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Effects of S-11701 on accumulation, release and metabolism of norepinephrine in isolated canine saphenous veins
Gen Pharmac Vol 23, No 6, pp 1079-1085, 1992 Printed m Great Britain All rights reserved
0306-3623/92 $5 00 + 0 00 Copyright © 1992 Pergamon Press Ltd
EFFECTS OF S-11701 ON ACCUMULATION, RELEASE A N D METABOLISM OF NOREPINEPHRINE IN ISOLATED CANINE SAPHENOUS VEINS HENG-Y! G u o , 1 ROBERT R LORENZ, l TONY J VERBEUREN 2 and PAUL M VANHOUTTE 1 * t Department of Medicine, Center for Experimental Therapeutics, Baylor College of Medicine, Houston, TX 77030, U S A [Tel (713) 798-6453, Fax (713) 700-2469] and 2Servler Research Institute, Department of Angiology, 11, rue des Mouhneaux, 92150 Suresnes, France
(Received 14 Aprd 1992) Abstract--1 The effects S-11701 ([morphohnyl-2)-methoxy]-8-tetrahydro-l,2,3,4 qmnoleme) on accumulation, overflow and metabolism of [3H]norepmephnne were investigated m Isolated camne saphenous veins 2 Saphenous veins were incubated wlth [3H]norepmephnne m the absence or the presence of S-11701, the drug caused a concentration-dependent inhibitionof the tissuecontent of [5H]noreplnephnne and its mctabohtes, except for 3-methoxy-4-hydroxymandehc acid ( V M A ) 3 In helicalstripsof canine saphenous veins previously incubated wlth [3H]norepmephnne and then suspended for isometric tension recording and measurement of the overflow of labelled transmitterand itsmetabohtes, S-I 1701 (30/~M ) significantlyincreased the spontaneous effiuxof total 3H, thlseffectwas almost exclusivelydue to an augmentation of the effiuxof [3H ] D O P E G 4 D u n n g electncal stimulation (9V, I Hz), S-I1701 at I/~M shghtly increased the overflow of extraneuronal norepmephnnc metabohtes vothout affecting the contractile response At the hlgher concentration (30 # M ) the compound increased the contractiveresponse and the overflow of 3H, the latter was duc mainly to an increase in [ 3 H ] D O P E G and, to a lesserextent, m [3H]norepinephnne 5 D M I (I/aM) dld not interferewlth the effectsof S-I1701 on D O P E ( } efflux 6 These expenments indicate that in the camne saphenous veln, S-I1701 causes a concentrationdependent inhibitionof neuronal accumulation of [3H]norepmephnne At higher concentrations,S-I 170 I enters the adrenerglc nerve terminals independently of the neuronal anune carrier and displaces [3H]norepmephnne from its storage sites
INTRODUCTION Prehrmnary observations suggested that S-11701 ([morphohnyl-2-methoxy]-8-tetrahydro- 1,2,3,4 quinoleme) is an inhibitor of the neuronal uptake of catecholammes The neuronal uptake lnlubltors cocaine and D M I have been extensively studied in tsolated blood vessels and these substances exert a variety of effects on the accumulation, the release and the metabolism of norepmephnne (Langer, 1980, Langer and Enero, 1974, Verbeuren and Vanhoutte, 1982b, see Trendelenburg, 1972, Vanhoutte et al, 1981) The present expenments were designed to explore the effects of S-11701, a putative neuronal uptake mlubltor, on the accumulation release and metabohsm of noreplnephnne at the adrenerglc neuroeffector junction m the canine saphenous vein The selection of this blood vessel was based on our past experience with this richly innervated blood vessel (Vanhoutte et al, 1973, Verbeuren et al, 1977, 1978, Verbeuren and Vanhoutte, 1982a, b) METHODS
All experiments were performed on isolated lateral saphenous veins taken from dogs (20-30 kg) anaesthetized vath *To w h o m all correspondence should be addressed
pentobarbltal (30 mg/kg I v ) The preparations were placed m Krebs-Rmger bicarbonate solution of the foUowmg composition (retool/l) NaC1, 118 3, KCI, 4 7, MgSO4, 1 2, KH2PO4, 1 2, CaC12, 2 5, NaHCO3, 25, glucose, 11 I, and Ca-EDTA, 0 026 The solution was maintained at 37°C and oxygenated voth a mixture of 95% 02-5% CO2 Accumulatwn of [3H]norepmephrme Paired strips of veins were equlhbrated for 30rmn in Krebs-Ranger solution, with or without S-11701 They were then incubated for I hr In the same soluuons contalmng (L)-7-[3H]norepmephnne (0 3/aM) After incubation, the preparations were nnsed repeatedly with fresh, ice-cold control solution, blotted dry and weighed The radzoactlvlty was then extracted voth acetic acad (1 b0 containing 0 03 mM Na2EDTA and 5 mM ascorblc acid (Verbeuren et al, 1978) The extract was used for the measurement of total radioactivity and subsequent chromatographic analysis (Verbeuren et al, 1978) Effiux of[3H]norepmephrme Hehcal strips of saphenous veins were Incubated for 2 hr in Krebs-Ranger solution containing (L)-7-[3H]norepmephnne (0 3 pM) After incubation, the stnps were nnsed vath control solution and mounted for superfuslon and isometric tension recording (Vanhoutte et al, 1973) The preparations were superfused by means of a roller pump at 3 ml/nun with control solution aerated vath 95% 02-5% CO2 and maintained at 37°C The adrenergm nerves could be stimulated electncaUy by means of two Pt wires placed parallel to and in contact wath the strips, both the strip and the electrodes were superfused continuously S-11701 could be infused upstream from the roller pump The superfusate was collected and lnvestagated under basal conditions, durmg electrical stimulation (square waves, 9 V, 2 mS, 1 Hz) and m the absence or presence of
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HENG-YI Guo et al
1080
desmethyllm~pramme (DMI) At the end of each experiment, the stnps were blotted dry, weighed and the total trmum extracted w~th 3 ml of Soluene-350 (Packard) The total tntmm content at the start of the experiment was determined by adding the radmoactw]ty collected m all the superfusate samples to the [3H] content m the Ussue at the end of the superfuslon The release of [3H] 1s expressed as fracUonal release Chromatographtc analysts Norepmephnne was separated from ~ts major metabohtes (3,4-d~hydroxyphenylglycol, DOPEG, 3,4-dlhydroxymandehc acid, DOMA, normetanephrlne, NMN, 3-methoxy 4-hydroxyphenylglycol, MOPEG, and 3-methoxy-4-hydroxymandehc acid, VMA) by column chromatography (Verbeuren et al, 1978) The samples obtained dunng the incubation expenments were separated into 6 fractions The samples of the superfus~on expenments were separated into norepmephnne, DOPEG and a fracuon containing the extraneuronal metabohtes Radwactw~ty measurements Ahquots (1 ml) of the superfusate and of the fractions obtained dunng the column chromatograptuc procedure were added to 10ml of Insta-Gel (Packard Instrument Corp, Downers Grove, IL) and the radmacUwty was measured m a hqmd scmtdlauon spectrometer (Beckman model 8600)
Drugs The follovang pharmacolog]cal agents were used S-11701 (Servler Research InstRute, Suresnes, France), desmethyhmlpramme (Sigma Chermcals, St Lores, MO) and (L)-7-[3H]norepmephnne (sp act 14 2 C]/nmol, New England Nuclear, Boston, MA) The concentrations of all drugs are expressed as final molar (M) concentrations m the incubation or the superfusmn medium Stattstzcal analysts and ealculatwns The data are expressed as means + SEM, n = the number of dogs from which the saphenous veto was taken In mcubatmn experiments, all data are expressed on the bas]s of veto weight to correct for vanat~ons m veto size In superfusmn expenments, total tntmm overflow was expressed as fracUonal release The data obtained after column chromatographic analysis of the extraction samples were corrected for recovenes and cross-contammahons, as descnbed prewously (Verbeuren et al, 1977) For statistical analysxs of the data, Student's t-test for pa~red or unpaired
observatmns was used, P-values less than 005 were considered to be slgmficant RESULTS
Accumulatwn o f norepmephrme Paired strips of the same saphenous veins were incubated m parallel wRh [3H]norepmephnne m control solution and m solutions contammg increasing concentrations of S-11701 (0 3-30 t~M) In the control tissues, intact [3H]norepmephnne represented the major fraction of the radioactivity retained S11701 caused a dose-dependent m h l b m o n of the tissue content of total tntmm, paralleled by progress~ve decreases m the tissue content of intact [3H]norepmephnne At the maximum concentration (30 #M), the m h l b m o n was 95% ( R g 1, left) Of the metabohtes, the tissue content of D O P E G , D O M A , N M N and M O P E G was also progressively reduced, the percentage contributions of D O P E G , D O M A and N M N to the total tritium recovered remained constant over the concentraUon range of S-11701 tested whde that of M O P E G increased shghtly In contrast, the tissue content of V M A remained constant over the concentration range of S-11701 tested and its percentage contribution increased markedly m the presence of increasing concentrations of S-11701 (Table 1) Thus, m addition to the decreases m norepmephnne and the mtraneuronal metabohte (DOPEG), the extraneuronal metabohtes ( D O M A , N M N , M O P E G plus V M A ) also decreased markedly (Fig 1, right)
Efftux o f norepmephrme Basal effiux Paired strips of saphenous veins labelled wRh [3H]norepmephnne were superfused in parallel with either control solution or solutaon containing 1 # M D M I , after 30 nun the first sample was collected S-11701 caused a small increase in resting tensmn and a marked increase in the overflow
• NE
• NE + DOPEG
0 total radioactivity
0 extraneuronal
o_a
0 3x10 "7
10 "0
3x10 "0
10 .0
3x10 "5
Concentration
0 3x10 "7 of S-11701,
104
3x10 "0
10 .0
3x10 "s
M
Ftg 1 Percentage mhlbmon of the tissue content of total tntaum and intact [3H]norepmephnne (NE) (left) and of norepmephnne ( N E ) + D O P E G and of the ¢xtraneuronal metabohtes (DOMA, NMN, MOPEG + VMA) (right) caused by S-11701 Data expressed as means + SEM The astensk denotes a statmtlcally slgmficant difference (P < 0 05) from control levels
S-11701 and adrenerglc neuroeffector mteractmn
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Table I Effect of S-11701 on ttssue content and relaUve dlstnbutmn of [3H]norepmephnne and its metabohtes m canine saphenous veins incubated for 60 nun vath [3H]norepmephnne (0 3/aM)" Tissue content
(103 DPM/mgw/w) Group n Control 20
Total radmacUwty 535±10
NE 449±08
DOPEG 15±01
DOMA 36±02
NMN 04±003
MOPEG 19±02
VMA 13±01
S-11701 03/~M lpM 3pM 10pM 30pM
307±10" 236±30* 134±24" 78±21" 40±05*
241±09 183±25" 91±18" 41±13" 16±02"
07±006* 06±006* 03±005* 02±002* 01±003"
22±04* 16±04" 10±003" 05±02* 02±006*
03±003 02±001" 11±02 005±001" 005±001"
17±03 ll±02 18_+02" 09±01 06±006*
16±03 18±03" 22±05* 14±002"
Relative dxstnbutmn (% of total radmacUwty) Control 20
100
837±49
27±02
68±04
08±01
35±04
25_+02
S-11701 03#M lpM 3pM 10pM 30pM
100 100 1~ 1~ 1~
786±08* 770±17" 672±18" 502±22* 390±08*
25±02 26±02 20±02 22±03 28±05
70±11 64±10 72±11 57±11 59±10
10±01 09±02 06±01 07±01 12±01"
57±11" 53±15 93±15" 129±33" 160±31"
53±09* 78±05* 137±12" 285±17" 351±17"
4 4 4 4 4
4 4 4 4 4
aData expressedas means+ SEM *S]gnxficantlydifferent(P < 0 05) from control levels of total tritium which was due mainly to an increase in [3H]DOPEG, intact [3H]norepmephnne also increased shghtly (Fig 2, left) In the presence of DMI 1 #M), the increase m tensmn was prevented, while the increases m the overflow of total tritium, [3H]norepmephnne and [3H]DOPEG remained (Fig 2, right) Electrwal snmulatwn In these experiments, the tissues were stimulated electrically for 40 mm, S11701 was refused 20mm after the onset of the electrical stimulation In control solutmn, and m presence of DMI (1 #M), S-l1701 (1 gM) did not sigmficantly affect the contractile response nor the overflow of total t n t m m [3H]norepinephrlne, [3H]DOPEG, and of the extraneuronal metabolites (Figs 3 and 4)
At 20 #M, S-11701, given during continued electrical stimulatmn, increased the contractde response and augmented the overflow of total tntmm, the latter was due to increases in [3H]norepmephnne and especmlly m [3H]DOPEG (Fig 5, left, Fig 6, left) Infusmn of 30 # M S-11701 dunng continued electncal stlmulatmn m the presence of DMI (1/~M) did not s~gnificantly affect the contractde response, but significantly augmented the overflow of total tntmm, the latter was caused by a marked increase m the efflux of [3H]DOPEG (Fig 5, nght, Ftg 6, nght) DISCUSSION
The major goal of the present invesUgatton was to determine the effects of the potentml neuronal uptake
S-11701, 30 pM Tension (g)
2 0 t.
) s 10 min
Fractlonai release ( xl0 "3)
DPM ( x103/10ml )
In the presence of DMI, l~xM S-11701, 30 pM
.N.
20 f •[3 DOPEG 30 NE i 10 o
£]
Fig 2 Effect of S-11701 (30/~M) on tsometnc tension, fractional release and overflow of [3H]norepmephnne (hiE) and [~H]DOPEG m qmesc~nt camne saphenous veins m the absence and the presence of DMI (1 ~uM) Data expressed as means :i: SEM The astensk denotes a stattst]cally significant difference (P < 0 05) from levels reached pnor to adrmmstratlon of S-11701 OP 2~16.--K
1082
HENo-YI Guo et al
in the presence of DMI, l p M S-11701, I pM
Tension (g)
i
~
] ~ - : - - : : 2~! ) ' ~ ~ l
::::-
30j" • OPM (x103/10ml)
S-11701, I pM
2 10
10 rain
¢
NE
,f
0
~
0 Electrical stimulation, 1 Hz
Electricalstimulation, 1 Hz
Fig 3 Effects of 1/~M S-11701 on lsometnc tension, fractional release of total [3H] and efflux of [3H]norepmephnne (NE) and [3H]DOPEG from canine saphenous veins dunng electncal stimulation m the absence and the presence of DMI (1 pM) Data expressed as means+ SEM The astensks denote slgmficant &fferences (P < 0 05) from basal condmons mhihitor, S-l1701, on the accumulation, the release and the metabohsm of noreplnephnne m the canine saphenous veto The important findings of our study can be summarized as follows (a) S-11701 causes a concentration-dependent mhibmon of the t~ssue accumulation of [3H]noreplnephnne, (b) S-11701 at high concentrations penetrates the adrenerg~c nerve terminals and displaces norepmephnne from ~ts storage rues,
(c) S-11701 also affects the extraneuronal accumulation of norepmephnne, and (d) S-11701 influences the release and metabohsm of norepmephnne differently than cocaine and DMI presumably because of Rs combined actions on pre- and post-juncUonal uptake sites S-11701 caused a concentration-dependent mhibmon of the tissue accumulation of [3H]norepmephnne, at the highest concentrat]on used, the drug nearly
In the presence of DMI 150
g 100 O I
) @
50
0
m
NE
DOPEG Extraneuronal
I
NE
DOPEG Extraneuronal
Fig 4 No changes m the overflow of intact [3H]norepmephnne (NE) and its metabohtes from canine saphenous veins were mused by 1 pM S-11701 dunng electncal sttmttlatlon m the absence of presence of 1 #M DMI Data expressed as means :t: SEM and shown as percentage from the levels reached pnor to adrmmstratmn of S-11701
S-11701 and adrenerglc neuroeffector mteractzon
1083
In the presence of DMI, 1 11M S-11701, 30 pM
Tension (g)
S-11701, 30 pM
10 rain
--::¢
Fractional release ( xlO "3)
301" I N S DPM 2 ( x103/10ml ) 10:
~
•
0
.f
~
0 Electrical stimulation, 1 Hz
Electrical stimulation, 1 Hz
Fig 5 Effects of 30 pM S-11701 on lsometnc tension, fractaonal release and efltux of [3H]norepmephnne (NE) and [3H]DOPEG from canine saphenous veins dunng electrical stlmulatmn m control conchUons (left) and m the presence of 1/aM DMI (right) Data expressed as means + SEM The asterisk denotes a slgmficant chfference (P < 0 05) from basal conditions The filled circles denote slgmficant differences (P < 0 05) from levels reached prior to adnumstratlon of S-11701 abohshed the accumulat]on of the labelled transrmtter This effect of S-11701 is comparable to that of cocaine and DMI (e g Iversen, 1967, Trendelenburg, 1972, Verbeuren et al, 1978, Lorenz et al, 1980, Verbeuren and Vanhoutte, 1982b) An interesting observaUon was that the compound progresswely reduced both the accumulation of neuronal (IX)PEG) and extraneuronal (DOMA, NMN, MOPEG and VMA) metabohtes, this result chffers
~'~ ~
In the presence of DMI
±
6O0 '~'
from what has been described for cocoane m the same tzssue Indeed, m the presence of l~gh coneentrat~ons of cocaine, the actual Ussue accumulaUon of the extraneuronal metabohtes was larger than sn the control tissues (Verbeuren and Vanhoutte, 1982b) These data then suggest that, m addmon to mlub~tmg the neuronal accumulataon of norepmephnne, S11701 also blocks the passage of norepmephnne into the extraneuronal sites Consldenng the extraneuronal
500 400
=(n '0,~.
~c o
200 IO0 0
NE
DOPEG
Extaneuronal
LI/_j
NE
DOPEG
Extraneuronal
Fig 6 Changes m the overflow of intact [3H]norepmephnne (NE) and Its metabohtes from carane saphenous veins caused by 30pM S-11701 durmg electrical sUmulabon m control con&Uons (left) and m the presence of I/aM DMI (right) Data expressed as means + SEM and shown as percentage changes from the levels reached pnor to adrmmstratton of S-11701 The asterisks denote slgndicant differences (P < 0 05) from levels reached prior to adrmmstratlon of S-11701
1084
HENG-YI Guo et al
metabohtes of norepmephrlne, we noticed that the tissue accumulation of VMA remained constant over the concentration range of S-11701 tested, the percentage dlstnbutlon of th~s metabohte to the total tritium recovered increased progressively This observation confirms the fact that in the canine saphenous vein, the O-methylated-deammated metabohte, VMA, once formed, is retained much longer m the tissue than the other metabohtes (Verbeuren and Vanhoutte, 1982b, see also Trendelenburg et a l , 1980) In the canine saphenous veto as In other adrenergically Innervated tissues, prolonged inhlbmon of neuronal uptake w~th, e g cocaine or DMI does not influence the spontaneous efflux of [aH]norepmephrlne and ItS metabohtes (e g Langer, 1970, Cubeddu et a l , 1974, Langer and Enero, 1974, Endo et al , 1977, Muldoon et al , 1978, Verbeuren et a l , 1978, De Mey and Vanhoutte, 1980, Verbeuren and Vanhoutte, 1982b) Since S-11701 at 3 0 # M causes an increase in resting tension, in the efflux of [3H]norepmephnne and especially in the etflux of [3H]DOPEG, this compound, besides inhibiting neuronal uptake, must possess additional properties at the level of the adrenerglc nerve terminals The most hkely explanation for the important augmentation of the DOPEG-efftux is that S-11701 enters the adrenerglc nerve endings where it then displaces norepinephnne from its storage sites, most of the norepmephrlne is metabolized to DOPEG by the mtraneuronal monoamlne oxidase S-11701 shares this property with a vanety of other compounds such as etldocame, prazosin and labetoloi (see Vanhoutte et a l , 1981) Since a modest increase in the efflux of intact [3H]noreplnephrlne was noted with the drug, the data suggest that enough transmitter is displaced to reach the juncUonal cleft in the active form This then causes activation of the postjunctlonal ~adrenoceptors resulting In contraction It is obvious from Fig 2 that both the release of intact transmitter and the resulting contraction are small at the concentration of S-11701 used Since the increases in overflow of [3H]noreplnephrlne and [3H]DOPEG were not prevented by DMI, S-11701 does not seem to use the neuronal uptake carrier for noreplnephrlne to enter the adrenergtc nerve terminals DMI did, however, inhibit the shght increase m tension observed with S-11701, this effect probably results from a nonspeClfiC postjunctlonal acUon of DMI As illustrated in Figs 3 and 5, and m confirmation of earher studies (Vanhoutte et a l , 1973, Verbeuren et a l , 1977, 1978, Verbeuren and Vanhoutte, 1982a, b) continuous electrical stimulation of the saphenous vein strips caused a sustained contraction which Is accompamed by a substantial release of intact [3H]noreplnephrlne The efftux of the mtraneuronal metabohte DOPEG did not alter while that of the extraneuronal metabohtes increased m parallel with that of norepmephrlne (Verbeuren et a l , 1977, 1978, Verbeuren and Vanhoutte, 1982a, b) Dunng electneal stimulation, the lower concentrauon of S-11701 ( l g M ) , which inhibits the neuronal accumulaUon of norepmephnne by 60% (Fig 1) did not interfere with the contractile response or with the release and the dlsposmon of noreplnephnne With the higher concentration of S- 11701
(30/~M) an increase in the contractile response accompanied by an augmentaUon of the release of total 3H were observed As illustrated in Fig 6, the augmented 3H-release was for the greatest part due to and increased DOPEG efflux, there was also a modest increase in the overflow of norepmephrlne itself In this respect, S-11701 differs markedly from cocaine and DMI, these lnh,bltors, when added during electncal stimulation increase the norepmephnne overflow but decrease that of DOPEG (Muldoon et a l , 1978, Verbeuren et a l , 1978, De Mey and Vanhoutte, 1980, Rorle et a l , 1980, Verbeuren and Vanhoutte, 1982b) Another important d~fference between the action of S-11701 and that of cocaine and DMI is the absence of any slgmficant increase m the overflow of extraneuronal metabohtes dunng the infusion of S-11701 Both cocaine and DMI augmented the extraneuronal metabohte fractions during nerve actwatlon (Verbeuren and Vanhoutte, 1982b) This observation most likely reflects the potency of S-11701 to interfere with the extraneuronal uptake of the adrenerglc transmitter In the presence of the neuronal uptake inhibitor DMI some of the effects of S-11701 remain unaltered, thus the higher concentration of the compound stdl increases the overflow of total 3H and [3H]DOPEG indicating that DMI does not prevent the uptake of S-11701 by the adrenerglc nerves DMI does, however, prevent the contractde response and the increased overflow of [aH]norepmephrlne caused by S-11701 (see Fig 5) The latter effects of S-11701 thus seem to be the result of the mhlblUon of the neuronal uptake system In conclusion, in the canine saphenous vein, S11701 ~s a powerful inhibitor of the neuronal accumulation of noreplnephrlne The compound also enters the adrenerglc nerve terminals to cause displacement of noreplnephrlne from the storage vesicles, this norepmephrlne is nearly completely deammated by monoamlne oxldase resulting in an Increased etflux of DOPEG The entry of S-11701 into the adrenergic terminals occurs mdependently of the neuronal amine carrier since it is not prevented by DMI However, most hkely due to its inhibitory effect on the neuronal uptake mechanism, S-11701 augments the overflow of intact norepmephnne caused by nerve stimulation S- 11701 does not share the local anesthetic action and the direct stimulation effect on the smooth muscle cells reported for cocaine, nor the inhibitory effect on monoamme oxldase reported for DMI (Verbeuren and Vanhoutte, 1982a b) Acknowledgements--The authors thank Mrs Cindy Camrud, Ms Kathleen Kros, Mrs Kay Shaw and Ms Karmc Baudelocq for their secretanal assistance Dr Guo was supported by International Research Fellowship Award FO5 TW 03955, from the Fogarty International Center, NIH REFERENCES
Cubeddu L X, BarnesE M , Langer S Z and Weiner N (1974) Release of noreplnephnne and dopamme-/Ihydroxylase by nerve sUmulatlon I Role of neuronal and extraneuronal uptake and alpha-presynaptlc receptors J Pharmac Expl Ther 190, 431-450
S-11701 and adrenergdc neuroeffector interaction De Mey J G and Vanhoutte P M (1980) Companson of the reponslveness of cutaneous veins of dog and rabbit to adrenergic and chohnerglc stimulation Blood Vess 17, 27-43 Endo T , Starke K , Bangerter A and Taube H D (1977) Presynapt~c receptor systems on the noradrenerglc neurons of the rabbit pulmonary artery NaunynSchme~deberg's Arch Pharmac 296, 229-274 Iversen L L (1987) The Uptake and Storage of Noradrenahne m Sympathetw Nerves, p 253 Cambridge Umverslty Press, Cambridge Langer S Z (1970) The metabohsm of [3H]norepmephnne released by electrical stimulation from the isolated mctltatmg membrane of the cat and from the vas deferens of the rat J Phystol (Lond)208, 515-546 Langer S Z and Enero M A (1974) The potentmtlon of responses to adrenerg~c nerve stamulatmn in the presence of cocaine its relationship to the metabolic fate of released norepmephnne J Pharmac Expl Ther 191, 431-443 Lorenz R R , Powls D A , Vanhoutte P M and Shepherd J T (1980) The effects of acetylstrophantldln and ouabarn on the sympathetic adrenerglc neuroeffector junction m canine vascular smooth muscle Clrc Res 47, 845-854 Muldoon S M , Vanhoutte P M and Tyce G M (1978) Norepmephnne metabohsm in camne saphenous veto prevalence of glycol metabohtes Am J Phystol 234, H235-H243 Rone D K , Muldoon S M and Tyce G M (1980) The specific actwlty of retained and released norepmephnne in dog saphenous veto pre-labelled wtth ra&oactwe norepmephnne Life Sci 26, 707-714
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Trendelenburg U (1972) Classification of sympathomlmeUc attunes In Catecholammes (Blaschko H and Muschoh E , Eds) Spnnger, Berhn Handbook Expl Pbarmac 33, 336-362 Trenddenburg U , Bonlsch H , Graefe K H and Henschng M (1980) The rate constants for the efflux of metabohtes of catecholamlnes and phenethylamlnes Pbarmac Rev 31, 179-203 Vanhoutte P M , Lorenz R R and Tyce G M (1973) Inhlbmon of norepmephnne-[3H]-release from sympathetic nerve endings m veins by acetylchohne J Pharmac Expl Ther 185, 386-394 Vanhoutte P M , Verbeuren T J and Webb R C (1981) Local modulation of the adrenergac neuroeffector interaction m the blood vessel wall Phystol Rev 61, 151-247 Verbeuren T J and Vanhoutte P M (1982a) Deammatlon of released [3H]noradrenahne m the canine saphenous vein Naunyn Schmwdeberg's Arch Pharmac 318, 148-157 Verbeuren T J and Vanhoutte P M (1982b) Cocaine and neuronal uptake m the canine saphenous vein Naunyn Schmwdeberg' s Arch Pharmac 321, 207-212 Verbeuren T J , Coen E and Vanhoutte P M (1977) Determination of [3H]norepmephnne and Its metabohtes m superfusate from isolated blood vessels Arch Int Pharmacodyn Ther 227, 171-174 Verbeuren T J , Janssens W J and Vanhoutte P M (1978) Effects of moderate acidosis on adrenerg~c neurotranso mission in canine saphenous vems J Pharmac Expl Ther 206, 105-114
0306-3623/92 $5 00 + 0 00 Copyright © 1992 Pergamon Press Ltd
EFFECTS OF S-11701 ON ACCUMULATION, RELEASE A N D METABOLISM OF NOREPINEPHRINE IN ISOLATED CANINE SAPHENOUS VEINS HENG-Y! G u o , 1 ROBERT R LORENZ, l TONY J VERBEUREN 2 and PAUL M VANHOUTTE 1 * t Department of Medicine, Center for Experimental Therapeutics, Baylor College of Medicine, Houston, TX 77030, U S A [Tel (713) 798-6453, Fax (713) 700-2469] and 2Servler Research Institute, Department of Angiology, 11, rue des Mouhneaux, 92150 Suresnes, France
(Received 14 Aprd 1992) Abstract--1 The effects S-11701 ([morphohnyl-2)-methoxy]-8-tetrahydro-l,2,3,4 qmnoleme) on accumulation, overflow and metabolism of [3H]norepmephnne were investigated m Isolated camne saphenous veins 2 Saphenous veins were incubated wlth [3H]norepmephnne m the absence or the presence of S-11701, the drug caused a concentration-dependent inhibitionof the tissuecontent of [5H]noreplnephnne and its mctabohtes, except for 3-methoxy-4-hydroxymandehc acid ( V M A ) 3 In helicalstripsof canine saphenous veins previously incubated wlth [3H]norepmephnne and then suspended for isometric tension recording and measurement of the overflow of labelled transmitterand itsmetabohtes, S-I 1701 (30/~M ) significantlyincreased the spontaneous effiuxof total 3H, thlseffectwas almost exclusivelydue to an augmentation of the effiuxof [3H ] D O P E G 4 D u n n g electncal stimulation (9V, I Hz), S-I1701 at I/~M shghtly increased the overflow of extraneuronal norepmephnnc metabohtes vothout affecting the contractile response At the hlgher concentration (30 # M ) the compound increased the contractiveresponse and the overflow of 3H, the latter was duc mainly to an increase in [ 3 H ] D O P E G and, to a lesserextent, m [3H]norepinephnne 5 D M I (I/aM) dld not interferewlth the effectsof S-I1701 on D O P E ( } efflux 6 These expenments indicate that in the camne saphenous veln, S-I1701 causes a concentrationdependent inhibitionof neuronal accumulation of [3H]norepmephnne At higher concentrations,S-I 170 I enters the adrenerglc nerve terminals independently of the neuronal anune carrier and displaces [3H]norepmephnne from its storage sites
INTRODUCTION Prehrmnary observations suggested that S-11701 ([morphohnyl-2-methoxy]-8-tetrahydro- 1,2,3,4 quinoleme) is an inhibitor of the neuronal uptake of catecholammes The neuronal uptake lnlubltors cocaine and D M I have been extensively studied in tsolated blood vessels and these substances exert a variety of effects on the accumulation, the release and the metabolism of norepmephnne (Langer, 1980, Langer and Enero, 1974, Verbeuren and Vanhoutte, 1982b, see Trendelenburg, 1972, Vanhoutte et al, 1981) The present expenments were designed to explore the effects of S-11701, a putative neuronal uptake mlubltor, on the accumulation release and metabohsm of noreplnephnne at the adrenerglc neuroeffector junction m the canine saphenous vein The selection of this blood vessel was based on our past experience with this richly innervated blood vessel (Vanhoutte et al, 1973, Verbeuren et al, 1977, 1978, Verbeuren and Vanhoutte, 1982a, b) METHODS
All experiments were performed on isolated lateral saphenous veins taken from dogs (20-30 kg) anaesthetized vath *To w h o m all correspondence should be addressed
pentobarbltal (30 mg/kg I v ) The preparations were placed m Krebs-Rmger bicarbonate solution of the foUowmg composition (retool/l) NaC1, 118 3, KCI, 4 7, MgSO4, 1 2, KH2PO4, 1 2, CaC12, 2 5, NaHCO3, 25, glucose, 11 I, and Ca-EDTA, 0 026 The solution was maintained at 37°C and oxygenated voth a mixture of 95% 02-5% CO2 Accumulatwn of [3H]norepmephrme Paired strips of veins were equlhbrated for 30rmn in Krebs-Ranger solution, with or without S-11701 They were then incubated for I hr In the same soluuons contalmng (L)-7-[3H]norepmephnne (0 3/aM) After incubation, the preparations were nnsed repeatedly with fresh, ice-cold control solution, blotted dry and weighed The radzoactlvlty was then extracted voth acetic acad (1 b0 containing 0 03 mM Na2EDTA and 5 mM ascorblc acid (Verbeuren et al, 1978) The extract was used for the measurement of total radioactivity and subsequent chromatographic analysis (Verbeuren et al, 1978) Effiux of[3H]norepmephrme Hehcal strips of saphenous veins were Incubated for 2 hr in Krebs-Ranger solution containing (L)-7-[3H]norepmephnne (0 3 pM) After incubation, the stnps were nnsed vath control solution and mounted for superfuslon and isometric tension recording (Vanhoutte et al, 1973) The preparations were superfused by means of a roller pump at 3 ml/nun with control solution aerated vath 95% 02-5% CO2 and maintained at 37°C The adrenergm nerves could be stimulated electncaUy by means of two Pt wires placed parallel to and in contact wath the strips, both the strip and the electrodes were superfused continuously S-11701 could be infused upstream from the roller pump The superfusate was collected and lnvestagated under basal conditions, durmg electrical stimulation (square waves, 9 V, 2 mS, 1 Hz) and m the absence or presence of
1079
HENG-YI Guo et al
1080
desmethyllm~pramme (DMI) At the end of each experiment, the stnps were blotted dry, weighed and the total trmum extracted w~th 3 ml of Soluene-350 (Packard) The total tntmm content at the start of the experiment was determined by adding the radmoactw]ty collected m all the superfusate samples to the [3H] content m the Ussue at the end of the superfuslon The release of [3H] 1s expressed as fracUonal release Chromatographtc analysts Norepmephnne was separated from ~ts major metabohtes (3,4-d~hydroxyphenylglycol, DOPEG, 3,4-dlhydroxymandehc acid, DOMA, normetanephrlne, NMN, 3-methoxy 4-hydroxyphenylglycol, MOPEG, and 3-methoxy-4-hydroxymandehc acid, VMA) by column chromatography (Verbeuren et al, 1978) The samples obtained dunng the incubation expenments were separated into 6 fractions The samples of the superfus~on expenments were separated into norepmephnne, DOPEG and a fracuon containing the extraneuronal metabohtes Radwactw~ty measurements Ahquots (1 ml) of the superfusate and of the fractions obtained dunng the column chromatograptuc procedure were added to 10ml of Insta-Gel (Packard Instrument Corp, Downers Grove, IL) and the radmacUwty was measured m a hqmd scmtdlauon spectrometer (Beckman model 8600)
Drugs The follovang pharmacolog]cal agents were used S-11701 (Servler Research InstRute, Suresnes, France), desmethyhmlpramme (Sigma Chermcals, St Lores, MO) and (L)-7-[3H]norepmephnne (sp act 14 2 C]/nmol, New England Nuclear, Boston, MA) The concentrations of all drugs are expressed as final molar (M) concentrations m the incubation or the superfusmn medium Stattstzcal analysts and ealculatwns The data are expressed as means + SEM, n = the number of dogs from which the saphenous veto was taken In mcubatmn experiments, all data are expressed on the bas]s of veto weight to correct for vanat~ons m veto size In superfusmn expenments, total tntmm overflow was expressed as fracUonal release The data obtained after column chromatographic analysis of the extraction samples were corrected for recovenes and cross-contammahons, as descnbed prewously (Verbeuren et al, 1977) For statistical analysxs of the data, Student's t-test for pa~red or unpaired
observatmns was used, P-values less than 005 were considered to be slgmficant RESULTS
Accumulatwn o f norepmephrme Paired strips of the same saphenous veins were incubated m parallel wRh [3H]norepmephnne m control solution and m solutions contammg increasing concentrations of S-11701 (0 3-30 t~M) In the control tissues, intact [3H]norepmephnne represented the major fraction of the radioactivity retained S11701 caused a dose-dependent m h l b m o n of the tissue content of total tntmm, paralleled by progress~ve decreases m the tissue content of intact [3H]norepmephnne At the maximum concentration (30 #M), the m h l b m o n was 95% ( R g 1, left) Of the metabohtes, the tissue content of D O P E G , D O M A , N M N and M O P E G was also progressively reduced, the percentage contributions of D O P E G , D O M A and N M N to the total tritium recovered remained constant over the concentraUon range of S-11701 tested whde that of M O P E G increased shghtly In contrast, the tissue content of V M A remained constant over the concentration range of S-11701 tested and its percentage contribution increased markedly m the presence of increasing concentrations of S-11701 (Table 1) Thus, m addition to the decreases m norepmephnne and the mtraneuronal metabohte (DOPEG), the extraneuronal metabohtes ( D O M A , N M N , M O P E G plus V M A ) also decreased markedly (Fig 1, right)
Efftux o f norepmephrme Basal effiux Paired strips of saphenous veins labelled wRh [3H]norepmephnne were superfused in parallel with either control solution or solutaon containing 1 # M D M I , after 30 nun the first sample was collected S-11701 caused a small increase in resting tensmn and a marked increase in the overflow
• NE
• NE + DOPEG
0 total radioactivity
0 extraneuronal
o_a
0 3x10 "7
10 "0
3x10 "0
10 .0
3x10 "5
Concentration
0 3x10 "7 of S-11701,
104
3x10 "0
10 .0
3x10 "s
M
Ftg 1 Percentage mhlbmon of the tissue content of total tntaum and intact [3H]norepmephnne (NE) (left) and of norepmephnne ( N E ) + D O P E G and of the ¢xtraneuronal metabohtes (DOMA, NMN, MOPEG + VMA) (right) caused by S-11701 Data expressed as means + SEM The astensk denotes a statmtlcally slgmficant difference (P < 0 05) from control levels
S-11701 and adrenerglc neuroeffector mteractmn
1081
Table I Effect of S-11701 on ttssue content and relaUve dlstnbutmn of [3H]norepmephnne and its metabohtes m canine saphenous veins incubated for 60 nun vath [3H]norepmephnne (0 3/aM)" Tissue content
(103 DPM/mgw/w) Group n Control 20
Total radmacUwty 535±10
NE 449±08
DOPEG 15±01
DOMA 36±02
NMN 04±003
MOPEG 19±02
VMA 13±01
S-11701 03/~M lpM 3pM 10pM 30pM
307±10" 236±30* 134±24" 78±21" 40±05*
241±09 183±25" 91±18" 41±13" 16±02"
07±006* 06±006* 03±005* 02±002* 01±003"
22±04* 16±04" 10±003" 05±02* 02±006*
03±003 02±001" 11±02 005±001" 005±001"
17±03 ll±02 18_+02" 09±01 06±006*
16±03 18±03" 22±05* 14±002"
Relative dxstnbutmn (% of total radmacUwty) Control 20
100
837±49
27±02
68±04
08±01
35±04
25_+02
S-11701 03#M lpM 3pM 10pM 30pM
100 100 1~ 1~ 1~
786±08* 770±17" 672±18" 502±22* 390±08*
25±02 26±02 20±02 22±03 28±05
70±11 64±10 72±11 57±11 59±10
10±01 09±02 06±01 07±01 12±01"
57±11" 53±15 93±15" 129±33" 160±31"
53±09* 78±05* 137±12" 285±17" 351±17"
4 4 4 4 4
4 4 4 4 4
aData expressedas means+ SEM *S]gnxficantlydifferent(P < 0 05) from control levels of total tritium which was due mainly to an increase in [3H]DOPEG, intact [3H]norepmephnne also increased shghtly (Fig 2, left) In the presence of DMI 1 #M), the increase m tensmn was prevented, while the increases m the overflow of total tritium, [3H]norepmephnne and [3H]DOPEG remained (Fig 2, right) Electrwal snmulatwn In these experiments, the tissues were stimulated electrically for 40 mm, S11701 was refused 20mm after the onset of the electrical stimulation In control solutmn, and m presence of DMI (1 #M), S-l1701 (1 gM) did not sigmficantly affect the contractile response nor the overflow of total t n t m m [3H]norepinephrlne, [3H]DOPEG, and of the extraneuronal metabolites (Figs 3 and 4)
At 20 #M, S-11701, given during continued electrical stimulatmn, increased the contractde response and augmented the overflow of total tntmm, the latter was due to increases in [3H]norepmephnne and especmlly m [3H]DOPEG (Fig 5, left, Fig 6, left) Infusmn of 30 # M S-11701 dunng continued electncal stlmulatmn m the presence of DMI (1/~M) did not s~gnificantly affect the contractde response, but significantly augmented the overflow of total tntmm, the latter was caused by a marked increase m the efflux of [3H]DOPEG (Fig 5, nght, Ftg 6, nght) DISCUSSION
The major goal of the present invesUgatton was to determine the effects of the potentml neuronal uptake
S-11701, 30 pM Tension (g)
2 0 t.
) s 10 min
Fractlonai release ( xl0 "3)
DPM ( x103/10ml )
In the presence of DMI, l~xM S-11701, 30 pM
.N.
20 f •[3 DOPEG 30 NE i 10 o
£]
Fig 2 Effect of S-11701 (30/~M) on tsometnc tension, fractional release and overflow of [3H]norepmephnne (hiE) and [~H]DOPEG m qmesc~nt camne saphenous veins m the absence and the presence of DMI (1 ~uM) Data expressed as means :i: SEM The astensk denotes a stattst]cally significant difference (P < 0 05) from levels reached pnor to adrmmstratlon of S-11701 OP 2~16.--K
1082
HENo-YI Guo et al
in the presence of DMI, l p M S-11701, I pM
Tension (g)
i
~
] ~ - : - - : : 2~! ) ' ~ ~ l
::::-
30j" • OPM (x103/10ml)
S-11701, I pM
2 10
10 rain
¢
NE
,f
0
~
0 Electrical stimulation, 1 Hz
Electricalstimulation, 1 Hz
Fig 3 Effects of 1/~M S-11701 on lsometnc tension, fractional release of total [3H] and efflux of [3H]norepmephnne (NE) and [3H]DOPEG from canine saphenous veins dunng electncal stimulation m the absence and the presence of DMI (1 pM) Data expressed as means+ SEM The astensks denote slgmficant &fferences (P < 0 05) from basal condmons mhihitor, S-l1701, on the accumulation, the release and the metabohsm of noreplnephnne m the canine saphenous veto The important findings of our study can be summarized as follows (a) S-11701 causes a concentration-dependent mhibmon of the t~ssue accumulation of [3H]noreplnephnne, (b) S-11701 at high concentrations penetrates the adrenerg~c nerve terminals and displaces norepmephnne from ~ts storage rues,
(c) S-11701 also affects the extraneuronal accumulation of norepmephnne, and (d) S-11701 influences the release and metabohsm of norepmephnne differently than cocaine and DMI presumably because of Rs combined actions on pre- and post-juncUonal uptake sites S-11701 caused a concentration-dependent mhibmon of the tissue accumulation of [3H]norepmephnne, at the highest concentrat]on used, the drug nearly
In the presence of DMI 150
g 100 O I
) @
50
0
m
NE
DOPEG Extraneuronal
I
NE
DOPEG Extraneuronal
Fig 4 No changes m the overflow of intact [3H]norepmephnne (NE) and its metabohtes from canine saphenous veins were mused by 1 pM S-11701 dunng electncal sttmttlatlon m the absence of presence of 1 #M DMI Data expressed as means :t: SEM and shown as percentage from the levels reached pnor to adrmmstratmn of S-11701
S-11701 and adrenerglc neuroeffector mteractzon
1083
In the presence of DMI, 1 11M S-11701, 30 pM
Tension (g)
S-11701, 30 pM
10 rain
--::¢
Fractional release ( xlO "3)
301" I N S DPM 2 ( x103/10ml ) 10:
~
•
0
.f
~
0 Electrical stimulation, 1 Hz
Electrical stimulation, 1 Hz
Fig 5 Effects of 30 pM S-11701 on lsometnc tension, fractaonal release and efltux of [3H]norepmephnne (NE) and [3H]DOPEG from canine saphenous veins dunng electrical stlmulatmn m control conchUons (left) and m the presence of 1/aM DMI (right) Data expressed as means + SEM The asterisk denotes a slgmficant chfference (P < 0 05) from basal conditions The filled circles denote slgmficant differences (P < 0 05) from levels reached prior to adnumstratlon of S-11701 abohshed the accumulat]on of the labelled transrmtter This effect of S-11701 is comparable to that of cocaine and DMI (e g Iversen, 1967, Trendelenburg, 1972, Verbeuren et al, 1978, Lorenz et al, 1980, Verbeuren and Vanhoutte, 1982b) An interesting observaUon was that the compound progresswely reduced both the accumulation of neuronal (IX)PEG) and extraneuronal (DOMA, NMN, MOPEG and VMA) metabohtes, this result chffers
~'~ ~
In the presence of DMI
±
6O0 '~'
from what has been described for cocoane m the same tzssue Indeed, m the presence of l~gh coneentrat~ons of cocaine, the actual Ussue accumulaUon of the extraneuronal metabohtes was larger than sn the control tissues (Verbeuren and Vanhoutte, 1982b) These data then suggest that, m addmon to mlub~tmg the neuronal accumulataon of norepmephnne, S11701 also blocks the passage of norepmephnne into the extraneuronal sites Consldenng the extraneuronal
500 400
=(n '0,~.
~c o
200 IO0 0
NE
DOPEG
Extaneuronal
LI/_j
NE
DOPEG
Extraneuronal
Fig 6 Changes m the overflow of intact [3H]norepmephnne (NE) and Its metabohtes from carane saphenous veins caused by 30pM S-11701 durmg electrical sUmulabon m control con&Uons (left) and m the presence of I/aM DMI (right) Data expressed as means + SEM and shown as percentage changes from the levels reached pnor to adrmmstratton of S-11701 The asterisks denote slgndicant differences (P < 0 05) from levels reached prior to adrmmstratlon of S-11701
1084
HENG-YI Guo et al
metabohtes of norepmephrlne, we noticed that the tissue accumulation of VMA remained constant over the concentration range of S-11701 tested, the percentage dlstnbutlon of th~s metabohte to the total tritium recovered increased progressively This observation confirms the fact that in the canine saphenous vein, the O-methylated-deammated metabohte, VMA, once formed, is retained much longer m the tissue than the other metabohtes (Verbeuren and Vanhoutte, 1982b, see also Trendelenburg et a l , 1980) In the canine saphenous veto as In other adrenergically Innervated tissues, prolonged inhlbmon of neuronal uptake w~th, e g cocaine or DMI does not influence the spontaneous efflux of [aH]norepmephrlne and ItS metabohtes (e g Langer, 1970, Cubeddu et a l , 1974, Langer and Enero, 1974, Endo et al , 1977, Muldoon et al , 1978, Verbeuren et a l , 1978, De Mey and Vanhoutte, 1980, Verbeuren and Vanhoutte, 1982b) Since S-11701 at 3 0 # M causes an increase in resting tension, in the efflux of [3H]norepmephnne and especially in the etflux of [3H]DOPEG, this compound, besides inhibiting neuronal uptake, must possess additional properties at the level of the adrenerglc nerve terminals The most hkely explanation for the important augmentation of the DOPEG-efftux is that S-11701 enters the adrenerglc nerve endings where it then displaces norepinephnne from its storage sites, most of the norepmephrlne is metabolized to DOPEG by the mtraneuronal monoamlne oxidase S-11701 shares this property with a vanety of other compounds such as etldocame, prazosin and labetoloi (see Vanhoutte et a l , 1981) Since a modest increase in the efflux of intact [3H]noreplnephrlne was noted with the drug, the data suggest that enough transmitter is displaced to reach the juncUonal cleft in the active form This then causes activation of the postjunctlonal ~adrenoceptors resulting In contraction It is obvious from Fig 2 that both the release of intact transmitter and the resulting contraction are small at the concentration of S-11701 used Since the increases in overflow of [3H]noreplnephrlne and [3H]DOPEG were not prevented by DMI, S-11701 does not seem to use the neuronal uptake carrier for noreplnephrlne to enter the adrenergtc nerve terminals DMI did, however, inhibit the shght increase m tension observed with S-11701, this effect probably results from a nonspeClfiC postjunctlonal acUon of DMI As illustrated in Figs 3 and 5, and m confirmation of earher studies (Vanhoutte et a l , 1973, Verbeuren et a l , 1977, 1978, Verbeuren and Vanhoutte, 1982a, b) continuous electrical stimulation of the saphenous vein strips caused a sustained contraction which Is accompamed by a substantial release of intact [3H]noreplnephrlne The efftux of the mtraneuronal metabohte DOPEG did not alter while that of the extraneuronal metabohtes increased m parallel with that of norepmephrlne (Verbeuren et a l , 1977, 1978, Verbeuren and Vanhoutte, 1982a, b) Dunng electneal stimulation, the lower concentrauon of S-11701 ( l g M ) , which inhibits the neuronal accumulaUon of norepmephnne by 60% (Fig 1) did not interfere with the contractile response or with the release and the dlsposmon of noreplnephnne With the higher concentration of S- 11701
(30/~M) an increase in the contractile response accompanied by an augmentaUon of the release of total 3H were observed As illustrated in Fig 6, the augmented 3H-release was for the greatest part due to and increased DOPEG efflux, there was also a modest increase in the overflow of norepmephrlne itself In this respect, S-11701 differs markedly from cocaine and DMI, these lnh,bltors, when added during electncal stimulation increase the norepmephnne overflow but decrease that of DOPEG (Muldoon et a l , 1978, Verbeuren et a l , 1978, De Mey and Vanhoutte, 1980, Rorle et a l , 1980, Verbeuren and Vanhoutte, 1982b) Another important d~fference between the action of S-11701 and that of cocaine and DMI is the absence of any slgmficant increase m the overflow of extraneuronal metabohtes dunng the infusion of S-11701 Both cocaine and DMI augmented the extraneuronal metabohte fractions during nerve actwatlon (Verbeuren and Vanhoutte, 1982b) This observation most likely reflects the potency of S-11701 to interfere with the extraneuronal uptake of the adrenerglc transmitter In the presence of the neuronal uptake inhibitor DMI some of the effects of S-11701 remain unaltered, thus the higher concentration of the compound stdl increases the overflow of total 3H and [3H]DOPEG indicating that DMI does not prevent the uptake of S-11701 by the adrenerglc nerves DMI does, however, prevent the contractde response and the increased overflow of [aH]norepmephrlne caused by S-11701 (see Fig 5) The latter effects of S-11701 thus seem to be the result of the mhlblUon of the neuronal uptake system In conclusion, in the canine saphenous vein, S11701 ~s a powerful inhibitor of the neuronal accumulation of noreplnephrlne The compound also enters the adrenerglc nerve terminals to cause displacement of noreplnephrlne from the storage vesicles, this norepmephrlne is nearly completely deammated by monoamlne oxldase resulting in an Increased etflux of DOPEG The entry of S-11701 into the adrenergic terminals occurs mdependently of the neuronal amine carrier since it is not prevented by DMI However, most hkely due to its inhibitory effect on the neuronal uptake mechanism, S-11701 augments the overflow of intact norepmephnne caused by nerve stimulation S- 11701 does not share the local anesthetic action and the direct stimulation effect on the smooth muscle cells reported for cocaine, nor the inhibitory effect on monoamme oxldase reported for DMI (Verbeuren and Vanhoutte, 1982a b) Acknowledgements--The authors thank Mrs Cindy Camrud, Ms Kathleen Kros, Mrs Kay Shaw and Ms Karmc Baudelocq for their secretanal assistance Dr Guo was supported by International Research Fellowship Award FO5 TW 03955, from the Fogarty International Center, NIH REFERENCES
Cubeddu L X, BarnesE M , Langer S Z and Weiner N (1974) Release of noreplnephnne and dopamme-/Ihydroxylase by nerve sUmulatlon I Role of neuronal and extraneuronal uptake and alpha-presynaptlc receptors J Pharmac Expl Ther 190, 431-450
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