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The mode of morphine uptake into brain slices
Brain Research, 77 (1974) 121-136 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
121
THE MODE OF MORPHINE UPTAKE INTO BRAIN SLICES
DAVID N. TELLER, TERESITA DE GUZMAN AND ABEL LAJTHA
New York State Research Institute for Neurochemistry and Drug Addiction, Ward's Island, New York, N.Y, 10035 (U.S.A.) (Accepted March 29th, 1974)
SUMMARY
The uptake of [N-14C methyl]morphine into rat, mouse, or guinea pig brain slices does not meet the criteria for an active transport process. (1) Methods are described for the extraction and TLC of morphine at 9 6 ~ recovery levels, and for the determination of 14C at 70-90 ~ counting efficiency with brain tissue samples. (2) After morphine had been in the tissue for 30 min, 96~ of the radioactivity was extractable, and 99~ of this was unmetabolized morphine. (3) The ratio of the concentration in the tissue to that in the medium was 2.22+ 0.16 e.s.e., from 3 × 10-8 M to 2 × 10-3 M. Therefore, the uptake is not saturable. (4) Strong metabolic inhibitors, e.g. cyanide, at concentrations that completely block amino acid transport, did not markedly reduce the uptake of morphine. (5) The uptake into and efflux from brain tissue was not affected by concentrated morphine, analogs, or narcotic antagonists. Therefore, the uptake of morphine showed no signs of the competitive inhibition that is typical of active transport processes. In addition, morphine uptake was not affected by acute or chronic morphine injections in the mouse or guinea pig. (6) The uptake of morphine into brain tissue was temperature dependent, but it occurred at 0.5 °C and also after the tissue was kept at 95 °C for 10 min.
INTRODUCTION
Morphine has been reported to be actively transported by brain slices18, by choroid plexusi,n, 19, and possibly in vivo from blood to brainlZ,15,23,25. Other reports appear to contradict some of these findings4,9,12,2a. There were technical differences between the various investigations, and some studies were primarily concerned with dihydromorphineX2, e2,z4. Although morphine and dihydromorphine are almost
122
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identical pharmacologically and structurally, there appear to be differences between these two drugs in their mechanism of uptake into brain slices. For an understanding of the distribution and fate of pharmacological agents, it would be important to know whether specific transport processes exist for drugs such as morphine, or if the transport mechanisms for endogenous compounds such as amino acids can also mediate the movement of drugs with related structure. Knowledge of the characteristics of transport processes for drugs might provide the possibility of facilitation or blockade of entry into the brain, thereby influencing drug action. In this study we observed that although morphine accumulates in brain slices, this does not occur via an active transport process. Indeed, although experimental criteria for the demonstration of active transport processes are not absolute, morphine uptake in brain slices meets none of the tests 17 satisfactorily. Morphine uptake in brain slices was not saturable, temperature dependent, competitively inhibited by structural analogs, or blocked by metabolic inhibitors. Heat treatment (95 ~C) that destroyed enzyme activity and stopped amino acid transport did not abolish morphine uptake. In contrast to the absence of demonstrable active transport of morphine in brain slices, kidney slices were shown to possess an active transport system for morphine :tl. In separate reports we have confirmed and extended these findings 2°,'1. MATERIALS A N D METHODS
Materials
Morphine, labeled with 14C in the N-methyl group was obtained from Amersham/Searle. Three batches were used, batches 8, 9, and 11, lot CFA 363, at 57 mCi/ mmole. Morphine hemisulfate, from Mallinckrodt Chemical Works, was converted to the hydrochloride by reaction with BaCls and CO2. The solution was dried under N2; the morphine HCI was recrystallized from ethanol and was assayed colorimetrically with the same reagents and procedure used for protein 4, with authentic morphine hemisulfate as a standard. Radioactive solutions of morphine were prepared in water, except where this would have diluted the incubation medium more than 5~. In such cases, the radioactive solution was prepared from a more concentrated aqueous stock by dilution with medium. The constriction pipets used to deliver the radioactive morphine into the incubation flasks were calibrated by delivery of water and dye to 3 significant figures. The average variance in delivery and counting of a nominal 10/zl volume was ~: 0.6~, when 10~ counts were recorded. Variance in tissue wet or dry weight measurement was -~ 0.06 mg/10 mg; estimated standard errors from counting the same sample or standard were less than 0.3~. Radioactive amino acids, uniformly labeled with 14C, were obtained from New England Nuclear Corp. and were diluted with non-radioactive carrier to approximately 2/~Ci/mmole. These solutions were prepared in media at 10 and 20 mM, and 0.5 ml was used per 4.5 ml + tissue in each flask. Non-radioactive compounds were prepared in water, buffer, medium, or solvent as indicated, and controls were added to the experimental protocol to measure the effects of the diluent alone on the uptake of the radioactive drug. All concentrated solutions of the narcotics and their analogs
MORPHINE UPTAKE INTO BRAIN SLICES
123
were kept dark and cold, or frozen, before use. With some drugs, e.g., levorphanol and nalorphine, the room was darkened during the entire procedure to avoid photodecomposition of the dilute compounds. Incubation. The 3 types of incubation media were: HEPES-2 (see refs. 6 and 8), the medium used by Scrafani and Hug 18, and that used by Hamberger 1°. HEPES-2 medium contains 119 mM NaC1, 5.0 mM KCI, 0.75 mM CaCI~, 1.2 mM MgSO4, 1.0 mM NaH~PO4, 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid (H EPE S) 12 mM NaOH, 10 mM glucose, pH 7.4 (with NaOH) at 25 °C (see in ref. 8, p. 190, footnote to Table II). The medium of Scrafani and Hug 18 contains 134 mM NaCI, 2.7 mM KCI, 0.48 mM MgC12, 0.92 mM CaClz, 14.8 mM NaHCO3, 3.6 mM Na2HPO4 (pH 7.4), and 5.5 mM glucose, with pH adjusted to 7.3-7.5 (with HC1). Hamberger's mediumTM contains 35 mM Tris-HCl (pH 7.6), 120 mM NaCI, 5 mM KCI, 5 mM Na3PO4 (pH 7.6), 20 mM glucose, 2.5 mM MgC12. Species, treatment in vivo, and tissue preparation. Mice, rats, and guinea pigs were inbred from strains derived from Swiss Webster, Wistar, and Cayley varieties, respectively. The mice were 6-10 weeks old, the rats were 6-8 months old and the guinea pigs were 9 months old when they were used for these experiments. Some mice and guinea pigs were injected with morphine, from 40 mg/kg, i.p., to more than 1 g/kg, s.c., to produce tolerance. Animals 'withdrawn' from morphine were given no injection for 18 h prior to being killed; 'chronic' animals received a last dose of 4~30 mg/kg, i.p., 2-3 h before use; 'acute' animals received a single dose of 40 mg/kg, i.p., 1 h before they were killed. In all cases, both sexes were used, because no consistent effects were observed that might be attributable to sex differences. All tissue slices were 416/~m (:kl8/*m, S.D.) transverse sections made with a Mcllwain-Mickle chopper. The sliced tissues were transferred to 4.5-5 ml of medium that had been oxygenated for 4 min and were equilibrated in the shaker bath for 20-30 min at the temperature for the particular experiment. During this time the Na + and K + levels in the tissue apparently regained some differential equilibrium with the medium, because when the tissues were freshly excised and in iced medium, the enzymes that maintain the intracellular ion levels were relatively inactive. Therefore, 20-30 min after the slices were added to the medium, radioactive morphine was added, and incubation was continued for an additional 2-120 min. Incubation was stopped by filtering the medium from the slicess, and the slices were frozen in powdered dry ice. The weighed frozen tissue pellet was homogenized in 39/0 perchloric acid (w/v), and the supernatant was saved for determination of radioactivity, extraction of morphine, ion and ATP determinations, etc. The pellet of perchloric acid-insoluble material was dissolved in 1 N NaOH and was prepared for counting as described below. In most cases the frozen tissue was simply digested in 1 ml of 1 N NaOH (overnight at room temperature, or for 10 min at 65 °C). Efflux of radioactive morphine was measured by filtering the radioactive medium from the slices through a Hirsch funnel, rinsing with 2 ml of warmed, oxygenated medium, and transferring the slices to fresh oxygenated media which contained no radioactivity. After an additional 2-120 min the slices were filtered from the washout medium and were frozen. In some cases non-radioactive morphine, naloxone, CN-,
t). N. TELLERel al.
124
ouabain, etc., were in the washout medium. In other experiments, the efflux temperature was varied, using a second shaker bath. Radioactivity was measured using an lntertechnique SL 30 liquid scintillation spectrometer with external Cesium standardization and high efficiency photodetectors. The counting efficiency varied for 14C from 92 to 83~, with a background of 45 counts/min, and was automatically corrected to disint./min (dpm) by a Multimat 8K-bit computer. Two liquid scintillation formulations were used: 16 ml of a modified Prockop-Ebert cocktail, toluene-ethyleneglycol monomethyl ether (10:6) carrying 5 g 2,5-diphenyloxazole (PPO) and 25 mg 1,4-bis-2-(5-phenyloxazolyl)-benzene (POPOP) per liter ~. However, only 0.5 ml of aqueous sample can be dissolved in this cocktail. For digested pellets, larger aqueous volumes, etc., a modified 'tT76' scintillation gel was used 16. The scintillator composition is 8 g of PPO and 150 mg of dimethylPOPOP (or bis-MSB)* per liter of toluene. To 700 ml of toluene scintillator, 600 ml of Rohm and Haas Triton X-100 was added (no silica gel purification is required with current batches of Triton X-100)to produce tT76. The digested tissue in 1 ml of I N N a O H was neutralized and acidified (to reduce luminescence) with 1.5 ml of 1 N HCI ; 2.0 ml of H~O was added, and 8.8 ml of tT76. The gel that formed at 18 °C was stable and thixotropic, and showed no change in counting efficiency for several weeks. Efficiencies ranged from 83 to 90~ with this mixture, and background varied from 39 to 44 counts/min. If the tissue was accidentally heated beyond 65 °C or contained blood, the yellow color was removed from the NaOH digest with 0.05 ml of n-octanol (to prevent subsequent foaming) and 1 ml of 30~ HeO2; after decolorization, crystalline ascorbic acid was added until effervescence ceased. HCI was added as before and the volume was reduced, if necessary, to give a final aqueous volume of 4.5 ml before tT76 was added. In such cases, counting efficiency for 14C dropped to 60-75~. All standards were prepared in quintuplicate, in both the Prockop-Ebert and tT76 scintillation mixtures. The corrected dpm varied more within the sets than between the scintillation mixtures. In addition, a dilution of the standard was added to each counted sample from the first incubation with new batches of [14C]morphine, and the results were analyzed with respect to counting efficiency and recovery of added dpm in comparison to [14C]toluene standards that were similarly employed. Calculations of the results entailed several assumptions with varying degrees of certainty: dry weight, extracellular/intracellular space, etc. The calculation of tissue/ medium (T/M) concentrations was: dpm in tissue/tissue wet weight/0.8 dpm/ml in medium at start of incubation When more than 10~ of the morphine was removed during the incubation (below 10 8 M) by the tissue, the final medium concentration was determined by counting samples after the tissue was removed.
* These are more soluble and less quenched by water than the POPOP originally used for tT76.
MORPHINE UPTAKE INTO BRAIN SLICES
125
The other factor calculated, concentrative uptake, has been reviewed elsewhere s. The assumptions of intracellular and extracellular space, water content, swelling, etc., have also been described at length 6. In this calculation, the concentration of morphine above that of the medium at the end of the incubation is expressed in terms of the calculated intracellular water of the tissue. After incubation without drug, or with less than 10-5 M morphine in the medium, the swelling was less than 8 mg/1130 mg dry weight in brain. No correction for swelling was used in any calculation reported here. The factor (wet weight × 0.8), was used in all cases to estimate the tissue volume of water (to calculate T in T/M calculations). All determinations were performed on triplicate samples, i.e., 3 separate incubation flasks. Occasionally, where there were differences greater than 10~ among the triplicate samples, the experiment was repeated until 6-12 individual data bits could be averaged with confidence. Estimated standard errors of means are reported where N = 6-15; standard deviation where N is greater than 15. Na +, K ÷ and ATP. Sodium and potassium concentrations were determined from diluted perchloric acid supernatants with an IL/343 flame photometer (Instrumentation Laboratories). ATP was determined with a DuPont Luminescence Biometer2. Extraction and thin-layer chromatography (TLC) of the radioactive material from tissue. Two types of extractions were performed: of 3~ perchloric acid supernatants of brain slices, and of slice homogenates (5-10~, w/v) in 1 N HC1. The procedures of Misra and Mule 14 were modified: homogenates and perchloric acid supernatants (4 ml portions) were neutralized with 2.5 N NaOH, brought to pH 10 i 0.2 using a pH meter, and 1.5 g of NaC1 and 1.0 ml of potassium phosphate buffer, pH 10.4 (350 g K2HPO4, 50 g K3PO4 per liter) was added to each basic solution. The basic solution, now more than 6 ml, was mixed thoroughly and then was shaken with 15 ml of 20~o isopropanol in dichloroethane (v/v) for 40 min using a wrist-action shaker. After phase separation by centrifugation at 613C0 ×gav, the supernatant organic phase (Uph- 1) was aspirated, leaving approximately 0.5 ml of Uph-1 in the 25 ml Corex tube. The lower phase was again extracted with 10~o isopropanol in dichloroethane. Samples of Uph-1, Uph-2, and final lower phase were counted. The combined upper phases were evaporated under N2 at 65 °C and dissolved in 1130 ~1 of methanol for TLC. Nominal 20 #1 samples (2-32 × 103 dpm) were applied to E. Merck silica gel plates (250/zm thick without indicator) using a stream of warm air to speed drying. Development was carried out for 2.3 h in ethyl acetate-methanol-water-NH4OH (85:10:3:2). The solvent rose 14 cm above the origin with morphine appearing at RE 0.31-(4.3/14) cm. The plates were sprayed with chloroform saturated with iodine, and after warming, with 0.5~o (w/v) platinic iodide in 2~o (w/v) potassium iodide. Morphine appeared blue-black. A second solvent system, ethanol-acetic acid-water (60:30:10), also produced good results in 4 h (RE morphine ----- 0.18); n-butanol-NH4OH-water (4:1:3) showed phase separation and tailing on the plates, although this last solvent system has been used by others for paper chromatograms. After development and spraying with indicator, the morphine spots and other colored, or previously fluorescent, spots were scraped into tT76 and counted. The residual iodine and platinum salts did not quench the scintillator gel below 78~o effi-
126
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Fig. 1. The uptake of [a4C]morphine by mouse brain slices is not saturable from 3 ~< 10 s M to 2 x 10 '~M. Each point is the average of 12-66 separate incubations, with the extent of the standard deviations shown by the bar. The over-all average of 402 control incubations is T/M = 2.216 ~0.163 (estimated standard error of the mean, e.s.e.). The T/M reported here is at equilibrium, 30 rain after morphine addition. From 3 × 10-8 M t o 10 7 M n o carrier morphine was added to the radioactive drug; from 2.5 x 10 7 M t o 2 x 10 4 M the carrier was morphine-HC1;from 5 >: 10 4 M to 2 x 10 -3 M the carrier was morphine hemisulfate, U.S.P. Fig. 2. Increasing the mass of mouse or rat brain incubated decreases the uptake of morphine. The ratio of the medium volume (in most experiments, 5 ml) divided by the tissue volume (in most experiments, 80-150 rag/0.8 ml g-1 water) x 100 provides integer abscissa numbers at the extreme left portion of the graph. However, reducing the mass incubated or increasing the volume of the medium provides ratios from 50 to 600. In the case of rat or mouse brain, an optimum ratio for utpake is present, and further 'dilution' of the tissue in medium does not increase the uptake. This can be seen both at low morphine concentrations with rat brain slices (upper data set), or with high morphine concentrations (lower data) for mouse brain slices. Two other concentrations with mouse and rat brain gave similar results and the optimum ratio is independent, for each species, of incubation time. In addition, this figure shows that the two species have different uptake characteristics for morphine. Each point is the result of an individual incubation, with the data from 4 separate days indicated by the 4 symbols.
ciency. The addition of 0.2 Fmoles of non-radioactive morphine as 'cold carrier' to homogenates or acid supernatants increased recovery from 89 to 96~o. Less than 1~ of the total radioactivity of brain homogenate extracts applied to the T L C plates appeared at positions other than that coincident with morphine. RESULTS
Brain slices took up morphine to a level higher than that of the medium, but this uptake was not saturable over a concentration range from 3 × l0 --8 to 2 x 16 -3 M (Fig. 1). The uptake of morphine did depend upon the amount of tissue in the medium. Results of varying the M/T volumes from approximately 30 to 6(;0 are shown in Fig. 2. When the morphine content of rat brain slices is in equilibrium with the medium concentration (3.1 x 10 8 M) after 30 min incubation, the regression of uptake on tissue weight fits the equation T/M ~- (--0.1 X) + 4, where X = slice weight in rag, with a correlation coefficient -- 0.93. When the volume of the medium was 30-3C~fold greater (v = 30-360) than the tissue volume, the T/M increased by 0.034/106v.
MORPHINE UPTAKE INTO BRAIN SLICES I
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Fig. 3. The effect of increasing the cut surface area of mouse brain cortex slices on morphine uptake. The slices and prisms of the tissue (see text for method of preparation) were incubated at 37 °C for 20 min before addition of 2.5/~M [14C]morphine. At 2.5 # M morphine with mouse brain slices this effect increased to 1.18 (T/M)/100v (lower data set, Fig. 2). To determine whether or not material liberated from the incubated slice mass absorbed morphine and effectively lowered the concentration in the medium (thereby decreasing the T / M observed for the tissue mass) we 'conditioned' media by incubating large and small amounts of tissue in it for 30 min. After this, the medium was filtered from the slices and was reoxygenated for use with a second set of slices. Such conditioned media did not affect the morphine uptake by brain slices. Another experiment was performed to see if the cut surface area determined the rate of entry or tissue concentration of morphine at equilibrium with the medium. Here the slices were turned 90 ° and sliced again, producing rectangular prisms (416 sq. #m) × (ventricular-pial height). This increases the surface area of the tissue by 50-75~. As shown in Fig. 3, the rate of morphine entry was increased but the equilibrium concentration was the same as with standard slices. The uptake of morphine by brain slices from rat and guinea pig was similar to that of mouse brain slices. Efflux from the brain slices was rapid. In the case of rat and guinea pig brain cortex slices, 70-80~o of the radioactivity washed out in 10 min. Addition of a 10-fold excess of the narcotic antagonist, naloxone, to either the uptake or efflux medium did not alter the results significantly (Table I). Several methods were used to test whether the uptake of morphine by the brain slices was saturable. In one of these, the mouse cortex slices were loaded with nonradioactive morphine for 30 min during temperature equilibration at a concentration higher than that of the radioactive morphine to be added subsequently. Thus the slices were incubated in 2.5 or 5.3 # M morphine and were then transferred to 1 or 2.5 ~ M [t4C]morphine. No inhibition of uptake of the radioactivity was observed (Table 1I). Another similar procedure, in which the dilute (2 or 5 x 10 -7 M) radioactive morphine was added to the slices while they were still in the 'excess' (2.84 x 10 -4 M) cold
128
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TABLE I U P T A K E A N D El;FLUX OF M O R P H I N E FROM BRAIN SLICES OF R A T A N D G U I N E A PIG IN THE PRESENCE A N D ABSENCE OF NALOXONE
Species
Brain area
Morphine (M)
(n)
T I M ( ±e.s.e) after 30 min
Percent washed out in 10 rain
Rat
Cerebral cortex
Guinea pig
Cerebral cortex
1 × 10 6 + naloxone* 1 × 10-~ + naloxone 2.5 x 10 6 2.5 ~'< 10-~ + naloxone
4 4 6 6 6 6 6
2.23 _5_0.16 1.96 ± 0.39 2.03 ~z 0.08 1.70 ~ 0.09 2.03 3= 0.16 1.64 ~ 0.15 1.75 ~ 0.11
72.8 ~: 5.4 64.2:5 4.8 69.5 ± 7.2 65.9 :i~ 4.6 76.2 ~:: 6.6 ---
Thalamus + hypothalamus 2.5 × 10-6 + naloxone Pons + medulla 2.5 × 10-6 ~- naloxone
6 6 3 3
2.08 ± 0.14 1.97 i 0.07 1.88 1.86
----
Cerebellum
77.1 _~::8.4** 75.3 ~ 6.3 79.2 ± 7.1 79.1 :~- 5.5 68.6 ± 6.5 --
---
* Incubation medium contained 10-5 M naloxone. ** Washout medium contained 10 5 M naloxone.
m o r p h i n e , also showed no i n h i b i t i o n o f u p t a k e o f r a d i o a c t i v i t y by r a t b r a i n slices. T o e x a m i n e the effect o f the presence o f m o r p h i n e on the exit o f r a d i o a c t i v e l y l a b e l e d m o r p h i n e f r o m the slices, the slices were transferred after i n c u b a t i o n to fresh m e d i u m , with a n d w i t h o u t a d d e d m o r p h i n e . T h e r e was no difference in the efflux o f r a d i o activity when m o r p h i n e was in the w a s h o u t m e d i u m . T o test the possibility t h a t the m e d i u m currently used in o u r l a b o r a t o r i e s , b a s e d on H E P E S buffer, was less suitable for m o r p h i n e u p t a k e into brain t h a n the one r e p o r t e d by Scrafani a n d H u g 18, we p r e p a r e d their m e d i u m , a n d a n o t h e r described b y H a m b e r g e d 0, which was used to s u p p o r t isolated b r a i n cell types for investigations o f active t r a n s p o r t . T h e similar results we o b t a i n e d with these 3 m e d i a are shown in T a b l e III. I n a d d i t i o n , the extraction, recovery, a n d c h r o m a t o g r a p h y o f m o r p h i n e f r o m rat b r a i n a n d m o u s e b r a i n did n o t show t h a t a n y significant m e t a b o l i c transform a t i o n s o c c u r r e d in a 30-min i n c u b a t i o n period. T h e effects o f varying some m e d i u m constituents on the u p t a k e o f m o r p h i n e are shown in Fig. 4. T h e presence or absence o f C a 2+, M g 2+, or both, in H E P E S - 2 m e d i a h a d m i n o r effects on the u p t a k e o f m o r p h i n e b y b r a i n slices. T h e u p t a k e a n d efflux o f m o r p h i n e f r o m m o u s e b r a i n slices is t e m p e r a t u r e d e p e n d e n t . A t 0.5 °C u p t a k e was less t h a n at 37 °C. Efflux was also slower. A s shown in Fig. 5, the first samples for efflux were t a k e n 5 rain after the tissue was transferred to fresh m e d i u m . T h e time r e q u i r e d to wash o u t 1/2 o f the m o r p h i n e (Eft. t/2) was greater at 0.5 °C (27.5 rain) when 5.4/~moles/ml tissue v o l u m e were t a k e n up in 30 rain (at 0.5 °C, Eft. t/2 = 15 min). In c o m p a r i s o n , the efflux o f m o r p h i n e at 37 °C from slices i n c u b a t e d at 0.5 °C o r 37 °C, in 2 m M C N - o r after h e a t inactivation (see below) was a p p r o x i m a t e l y the same, with an Eft. t / 2 = 2.5-4 min.
129
MORPHINE UPTAKE INTO BRAIN SLICES TABLE II EXCHANGE AND EXCHANGE EFFLUX OF MORPHINE
Tissue
in vitro
Morphine concentration Preincuba- Incubation Efflux*** tion* (M) (M)** (M)
Rat brain§
Mouse brain§
0 2 x 10 - 7 _ 2.84 x 10 -4 ( + ) 2 x 10 - 7 _ 0 5 x 10 - 7 _ 2.84 × 10 4 ( + ) 5 x 10 - 7 _ 0 10 -6 -2.5 x 10 -e 10 -6 -5.3 x 10 -6 10 -6 -0 2.5 x 10 -8 - 2.5 x 10 -a 2.5 x 10 -6 - 5.3 z 10 -6 2.5 x 10 -6 - 0 2 . 5 x 10 -6 0 0 2 . 5 x 10 -a 2.5 x 10 -a
Time (min) and relative concentration of radioactive morphine in slices 5 1.77§§ 1.93 1.78 1.86 1.93 1.82 1.80 1.78 1.72 1.92 2.04/46.7§§§ 2.15/49.4
10
30
2.20 2.41 2.40 2.43 2.20 2.18 2.46 2.40 2.09 2.61 1.15/26.2 1.22/25.5
2.32 2.25 2.31 2.33 2.51 2.55 2.88 2.43 2.38 2.76 0.52/11.5 0.57/12.8
60 2.34 -2.53 2.37 2.37 2.59 2.56 2.53 2.34 2.69 0.42/9.6 0.41/9.2
* Period of temperature equilibration = 30 rain. ** Radioactive morphine, in place of; or ( + ) = in addition to the concentration already present. *** Non-radioactive m o r p h i n e in efflux medium. § Cortex. §§ T / M . §§§ (nmoles/ml tissue vol.)/percent of radioactive m o r p h i n e remaining in tissue, at each interval of the 30 min of uptake. At the start of the efflux period (t = '0' min) the tissue [14C]morphine content was 4.36 nmoles/ml tissue vol. ( = 100K).
Uptake of morphine from 2 to 10 min after addition to the brain slices was rapid, and linear with time. At various temperatures from 0.5 °C to 16 °C, the rate (in medium containing 1 ffM morphine) increased at 18 nmoles/h °C. From 20 to 43 °C the 'initial rate' was 63 nmoles/h °C. This relatively sharp change in 'initial rate' is observed with other substances 7, and has been called 'a temperature break'. Such temperature effects may not be strong evidence for active transport since they may instead reflect change in the thermodynamic properties of the tissue. TABLE III COMPARISON OF MORPHINE UPTAKE INTO BRAIN SLICES IN 3 INCUBATION MEDIA
Morphine (M)
C. U Medium I*
C. U Medium II**
5 x 10 -8 2.5 x 10 -8
16 9.7 4.2 2.0 0.38
15 7.4 3.6 1.6 0.36
1 x 10 - e
5 x 10 -7 1 x 10 - r
C. U Medium III 15 8.2 3.9 ---
* I n c u b a t i o n for 10 m i n at 37 °C; C . U = concentrative uptake, nmoles/ml intracellular water above the concentration in the m e d i u m at the end o f the incubation. ** M e d i u m I = Scrafani and Hugla; M e d i u m I I = H E P E S - 2 (see refs. 6 and 8); M e d i u m I I I = H a m b e r g e r 10.
130
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' MIN.
EFFLUX DURATION
Fig. 4. The effects of changing Ca "+, Mg 2~, or both, on the uptake of morphine by mouse brain slices are minimal. The abscissa is the ratio of the experimental ion concentration: normal medium concentration (q.v., see Methods). The slices were incubated for a total of 60 min in the various media; during the last 30-rain period 5 × 10--7 M [14C]morphine was present. Results from media in which Mg 2~ was varied are given by the solid line; Ca 2+ data are shown by the dotted line; and results from experiments with both Ca 2+ and Mg e+ varying, by the dash-dot line. Fig. 5. The efflux of morphine from brain slices is temperature-dependent. The two upper sets of data points are from efflux incubations at 0.5 °¢-. After the initial rinse of the tissue slices, the exit is logarithmic - - the straight lines upon which t,le circles and squares are plotted. However, the triangular data points show that efflux at 37 °C after incubations at 0.5 °C or 37 °C (0°/37°; 37°/37) is more rapid, with a Eft. t/2 of 2.5 min. The effiux of morphine from heat-inactivated (10 min at 95 °C) or metabolically poisoned tissue (2 mM NaCN) is shown by the dashed line, with a Eft. t/2 of 4 rain. The time required for 50% of the morphine to exit is given by the small numbers in parentheses on the graph. The identifying numbers on the right of each data set are: temperature for uptake/temperature for effiux.
V a r i o u s t r e a t m e n t s with m o r p h i n e in vivo h a d little effect on u p t a k e or exit in vitro. T y p i c a l results o b t a i n e d f r o m guinea pig b r a i n slices are shown in T a b l e IV. O n l y the animals which were m a d e t o l e r a n t to m o r p h i n e a n d were then n o t injected for 18 h showed a n y change in m o r p h i n e u p t a k e . T o d e t e r m i n e whether the m o r p h i n e u p t a k e was sensitive to (metabolic) inhibitors o f active t r a n s p o r t processes, cyanide was a d d e d to the i n c u b a t i o n m e d i u m , before the r a d i o a c t i v e m o r p h i n e . As shown in T a b l e V, c o n c e n t r a t i o n s o f C N - , which b l o c k the u p t a k e o f L-glutamic acid, a n d which deplete A T P a n d K ~, a n d increase N a t (see ref. 3), have little effect on m o r p h i n e uptake. Brain slices t o o k up m o r p h i n e to a level higher than t h a t o f the m e d i u m , but when these slices were heated to 95 °C for 10 rain in a boiling water bath, the u p t a k e did n o t stop. A f t e r heating the tissue the rate o f m o r p h i n e entry during i n c u b a t i o n at 37 °C was different f r o m that in the control, u n h e a t e d p r e p a r a t i o n , but the a c c u m u l a tion o f m o r p h i n e in the heated slices c o u l d exceed the level in the u n h e a t e d c o n t r o l after 30-90 min, as shown in Fig. 6. A f t e r heat treatment, the u p t a k e o f m o r p h i n e by the tissue at 0.5 °C was identical with that o f u n h e a t e d c o n t r o l s i n c u b a t e d at 0.5 °C. F o r c o m p a r i s o n , similar experiments were p e r f o r m e d with b r a i n slices a n d the a m i n o acids g l u t a m a t e a n d valine, which are actively t r a n s p o r t e d in brain. The boiling b l o c k e d a m i n o acid uptake, a n d indeed, the T / M did not exceed 2 (Table VI). The possibility
MORPHINE UPTAKE INTO BRAIN SLICES
131
TABLE IV MORPHINE UPTAKE AND EFFLUX BY BRAIN SLICES FROM GUINEA PIG CORTEX AFTER VARIOUS INJECTION SEQUENCES in vivo
Injection group*
TIM 4- e.s.e.**
n
P
% w.o.***
Control Tolerant Acute Withdrawn
1.98 2.06 1.84 2.38
10 8 4 11
-n.s. n.s. 0.05§
70.8 72.6 73.4 72.9
4± 44-
0.09 0.08 0.01 0.09
* 'Tolerant' animals were injected twice daily for 14 days with 50-500 mg/kg morphine hemisulfate in progressively increasing doses. ~Acute' animals were injected with 50 mg morphine, i.p., 1 h before the brains were removed. 'Withdrawn' animals were not injected for 18 h before the brains were removed, after 14 days of injections to produce tolerance. ** e.s.e. = estimated standard error; M = 10 -6 M. *** Percent of morphine that washes out in 10 min. § Probability of difference in uptake between Control and Withdrawn average arising by chance, using Student's t-test (F-test = not significant, n.s.). Addition of 50 # M naloxone to the incubation media had no effect on the T / M from any group.
that morphine was being bound investigated in comparison
differently in boiled and control preparations
to the distribution
of the amino
was
a c i d s i n b r a i n slice
p r e p a r a t i o n s . T h e r e s u l t s a r e s h o w n i n T a b l e V I a n d d o n o t i n d i c a t e a n y g r o s s difference in the retention of the acid extractable substances after the heat treatment
of
t h e slices. Various substances were added to the medium
a n d slices 30 r a i n b e f o r e t h e
m o r p h i n e t o see w h e t h e r t h e y w o u l d a f f e c t t h e u p t a k e o f t h e d r u g . A m o n g t h e s e s u b TABLE V COMPARISON OF THE EFFECTS OF C N GLUTAM1C ACID
CN Substrate (raM)
--0.5 0.5 2.0 2.0 * ** *** §
L-Glutamic acid 1 mM Morphine 1 # M Glutamic acid 1 mM Morphine 1 # M Glutamic acid 1 mM Morphine 1 # M
ON BRAIN SLICE N a + ~ K+~
TIM
Na +
ATP,
AND UPTAKE OF MORPHINE OR
K+
ATP
% C* #moles/ ml
%C
#moles**/ % C ml
#moles/ml % C
100 100
113 118
100 100
70 68
100 100
1.0 1.0
10.8 2.03
49 90
115 165
101 141
26 25
39 37
0.13 0.13
1.98 1.69
9 75
144 168
127 142
14 13
20 19
0.01 0.009
22.0*** 2.26§
~ C = percent of control; no N a C N present = 100K. # m o l e s / m l tissue volume. Incubation for 60 min; data taken from ref. 3. Incubation for 30 min.
100 100 13 13 1.0 0.9
132
p.N.
T_./'~ r ] ~
" i
i~
7
d
.
u
r
~ ~ ! ~ . : . . . . = ~ -
2b~ l - - ~ - - - e
%
I I/o
HEATED 1o M,V. A r
1.5{~ I ILLJ_L~
TELLER dl a/.
T
~--
I
95°
M: ,o-4M I
i
_ _.~
J
5 I0 20~ 30 60 90 DURATION OF INCUBATION AT 3 7 ° , MINUTES
I
12.0
Fig. 6. The uptake of morphine by heat-inactivated brain slices may exceed uptake by control (unheated) slices. In these experiments the morphine concentration was initially 10-4 M. The slice preparations were treated as described in the text. They were removed from the initial media that had been heated or iced, and were transferred to fresh, oxygenated media for the uptake incubation, which followed the standard procedure: 30 rain temperature equilibrium followed by varying durations of exposure to the radioactive morphine. Data points are from individual incubations. Several other drugs, e.g., barbiturates, salicylates, etc., also exhibit 'uptake' after heat-inactivation of enzymatic activity in brain slices. In particular, barbiturates show much higher uptake in slices boiled for 10 min than in unheated controls '°. stances were other drugs, a m i n o acids, m e t a b o l i c poisons, an d narcotic antagonists. N o n e o f these m a r k e d l y inhibited ( > 25%) or stimulated ( > 20%) the u p t a k e o f [14C]m o r p h i n e . A list o f these substances a n d the c o n c e n t r a t i o n s tested for effects on m o r p h i n e u p t a k e are given in T a b l e VII. Similar results were o b t a i n e d with these c o m p o u n d s when tested with slices f r o m rat an d guinea pig brain (cortex). N a l o x o n e in vitro h ad no effect on the uptake o f m o r p h i n e into, or efflux f r o m m o u s e or rat brain
TABLE VI DISTRIBUTION OF RADIOACTIVE SUBSTRATES IN MOUSE BRAIN SLICE ACID EXTRACTS" EFFECT OF HEAT TREATMENT
Radioactive substrate and initial concentration in medium
Tissue treatment*
Concentration o f radioactive substrate in acid extract** ( ff moles/ ml )
L-Glutamic acid§, 1 mM
I, 5 min I, 60 min B, 5 rain B, 60 min I, 5 rain I, 60 min B, 5 min B, 60 min I, 5 rain I, 60 min B, 5 min B, 60 min
7.26 22.0 1.47 1.95 3.60 7.65 0.81 1.62 0.20 0.24 0.15 0.23
L-Valine §§, 2 mM
Morphine§§§, 0.1 mM
* I = iced at 0.5°C for 10 min after slicing, before incubation for 30 + 5 (or 60)rain with radioactive compound. B = heated to 95 °C for 10 rain. ** #moles/ml tissue vol.; duplicate incubations. From centrifugation of 3% HC104 homogenate at 15,000 rcf x gmax/10 rain at 4 °C. § Uniformly labeled with 1~C; carrier = 10 mM; S.A. - 25,400 dpm/ffmole. §§ Uniformly labeled with 14C; carrier = 20 mM; S.A. = 32,750 dpm/ffmole. §§§ n-methyl 14C; carrier = 10 mM; S.A. = 63,300 dpm/ffmole.
MORPHINE UPTAKE INTO BRAIN SLICES
133
TABLE VII SUBSTANCES TESTED FOR EFFECT ON
1-2,5 X 10-8 M
MORPHINE UPTAKE INTO BRAIN SLICES
Concn. (mM) Endogenous compounds L-Lysine Arginine Leucine Valine Glutamic acid Aspartic acid Cadaverine D-Glucose D,L-Lactate Pyruvate Succinate
2 2 2 1-2 1-2 1-2 2 0.5-20 0.1-10 0.05-10 0.05-10
Poisons Cyanide Rotenone Iodoacetate Fluoride
0.1-2.0 0.0001-0.01 0.1-2.0 0.1-2.0
Concn. (mM) Drugs--Non-narcotic Amytal Phenobarbital Ouabain Probenecid Ethanol
0.05-5 0.05-5 0.1-2,0 0.4-5 1-10
Narcotic analogs or antagonists Naloxone Levorphanol Nalorphine Methadone Codeine
0.002-1 0.002-0.1 0.002-0.1 0.022-0.1 0.002-0.1
Others a-Aminoisobutyric acid
slices, or w h e n tested with b r a i n slices f r o m mice t o l e r a n t to, d e p e n d e n t on, or acutely injected with, m o r p h i n e . Des p it e the a p p a r e n t lack o f sensitivity o f the m o r p h i n e u p t a k e to v ar i o u s treatments, we were able to c o n f ir m t h a t the i n h i b i t i o n o f m o r p h i n e u p t a k e by o u a b a i n or cy an i d e in the m e d i u m o f Scrafani a n d H u g is is greater t h a n that in H E P E S - 2 . Th ese results are s h o w n in T a b l e V I I I , a n d in c o n j u n c t i o n with the other results described above, m a y be a reflection m o r e o f the different abilities o f the m e d i a to m a i n t a i n the
TABLE VIII A COMPARISON OF THE EFFECTS OF CYANIDE AND OUABA1N ON T I l E UPTAKE OF MORPHINE BY MOUSE BRAIN SLICES IN T W O DIFFERENT MEDIA
Medium
Inhibitor
Concentration TIM at 10' (M)
%C
I*
Ouabain
i
2.18"*
100
1 x 10-a --
1.68 2.13
1 x 10 -a
1.95
-5 x 2 x i 5 x 2 x
2.61"** 1.67 1.49 2.40 1.92 1.65
II I
II
Cyanide
10-4 10-a 10-4 10-a
76.8 100
91.7 100 64.1 57.2 100 77.5 66.7
* Medium I = Scrafani and HuglS; II = HEPES-2 (see refs. 6 and 8). ** Morphine = 5 x 10-e M in experiments with ouabain. *** Morphine = 10-8 M in experiments with cyanide.
%I
0
23.2 0
8.3 0 35.8 42.8 0 22.4 33.3
134
,~. N. ~IELLER ~'l (I[.
viability of the tissue than of the effects of the inhibitors on ally narcotic 'trans!~ort' processes. DISCUSS[ON
We have observed that the following conditions did not affect the uptake of morphine into mouse, rat, or guinea pig brain slices: treatment in vivo with morphine or naloxone; presence of similar (narcotic), antagonist, or unrelated drugs, general metabolic poisons; prior incubation in 1-1000-fold higher morphine concentrations; alteration of either Ca 2+ or Mg 2~, or both, in the medium. The following did affect morphine uptake to some degree: varying the mass of tissue in a fixed volume of medium; boiling the tissue for 10 min; incubating at 0.5 °C, or under N2, or in the presence of concentrated cyanide ( ~ 1 mM). The maximum inhibition of uptake that we achieved was less than 50%, with a concentration of morphine in the tissue still well above the level in the medium. Because the criteria for active transport are not absolute, we believe that a conservative conclusion is justified by these results: if active transport of morphine into brain slices occurs, it is not :responsive to various treatments that modify the active transport of amino acids3; secondly, those treatments that effectively reduce the morphine uptake also do so with heat inactivated tissue, or alter only very slightly the concentrating ability of the tissue; in addition, the lack of effects of various treatments on the efflux of morphine from brain slices would suggest that this process also, is primarily passive. These conclusions, and particularly some of the results of the experimental procedures, do not agree with those reported by Scrafani and Hug is. Craig et at2 reported only two T/M values of 1.07 and 2.63 at 10-3 and at 10-6 M, with rat cerebral cortex slices, but did [lot extend the concentration range to see whether the T/M increased with lower morphine levels in their medium. As in this study, they did not find differences in uptake caused by treatment with morphine in vivo; or by K 4 stimulation in vitro. The majority of the differences may be explained by the relative resistance of the HEPES-2 medium to pH change, in comparison to phosphate buffers at pH 7.27.6. For example, the pH of the medium of Hug and Scrafani 18 is markedly affected during incubation by the addition of more than 10`-4 M morphine. This observation had also been made earlier by BelD, who described in great detail the relative values of bicarbonate and phosphate-buffered media for drug-uptake studies (HEPES was not available then). In addition, possible variations in the size of the tissue mass/ volume of the incubation medium may also contribute to experimental differences. The major discrepancy appears in our finding an absence of saturability of the uptake between 10 s and 10-3 M. Because we observed this discrepancy early in these investigations, we went to extreme lengths to document this finding. Unless the criteria that apply to drugs are different from those used to assess the active transport of endogenous compounds, i.e., amino acids, morphine uptake by brain slices clearly does not occur by active transport. We do not extrapolate these findings to the possibility of active transport of dihydromorphine, for which many supporting, and contesting, reports have appearedlZ,'~, 24.
MORPHINE UPTAKE INTO BRAIN SLICES
135
ACKNOWLEDGEMENTS P o r t i o n s of this work were supported by U.S. Public Health Service G r a n t N S 03226. The a u t h o r s t h a n k Dr. A. Neidle for advice a n d suggestions regarding extract i o n a n d thin-layer c h r o m a t o g r a p h y of m o r p h i n e , a n d Dr. M. Banay-Schwartz for N a ÷, K +, a n d A T P determinations.
REFERENCES 1 ASHGAR, K., AND WAY, E. L., Active removal of morphine from the cerebral ventricles, J. Pharmacol. exp. Ther., 175 (1970) 75-83. 2 BANAV-SCHWARTZ,M., GERGELY,A., ANDLAJTHA,A., Independence of amino acid uptake from tissue swelling in incubated slices of brain, Brain Research, 65 (1974) 265-276. 3 BANAY-SCHWARTZ,M., TELLER,D. N., GERGELY,A., AND LAJTHA, m., The effects of metabolic inhibitors on amino acid uptake and levels of ATP, Na + and K ÷ in incubated slices of mouse brain, Brain Research, 71 (1974) 117-131. 4 BELL,J. L., Concentration of some analgesic compounds and their analogues in tissues surviving in vitro: relation to metabolism and in vivo effect, J. Neurochem., 2 (1958) 265-282. 5 BLASBERG,R., AND LAJTHA, A., Substrate specificity of steady state amino acid transport in mouse brain slices, Arch. Biochem., 112 (1965) 361-377. 6 COHEN, S. R., The estimation of extracellular space of brain tissue in vitro. In N. MARKSAND R. RODNIGHT(Eds.), Research Methods in Neurochemistry, Vol. 1, Plenum Press, New York, 1972, pp. 179-219. 7 COHEN, S. R., The rate of respiration of mouse brain tissue in physiological and non-physiological media as a function of temperature, Comp. Biochem. Physiol., 45 (1973) 1031-1045. 8 COHEN,S. R., STAMPLEMAN,P. F., AND LAJTHA, A., The temperature-dependent compartmentation of the extracellular space in mouse brain slices as revealed by the markers: inulin, sucrose, D-mannitol, D-sorbitol and sulfate, Brain Research, 21 (1970) 419--434. 9 CRAXG,A. L., O'DEA, R. F., AND TAKEMORI,A. E., The uptake of morphine by the choroid plexus and cerebral cortical slices of animals chronically treated with morphine, Neuropharmacology, 10 (1971) 709-714. 10 HAMBER~ER,A., Amino acid uptake in neuronal and glial cell fractions from rabbit cerebral cortex, Brain Research, 31 (1971) 169-178. 11 Hue, JR., C. C., Transport of narcotic analgesics by choroid plexus and kidney tissue in vitro, Biochem. Pharmacol., 16 (1967) 345-359. 12 KAYAN,S., MISRA, A. L., ANDWOODS, L. A., Uptake of [7,8-aH]dihydromorphine by rat cerebral cortical slices and eye tissue, J. Pharmacol. (Kyoto), 22 (1970) 941-943. 13 LOll, H. H., SHEN,F.-H., ANDWAY,E. L., Effect of dactinomycin on the acute toxicity and brain uptake of morphine, J. Pharmacol. exp. Ther., 177 (1971) 326-331. 14 MISRA,A. L., AND MUL~, S. J., Disposition and metabolism of [3H]pseudomorphine in the rat, Biochem. Pharmacol., 21 (1972) 193-207. 15 OLDENDORF,W. H., HYMAN,S., BRAUN,L., ANDOLDENDORF,S. Z., Blood-brain barrier: Penetration of morphine, codeine, heroin and methadone after carotid injection, Science, 178 (1972) 984-986. 16 PATTERSON,M. S., AND GREENE, R. C., Measurement of low energy beta emitters in aqueous solution by liquid scintillation counting of emulsions, Anal. Chem., 37 (1965) 854-857. 17 SCHANKER,L. S., Physiological transport of drugs, Advanc. Drug Res., 1 (1964) 72-106. 18 SCRAFANI,J. T., AND HUG, JR., C. C., Active uptake of dihydromorphine and other narcotic analgesics by cerebral cortical slices, Biochem. Pharmacol., 17 (1968) 1555-1566. 19 TArEMORI,A. E., AND STENWICK, M. W., Studies on the uptake of morphine by the choroid plexus in vitro, J. Pharmacol. exp. Ther., 154 (1966) 586-594. 20 TELLER,O. N., DE GUZMAN,T., AND LAJTHA,A., Transport of morphine and barbiturates into brain, liver and kidney in vitro, Psychopharmacologia (Berl.), 26, Suppl. CINP (1972) 120.
136
l). N. TELLER Ct a[.
21 TELLER, D. N., DE GUZMAN, T., AND LAJTHA, A., Energy requirements for the transport of morphine into tissue slices, Trans. Amer. Soc. Neurochem., 3 (1973) 130. 22 VASKO,M. R., AND HUG, JR., C. C., Is the uptake of narcotic analgesics by cerebral slices mediated in part by active transport?, J. Pharm. Pharmacol., 25 (1973) 180-183. 23 WANG, J. H., AND TAKEMORI,A. E., The exchange of morphine (M) between blood and CSF in rabbits, Fed. Proc., 30 (1971) 501 (abstr. 1705). 24 WAN6, J. H., AND TAKEMORI,A. E., Studies on the transport of morphine out of the perfused cerebral ventricles of rabbits, J. Pharmacol. exp. Ther., 181 (1972) 46-52. 25 WOODS, L. A., AND MULI~, S. J., Distribution of N-C14-methyl labeled morphine: I. In central nervous system of nontolerant and tolerant dogs, J. Pharmacol. exp. Ther., 136 (1963) 232-241.
121
THE MODE OF MORPHINE UPTAKE INTO BRAIN SLICES
DAVID N. TELLER, TERESITA DE GUZMAN AND ABEL LAJTHA
New York State Research Institute for Neurochemistry and Drug Addiction, Ward's Island, New York, N.Y, 10035 (U.S.A.) (Accepted March 29th, 1974)
SUMMARY
The uptake of [N-14C methyl]morphine into rat, mouse, or guinea pig brain slices does not meet the criteria for an active transport process. (1) Methods are described for the extraction and TLC of morphine at 9 6 ~ recovery levels, and for the determination of 14C at 70-90 ~ counting efficiency with brain tissue samples. (2) After morphine had been in the tissue for 30 min, 96~ of the radioactivity was extractable, and 99~ of this was unmetabolized morphine. (3) The ratio of the concentration in the tissue to that in the medium was 2.22+ 0.16 e.s.e., from 3 × 10-8 M to 2 × 10-3 M. Therefore, the uptake is not saturable. (4) Strong metabolic inhibitors, e.g. cyanide, at concentrations that completely block amino acid transport, did not markedly reduce the uptake of morphine. (5) The uptake into and efflux from brain tissue was not affected by concentrated morphine, analogs, or narcotic antagonists. Therefore, the uptake of morphine showed no signs of the competitive inhibition that is typical of active transport processes. In addition, morphine uptake was not affected by acute or chronic morphine injections in the mouse or guinea pig. (6) The uptake of morphine into brain tissue was temperature dependent, but it occurred at 0.5 °C and also after the tissue was kept at 95 °C for 10 min.
INTRODUCTION
Morphine has been reported to be actively transported by brain slices18, by choroid plexusi,n, 19, and possibly in vivo from blood to brainlZ,15,23,25. Other reports appear to contradict some of these findings4,9,12,2a. There were technical differences between the various investigations, and some studies were primarily concerned with dihydromorphineX2, e2,z4. Although morphine and dihydromorphine are almost
122
~). N. TELLER cl a[.
identical pharmacologically and structurally, there appear to be differences between these two drugs in their mechanism of uptake into brain slices. For an understanding of the distribution and fate of pharmacological agents, it would be important to know whether specific transport processes exist for drugs such as morphine, or if the transport mechanisms for endogenous compounds such as amino acids can also mediate the movement of drugs with related structure. Knowledge of the characteristics of transport processes for drugs might provide the possibility of facilitation or blockade of entry into the brain, thereby influencing drug action. In this study we observed that although morphine accumulates in brain slices, this does not occur via an active transport process. Indeed, although experimental criteria for the demonstration of active transport processes are not absolute, morphine uptake in brain slices meets none of the tests 17 satisfactorily. Morphine uptake in brain slices was not saturable, temperature dependent, competitively inhibited by structural analogs, or blocked by metabolic inhibitors. Heat treatment (95 ~C) that destroyed enzyme activity and stopped amino acid transport did not abolish morphine uptake. In contrast to the absence of demonstrable active transport of morphine in brain slices, kidney slices were shown to possess an active transport system for morphine :tl. In separate reports we have confirmed and extended these findings 2°,'1. MATERIALS A N D METHODS
Materials
Morphine, labeled with 14C in the N-methyl group was obtained from Amersham/Searle. Three batches were used, batches 8, 9, and 11, lot CFA 363, at 57 mCi/ mmole. Morphine hemisulfate, from Mallinckrodt Chemical Works, was converted to the hydrochloride by reaction with BaCls and CO2. The solution was dried under N2; the morphine HCI was recrystallized from ethanol and was assayed colorimetrically with the same reagents and procedure used for protein 4, with authentic morphine hemisulfate as a standard. Radioactive solutions of morphine were prepared in water, except where this would have diluted the incubation medium more than 5~. In such cases, the radioactive solution was prepared from a more concentrated aqueous stock by dilution with medium. The constriction pipets used to deliver the radioactive morphine into the incubation flasks were calibrated by delivery of water and dye to 3 significant figures. The average variance in delivery and counting of a nominal 10/zl volume was ~: 0.6~, when 10~ counts were recorded. Variance in tissue wet or dry weight measurement was -~ 0.06 mg/10 mg; estimated standard errors from counting the same sample or standard were less than 0.3~. Radioactive amino acids, uniformly labeled with 14C, were obtained from New England Nuclear Corp. and were diluted with non-radioactive carrier to approximately 2/~Ci/mmole. These solutions were prepared in media at 10 and 20 mM, and 0.5 ml was used per 4.5 ml + tissue in each flask. Non-radioactive compounds were prepared in water, buffer, medium, or solvent as indicated, and controls were added to the experimental protocol to measure the effects of the diluent alone on the uptake of the radioactive drug. All concentrated solutions of the narcotics and their analogs
MORPHINE UPTAKE INTO BRAIN SLICES
123
were kept dark and cold, or frozen, before use. With some drugs, e.g., levorphanol and nalorphine, the room was darkened during the entire procedure to avoid photodecomposition of the dilute compounds. Incubation. The 3 types of incubation media were: HEPES-2 (see refs. 6 and 8), the medium used by Scrafani and Hug 18, and that used by Hamberger 1°. HEPES-2 medium contains 119 mM NaC1, 5.0 mM KCI, 0.75 mM CaCI~, 1.2 mM MgSO4, 1.0 mM NaH~PO4, 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid (H EPE S) 12 mM NaOH, 10 mM glucose, pH 7.4 (with NaOH) at 25 °C (see in ref. 8, p. 190, footnote to Table II). The medium of Scrafani and Hug 18 contains 134 mM NaCI, 2.7 mM KCI, 0.48 mM MgC12, 0.92 mM CaClz, 14.8 mM NaHCO3, 3.6 mM Na2HPO4 (pH 7.4), and 5.5 mM glucose, with pH adjusted to 7.3-7.5 (with HC1). Hamberger's mediumTM contains 35 mM Tris-HCl (pH 7.6), 120 mM NaCI, 5 mM KCI, 5 mM Na3PO4 (pH 7.6), 20 mM glucose, 2.5 mM MgC12. Species, treatment in vivo, and tissue preparation. Mice, rats, and guinea pigs were inbred from strains derived from Swiss Webster, Wistar, and Cayley varieties, respectively. The mice were 6-10 weeks old, the rats were 6-8 months old and the guinea pigs were 9 months old when they were used for these experiments. Some mice and guinea pigs were injected with morphine, from 40 mg/kg, i.p., to more than 1 g/kg, s.c., to produce tolerance. Animals 'withdrawn' from morphine were given no injection for 18 h prior to being killed; 'chronic' animals received a last dose of 4~30 mg/kg, i.p., 2-3 h before use; 'acute' animals received a single dose of 40 mg/kg, i.p., 1 h before they were killed. In all cases, both sexes were used, because no consistent effects were observed that might be attributable to sex differences. All tissue slices were 416/~m (:kl8/*m, S.D.) transverse sections made with a Mcllwain-Mickle chopper. The sliced tissues were transferred to 4.5-5 ml of medium that had been oxygenated for 4 min and were equilibrated in the shaker bath for 20-30 min at the temperature for the particular experiment. During this time the Na + and K + levels in the tissue apparently regained some differential equilibrium with the medium, because when the tissues were freshly excised and in iced medium, the enzymes that maintain the intracellular ion levels were relatively inactive. Therefore, 20-30 min after the slices were added to the medium, radioactive morphine was added, and incubation was continued for an additional 2-120 min. Incubation was stopped by filtering the medium from the slicess, and the slices were frozen in powdered dry ice. The weighed frozen tissue pellet was homogenized in 39/0 perchloric acid (w/v), and the supernatant was saved for determination of radioactivity, extraction of morphine, ion and ATP determinations, etc. The pellet of perchloric acid-insoluble material was dissolved in 1 N NaOH and was prepared for counting as described below. In most cases the frozen tissue was simply digested in 1 ml of 1 N NaOH (overnight at room temperature, or for 10 min at 65 °C). Efflux of radioactive morphine was measured by filtering the radioactive medium from the slices through a Hirsch funnel, rinsing with 2 ml of warmed, oxygenated medium, and transferring the slices to fresh oxygenated media which contained no radioactivity. After an additional 2-120 min the slices were filtered from the washout medium and were frozen. In some cases non-radioactive morphine, naloxone, CN-,
t). N. TELLERel al.
124
ouabain, etc., were in the washout medium. In other experiments, the efflux temperature was varied, using a second shaker bath. Radioactivity was measured using an lntertechnique SL 30 liquid scintillation spectrometer with external Cesium standardization and high efficiency photodetectors. The counting efficiency varied for 14C from 92 to 83~, with a background of 45 counts/min, and was automatically corrected to disint./min (dpm) by a Multimat 8K-bit computer. Two liquid scintillation formulations were used: 16 ml of a modified Prockop-Ebert cocktail, toluene-ethyleneglycol monomethyl ether (10:6) carrying 5 g 2,5-diphenyloxazole (PPO) and 25 mg 1,4-bis-2-(5-phenyloxazolyl)-benzene (POPOP) per liter ~. However, only 0.5 ml of aqueous sample can be dissolved in this cocktail. For digested pellets, larger aqueous volumes, etc., a modified 'tT76' scintillation gel was used 16. The scintillator composition is 8 g of PPO and 150 mg of dimethylPOPOP (or bis-MSB)* per liter of toluene. To 700 ml of toluene scintillator, 600 ml of Rohm and Haas Triton X-100 was added (no silica gel purification is required with current batches of Triton X-100)to produce tT76. The digested tissue in 1 ml of I N N a O H was neutralized and acidified (to reduce luminescence) with 1.5 ml of 1 N HCI ; 2.0 ml of H~O was added, and 8.8 ml of tT76. The gel that formed at 18 °C was stable and thixotropic, and showed no change in counting efficiency for several weeks. Efficiencies ranged from 83 to 90~ with this mixture, and background varied from 39 to 44 counts/min. If the tissue was accidentally heated beyond 65 °C or contained blood, the yellow color was removed from the NaOH digest with 0.05 ml of n-octanol (to prevent subsequent foaming) and 1 ml of 30~ HeO2; after decolorization, crystalline ascorbic acid was added until effervescence ceased. HCI was added as before and the volume was reduced, if necessary, to give a final aqueous volume of 4.5 ml before tT76 was added. In such cases, counting efficiency for 14C dropped to 60-75~. All standards were prepared in quintuplicate, in both the Prockop-Ebert and tT76 scintillation mixtures. The corrected dpm varied more within the sets than between the scintillation mixtures. In addition, a dilution of the standard was added to each counted sample from the first incubation with new batches of [14C]morphine, and the results were analyzed with respect to counting efficiency and recovery of added dpm in comparison to [14C]toluene standards that were similarly employed. Calculations of the results entailed several assumptions with varying degrees of certainty: dry weight, extracellular/intracellular space, etc. The calculation of tissue/ medium (T/M) concentrations was: dpm in tissue/tissue wet weight/0.8 dpm/ml in medium at start of incubation When more than 10~ of the morphine was removed during the incubation (below 10 8 M) by the tissue, the final medium concentration was determined by counting samples after the tissue was removed.
* These are more soluble and less quenched by water than the POPOP originally used for tT76.
MORPHINE UPTAKE INTO BRAIN SLICES
125
The other factor calculated, concentrative uptake, has been reviewed elsewhere s. The assumptions of intracellular and extracellular space, water content, swelling, etc., have also been described at length 6. In this calculation, the concentration of morphine above that of the medium at the end of the incubation is expressed in terms of the calculated intracellular water of the tissue. After incubation without drug, or with less than 10-5 M morphine in the medium, the swelling was less than 8 mg/1130 mg dry weight in brain. No correction for swelling was used in any calculation reported here. The factor (wet weight × 0.8), was used in all cases to estimate the tissue volume of water (to calculate T in T/M calculations). All determinations were performed on triplicate samples, i.e., 3 separate incubation flasks. Occasionally, where there were differences greater than 10~ among the triplicate samples, the experiment was repeated until 6-12 individual data bits could be averaged with confidence. Estimated standard errors of means are reported where N = 6-15; standard deviation where N is greater than 15. Na +, K ÷ and ATP. Sodium and potassium concentrations were determined from diluted perchloric acid supernatants with an IL/343 flame photometer (Instrumentation Laboratories). ATP was determined with a DuPont Luminescence Biometer2. Extraction and thin-layer chromatography (TLC) of the radioactive material from tissue. Two types of extractions were performed: of 3~ perchloric acid supernatants of brain slices, and of slice homogenates (5-10~, w/v) in 1 N HC1. The procedures of Misra and Mule 14 were modified: homogenates and perchloric acid supernatants (4 ml portions) were neutralized with 2.5 N NaOH, brought to pH 10 i 0.2 using a pH meter, and 1.5 g of NaC1 and 1.0 ml of potassium phosphate buffer, pH 10.4 (350 g K2HPO4, 50 g K3PO4 per liter) was added to each basic solution. The basic solution, now more than 6 ml, was mixed thoroughly and then was shaken with 15 ml of 20~o isopropanol in dichloroethane (v/v) for 40 min using a wrist-action shaker. After phase separation by centrifugation at 613C0 ×gav, the supernatant organic phase (Uph- 1) was aspirated, leaving approximately 0.5 ml of Uph-1 in the 25 ml Corex tube. The lower phase was again extracted with 10~o isopropanol in dichloroethane. Samples of Uph-1, Uph-2, and final lower phase were counted. The combined upper phases were evaporated under N2 at 65 °C and dissolved in 1130 ~1 of methanol for TLC. Nominal 20 #1 samples (2-32 × 103 dpm) were applied to E. Merck silica gel plates (250/zm thick without indicator) using a stream of warm air to speed drying. Development was carried out for 2.3 h in ethyl acetate-methanol-water-NH4OH (85:10:3:2). The solvent rose 14 cm above the origin with morphine appearing at RE 0.31-(4.3/14) cm. The plates were sprayed with chloroform saturated with iodine, and after warming, with 0.5~o (w/v) platinic iodide in 2~o (w/v) potassium iodide. Morphine appeared blue-black. A second solvent system, ethanol-acetic acid-water (60:30:10), also produced good results in 4 h (RE morphine ----- 0.18); n-butanol-NH4OH-water (4:1:3) showed phase separation and tailing on the plates, although this last solvent system has been used by others for paper chromatograms. After development and spraying with indicator, the morphine spots and other colored, or previously fluorescent, spots were scraped into tT76 and counted. The residual iodine and platinum salts did not quench the scintillator gel below 78~o effi-
126
~). N. TELtJ~:R e/ al. I
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[ 1 3 4 LOG ( I / M )
-~
-
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/ 2 .5 4 5 MEDIUM VOL./(TISSUE VOL. x I00)
[ ~ 6
Fig. 1. The uptake of [a4C]morphine by mouse brain slices is not saturable from 3 ~< 10 s M to 2 x 10 '~M. Each point is the average of 12-66 separate incubations, with the extent of the standard deviations shown by the bar. The over-all average of 402 control incubations is T/M = 2.216 ~0.163 (estimated standard error of the mean, e.s.e.). The T/M reported here is at equilibrium, 30 rain after morphine addition. From 3 × 10-8 M t o 10 7 M n o carrier morphine was added to the radioactive drug; from 2.5 x 10 7 M t o 2 x 10 4 M the carrier was morphine-HC1;from 5 >: 10 4 M to 2 x 10 -3 M the carrier was morphine hemisulfate, U.S.P. Fig. 2. Increasing the mass of mouse or rat brain incubated decreases the uptake of morphine. The ratio of the medium volume (in most experiments, 5 ml) divided by the tissue volume (in most experiments, 80-150 rag/0.8 ml g-1 water) x 100 provides integer abscissa numbers at the extreme left portion of the graph. However, reducing the mass incubated or increasing the volume of the medium provides ratios from 50 to 600. In the case of rat or mouse brain, an optimum ratio for utpake is present, and further 'dilution' of the tissue in medium does not increase the uptake. This can be seen both at low morphine concentrations with rat brain slices (upper data set), or with high morphine concentrations (lower data) for mouse brain slices. Two other concentrations with mouse and rat brain gave similar results and the optimum ratio is independent, for each species, of incubation time. In addition, this figure shows that the two species have different uptake characteristics for morphine. Each point is the result of an individual incubation, with the data from 4 separate days indicated by the 4 symbols.
ciency. The addition of 0.2 Fmoles of non-radioactive morphine as 'cold carrier' to homogenates or acid supernatants increased recovery from 89 to 96~o. Less than 1~ of the total radioactivity of brain homogenate extracts applied to the T L C plates appeared at positions other than that coincident with morphine. RESULTS
Brain slices took up morphine to a level higher than that of the medium, but this uptake was not saturable over a concentration range from 3 × l0 --8 to 2 x 16 -3 M (Fig. 1). The uptake of morphine did depend upon the amount of tissue in the medium. Results of varying the M/T volumes from approximately 30 to 6(;0 are shown in Fig. 2. When the morphine content of rat brain slices is in equilibrium with the medium concentration (3.1 x 10 8 M) after 30 min incubation, the regression of uptake on tissue weight fits the equation T/M ~- (--0.1 X) + 4, where X = slice weight in rag, with a correlation coefficient -- 0.93. When the volume of the medium was 30-3C~fold greater (v = 30-360) than the tissue volume, the T/M increased by 0.034/106v.
MORPHINE UPTAKE INTO BRAIN SLICES I
I
127
I
TIM PRISM zo-
/,/
•
~SLICE
1.0 / M = 2.5 x IO-6M
0
t I i 0 5 10 20 INCUBATION TIME, MIN.
Fig. 3. The effect of increasing the cut surface area of mouse brain cortex slices on morphine uptake. The slices and prisms of the tissue (see text for method of preparation) were incubated at 37 °C for 20 min before addition of 2.5/~M [14C]morphine. At 2.5 # M morphine with mouse brain slices this effect increased to 1.18 (T/M)/100v (lower data set, Fig. 2). To determine whether or not material liberated from the incubated slice mass absorbed morphine and effectively lowered the concentration in the medium (thereby decreasing the T / M observed for the tissue mass) we 'conditioned' media by incubating large and small amounts of tissue in it for 30 min. After this, the medium was filtered from the slices and was reoxygenated for use with a second set of slices. Such conditioned media did not affect the morphine uptake by brain slices. Another experiment was performed to see if the cut surface area determined the rate of entry or tissue concentration of morphine at equilibrium with the medium. Here the slices were turned 90 ° and sliced again, producing rectangular prisms (416 sq. #m) × (ventricular-pial height). This increases the surface area of the tissue by 50-75~. As shown in Fig. 3, the rate of morphine entry was increased but the equilibrium concentration was the same as with standard slices. The uptake of morphine by brain slices from rat and guinea pig was similar to that of mouse brain slices. Efflux from the brain slices was rapid. In the case of rat and guinea pig brain cortex slices, 70-80~o of the radioactivity washed out in 10 min. Addition of a 10-fold excess of the narcotic antagonist, naloxone, to either the uptake or efflux medium did not alter the results significantly (Table I). Several methods were used to test whether the uptake of morphine by the brain slices was saturable. In one of these, the mouse cortex slices were loaded with nonradioactive morphine for 30 min during temperature equilibration at a concentration higher than that of the radioactive morphine to be added subsequently. Thus the slices were incubated in 2.5 or 5.3 # M morphine and were then transferred to 1 or 2.5 ~ M [t4C]morphine. No inhibition of uptake of the radioactivity was observed (Table 1I). Another similar procedure, in which the dilute (2 or 5 x 10 -7 M) radioactive morphine was added to the slices while they were still in the 'excess' (2.84 x 10 -4 M) cold
128
I). N. TELLER ~'l a[.
TABLE I U P T A K E A N D El;FLUX OF M O R P H I N E FROM BRAIN SLICES OF R A T A N D G U I N E A PIG IN THE PRESENCE A N D ABSENCE OF NALOXONE
Species
Brain area
Morphine (M)
(n)
T I M ( ±e.s.e) after 30 min
Percent washed out in 10 rain
Rat
Cerebral cortex
Guinea pig
Cerebral cortex
1 × 10 6 + naloxone* 1 × 10-~ + naloxone 2.5 x 10 6 2.5 ~'< 10-~ + naloxone
4 4 6 6 6 6 6
2.23 _5_0.16 1.96 ± 0.39 2.03 ~z 0.08 1.70 ~ 0.09 2.03 3= 0.16 1.64 ~ 0.15 1.75 ~ 0.11
72.8 ~: 5.4 64.2:5 4.8 69.5 ± 7.2 65.9 :i~ 4.6 76.2 ~:: 6.6 ---
Thalamus + hypothalamus 2.5 × 10-6 + naloxone Pons + medulla 2.5 × 10-6 ~- naloxone
6 6 3 3
2.08 ± 0.14 1.97 i 0.07 1.88 1.86
----
Cerebellum
77.1 _~::8.4** 75.3 ~ 6.3 79.2 ± 7.1 79.1 :~- 5.5 68.6 ± 6.5 --
---
* Incubation medium contained 10-5 M naloxone. ** Washout medium contained 10 5 M naloxone.
m o r p h i n e , also showed no i n h i b i t i o n o f u p t a k e o f r a d i o a c t i v i t y by r a t b r a i n slices. T o e x a m i n e the effect o f the presence o f m o r p h i n e on the exit o f r a d i o a c t i v e l y l a b e l e d m o r p h i n e f r o m the slices, the slices were transferred after i n c u b a t i o n to fresh m e d i u m , with a n d w i t h o u t a d d e d m o r p h i n e . T h e r e was no difference in the efflux o f r a d i o activity when m o r p h i n e was in the w a s h o u t m e d i u m . T o test the possibility t h a t the m e d i u m currently used in o u r l a b o r a t o r i e s , b a s e d on H E P E S buffer, was less suitable for m o r p h i n e u p t a k e into brain t h a n the one r e p o r t e d by Scrafani a n d H u g 18, we p r e p a r e d their m e d i u m , a n d a n o t h e r described b y H a m b e r g e d 0, which was used to s u p p o r t isolated b r a i n cell types for investigations o f active t r a n s p o r t . T h e similar results we o b t a i n e d with these 3 m e d i a are shown in T a b l e III. I n a d d i t i o n , the extraction, recovery, a n d c h r o m a t o g r a p h y o f m o r p h i n e f r o m rat b r a i n a n d m o u s e b r a i n did n o t show t h a t a n y significant m e t a b o l i c transform a t i o n s o c c u r r e d in a 30-min i n c u b a t i o n period. T h e effects o f varying some m e d i u m constituents on the u p t a k e o f m o r p h i n e are shown in Fig. 4. T h e presence or absence o f C a 2+, M g 2+, or both, in H E P E S - 2 m e d i a h a d m i n o r effects on the u p t a k e o f m o r p h i n e b y b r a i n slices. T h e u p t a k e a n d efflux o f m o r p h i n e f r o m m o u s e b r a i n slices is t e m p e r a t u r e d e p e n d e n t . A t 0.5 °C u p t a k e was less t h a n at 37 °C. Efflux was also slower. A s shown in Fig. 5, the first samples for efflux were t a k e n 5 rain after the tissue was transferred to fresh m e d i u m . T h e time r e q u i r e d to wash o u t 1/2 o f the m o r p h i n e (Eft. t/2) was greater at 0.5 °C (27.5 rain) when 5.4/~moles/ml tissue v o l u m e were t a k e n up in 30 rain (at 0.5 °C, Eft. t/2 = 15 min). In c o m p a r i s o n , the efflux o f m o r p h i n e at 37 °C from slices i n c u b a t e d at 0.5 °C o r 37 °C, in 2 m M C N - o r after h e a t inactivation (see below) was a p p r o x i m a t e l y the same, with an Eft. t / 2 = 2.5-4 min.
129
MORPHINE UPTAKE INTO BRAIN SLICES TABLE II EXCHANGE AND EXCHANGE EFFLUX OF MORPHINE
Tissue
in vitro
Morphine concentration Preincuba- Incubation Efflux*** tion* (M) (M)** (M)
Rat brain§
Mouse brain§
0 2 x 10 - 7 _ 2.84 x 10 -4 ( + ) 2 x 10 - 7 _ 0 5 x 10 - 7 _ 2.84 × 10 4 ( + ) 5 x 10 - 7 _ 0 10 -6 -2.5 x 10 -e 10 -6 -5.3 x 10 -6 10 -6 -0 2.5 x 10 -8 - 2.5 x 10 -a 2.5 x 10 -6 - 5.3 z 10 -6 2.5 x 10 -6 - 0 2 . 5 x 10 -6 0 0 2 . 5 x 10 -a 2.5 x 10 -a
Time (min) and relative concentration of radioactive morphine in slices 5 1.77§§ 1.93 1.78 1.86 1.93 1.82 1.80 1.78 1.72 1.92 2.04/46.7§§§ 2.15/49.4
10
30
2.20 2.41 2.40 2.43 2.20 2.18 2.46 2.40 2.09 2.61 1.15/26.2 1.22/25.5
2.32 2.25 2.31 2.33 2.51 2.55 2.88 2.43 2.38 2.76 0.52/11.5 0.57/12.8
60 2.34 -2.53 2.37 2.37 2.59 2.56 2.53 2.34 2.69 0.42/9.6 0.41/9.2
* Period of temperature equilibration = 30 rain. ** Radioactive morphine, in place of; or ( + ) = in addition to the concentration already present. *** Non-radioactive m o r p h i n e in efflux medium. § Cortex. §§ T / M . §§§ (nmoles/ml tissue vol.)/percent of radioactive m o r p h i n e remaining in tissue, at each interval of the 30 min of uptake. At the start of the efflux period (t = '0' min) the tissue [14C]morphine content was 4.36 nmoles/ml tissue vol. ( = 100K).
Uptake of morphine from 2 to 10 min after addition to the brain slices was rapid, and linear with time. At various temperatures from 0.5 °C to 16 °C, the rate (in medium containing 1 ffM morphine) increased at 18 nmoles/h °C. From 20 to 43 °C the 'initial rate' was 63 nmoles/h °C. This relatively sharp change in 'initial rate' is observed with other substances 7, and has been called 'a temperature break'. Such temperature effects may not be strong evidence for active transport since they may instead reflect change in the thermodynamic properties of the tissue. TABLE III COMPARISON OF MORPHINE UPTAKE INTO BRAIN SLICES IN 3 INCUBATION MEDIA
Morphine (M)
C. U Medium I*
C. U Medium II**
5 x 10 -8 2.5 x 10 -8
16 9.7 4.2 2.0 0.38
15 7.4 3.6 1.6 0.36
1 x 10 - e
5 x 10 -7 1 x 10 - r
C. U Medium III 15 8.2 3.9 ---
* I n c u b a t i o n for 10 m i n at 37 °C; C . U = concentrative uptake, nmoles/ml intracellular water above the concentration in the m e d i u m at the end o f the incubation. ** M e d i u m I = Scrafani and Hugla; M e d i u m I I = H E P E S - 2 (see refs. 6 and 8); M e d i u m I I I = H a m b e r g e r 10.
130
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Fig. 4. The effects of changing Ca "+, Mg 2~, or both, on the uptake of morphine by mouse brain slices are minimal. The abscissa is the ratio of the experimental ion concentration: normal medium concentration (q.v., see Methods). The slices were incubated for a total of 60 min in the various media; during the last 30-rain period 5 × 10--7 M [14C]morphine was present. Results from media in which Mg 2~ was varied are given by the solid line; Ca 2+ data are shown by the dotted line; and results from experiments with both Ca 2+ and Mg e+ varying, by the dash-dot line. Fig. 5. The efflux of morphine from brain slices is temperature-dependent. The two upper sets of data points are from efflux incubations at 0.5 °¢-. After the initial rinse of the tissue slices, the exit is logarithmic - - the straight lines upon which t,le circles and squares are plotted. However, the triangular data points show that efflux at 37 °C after incubations at 0.5 °C or 37 °C (0°/37°; 37°/37) is more rapid, with a Eft. t/2 of 2.5 min. The effiux of morphine from heat-inactivated (10 min at 95 °C) or metabolically poisoned tissue (2 mM NaCN) is shown by the dashed line, with a Eft. t/2 of 4 rain. The time required for 50% of the morphine to exit is given by the small numbers in parentheses on the graph. The identifying numbers on the right of each data set are: temperature for uptake/temperature for effiux.
V a r i o u s t r e a t m e n t s with m o r p h i n e in vivo h a d little effect on u p t a k e or exit in vitro. T y p i c a l results o b t a i n e d f r o m guinea pig b r a i n slices are shown in T a b l e IV. O n l y the animals which were m a d e t o l e r a n t to m o r p h i n e a n d were then n o t injected for 18 h showed a n y change in m o r p h i n e u p t a k e . T o d e t e r m i n e whether the m o r p h i n e u p t a k e was sensitive to (metabolic) inhibitors o f active t r a n s p o r t processes, cyanide was a d d e d to the i n c u b a t i o n m e d i u m , before the r a d i o a c t i v e m o r p h i n e . As shown in T a b l e V, c o n c e n t r a t i o n s o f C N - , which b l o c k the u p t a k e o f L-glutamic acid, a n d which deplete A T P a n d K ~, a n d increase N a t (see ref. 3), have little effect on m o r p h i n e uptake. Brain slices t o o k up m o r p h i n e to a level higher than t h a t o f the m e d i u m , but when these slices were heated to 95 °C for 10 rain in a boiling water bath, the u p t a k e did n o t stop. A f t e r heating the tissue the rate o f m o r p h i n e entry during i n c u b a t i o n at 37 °C was different f r o m that in the control, u n h e a t e d p r e p a r a t i o n , but the a c c u m u l a tion o f m o r p h i n e in the heated slices c o u l d exceed the level in the u n h e a t e d c o n t r o l after 30-90 min, as shown in Fig. 6. A f t e r heat treatment, the u p t a k e o f m o r p h i n e by the tissue at 0.5 °C was identical with that o f u n h e a t e d c o n t r o l s i n c u b a t e d at 0.5 °C. F o r c o m p a r i s o n , similar experiments were p e r f o r m e d with b r a i n slices a n d the a m i n o acids g l u t a m a t e a n d valine, which are actively t r a n s p o r t e d in brain. The boiling b l o c k e d a m i n o acid uptake, a n d indeed, the T / M did not exceed 2 (Table VI). The possibility
MORPHINE UPTAKE INTO BRAIN SLICES
131
TABLE IV MORPHINE UPTAKE AND EFFLUX BY BRAIN SLICES FROM GUINEA PIG CORTEX AFTER VARIOUS INJECTION SEQUENCES in vivo
Injection group*
TIM 4- e.s.e.**
n
P
% w.o.***
Control Tolerant Acute Withdrawn
1.98 2.06 1.84 2.38
10 8 4 11
-n.s. n.s. 0.05§
70.8 72.6 73.4 72.9
4± 44-
0.09 0.08 0.01 0.09
* 'Tolerant' animals were injected twice daily for 14 days with 50-500 mg/kg morphine hemisulfate in progressively increasing doses. ~Acute' animals were injected with 50 mg morphine, i.p., 1 h before the brains were removed. 'Withdrawn' animals were not injected for 18 h before the brains were removed, after 14 days of injections to produce tolerance. ** e.s.e. = estimated standard error; M = 10 -6 M. *** Percent of morphine that washes out in 10 min. § Probability of difference in uptake between Control and Withdrawn average arising by chance, using Student's t-test (F-test = not significant, n.s.). Addition of 50 # M naloxone to the incubation media had no effect on the T / M from any group.
that morphine was being bound investigated in comparison
differently in boiled and control preparations
to the distribution
of the amino
was
a c i d s i n b r a i n slice
p r e p a r a t i o n s . T h e r e s u l t s a r e s h o w n i n T a b l e V I a n d d o n o t i n d i c a t e a n y g r o s s difference in the retention of the acid extractable substances after the heat treatment
of
t h e slices. Various substances were added to the medium
a n d slices 30 r a i n b e f o r e t h e
m o r p h i n e t o see w h e t h e r t h e y w o u l d a f f e c t t h e u p t a k e o f t h e d r u g . A m o n g t h e s e s u b TABLE V COMPARISON OF THE EFFECTS OF C N GLUTAM1C ACID
CN Substrate (raM)
--0.5 0.5 2.0 2.0 * ** *** §
L-Glutamic acid 1 mM Morphine 1 # M Glutamic acid 1 mM Morphine 1 # M Glutamic acid 1 mM Morphine 1 # M
ON BRAIN SLICE N a + ~ K+~
TIM
Na +
ATP,
AND UPTAKE OF MORPHINE OR
K+
ATP
% C* #moles/ ml
%C
#moles**/ % C ml
#moles/ml % C
100 100
113 118
100 100
70 68
100 100
1.0 1.0
10.8 2.03
49 90
115 165
101 141
26 25
39 37
0.13 0.13
1.98 1.69
9 75
144 168
127 142
14 13
20 19
0.01 0.009
22.0*** 2.26§
~ C = percent of control; no N a C N present = 100K. # m o l e s / m l tissue volume. Incubation for 60 min; data taken from ref. 3. Incubation for 30 min.
100 100 13 13 1.0 0.9
132
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I
12.0
Fig. 6. The uptake of morphine by heat-inactivated brain slices may exceed uptake by control (unheated) slices. In these experiments the morphine concentration was initially 10-4 M. The slice preparations were treated as described in the text. They were removed from the initial media that had been heated or iced, and were transferred to fresh, oxygenated media for the uptake incubation, which followed the standard procedure: 30 rain temperature equilibrium followed by varying durations of exposure to the radioactive morphine. Data points are from individual incubations. Several other drugs, e.g., barbiturates, salicylates, etc., also exhibit 'uptake' after heat-inactivation of enzymatic activity in brain slices. In particular, barbiturates show much higher uptake in slices boiled for 10 min than in unheated controls '°. stances were other drugs, a m i n o acids, m e t a b o l i c poisons, an d narcotic antagonists. N o n e o f these m a r k e d l y inhibited ( > 25%) or stimulated ( > 20%) the u p t a k e o f [14C]m o r p h i n e . A list o f these substances a n d the c o n c e n t r a t i o n s tested for effects on m o r p h i n e u p t a k e are given in T a b l e VII. Similar results were o b t a i n e d with these c o m p o u n d s when tested with slices f r o m rat an d guinea pig brain (cortex). N a l o x o n e in vitro h ad no effect on the uptake o f m o r p h i n e into, or efflux f r o m m o u s e or rat brain
TABLE VI DISTRIBUTION OF RADIOACTIVE SUBSTRATES IN MOUSE BRAIN SLICE ACID EXTRACTS" EFFECT OF HEAT TREATMENT
Radioactive substrate and initial concentration in medium
Tissue treatment*
Concentration o f radioactive substrate in acid extract** ( ff moles/ ml )
L-Glutamic acid§, 1 mM
I, 5 min I, 60 min B, 5 rain B, 60 min I, 5 rain I, 60 min B, 5 min B, 60 min I, 5 rain I, 60 min B, 5 min B, 60 min
7.26 22.0 1.47 1.95 3.60 7.65 0.81 1.62 0.20 0.24 0.15 0.23
L-Valine §§, 2 mM
Morphine§§§, 0.1 mM
* I = iced at 0.5°C for 10 min after slicing, before incubation for 30 + 5 (or 60)rain with radioactive compound. B = heated to 95 °C for 10 rain. ** #moles/ml tissue vol.; duplicate incubations. From centrifugation of 3% HC104 homogenate at 15,000 rcf x gmax/10 rain at 4 °C. § Uniformly labeled with 1~C; carrier = 10 mM; S.A. - 25,400 dpm/ffmole. §§ Uniformly labeled with 14C; carrier = 20 mM; S.A. = 32,750 dpm/ffmole. §§§ n-methyl 14C; carrier = 10 mM; S.A. = 63,300 dpm/ffmole.
MORPHINE UPTAKE INTO BRAIN SLICES
133
TABLE VII SUBSTANCES TESTED FOR EFFECT ON
1-2,5 X 10-8 M
MORPHINE UPTAKE INTO BRAIN SLICES
Concn. (mM) Endogenous compounds L-Lysine Arginine Leucine Valine Glutamic acid Aspartic acid Cadaverine D-Glucose D,L-Lactate Pyruvate Succinate
2 2 2 1-2 1-2 1-2 2 0.5-20 0.1-10 0.05-10 0.05-10
Poisons Cyanide Rotenone Iodoacetate Fluoride
0.1-2.0 0.0001-0.01 0.1-2.0 0.1-2.0
Concn. (mM) Drugs--Non-narcotic Amytal Phenobarbital Ouabain Probenecid Ethanol
0.05-5 0.05-5 0.1-2,0 0.4-5 1-10
Narcotic analogs or antagonists Naloxone Levorphanol Nalorphine Methadone Codeine
0.002-1 0.002-0.1 0.002-0.1 0.022-0.1 0.002-0.1
Others a-Aminoisobutyric acid
slices, or w h e n tested with b r a i n slices f r o m mice t o l e r a n t to, d e p e n d e n t on, or acutely injected with, m o r p h i n e . Des p it e the a p p a r e n t lack o f sensitivity o f the m o r p h i n e u p t a k e to v ar i o u s treatments, we were able to c o n f ir m t h a t the i n h i b i t i o n o f m o r p h i n e u p t a k e by o u a b a i n or cy an i d e in the m e d i u m o f Scrafani a n d H u g is is greater t h a n that in H E P E S - 2 . Th ese results are s h o w n in T a b l e V I I I , a n d in c o n j u n c t i o n with the other results described above, m a y be a reflection m o r e o f the different abilities o f the m e d i a to m a i n t a i n the
TABLE VIII A COMPARISON OF THE EFFECTS OF CYANIDE AND OUABA1N ON T I l E UPTAKE OF MORPHINE BY MOUSE BRAIN SLICES IN T W O DIFFERENT MEDIA
Medium
Inhibitor
Concentration TIM at 10' (M)
%C
I*
Ouabain
i
2.18"*
100
1 x 10-a --
1.68 2.13
1 x 10 -a
1.95
-5 x 2 x i 5 x 2 x
2.61"** 1.67 1.49 2.40 1.92 1.65
II I
II
Cyanide
10-4 10-a 10-4 10-a
76.8 100
91.7 100 64.1 57.2 100 77.5 66.7
* Medium I = Scrafani and HuglS; II = HEPES-2 (see refs. 6 and 8). ** Morphine = 5 x 10-e M in experiments with ouabain. *** Morphine = 10-8 M in experiments with cyanide.
%I
0
23.2 0
8.3 0 35.8 42.8 0 22.4 33.3
134
,~. N. ~IELLER ~'l (I[.
viability of the tissue than of the effects of the inhibitors on ally narcotic 'trans!~ort' processes. DISCUSS[ON
We have observed that the following conditions did not affect the uptake of morphine into mouse, rat, or guinea pig brain slices: treatment in vivo with morphine or naloxone; presence of similar (narcotic), antagonist, or unrelated drugs, general metabolic poisons; prior incubation in 1-1000-fold higher morphine concentrations; alteration of either Ca 2+ or Mg 2~, or both, in the medium. The following did affect morphine uptake to some degree: varying the mass of tissue in a fixed volume of medium; boiling the tissue for 10 min; incubating at 0.5 °C, or under N2, or in the presence of concentrated cyanide ( ~ 1 mM). The maximum inhibition of uptake that we achieved was less than 50%, with a concentration of morphine in the tissue still well above the level in the medium. Because the criteria for active transport are not absolute, we believe that a conservative conclusion is justified by these results: if active transport of morphine into brain slices occurs, it is not :responsive to various treatments that modify the active transport of amino acids3; secondly, those treatments that effectively reduce the morphine uptake also do so with heat inactivated tissue, or alter only very slightly the concentrating ability of the tissue; in addition, the lack of effects of various treatments on the efflux of morphine from brain slices would suggest that this process also, is primarily passive. These conclusions, and particularly some of the results of the experimental procedures, do not agree with those reported by Scrafani and Hug is. Craig et at2 reported only two T/M values of 1.07 and 2.63 at 10-3 and at 10-6 M, with rat cerebral cortex slices, but did [lot extend the concentration range to see whether the T/M increased with lower morphine levels in their medium. As in this study, they did not find differences in uptake caused by treatment with morphine in vivo; or by K 4 stimulation in vitro. The majority of the differences may be explained by the relative resistance of the HEPES-2 medium to pH change, in comparison to phosphate buffers at pH 7.27.6. For example, the pH of the medium of Hug and Scrafani 18 is markedly affected during incubation by the addition of more than 10`-4 M morphine. This observation had also been made earlier by BelD, who described in great detail the relative values of bicarbonate and phosphate-buffered media for drug-uptake studies (HEPES was not available then). In addition, possible variations in the size of the tissue mass/ volume of the incubation medium may also contribute to experimental differences. The major discrepancy appears in our finding an absence of saturability of the uptake between 10 s and 10-3 M. Because we observed this discrepancy early in these investigations, we went to extreme lengths to document this finding. Unless the criteria that apply to drugs are different from those used to assess the active transport of endogenous compounds, i.e., amino acids, morphine uptake by brain slices clearly does not occur by active transport. We do not extrapolate these findings to the possibility of active transport of dihydromorphine, for which many supporting, and contesting, reports have appearedlZ,'~, 24.
MORPHINE UPTAKE INTO BRAIN SLICES
135
ACKNOWLEDGEMENTS P o r t i o n s of this work were supported by U.S. Public Health Service G r a n t N S 03226. The a u t h o r s t h a n k Dr. A. Neidle for advice a n d suggestions regarding extract i o n a n d thin-layer c h r o m a t o g r a p h y of m o r p h i n e , a n d Dr. M. Banay-Schwartz for N a ÷, K +, a n d A T P determinations.
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l). N. TELLER Ct a[.
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