONARD and W .F . KAFOE, Biochem . Pharmacol . 25 : 1939B 1942 (1976) W .F . KAFOE and B .E . LEONARD, Biochem . Pharmacol . 26 : 10831084 (1977) . A . CARLSSON, Handb . Exp . Pharmacol . 19 : 529-592 (1965) . T .A . SLOTKIN, in Neuropoisons : Their Pathophysiological Actions" vol . 2 (Ed . L . L . Simpson and D .R . Curtis) pp . 1-60, Plenum, New York (1974) . R .E . STITZEL, Pharmacol . Rev . 28 : 179-205 (1976) . U . S . v . EULER, Handb . Exp . Pharmacol . 33 : 186-230 (1972) . S .B . ROSS, in "The Mechanism of Neuronaland Extraneuronal Transport of Catepholamines" (Ed . D .M . Paton) pp . 67-93, Raven Press, New York (1976) . K . FUXE and U . UNGERSTEDT, Histochemie 13 : 16-28 (1968) . L .L . IVERSEN, in "Advances in Biochemical Psychopharmacology" vol . 2 (Ed . E . Costa and E . Giacobini) pp . 109-132, Raven Press, New York (1969) . M .J . KUHAR, E .G . SHASKAN and S .H . SNYDER, J . Neurochem . 18 : 333-343 (1971) . TT THURESON-KLEIN, R .L . KLEIN and H . LAGERCRANTZ, J_ . Neuroc to~l . 2 : 13-27 (1973) B .L . MIRK N, N .J . GIARMAN and D .X . FREEDMAN, Biochem . Pharmacol . 13 : 1027-1035 (1964) . A . IFH=PUand J . Naun n-Schmiedeber 's Arch . Pharmacol . 278 : 387-402 BEYER4973) . A.-=L-ITPU,H. BECKE and A . BURGER . Eur . J . Pharmacol . 6 : 96-101 (1969) 1. PHILIPPU, H . MATTHAEI and H . LENTZEN, Naun nSchmiedeberg's Arch . Pharmacol . 287 : 181-190 1975) " A . PHILIPPU and H . MATTHAEI, Naun n-Schmiedeber 's Arch . Pharmacol . 287 : 191-204 (1975) . R .L . KLEIN and H . LAGERCRANTZ, Acta Physiol . Scand . 83 : 179-190 (1971) . R .M . KOSTRZEWA and D .M . JACOBOWITZ, Pharmacol . Rev . _26 : 199288 (1974) .
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23 . 24 . 25 . 26 . 27 . 28 . 29 .
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H .G . BAUMGARTEN, A . BJÖRKLUND, A . NOBIN, E . ROSENGREN and H .G . SCHLOSSBERGER, Acta Physiol . Scand . Suppl . 429, pp . 1-27 (1975) . P .V . THADANI, C . LAU, T .A . SLOTKIN and S .M . SCHANBERG, J . Pharmacol . Exp . Ther . 200 : 292-297 (1977) " T .A . SLOTKIN, T .R . ANDERSON, F .J . SEIDLER and C . LAU, Biochem . Pharmacol . 24 : 1413-1419 (1975) " P . LUNDBORG, Acta Physiol . Scand . 423-429 (1966) . J . JONASSON, E . ROSENGREN and B . WALDECK, Acta Physiol . Scand . 60 : 136-140 (1964) . O .H . VIVEROS, L . ARQUEROS, R .J . CONNETT and N . KIRSHNER, Mol . Pharmacol . 5 : 69-82 (1969) . H . 0 . GREEN and T.A . SLOTKIN, Mol . Pharmacol . 9 : 748-755 (1973) .
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