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Alteration by methadone of catecholamine uptake and release in isolated rat adrenomedullary storage vesicles
Life Sciences Vol . 19, pp . 483-492, 1976 . Printed in thë II .S .A .
Pergamon Preee
ALTERATION BY METHADONE OF CATECHOLAMINE UPTAI~ AND RELEASE IN ISOLATED RAT ADRENOMEDULLARY STORAGE VESICLES Theodore A . Slotkin, Christopher Lau, Maria Bartolomé and Frederic J . Seidler Department of Physiology and Pharmacology Duke University Medical Center Durham, North Carolina 27710 (Received in final form April 30, 1976) Incubation of isolated rat adreno ullary storage vesicles with methadone produced inhibition of H-epinephrine uptake and promotion of release of endogenous catecholamines . Neither effect was seen using morphine, nor could morphine antagonize methadone-induced catecholamine release, suggesting that these actions are not mediated by opiate receptors . Inhibition of uptake by methadone appeared to contain a competitive com~onent with a lower Ki for methadone compared to the Km for H-epinephrine . Despite competitive inhibition by methadone, the maximal uptake capacity analogous to Vmax) as determined by double-reciprocal plots, was increased by the drug, probably as a result of greater availability of intravesicular storage sites because of the drug-induced release of endogenous catecholamines . Agents which enhance or block catecholamine transport into vesicles had no effect on the catecholamine release by methadone, indicating that the latter is separable from the action on uptake . These alterations of catecholamine uptake and release may play a role in the effects of methadone on the adrenal medulla in vivo . The actions of opiates on biogenic amines of the central and peripheral adrenergic system involve effects on synthesis, uptake, storage and release (1-6) . Acute and chronic administration of morphine or methadone in vivo has been shown not only to evoke secretion of catecholamines and to increase levels of catecholamine biosynthetic enzymes, but also to inhibit the uptake of amines into either neurons or storage vesicles (7-9) . In cardiac adrenergic neurons, Montel and Starke (7)found that morphine in vitro can block catecholamine reuptake across the neuronal membrane and in the brain, Ciofalo (B) has demonstrated inhibition of serotonin uptake into synaptosomal preparations by methadone . While morphine administration in vivo also produces inhibition of catecholamine uptake into storage vesicles of the adrenal medulla, in contrast to the uptake studies in neurons, this action of morphine adminis tration in vivo is not observed with drug exposure in vitro (3,9), suggesting 483
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Methadone on Catecholamine Uptake, Release
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an indirect action possibly mediated by physiological or cellular actions of the drug . Similarly, the rate of spontaneous release of catecholamines from adrenal vesicle preparations is slowed after morphine administration ~n vivo . but not by direct exposure to drug ~_n vitro (3) . However, experiments with methadone suggest that this opiate may have a direct action ~ vitro on amine uptake into adrenomedullary vesicles (10) , indicating a different action from morphine ; administration of even a single dose of methadone a.5 mg/kg) to rats produced significant inhibition of the vesicular amine pump, and the effect was more pronounced on chronic treatment . In the current study, the mechanisms by which exposure of isolated adrenomedullary vesicles to methadone in vitro alters both uptake and release of catecholamines have been examined and contrasted with the effects of morphine . Method s Release of endogenous catecholamines . Male Sprague-Dawley rats (Zivic-Miller) weighing 200-250 g were decapitated and their adrenals removed, pooled and homogenized in 2 .5 ml/ 5gland of 0 .3 M sucrose buffered at pH 7 .4 M iproniazid to inhibit monoamine oaidase . with 0 .025 M Tris, containing 10 After centrifugation for 10 min at 800 g to remove debris, 0 .5 ml aliquots of the catediolamine storage vesicle containing supernatant were added to 0 .5 ml of sucrose-Tris containing a vari~v of agents singly or in combination, including methadone, morphine, ATP-Mq or reserpLne . Samples were incubated for 0, 10, 20, 30, 40 or 60 min at 30 °C, at which times 2 ml of ice-cold sucrose-Tris were added . After centrifugation for 10 min at 26 , 000 g to sediment the storage vesicles, both supernatant and particulate fractions were deproteinized with perchloric acid and analyzed for catecholamines by the trihydroxyindole method, utilizing an autoanalyzer (11) ; release of endogenous catecholamines was calculated as the amount remaining in the vesicles vs . time (12) . Uptake of 3H-epinephrine . Adrenal homogenates were centrifuged at 800 g and 0 .5 ml aliquots of the supernatant fraction were~ixed with equal y~olumes of sucrose-Tris containing 5 .0 wmoles of ATP-Mg , 5 .0 wCi of H-epinephrine and varying amounts of unlabeled epinephrine (0 .01-0 .05 Wmole), with and without addition of 0 .1 Wmole of methadone . Samples were incubated 30 min at 30 °C while duplicate tubes were kept on ice to same as blanks . Uptake was stopped by the addition of 2 ml of ice-cold sucrose-Tris and all samples were centrifuged 10 min at 26, 000 g to sediment the labeled vesicles . The supernatant was deproteinized and analyzed for catecholamines and radioactivity to permit determination of the specific activity of the incubation medium ; the vesicles were washed and recentrifuged twice, deproteinized and analyzed for catecholamines and radioactivity . Although contaminating organelles are present, under these conditions labeling occurs solely in storage vesicles (8, 14) . Detailed descriptions of the methodology and calculations have appeared previously (12-15) . Release _of newly-incorporated 3H-epinephrine . Labeled vesicles were prepared as described above, except that all samples contained 0 .05 Wmole of unlabeled epinephrine instead of varying amounts . The twice-washed vesicles were resuspended in fresh sucrose-iris and release
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Methadone on Catacholamiae Uptake, Release
48 5
of 3H-epinephrine determined in the presence and absence of 1 .0 mM methadone (final concentration) with or without addition of various concentrations of unlabeled epinephrine . In other studies, methadone in several concentrations was present only during the labeling of the vesicles and after washing twice with fresh sucrose-Tris, the subsequent release of 3H-epinephrine was determined without methadone . Statistics . Release curves were calculated by least squares analysis utilizing a two-compartment model as described previously (12,13), and each represents the average of 4 runs with 6 time points . Uptake data, presented as double reciprocal plots, were fitted by least squares analysis and each uptake point is the mean of 13-15 determinations at each concentration . Slopes and intercepts of release or uptake curves were compared utilizing the Student's t-test a Materials . Epinephrine-7- 3H (10 Ci./mmole) was obtained from New England Nuclear Corp ., epinephrine bitartrata from Winthrop Laboratories, reserpine from C1ba Pharmaceuticals and methadone HCl and morphine HCl from Merck, Sharp & Dohme . Results Prior to incubation at 30 °, approximately 7596 of the endogenous catecholamines were in the storage vesicle fraction, and about 2596 in the supernatant, the latter probably because of lysis of some veslcles .during preparation (3,16,17) (Fig . 1) . The subsequent, temperature-dependent release of catecholamines was bi-phasic, with an initially rapid (half-time < 10 min) loss of 1096 of the original amount followed by a slower rate of decline (half-time > 200 min) . Morphine in concentrations up to 10 -3 M, and methadone in concentrations up to 10 -4 M, had no effect on release . However, with 10 -3 M methadone there was a marked loss of amines even prior to incubation at 30 ° , and the temperature-dependent release of the remainder was accelerated ßialf-time = 28 min for slow release phase) . Since methadone inhibits catecholamine incorporation into storage vesicles (10), it was possible that the methadone-induced release could actually represent an effect on amine reuptake; hence, the action~~f agents known to enhance or inhibit uptake were tested . Neither ATP-Mg , which stimulates uptake, nor reserpine, which inhibits uptake, had any effect in themselves on the temperature-independent (Oo, zero-time) value for initial levels of endogenous catecholamines, and neither agent could~~verse this component of the methadone-induced release (Table 1) . ATP-Mg alone produced a lengthening of the time course of the late release phase and was able similarly to increase the half-time in the presence of methadone; however, the combined effects of ATP-Mq2+ + methadone appeared to be only additive . Reserpine had no effect on the half-time of the temperature-dependent phase and did not alter the methadone-induced acceleration of this component of release; a similar lack of effectiveness was seen with morphine . To evaluate a possible relationship between methadone-induced release and exogenous catecholamine levels, experiments were done with
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FIG . 1
2r
Z a 11~, N ZW
s
~CONTiàOI oQ1mM METHADONE e1mM METHADONE X1mM MORPHINE
U W
TIME (mln~
Release of endogenous catecholamines in the presence of morphine or methadone . Intercepts on the ordinate are: control, 1 .33 t 0 .10 wg ; 10 -4 M methadone, 1 .33 t 0 .08 -3 M methadone, 0 . 90 f 0 . 06 ~g (P < 0 . O1); Wq (N . S .); 10 morphine, 1 .36 f 0 .05 wg (N .S .) . Half-times of M 10 late release phase (20-60 min) are: control, 226 t 10 min; -3 M methadone, 10 -4 M methadone, 234 f 12 min (N .S .) ; 10 3 28 f 3 min (P < 0 .001), M morphine, 213 f 10 min -4 M morphine (N .S .) . Release with 10~ M methadone or 10 (data not shown) was the same as control . Each sample contained a total of 2 wg of catecholaminea . Ordinate is logarithmic . high concentrations of epinephrine added to the medium ; since under these conditions release of endogenous amines could not be followed, the vesicles were pre-labeled with 3 H-epinephrine and release was determined radioactivity . Methadone alone produced the expected initial loss of 3y H-amine and the decreased half-time for the temperature-~ependent component of release ('Table 2) . Addition to the medium of 10 or 10 -3 M epinephrine had no effect in itself and did not alter the methadone-induced change in half-time, but there was a concentration-dependent antagonism of methadone's effect on the initial (0 ° , zero-time) 3 H-epinephrine level . In order to compare directly the effects of methadone on uptake vs . release, studies were done in which methadone was present during the labeling with 3H-epinephrine, but not during the subsequent release period (Fig . 2) . Increa3sinq concentrations of methadone produced a progressive decline in H-epinephrine uptake to as little as 2400 counts per minute (10 -3 M methadone} vs . 14 , 000 counts per minute in controls . There appeared to be little effect of methadone on the half-time of the subsequent late phase of release, but there was a shift in the proportion of incorporated 3H-amine displaying the characteristically short vs . long half-time
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Methadone on Catecholamine IIptake, Release
48 7
TABLE 1 Effects of ATP-Mq 2+ , Reserpine or Morphine on Methadone-Induced Catecholamine Release in Rat Adrenomedullary Storage Vesicles % of Amine Remaining in Vesicles at Zero Time
Additions None
70
5 .0 mM ATP-Mg
2+
f
5
69 t 4
f
181 t
8 S*
f
24
f
6***
4t
88
.f
g tt
f
5#
37
f
7
72
f
5
52
f
3~ °
73
1 .0 mM Methadone
46 t 3 **
ATP-Mg2+ + Methadone
50
f
Reserpine + Methadone
50
1 .0 mM Morphine Morphine + Methadone <0 .05 < 0 .01 < 0 .001 <0 .02 <0 .001 <0 .02 <0 .001 <0 .02 <0 .001
f
143
131
0 .01 mM Reserpine
*P ** p *** p tP it P #p
Half-time of. Late Efflux (20-60 min) (min)
4
12
150 t 10 26
f
5 °°'
vs . v~ . v~ . y3 " g,& . yg " yg "
control . control . control . ATP-Mq 2+ , not significant ~. methadone . ATP-Mg t ; P < 0 .002 v~r . methadone . reserpine; not significant_v,~ . methadone . reserpine; mt significanty,~ . . methadone . vs . morphine ; not significant vs . methadone . vs . morphine ; not significantyg . methadone .
for temperature-dependent release: while control samples released only 9 _~ 2% of the amine with a half-time < 10 min, samples incubated with 10 M methadone during labeling showed nearly a 3-fold increase (25 t 4%, P < 0 .02) in the proportion of readily-released 3 H-epinephrine . To determine whether the reduction of 3H-epinephrine uptake by methadone might represent a competitive action at the transport site in the vesicle membrane, the kinetics of inhibition were examined (Fig . 3) . Whlle metha done increased the Km for epinephrine, it also increased the maximal uptake capacity (analogous to Vmax); the magnitude of the shift in Km for epinephrine indicated that the affinity for methadone was greater than that for the substrate (Ki for methadone < Km for epinephrine) .
488
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Methadone on Catecholamiae Uptake, Release
TABLE 2 Effec~s of Exogenous EpinepY~rine on Methadone-Induced Release of H-Epinephrine from Rat Adrenomedullary Storage Vesicles % of H-Epinephrine Remaining in Vesicles at Zero-Time
Additions None
78 f 4
1 .0 mM Methadone
27 f 5
0 .1 mM Epinephrine
Half-Time of Late Efflux (20-60 min) (min) 126 f
10
45 f
5
77 f 6
112 t
8
0 .1 mM Epinephrine + Methadone
44 f 4 t
42 t
1 .0 mM Epinephrine
84 f 3
1 . 0 mM Epinephrine + Methadone
65 f 5 #
*P <
t p tt p
< <
#P < P <
0 .001 0,005 0 .001 0 . 02 0 .001
vs .
vs .
vs . vs .
vs . FIG . 2
control . 0 .1 mM 0 .1 mM 1 . 0 mM 1 .0 mM
*
113 f
epinephrine; epinephrine; epinephrine; epinephrine ;
31 t
*
4 tt 12 8 ##
P < 0 .05 vs . methadone . not significant vs . methadone . P < 0 , 002 vs . mettiador4e . not significant vs . methadone .
Release in drug-free medium of Hepinephrine with methadone present during prior labeling of vesicles . Intercepts on the ordinate are: control, 14300 f 300 CPM; 10 -4 methadone, 8410 f 320 CPM (P < 0 .001); 3 x 10 -4 M methadone, 5030 f 250 CPM (P< 0,001) ; 10 -3 M methadone, 2390 t 150 CPM (P < 0 .001) . Half-times of late release phase (20-60 min) are : control, 80 t 4 min; 10 -4 M methadone, 92 f 6 min (N .S .), 3 x 10 -4 M methadone, 90 t 5 min (N .S .) ; 10 -3 M methadone, 86 f 4 min (N . S . ) . Ordinate is logarithmic .
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Methadone on Catecholamine Uptake, Release
48 9
FIG . 3
C
~4 EPINEPFR~~ (ftA~~)
ü~
Double-reciprocal plot of uptake of 3H-epinephrine in the presence and absence of methadone . Intercepts on both ordinate and abscissa are different from control (P< 0 .001) . For control, Km = 39 .5 f 2 .7 ~M, maximal uptake = 0 . 80 f 0 . 05 nmols/30 min; with methadone, Km = 275 f 12 WM, maximal uptake = 1 .92 f 0 .08 nmols/30 min . Washed vesicle pellets contained an average of 2 .61 wq of endogenous catecholamines for control, 2 .14 Wg for methadone .
Discussion The uptake of çatecholamines into isolated adrenomedullary storage vesicles is a multi-step process, consisting of three separable components (12,13,15,18) : ~~inwaid transport across the vesicle membrane, stimuand blocked byreserpine (19,20) ; 2) subsequent storage lated by ATP-Mq of amines inside the vesicle in either a stable or readily-released pool (12,13); 3) release of amines from vesicle to medium with a characteristically short half-time for the ready-release pool and a long half-time for the stable pool (12,13) . Despite the complexity of this system, mathematical analysis of kinetic data reveals a close fit to Michaelis-Menten kinetics (15 ,17) . The apparent Km reflects primarily the affinity of the substrate for the membrane transport system, while maximal uptake depends to a large extent upon the availability of storage sites for the newly-incorporated amines and upon the rate of release (15) . Incubation of vesicles with methadone produced inhibition of 3Hepinephrine uptake of a competitive nature, indicated by an increase in the Km for substrate . The affinity of the opiate for the transport site appeared to be greater than that of epinephrine; however, it is unlikely that this action of methadone is mediated by opiate receptors, since morphine was itself ineffective and could not alter the action of methadone . A similar result has been obtained with serotonin uptake in synaptosomes, namely, competitive inhibition by methadone but not by morphine, lack of imrolvement of opiate receptors, and higher affinity for methadone than for substrate (8); interestingly, the high affinity and specificity for methadone is not observed for catecholamine uptake into synaptosomes (21) . It should be noted that the storage vesicles of the adrenal medulla actually show preferential uptake of serotonin rather than their natural catecholamine substrates (13,17, 22), suggesting that the competitive inhibitory effect of methadone may be related generally to transport sites with high affinities for indoleamines .
490
Methadone on Catecholamine Uptake, Release
Vol . 19, No . 4
In addition to increasing the Km for epinephrine, methadone caused an apparent increase in the maximal capacity for amine uptake . Because the latter parameter reflects both storage and release phenomena (15 ,17, 22), it was important to establish whether methadone could exert an effect exclusive of that on inward transport . Incubation of vesicles with methadpne produced an acceleration of endogenous catecholamine release, which, as with uptake, could not be achieved with morphine . The releasing action of the drug could be differentiated from that on inward transport (which by its insensitivity to ATP-Mg 2 stimulates amine uptake) or reserpine (which inhibits uptake) . Thus, by mobilizing endogenous catecholamines from intravesicular storage sites, methadone may be able to increase the maximal uptake capacity . In addition to enhanced exchangeability of the stable storage pool, indicated. by the lowered half-time of the late release phase, methadone also might effect a shift in the proportion of amine in the re~dy-release pool . This hypothesis was tested by measuring the release of H-epinephrine after prior incorporation into the vesicles in the presence of methadone ; concurrently with the competitive inhibition of uptake, drug exposure during labeling produced an increase in the proportion of newly-incorporated amine in the rapid-release pool, indicating again an increase in the ability of stored amines to exchange with newly-incorporated 3H-epinephrine . Thus, while promoting release of endogenous catecholamines, methadone may thereby enhance the maximal capacity to incorporate exogenous amines . It should be noted that the uptake of exogenous amines represents only a few per cent of the total content of the vesicle (13), hence, even a small change in the number of available sites could cause a significant shift in capacity for newlyincorporated 3H-epinephrine . There is some evidence that the effects of methadone on temperature-dependent release and those on temperature-independent (0 °, zerotime) release are separable . High concentrations of epinephrine in the medium were able to antagonize the 0° release by methadone, but not the release occurring at 30 °. The significance of this dual action is unclear, and the low-temperature process may represent simply lysis of some of the vesicles . In conclusion, methadone produces both inhibition of catecholamine uptake and enhancement of catecholamine release in isolated adrenomedullary vesicles . Despite the high concentrations of methadone required for uptake inhibition in vitro , the same effect has been seen with administration ~v~vo (10) . Thus, direct actions of the drug may play some role in its systemic effects . Açknowledc~e-m~nks This work was supported by U . S . Public Health Service Grant No . DA-00465 . Dr . Slotkin is the recipient of Research Scientist Development Award No . DA-00006 from the National Institute on Drug Abuse . Referen~s 1.
E . L . WAY and F . H . SHEN, Narcotic Druas : Biochemical Pharma colo~nr (Ed ., D . Clouet) pp . 229-253, Plenum, New York (1971) .
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2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 .
Methadone on Catecholamine Uptake, Belasse
491
T . YOSHIZAKI, Jap . J. Pharmacol . ~: 695-699 (1973) . T . R. ANDERSON and T . A . SLOTKIN, Biochem . Pharmacol . 24 : 671-679 (1975) . H . MONTEL, K . STA.RI~ and H . D . TAUBE, Naunyn-Schmiedebergs Arch . Pharmacol . ~: 415-426 (1975) . H . MONTEL, K. STARKE and H. D . TAUBE, Naunyn-Schmiedebergs Arch . Pharmacol . 288: 427-433 (1975) . T . R. ANDERSON and T . A . SLOTKIN, Biochem . Pharmacol . 75 : 1071-1074 (1976) . H . MONTEL and K. STARKE, Brit . J . Pharmacol . 49 : 628-641 (1973) . F . R . CIOFALO, J. Pharmacol . Exp . Then . 189: 83-89 (1974) . T . A. SLOTKIN and T . R. ANDERSON, Neuropharmacol . 14 : 159-161 (1975) . T . A . SLOTKIN, C . LAU, M . BARTOLOMÉ and F . J . SEII~LER, Biochem . Pharmacol .,In Press (1976) . R. J . MERRILLS, Analyt . Biochem . 6 : 272-282 (1963) . T . A.~ SLOTKIN, R . M . FERRIS and N. KIRSHNER, Mol . Pharmacol . 7: 308-316 (1971) . T . A. SLOTKIN and N . KIRSHNER, Mol . Pharmacol . 7: 581-592 (1971) . T . A . SLOTKIN and N . KIRSHNER, Mol . Pharmacol . 9 : 105-116 (1973) . T . A . SLOTKIN, Biochem . Pharmacol . 24 : 89-97 (1975) . T . A. SLOTKIN and N . KIRSHNER, Biochem . Pharmacol . 22 : 205-219 (1973) . T . A. SLOTKIN and H . O . GREEN, Biochem . Pharmacol . 24 : 173-180 (1975) . T . A. SLOTKIN, Life Sci . lei : 673-683 (1973) . N . KIRSHNER, J. Biol . Chem . 237: 2311-2317 (1962) . A . CARLSSON, NA . HILLARP and B . WALDECK, Med . Exp . (Basel) 6 : 47-53 (1962) . F . R . CIOFA.LO, Life Sci . 11 : 573-580 (1972) . T . A . SLOTKIN, T . R. ANDERSON, F . J. SEIDLER and C . LAU, Biochem . Pharmacol . 24 : 1413-1419 (1975) .
Pergamon Preee
ALTERATION BY METHADONE OF CATECHOLAMINE UPTAI~ AND RELEASE IN ISOLATED RAT ADRENOMEDULLARY STORAGE VESICLES Theodore A . Slotkin, Christopher Lau, Maria Bartolomé and Frederic J . Seidler Department of Physiology and Pharmacology Duke University Medical Center Durham, North Carolina 27710 (Received in final form April 30, 1976) Incubation of isolated rat adreno ullary storage vesicles with methadone produced inhibition of H-epinephrine uptake and promotion of release of endogenous catecholamines . Neither effect was seen using morphine, nor could morphine antagonize methadone-induced catecholamine release, suggesting that these actions are not mediated by opiate receptors . Inhibition of uptake by methadone appeared to contain a competitive com~onent with a lower Ki for methadone compared to the Km for H-epinephrine . Despite competitive inhibition by methadone, the maximal uptake capacity analogous to Vmax) as determined by double-reciprocal plots, was increased by the drug, probably as a result of greater availability of intravesicular storage sites because of the drug-induced release of endogenous catecholamines . Agents which enhance or block catecholamine transport into vesicles had no effect on the catecholamine release by methadone, indicating that the latter is separable from the action on uptake . These alterations of catecholamine uptake and release may play a role in the effects of methadone on the adrenal medulla in vivo . The actions of opiates on biogenic amines of the central and peripheral adrenergic system involve effects on synthesis, uptake, storage and release (1-6) . Acute and chronic administration of morphine or methadone in vivo has been shown not only to evoke secretion of catecholamines and to increase levels of catecholamine biosynthetic enzymes, but also to inhibit the uptake of amines into either neurons or storage vesicles (7-9) . In cardiac adrenergic neurons, Montel and Starke (7)found that morphine in vitro can block catecholamine reuptake across the neuronal membrane and in the brain, Ciofalo (B) has demonstrated inhibition of serotonin uptake into synaptosomal preparations by methadone . While morphine administration in vivo also produces inhibition of catecholamine uptake into storage vesicles of the adrenal medulla, in contrast to the uptake studies in neurons, this action of morphine adminis tration in vivo is not observed with drug exposure in vitro (3,9), suggesting 483
484
Methadone on Catecholamine Uptake, Release
Vol . 19, No . 4
an indirect action possibly mediated by physiological or cellular actions of the drug . Similarly, the rate of spontaneous release of catecholamines from adrenal vesicle preparations is slowed after morphine administration ~n vivo . but not by direct exposure to drug ~_n vitro (3) . However, experiments with methadone suggest that this opiate may have a direct action ~ vitro on amine uptake into adrenomedullary vesicles (10) , indicating a different action from morphine ; administration of even a single dose of methadone a.5 mg/kg) to rats produced significant inhibition of the vesicular amine pump, and the effect was more pronounced on chronic treatment . In the current study, the mechanisms by which exposure of isolated adrenomedullary vesicles to methadone in vitro alters both uptake and release of catecholamines have been examined and contrasted with the effects of morphine . Method s Release of endogenous catecholamines . Male Sprague-Dawley rats (Zivic-Miller) weighing 200-250 g were decapitated and their adrenals removed, pooled and homogenized in 2 .5 ml/ 5gland of 0 .3 M sucrose buffered at pH 7 .4 M iproniazid to inhibit monoamine oaidase . with 0 .025 M Tris, containing 10 After centrifugation for 10 min at 800 g to remove debris, 0 .5 ml aliquots of the catediolamine storage vesicle containing supernatant were added to 0 .5 ml of sucrose-Tris containing a vari~v of agents singly or in combination, including methadone, morphine, ATP-Mq or reserpLne . Samples were incubated for 0, 10, 20, 30, 40 or 60 min at 30 °C, at which times 2 ml of ice-cold sucrose-Tris were added . After centrifugation for 10 min at 26 , 000 g to sediment the storage vesicles, both supernatant and particulate fractions were deproteinized with perchloric acid and analyzed for catecholamines by the trihydroxyindole method, utilizing an autoanalyzer (11) ; release of endogenous catecholamines was calculated as the amount remaining in the vesicles vs . time (12) . Uptake of 3H-epinephrine . Adrenal homogenates were centrifuged at 800 g and 0 .5 ml aliquots of the supernatant fraction were~ixed with equal y~olumes of sucrose-Tris containing 5 .0 wmoles of ATP-Mg , 5 .0 wCi of H-epinephrine and varying amounts of unlabeled epinephrine (0 .01-0 .05 Wmole), with and without addition of 0 .1 Wmole of methadone . Samples were incubated 30 min at 30 °C while duplicate tubes were kept on ice to same as blanks . Uptake was stopped by the addition of 2 ml of ice-cold sucrose-Tris and all samples were centrifuged 10 min at 26, 000 g to sediment the labeled vesicles . The supernatant was deproteinized and analyzed for catecholamines and radioactivity to permit determination of the specific activity of the incubation medium ; the vesicles were washed and recentrifuged twice, deproteinized and analyzed for catecholamines and radioactivity . Although contaminating organelles are present, under these conditions labeling occurs solely in storage vesicles (8, 14) . Detailed descriptions of the methodology and calculations have appeared previously (12-15) . Release _of newly-incorporated 3H-epinephrine . Labeled vesicles were prepared as described above, except that all samples contained 0 .05 Wmole of unlabeled epinephrine instead of varying amounts . The twice-washed vesicles were resuspended in fresh sucrose-iris and release
Vol . 19, No . 4
Methadone on Catacholamiae Uptake, Release
48 5
of 3H-epinephrine determined in the presence and absence of 1 .0 mM methadone (final concentration) with or without addition of various concentrations of unlabeled epinephrine . In other studies, methadone in several concentrations was present only during the labeling of the vesicles and after washing twice with fresh sucrose-Tris, the subsequent release of 3H-epinephrine was determined without methadone . Statistics . Release curves were calculated by least squares analysis utilizing a two-compartment model as described previously (12,13), and each represents the average of 4 runs with 6 time points . Uptake data, presented as double reciprocal plots, were fitted by least squares analysis and each uptake point is the mean of 13-15 determinations at each concentration . Slopes and intercepts of release or uptake curves were compared utilizing the Student's t-test a Materials . Epinephrine-7- 3H (10 Ci./mmole) was obtained from New England Nuclear Corp ., epinephrine bitartrata from Winthrop Laboratories, reserpine from C1ba Pharmaceuticals and methadone HCl and morphine HCl from Merck, Sharp & Dohme . Results Prior to incubation at 30 °, approximately 7596 of the endogenous catecholamines were in the storage vesicle fraction, and about 2596 in the supernatant, the latter probably because of lysis of some veslcles .during preparation (3,16,17) (Fig . 1) . The subsequent, temperature-dependent release of catecholamines was bi-phasic, with an initially rapid (half-time < 10 min) loss of 1096 of the original amount followed by a slower rate of decline (half-time > 200 min) . Morphine in concentrations up to 10 -3 M, and methadone in concentrations up to 10 -4 M, had no effect on release . However, with 10 -3 M methadone there was a marked loss of amines even prior to incubation at 30 ° , and the temperature-dependent release of the remainder was accelerated ßialf-time = 28 min for slow release phase) . Since methadone inhibits catecholamine incorporation into storage vesicles (10), it was possible that the methadone-induced release could actually represent an effect on amine reuptake; hence, the action~~f agents known to enhance or inhibit uptake were tested . Neither ATP-Mg , which stimulates uptake, nor reserpine, which inhibits uptake, had any effect in themselves on the temperature-independent (Oo, zero-time) value for initial levels of endogenous catecholamines, and neither agent could~~verse this component of the methadone-induced release (Table 1) . ATP-Mg alone produced a lengthening of the time course of the late release phase and was able similarly to increase the half-time in the presence of methadone; however, the combined effects of ATP-Mq2+ + methadone appeared to be only additive . Reserpine had no effect on the half-time of the temperature-dependent phase and did not alter the methadone-induced acceleration of this component of release; a similar lack of effectiveness was seen with morphine . To evaluate a possible relationship between methadone-induced release and exogenous catecholamine levels, experiments were done with
48 6
Methadone on Catecholsmiae IIptake, Release
Vol . 19, No . 4
FIG . 1
2r
Z a 11~, N ZW
s
~CONTiàOI oQ1mM METHADONE e1mM METHADONE X1mM MORPHINE
U W
TIME (mln~
Release of endogenous catecholamines in the presence of morphine or methadone . Intercepts on the ordinate are: control, 1 .33 t 0 .10 wg ; 10 -4 M methadone, 1 .33 t 0 .08 -3 M methadone, 0 . 90 f 0 . 06 ~g (P < 0 . O1); Wq (N . S .); 10 morphine, 1 .36 f 0 .05 wg (N .S .) . Half-times of M 10 late release phase (20-60 min) are: control, 226 t 10 min; -3 M methadone, 10 -4 M methadone, 234 f 12 min (N .S .) ; 10 3 28 f 3 min (P < 0 .001), M morphine, 213 f 10 min -4 M morphine (N .S .) . Release with 10~ M methadone or 10 (data not shown) was the same as control . Each sample contained a total of 2 wg of catecholaminea . Ordinate is logarithmic . high concentrations of epinephrine added to the medium ; since under these conditions release of endogenous amines could not be followed, the vesicles were pre-labeled with 3 H-epinephrine and release was determined radioactivity . Methadone alone produced the expected initial loss of 3y H-amine and the decreased half-time for the temperature-~ependent component of release ('Table 2) . Addition to the medium of 10 or 10 -3 M epinephrine had no effect in itself and did not alter the methadone-induced change in half-time, but there was a concentration-dependent antagonism of methadone's effect on the initial (0 ° , zero-time) 3 H-epinephrine level . In order to compare directly the effects of methadone on uptake vs . release, studies were done in which methadone was present during the labeling with 3H-epinephrine, but not during the subsequent release period (Fig . 2) . Increa3sinq concentrations of methadone produced a progressive decline in H-epinephrine uptake to as little as 2400 counts per minute (10 -3 M methadone} vs . 14 , 000 counts per minute in controls . There appeared to be little effect of methadone on the half-time of the subsequent late phase of release, but there was a shift in the proportion of incorporated 3H-amine displaying the characteristically short vs . long half-time
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Methadone on Catecholamine IIptake, Release
48 7
TABLE 1 Effects of ATP-Mq 2+ , Reserpine or Morphine on Methadone-Induced Catecholamine Release in Rat Adrenomedullary Storage Vesicles % of Amine Remaining in Vesicles at Zero Time
Additions None
70
5 .0 mM ATP-Mg
2+
f
5
69 t 4
f
181 t
8 S*
f
24
f
6***
4t
88
.f
g tt
f
5#
37
f
7
72
f
5
52
f
3~ °
73
1 .0 mM Methadone
46 t 3 **
ATP-Mg2+ + Methadone
50
f
Reserpine + Methadone
50
1 .0 mM Morphine Morphine + Methadone <0 .05 < 0 .01 < 0 .001 <0 .02 <0 .001 <0 .02 <0 .001 <0 .02 <0 .001
f
143
131
0 .01 mM Reserpine
*P ** p *** p tP it P #p
Half-time of. Late Efflux (20-60 min) (min)
4
12
150 t 10 26
f
5 °°'
vs . v~ . v~ . y3 " g,& . yg " yg "
control . control . control . ATP-Mq 2+ , not significant ~. methadone . ATP-Mg t ; P < 0 .002 v~r . methadone . reserpine; not significant_v,~ . methadone . reserpine; mt significanty,~ . . methadone . vs . morphine ; not significant vs . methadone . vs . morphine ; not significantyg . methadone .
for temperature-dependent release: while control samples released only 9 _~ 2% of the amine with a half-time < 10 min, samples incubated with 10 M methadone during labeling showed nearly a 3-fold increase (25 t 4%, P < 0 .02) in the proportion of readily-released 3 H-epinephrine . To determine whether the reduction of 3H-epinephrine uptake by methadone might represent a competitive action at the transport site in the vesicle membrane, the kinetics of inhibition were examined (Fig . 3) . Whlle metha done increased the Km for epinephrine, it also increased the maximal uptake capacity (analogous to Vmax); the magnitude of the shift in Km for epinephrine indicated that the affinity for methadone was greater than that for the substrate (Ki for methadone < Km for epinephrine) .
488
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Methadone on Catecholamiae Uptake, Release
TABLE 2 Effec~s of Exogenous EpinepY~rine on Methadone-Induced Release of H-Epinephrine from Rat Adrenomedullary Storage Vesicles % of H-Epinephrine Remaining in Vesicles at Zero-Time
Additions None
78 f 4
1 .0 mM Methadone
27 f 5
0 .1 mM Epinephrine
Half-Time of Late Efflux (20-60 min) (min) 126 f
10
45 f
5
77 f 6
112 t
8
0 .1 mM Epinephrine + Methadone
44 f 4 t
42 t
1 .0 mM Epinephrine
84 f 3
1 . 0 mM Epinephrine + Methadone
65 f 5 #
*P <
t p tt p
< <
#P < P <
0 .001 0,005 0 .001 0 . 02 0 .001
vs .
vs .
vs . vs .
vs . FIG . 2
control . 0 .1 mM 0 .1 mM 1 . 0 mM 1 .0 mM
*
113 f
epinephrine; epinephrine; epinephrine; epinephrine ;
31 t
*
4 tt 12 8 ##
P < 0 .05 vs . methadone . not significant vs . methadone . P < 0 , 002 vs . mettiador4e . not significant vs . methadone .
Release in drug-free medium of Hepinephrine with methadone present during prior labeling of vesicles . Intercepts on the ordinate are: control, 14300 f 300 CPM; 10 -4 methadone, 8410 f 320 CPM (P < 0 .001); 3 x 10 -4 M methadone, 5030 f 250 CPM (P< 0,001) ; 10 -3 M methadone, 2390 t 150 CPM (P < 0 .001) . Half-times of late release phase (20-60 min) are : control, 80 t 4 min; 10 -4 M methadone, 92 f 6 min (N .S .), 3 x 10 -4 M methadone, 90 t 5 min (N .S .) ; 10 -3 M methadone, 86 f 4 min (N . S . ) . Ordinate is logarithmic .
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Methadone on Catecholamine Uptake, Release
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FIG . 3
C
~4 EPINEPFR~~ (ftA~~)
ü~
Double-reciprocal plot of uptake of 3H-epinephrine in the presence and absence of methadone . Intercepts on both ordinate and abscissa are different from control (P< 0 .001) . For control, Km = 39 .5 f 2 .7 ~M, maximal uptake = 0 . 80 f 0 . 05 nmols/30 min; with methadone, Km = 275 f 12 WM, maximal uptake = 1 .92 f 0 .08 nmols/30 min . Washed vesicle pellets contained an average of 2 .61 wq of endogenous catecholamines for control, 2 .14 Wg for methadone .
Discussion The uptake of çatecholamines into isolated adrenomedullary storage vesicles is a multi-step process, consisting of three separable components (12,13,15,18) : ~~inwaid transport across the vesicle membrane, stimuand blocked byreserpine (19,20) ; 2) subsequent storage lated by ATP-Mq of amines inside the vesicle in either a stable or readily-released pool (12,13); 3) release of amines from vesicle to medium with a characteristically short half-time for the ready-release pool and a long half-time for the stable pool (12,13) . Despite the complexity of this system, mathematical analysis of kinetic data reveals a close fit to Michaelis-Menten kinetics (15 ,17) . The apparent Km reflects primarily the affinity of the substrate for the membrane transport system, while maximal uptake depends to a large extent upon the availability of storage sites for the newly-incorporated amines and upon the rate of release (15) . Incubation of vesicles with methadone produced inhibition of 3Hepinephrine uptake of a competitive nature, indicated by an increase in the Km for substrate . The affinity of the opiate for the transport site appeared to be greater than that of epinephrine; however, it is unlikely that this action of methadone is mediated by opiate receptors, since morphine was itself ineffective and could not alter the action of methadone . A similar result has been obtained with serotonin uptake in synaptosomes, namely, competitive inhibition by methadone but not by morphine, lack of imrolvement of opiate receptors, and higher affinity for methadone than for substrate (8); interestingly, the high affinity and specificity for methadone is not observed for catecholamine uptake into synaptosomes (21) . It should be noted that the storage vesicles of the adrenal medulla actually show preferential uptake of serotonin rather than their natural catecholamine substrates (13,17, 22), suggesting that the competitive inhibitory effect of methadone may be related generally to transport sites with high affinities for indoleamines .
490
Methadone on Catecholamine Uptake, Release
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In addition to increasing the Km for epinephrine, methadone caused an apparent increase in the maximal capacity for amine uptake . Because the latter parameter reflects both storage and release phenomena (15 ,17, 22), it was important to establish whether methadone could exert an effect exclusive of that on inward transport . Incubation of vesicles with methadpne produced an acceleration of endogenous catecholamine release, which, as with uptake, could not be achieved with morphine . The releasing action of the drug could be differentiated from that on inward transport (which by its insensitivity to ATP-Mg 2 stimulates amine uptake) or reserpine (which inhibits uptake) . Thus, by mobilizing endogenous catecholamines from intravesicular storage sites, methadone may be able to increase the maximal uptake capacity . In addition to enhanced exchangeability of the stable storage pool, indicated. by the lowered half-time of the late release phase, methadone also might effect a shift in the proportion of amine in the re~dy-release pool . This hypothesis was tested by measuring the release of H-epinephrine after prior incorporation into the vesicles in the presence of methadone ; concurrently with the competitive inhibition of uptake, drug exposure during labeling produced an increase in the proportion of newly-incorporated amine in the rapid-release pool, indicating again an increase in the ability of stored amines to exchange with newly-incorporated 3H-epinephrine . Thus, while promoting release of endogenous catecholamines, methadone may thereby enhance the maximal capacity to incorporate exogenous amines . It should be noted that the uptake of exogenous amines represents only a few per cent of the total content of the vesicle (13), hence, even a small change in the number of available sites could cause a significant shift in capacity for newlyincorporated 3H-epinephrine . There is some evidence that the effects of methadone on temperature-dependent release and those on temperature-independent (0 °, zerotime) release are separable . High concentrations of epinephrine in the medium were able to antagonize the 0° release by methadone, but not the release occurring at 30 °. The significance of this dual action is unclear, and the low-temperature process may represent simply lysis of some of the vesicles . In conclusion, methadone produces both inhibition of catecholamine uptake and enhancement of catecholamine release in isolated adrenomedullary vesicles . Despite the high concentrations of methadone required for uptake inhibition in vitro , the same effect has been seen with administration ~v~vo (10) . Thus, direct actions of the drug may play some role in its systemic effects . Açknowledc~e-m~nks This work was supported by U . S . Public Health Service Grant No . DA-00465 . Dr . Slotkin is the recipient of Research Scientist Development Award No . DA-00006 from the National Institute on Drug Abuse . Referen~s 1.
E . L . WAY and F . H . SHEN, Narcotic Druas : Biochemical Pharma colo~nr (Ed ., D . Clouet) pp . 229-253, Plenum, New York (1971) .
Vol . 19, No . 4
2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 .
Methadone on Catecholamine Uptake, Belasse
491
T . YOSHIZAKI, Jap . J. Pharmacol . ~: 695-699 (1973) . T . R. ANDERSON and T . A . SLOTKIN, Biochem . Pharmacol . 24 : 671-679 (1975) . H . MONTEL, K . STA.RI~ and H . D . TAUBE, Naunyn-Schmiedebergs Arch . Pharmacol . ~: 415-426 (1975) . H . MONTEL, K. STARKE and H. D . TAUBE, Naunyn-Schmiedebergs Arch . Pharmacol . 288: 427-433 (1975) . T . R. ANDERSON and T . A . SLOTKIN, Biochem . Pharmacol . 75 : 1071-1074 (1976) . H . MONTEL and K. STARKE, Brit . J . Pharmacol . 49 : 628-641 (1973) . F . R . CIOFALO, J. Pharmacol . Exp . Then . 189: 83-89 (1974) . T . A. SLOTKIN and T . R. ANDERSON, Neuropharmacol . 14 : 159-161 (1975) . T . A . SLOTKIN, C . LAU, M . BARTOLOMÉ and F . J . SEII~LER, Biochem . Pharmacol .,In Press (1976) . R. J . MERRILLS, Analyt . Biochem . 6 : 272-282 (1963) . T . A.~ SLOTKIN, R . M . FERRIS and N. KIRSHNER, Mol . Pharmacol . 7: 308-316 (1971) . T . A. SLOTKIN and N . KIRSHNER, Mol . Pharmacol . 7: 581-592 (1971) . T . A . SLOTKIN and N . KIRSHNER, Mol . Pharmacol . 9 : 105-116 (1973) . T . A . SLOTKIN, Biochem . Pharmacol . 24 : 89-97 (1975) . T . A. SLOTKIN and N . KIRSHNER, Biochem . Pharmacol . 22 : 205-219 (1973) . T . A. SLOTKIN and H . O . GREEN, Biochem . Pharmacol . 24 : 173-180 (1975) . T . A. SLOTKIN, Life Sci . lei : 673-683 (1973) . N . KIRSHNER, J. Biol . Chem . 237: 2311-2317 (1962) . A . CARLSSON, NA . HILLARP and B . WALDECK, Med . Exp . (Basel) 6 : 47-53 (1962) . F . R . CIOFA.LO, Life Sci . 11 : 573-580 (1972) . T . A . SLOTKIN, T . R. ANDERSON, F . J. SEIDLER and C . LAU, Biochem . Pharmacol . 24 : 1413-1419 (1975) .