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Morphine tolerance and naloxone receptor binding
Life Sciences Vol. 16, pp . 1831-1836 Printed in the ü.S .A.
Pergamon Prese
I+lORPHINE TOLERANCE AND NALOXONE RECEPTOR BINDING Joseph Harris and Dolores T. Razmierowski Division of Neurobiology, Barrow Neurological Institute of St . Joseph's Hospital ~ Medical Center, Phoenia, Arizona 85013 . (Received in final form May 24, 1975)
3H-naloxone specific binding studies have confirmed the induction of receptor expansion after an acute injection of morphine, as reported bq Pert et al (3) as well as the lack of expansion in chronically morphinized rate show by Rlee and Streaty (4) using dihydramorphine. With a challenging test dose of morphine given to rats maintained drug free after acute and chronic regimens of morphine, the lack of ezpansion as measured by 3H-naloxone specific binding persisted up to at least 4 weeks . Between 4-8 weèks réceptor expansion can be re-induced `with a challenging teat dose . This "physical binding tolerance" ie dose related. That this persistant "tolerance" is not attributable to the presence of dissociable morphine remaining after the drug regimen or challenge dose caa be shown by detergent extraction and exhaustive dialysis of the standard buffer homogenate preparation as well as with fresh excised tissue . Many investigators of opiate receptors base their approach on the principle of stereospecificity of opiate-receptor binding, laid down by Goldstein et al (1), and the procedure of Pert and Snyder (2) which uses 3H-naloxone for studying receptor binding . Pert et al (3) reported that acute in vivo administration of opiates of their antagonists produced enhanced receptor binding, that is, the number of opiate binding sites appeared to have increased, an increase they consider not related to the phenomena of tolerance sad dependence . Rlee and Streaty (4) did not find a comparable enhancement of receptor binding to 3 H-dihydromorphine in morphine-dependent rats, an observation they believe, "provide (s) ev dance that morphine dependence (and therefore also toleraace) is not the result of an alteration in the number, or the nature of, the specific receptor sits ." In accordance with Rlee and Streaty we have observed, using H-naloaoae, a lack of expansion of receptor binding in brains of rate chronically treated with morphine over a period of seven days, but we have also confirmed the increase in naloxone receptor binding after as acute single opiate injection in the naive rat . In addition we have used cha11en~ing dose injections to teat for receptor eapansion under conditions of 1) a single acute injection previously administered 2) chronic morphinization and 3) poet-drug free state up to 8 weeks after the last dose of opiate . We now present data showin that : a) The lack of expaasion in a chronically morphinized anal when assayed by the Pert et
1832
Tolerance/Naloxone Receptor Binding
Vol . 16, No . 12
al (3~ technique reflects the presence of induced tolerance ; b) This 'physical binding tolerance" is induced after injection of morphine and persists for a period of time ; c) In situations showing a lack of receptor expansion tolerance may be demonstrated by the acute in~ection of a higher dose of the drug ; d) The physical binding to stance is dose related. Methods Preparation of Animals Groups of 10 adult Long-Evens male rata with an initial weight 150-200 gms were used . Acute experiments were performed employing a single intra-peritoneal injection of morphine sulfate 20 mg/Rg For chronic experiments two injections were given per day . The initial dose was 5 mg/Kg/day, doubling progressively to 20 mg/Kg/day and maintained at the 20 mg/Rg level for an additional 4 days . In experiments to teat for persistence of tolerance, groups of 10 rats were placed on the acute or chronic regimen respectively, then maintained for 8 weeks without further treatment . At weekly intervals after drug withdrawal, the rata were challenged with an acute intra-peritoneal injection of morphine sulfate, 20 mg/Rg, and decapitated 15 minutes after the acute drug test treatment . For experiments designed to show a dose relationship to physical binding tolerance, groups of naive and chronically morphinized rats were injected with increasing concentrations of morphine sulfate ; 15 minutes later the animals were sacrificed and binding assays performed . Preparation of Tissue Brains without the cerebellum, were homogenized with cold 10 volumes of 0 .05 M Tris-HC1 buffer, (pH 7 .4 at 35 °C) using a Teflon glass homogenizer (1800 rpm) . Before assaying for "naloxone receptor binding", the injected morphine was removed by the exhaustive washing procedure of Pert et al (3) . The homogenates were centrifuged at 180008 for 10 minutes ; the supernatants discarded and the pellets re-suspended in 14 ml of cold Tris-buffer . This washi rocedure was repeated three times and, as shown by Pert et a1~3~, removed virtually all of the opiate present . The washed homogenate was diluted to 120 volumes of original brain weight with cold Tris-buffer . A detergent extraction and exhaustive dialysis was used to assure removal of any dissociable morphine . Goldatein et al (1) reported that stereospecific-binding capacity is retained after extraction by 0 .5% Triton R-100 or O .1X sodium dodecyl sulfate, provided the detergent is removed by dialysis . A modified procedure of Swislocki and Tierneq (5) was used . Excised tissue (10 gm/100m1) was homogenized in 0 .2M Tris-Hcl (pH 7 .4) containing 0 .2M sucrose and Lubrol-PR (O .1M) . The homogenate was centrifuged at 27,000g for 10 min . The supernatant was recentrifuged at 165,0008 for 2 hr . and the non-sedimentable fraction was recentrifuged at 165,0008 for 2 hr . resulting in a soluble clear preparation . Tissue homogenates (1 :10) prepared by the standard Tria-HC1 (pH 7 .4) buffer method (3) were subjected to extraction with detergent (1 :10), centrifugation and dialysis (5) . Protein determinations were made by the method of Lowry et al (6) .
Vol . 16, No . 12
Tolerance/Naloxone Receptor Binding
183 3
3-Nalo$one Receptor Binding Specific binding of 3 H-naloxone (23 .6 Ci/mM, 98°L purity, New En land Nuclear Corp .) was assayed as described by Pert and Snyder (2~. Aliquots of homogenate were incubated in triplicate at 35°C for 10 minutes in the presence of dextrorphaa (0 .1 uM) or levorphanol (1 .0 uM), followed by the ad~ition of varying concentrations to give saturation levels of H-naloxone (16 nM/mg protein, 120,000 cpm) . The incubation was terminated by filtration of the chilled samples through Whatman GF/B paper pads and washed 3 times with 4 ml cold Tris buffer . The filter pads, transferred to count ing vials containing 1 ml of 10~ sodium dodecyl sulfate, were shaken for 30 minutes ; 10 ml of Packard "Insta-gel" was then added . After standing overnight, radioactivity was measured in a Tri-Carb Liquid Scintillation Spectrometer with counting efficiency at 30~ . Specific nalox~ne binding was taken to be the difference between the amount of H-naloxone bound in the absence and in the presence of excess levorphanol . Student's T-test (two-tailed) was used when applicable to analyze the data . All values shown represent the mean - S . E . M . Results and Discussion We confirm the expansion of receptor after an acute injection of morphine as reported by Pert et al (3) as well as the lack of expansion in the chronically morphinized animal as shown by Rlee and Streaty (4) . We also s ow e absence of any effect on specific binding in both the acute and chronic rats when challenged by a 20 mg/Rg dose of morphine . Enhanced receptor binding, seen by Pert et al (3) as early as 5 minutes after morphine administration, disappeared after 2 hours (Fig . 1) . FIG . 1
V~~iiiill~ni~
y r-~ w. w. T1~ AfTl11 L"~T o0~! IILAOT1011
Further, if acute or chronically morphinized rats are tested with the same challenging dose of morphine up to 4 weeks after their last drug administration, no racepfor expansion is obtained until 4-8 weeks . This condition, ae believe, reflects a physical binding tolerance related to pharmacological tolerance rather than to behavioral tolerance, since our animals have not been subjected to any test procedure . The question re®aina ~ghether a single dose of opiate can induce tolerance . While Cochin and Rornetaky (7) as well as Goldstein and Sheehen (8) have demonstrated pharmacological tolerance after a single injection of opiate, Rayan and
1834
Tolerance/Naloxone Receptor Binding
Vol . 16, No . 12
Mitchell (9) consider this to be behavioral tolerance, despite evidence of Migra et al (10) of a prolonged, firm and persistent association of morphine after a single injection . The physical binding assay of homygenatea previouely acutely or chronically morphinized, ceptor enhancement at a challenging acute test will do so at a higher acute challenging doses
from an animal, exhibiti no redose of ~ mg/Kg, (Fig . 2) .
FIG . 2
un N110111C S N
~4 DOR i .Wl . 1. .,wll4 ~ .-..,Imew! .y- Y.W .~..eKRW,Y.-IUll1l~ ! 1., r.- ü -./~1. ..
That our results are not attributable to the presence of dissociable morphine are supported by data obtained with detergent extraction and exhaustive dialysis of a standard buffer homogenate preparation or of freshly excised brain tissue (Fig . 3) . Rats tolerant to morphine gave specific binding measurements similar to their naive counter-parts . FIG . 3
r. . r sl..`~ .e m~ a. r.'r b er ~ rrt.-st, (~f j.~ r~,-e`~: M , .w .k. d~
We have observed that the injection of a single dose of morphine did not prevent receptor expansion induced by naloxone given one week later nor did an inj ection of naloxone prevent the induced receptor expansion when morphine was given a week later . Independent action thus appear to exist between naloxone and morphine . In this connection, we previously obtained differences between
Vol . 16, No . 12
Tolerancn/Nalozone Beceptor Binding
183 5
morphine and naloaone in their respective effects on calcium and catecholamine uptakes in rat brain striatal slices (11) differences suggesting that morphine and naloxone may react w~th a receptor at adjacent but overlapping sites . Such differences in binding sites on the opiate receptor for agonist and antagonist have been found recently by Wilson et al (12), This may contribute to the increased pharmacological sensitivity to naloxone that has been reported to occur with the development of dependence (13, 14) . That morphine has induced same type of structural change in the analgesic receptor or that tao types of analgesic receptors are present, has been discussed by Takemori and his group (15) . Further support for structural chaagea come from our detergentdialgsis treatment of brain preparations and in liver tissue used as a contr~l, The detergent treatment of brain tissue slightly increased H-naloxone specific binding over that of the standard brain preparation to a degree suggestive of induced receptor eapaasion, The detergent treatment of liver converted the standard liver hamogeaates from one lacking in binding into one possessing specific naloxone binding characteristics . Deter ents alter membrane structure by removal of phospholipids (16~, which could account for the transformation of liver having apecific receptor binding properties . A possible role of phoap lipid in the naloxone receptor binding is in keeping with the phospholipid changes produced by morphine (17) . The possibility of an artifact resulting from the detergent treatment cannot be discounted although retention of specific binding properties of brain preparation has been shown by Goldstein and Sheehan (1) also . Artifactual receptor-ligand binding does occur under certain circumstances as has been reported in experiments using the lipophilic Sephadex LH-20 in the isolation and purification of acetylcholine receptors (18) . Loh et al (19) found cerebroside sulfate devoid of protein to resemble the opiate recepfor with respect to its elution pattern on Sephadex LH-20 with chlorofo=mmethanol solvents . Our in vitro decreased responsiveness of the naloxone receptor system to morphine and the persistence of this does not fit current notions on tolerance and dependence . We, therefore, aug gent a modified view of these phenomena . If the pharmacological receptor is considered as a subuait of a membrane system of enzymes (20), then opiate interaction with the drug receptor site initiates allosteric conformation chasges si ificantly modulating the activity of the pharmacological aubunit receptor) which maq be reflected in a dose-response relationship . The initial drug effect on the subunit produces a change as well in adjacent components of the system, which then leads to a number of secondary changes in metabolic processes . Among these changes are alterations in level of neurotransmitter, ion transport, adenine nucleotide derivatives, energy transduction, oxidative metabolism, etc . Any or all o¬ these changes, some probably acting in concert, are capable of effecting a stable configurational change in the receptor . Our data on the binding of morphine antagonists are comp atible with Collier's hypothesis for the drug-induced changes in the number or kinds of receptore (21), The significance of our results is that we have shown tolerance by means of a physical binding procedure, which persists for longer periods of time than is
1836
Tolerance/Naloaone ßeceptor Binding
Vol . 16, No . 12
currently believed . We note that the disappearance of this physical binding tolerance between 4-8 weeks follows a time course similar to the disappearance of the morphine-induced repression of brain RNA transcriptase reported by Hodgson et al (22) . Aclmowle~ement This work was supported in part by a grant from the US Public Health Service . We thank Arthur Schwartz, Patricia Marchok and Jay Daviea for technical assistance . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 .
A. GOLDSTEIN L. L. LOWNEY, and B . R . PAL, Proc . U .S . Nat . Acad . Sci . 6~ 1742 (1971) . T and S . H . SNYDER, Proc . U .S . Nat . Acad . Sci . 70 2242 (1973j . C . B . PERT, G . PASTERNAR, and S . H. SNYDER, Science 182 1359 (1973) . W . A . RLEE, and R. H . STREATY, Nature 248 61 (1974) N. SWISIACRI and J . TIERNEY, BiocT 12 1862 (1973) 0. H. LOWRY, N. J . ROSEBROUGi~~ FARR, and R. J. RANDALL, J. Biol . Chem . 193 765 (1951j . an C . RORNE'PSRY, J . Pharmac . exp . Ther . 14 5 1 (1964) . A. GOLDSTEIN and P . SHEEHAN, J . Pharmac . exp . Ther . 16 9 175 (1969) . S . RAYAN and C . L . MITCHELL, Arch . int . Pharmacodvn. 199 407 (1972) . A. L . MISRA, C . L . MITCHELL, and L. A . WOODS, Nature 232 48 (1971) . S . L . MILLER and J . HARRIS, Traps . Soc . for Neuroeci . 4 340 (1974) . H. A, WILSON, G . W. PASTERNAR, and S . H. SNYDER, Nature 253 448 (1975) . E . L . WAY H . H. LOH, and R. H. SHEN, J . Pharmacol . exp . Ther . 16 7 1 (169) . R. J . HIT2MAIai, B . A . HITZMANN, and H . H. LOH, Life Sci . 14 2392 (1974) . A, E . TAREMORI, G . HAYASRI, and S . E . SMITS, Eur . J . Pharmacol. 20 85 (1972) . R . TANAHA and R . P . STRICRIAND, Arch . Biochem . Biovhvs . 111 583 (1965) . S . J . MULE, Biochem. Pharmacol . 19 581 (1970) . S . R . LEVINS an S, Biochem, Biophva . Acta . 288 241 (1972) . H . H . LOH, T . M. CHO, Y . C . WU, and E . L . WAY, Life Sciences 14 2231 (1974) . B . LIBET and T . TOSARA, Proc . U . S . Nat . Acad . Sci. 6 7 667 (1970) . H. 0 . J . COLLIER, Nature 205 181 (1965) . J . R . HODGSON, R. L-81fISTOW, and T . R. CASTLES, Nature 248 671 (1974) .
Pergamon Prese
I+lORPHINE TOLERANCE AND NALOXONE RECEPTOR BINDING Joseph Harris and Dolores T. Razmierowski Division of Neurobiology, Barrow Neurological Institute of St . Joseph's Hospital ~ Medical Center, Phoenia, Arizona 85013 . (Received in final form May 24, 1975)
3H-naloxone specific binding studies have confirmed the induction of receptor expansion after an acute injection of morphine, as reported bq Pert et al (3) as well as the lack of expansion in chronically morphinized rate show by Rlee and Streaty (4) using dihydramorphine. With a challenging test dose of morphine given to rats maintained drug free after acute and chronic regimens of morphine, the lack of ezpansion as measured by 3H-naloxone specific binding persisted up to at least 4 weeks . Between 4-8 weèks réceptor expansion can be re-induced `with a challenging teat dose . This "physical binding tolerance" ie dose related. That this persistant "tolerance" is not attributable to the presence of dissociable morphine remaining after the drug regimen or challenge dose caa be shown by detergent extraction and exhaustive dialysis of the standard buffer homogenate preparation as well as with fresh excised tissue . Many investigators of opiate receptors base their approach on the principle of stereospecificity of opiate-receptor binding, laid down by Goldstein et al (1), and the procedure of Pert and Snyder (2) which uses 3H-naloxone for studying receptor binding . Pert et al (3) reported that acute in vivo administration of opiates of their antagonists produced enhanced receptor binding, that is, the number of opiate binding sites appeared to have increased, an increase they consider not related to the phenomena of tolerance sad dependence . Rlee and Streaty (4) did not find a comparable enhancement of receptor binding to 3 H-dihydromorphine in morphine-dependent rats, an observation they believe, "provide (s) ev dance that morphine dependence (and therefore also toleraace) is not the result of an alteration in the number, or the nature of, the specific receptor sits ." In accordance with Rlee and Streaty we have observed, using H-naloaoae, a lack of expansion of receptor binding in brains of rate chronically treated with morphine over a period of seven days, but we have also confirmed the increase in naloxone receptor binding after as acute single opiate injection in the naive rat . In addition we have used cha11en~ing dose injections to teat for receptor eapansion under conditions of 1) a single acute injection previously administered 2) chronic morphinization and 3) poet-drug free state up to 8 weeks after the last dose of opiate . We now present data showin that : a) The lack of expaasion in a chronically morphinized anal when assayed by the Pert et
1832
Tolerance/Naloxone Receptor Binding
Vol . 16, No . 12
al (3~ technique reflects the presence of induced tolerance ; b) This 'physical binding tolerance" is induced after injection of morphine and persists for a period of time ; c) In situations showing a lack of receptor expansion tolerance may be demonstrated by the acute in~ection of a higher dose of the drug ; d) The physical binding to stance is dose related. Methods Preparation of Animals Groups of 10 adult Long-Evens male rata with an initial weight 150-200 gms were used . Acute experiments were performed employing a single intra-peritoneal injection of morphine sulfate 20 mg/Rg For chronic experiments two injections were given per day . The initial dose was 5 mg/Kg/day, doubling progressively to 20 mg/Kg/day and maintained at the 20 mg/Rg level for an additional 4 days . In experiments to teat for persistence of tolerance, groups of 10 rats were placed on the acute or chronic regimen respectively, then maintained for 8 weeks without further treatment . At weekly intervals after drug withdrawal, the rata were challenged with an acute intra-peritoneal injection of morphine sulfate, 20 mg/Rg, and decapitated 15 minutes after the acute drug test treatment . For experiments designed to show a dose relationship to physical binding tolerance, groups of naive and chronically morphinized rats were injected with increasing concentrations of morphine sulfate ; 15 minutes later the animals were sacrificed and binding assays performed . Preparation of Tissue Brains without the cerebellum, were homogenized with cold 10 volumes of 0 .05 M Tris-HC1 buffer, (pH 7 .4 at 35 °C) using a Teflon glass homogenizer (1800 rpm) . Before assaying for "naloxone receptor binding", the injected morphine was removed by the exhaustive washing procedure of Pert et al (3) . The homogenates were centrifuged at 180008 for 10 minutes ; the supernatants discarded and the pellets re-suspended in 14 ml of cold Tris-buffer . This washi rocedure was repeated three times and, as shown by Pert et a1~3~, removed virtually all of the opiate present . The washed homogenate was diluted to 120 volumes of original brain weight with cold Tris-buffer . A detergent extraction and exhaustive dialysis was used to assure removal of any dissociable morphine . Goldatein et al (1) reported that stereospecific-binding capacity is retained after extraction by 0 .5% Triton R-100 or O .1X sodium dodecyl sulfate, provided the detergent is removed by dialysis . A modified procedure of Swislocki and Tierneq (5) was used . Excised tissue (10 gm/100m1) was homogenized in 0 .2M Tris-Hcl (pH 7 .4) containing 0 .2M sucrose and Lubrol-PR (O .1M) . The homogenate was centrifuged at 27,000g for 10 min . The supernatant was recentrifuged at 165,0008 for 2 hr . and the non-sedimentable fraction was recentrifuged at 165,0008 for 2 hr . resulting in a soluble clear preparation . Tissue homogenates (1 :10) prepared by the standard Tria-HC1 (pH 7 .4) buffer method (3) were subjected to extraction with detergent (1 :10), centrifugation and dialysis (5) . Protein determinations were made by the method of Lowry et al (6) .
Vol . 16, No . 12
Tolerance/Naloxone Receptor Binding
183 3
3-Nalo$one Receptor Binding Specific binding of 3 H-naloxone (23 .6 Ci/mM, 98°L purity, New En land Nuclear Corp .) was assayed as described by Pert and Snyder (2~. Aliquots of homogenate were incubated in triplicate at 35°C for 10 minutes in the presence of dextrorphaa (0 .1 uM) or levorphanol (1 .0 uM), followed by the ad~ition of varying concentrations to give saturation levels of H-naloxone (16 nM/mg protein, 120,000 cpm) . The incubation was terminated by filtration of the chilled samples through Whatman GF/B paper pads and washed 3 times with 4 ml cold Tris buffer . The filter pads, transferred to count ing vials containing 1 ml of 10~ sodium dodecyl sulfate, were shaken for 30 minutes ; 10 ml of Packard "Insta-gel" was then added . After standing overnight, radioactivity was measured in a Tri-Carb Liquid Scintillation Spectrometer with counting efficiency at 30~ . Specific nalox~ne binding was taken to be the difference between the amount of H-naloxone bound in the absence and in the presence of excess levorphanol . Student's T-test (two-tailed) was used when applicable to analyze the data . All values shown represent the mean - S . E . M . Results and Discussion We confirm the expansion of receptor after an acute injection of morphine as reported by Pert et al (3) as well as the lack of expansion in the chronically morphinized animal as shown by Rlee and Streaty (4) . We also s ow e absence of any effect on specific binding in both the acute and chronic rats when challenged by a 20 mg/Rg dose of morphine . Enhanced receptor binding, seen by Pert et al (3) as early as 5 minutes after morphine administration, disappeared after 2 hours (Fig . 1) . FIG . 1
V~~iiiill~ni~
y r-~ w. w. T1~ AfTl11 L"~T o0~! IILAOT1011
Further, if acute or chronically morphinized rats are tested with the same challenging dose of morphine up to 4 weeks after their last drug administration, no racepfor expansion is obtained until 4-8 weeks . This condition, ae believe, reflects a physical binding tolerance related to pharmacological tolerance rather than to behavioral tolerance, since our animals have not been subjected to any test procedure . The question re®aina ~ghether a single dose of opiate can induce tolerance . While Cochin and Rornetaky (7) as well as Goldstein and Sheehen (8) have demonstrated pharmacological tolerance after a single injection of opiate, Rayan and
1834
Tolerance/Naloxone Receptor Binding
Vol . 16, No . 12
Mitchell (9) consider this to be behavioral tolerance, despite evidence of Migra et al (10) of a prolonged, firm and persistent association of morphine after a single injection . The physical binding assay of homygenatea previouely acutely or chronically morphinized, ceptor enhancement at a challenging acute test will do so at a higher acute challenging doses
from an animal, exhibiti no redose of ~ mg/Kg, (Fig . 2) .
FIG . 2
un N110111C S N
~4 DOR i .Wl . 1. .,wll4 ~ .-..,Imew! .y- Y.W .~..eKRW,Y.-IUll1l~ ! 1., r.- ü -./~1. ..
That our results are not attributable to the presence of dissociable morphine are supported by data obtained with detergent extraction and exhaustive dialysis of a standard buffer homogenate preparation or of freshly excised brain tissue (Fig . 3) . Rats tolerant to morphine gave specific binding measurements similar to their naive counter-parts . FIG . 3
r. . r sl..`~ .e m~ a. r.'r b er ~ rrt.-st, (~f j.~ r~,-e`~: M , .w .k. d~
We have observed that the injection of a single dose of morphine did not prevent receptor expansion induced by naloxone given one week later nor did an inj ection of naloxone prevent the induced receptor expansion when morphine was given a week later . Independent action thus appear to exist between naloxone and morphine . In this connection, we previously obtained differences between
Vol . 16, No . 12
Tolerancn/Nalozone Beceptor Binding
183 5
morphine and naloaone in their respective effects on calcium and catecholamine uptakes in rat brain striatal slices (11) differences suggesting that morphine and naloxone may react w~th a receptor at adjacent but overlapping sites . Such differences in binding sites on the opiate receptor for agonist and antagonist have been found recently by Wilson et al (12), This may contribute to the increased pharmacological sensitivity to naloxone that has been reported to occur with the development of dependence (13, 14) . That morphine has induced same type of structural change in the analgesic receptor or that tao types of analgesic receptors are present, has been discussed by Takemori and his group (15) . Further support for structural chaagea come from our detergentdialgsis treatment of brain preparations and in liver tissue used as a contr~l, The detergent treatment of brain tissue slightly increased H-naloxone specific binding over that of the standard brain preparation to a degree suggestive of induced receptor eapaasion, The detergent treatment of liver converted the standard liver hamogeaates from one lacking in binding into one possessing specific naloxone binding characteristics . Deter ents alter membrane structure by removal of phospholipids (16~, which could account for the transformation of liver having apecific receptor binding properties . A possible role of phoap lipid in the naloxone receptor binding is in keeping with the phospholipid changes produced by morphine (17) . The possibility of an artifact resulting from the detergent treatment cannot be discounted although retention of specific binding properties of brain preparation has been shown by Goldstein and Sheehan (1) also . Artifactual receptor-ligand binding does occur under certain circumstances as has been reported in experiments using the lipophilic Sephadex LH-20 in the isolation and purification of acetylcholine receptors (18) . Loh et al (19) found cerebroside sulfate devoid of protein to resemble the opiate recepfor with respect to its elution pattern on Sephadex LH-20 with chlorofo=mmethanol solvents . Our in vitro decreased responsiveness of the naloxone receptor system to morphine and the persistence of this does not fit current notions on tolerance and dependence . We, therefore, aug gent a modified view of these phenomena . If the pharmacological receptor is considered as a subuait of a membrane system of enzymes (20), then opiate interaction with the drug receptor site initiates allosteric conformation chasges si ificantly modulating the activity of the pharmacological aubunit receptor) which maq be reflected in a dose-response relationship . The initial drug effect on the subunit produces a change as well in adjacent components of the system, which then leads to a number of secondary changes in metabolic processes . Among these changes are alterations in level of neurotransmitter, ion transport, adenine nucleotide derivatives, energy transduction, oxidative metabolism, etc . Any or all o¬ these changes, some probably acting in concert, are capable of effecting a stable configurational change in the receptor . Our data on the binding of morphine antagonists are comp atible with Collier's hypothesis for the drug-induced changes in the number or kinds of receptore (21), The significance of our results is that we have shown tolerance by means of a physical binding procedure, which persists for longer periods of time than is
1836
Tolerance/Naloaone ßeceptor Binding
Vol . 16, No . 12
currently believed . We note that the disappearance of this physical binding tolerance between 4-8 weeks follows a time course similar to the disappearance of the morphine-induced repression of brain RNA transcriptase reported by Hodgson et al (22) . Aclmowle~ement This work was supported in part by a grant from the US Public Health Service . We thank Arthur Schwartz, Patricia Marchok and Jay Daviea for technical assistance . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 .
A. GOLDSTEIN L. L. LOWNEY, and B . R . PAL, Proc . U .S . Nat . Acad . Sci . 6~ 1742 (1971) . T and S . H . SNYDER, Proc . U .S . Nat . Acad . Sci . 70 2242 (1973j . C . B . PERT, G . PASTERNAR, and S . H. SNYDER, Science 182 1359 (1973) . W . A . RLEE, and R. H . STREATY, Nature 248 61 (1974) N. SWISIACRI and J . TIERNEY, BiocT 12 1862 (1973) 0. H. LOWRY, N. J . ROSEBROUGi~~ FARR, and R. J. RANDALL, J. Biol . Chem . 193 765 (1951j . an C . RORNE'PSRY, J . Pharmac . exp . Ther . 14 5 1 (1964) . A. GOLDSTEIN and P . SHEEHAN, J . Pharmac . exp . Ther . 16 9 175 (1969) . S . RAYAN and C . L . MITCHELL, Arch . int . Pharmacodvn. 199 407 (1972) . A. L . MISRA, C . L . MITCHELL, and L. A . WOODS, Nature 232 48 (1971) . S . L . MILLER and J . HARRIS, Traps . Soc . for Neuroeci . 4 340 (1974) . H. A, WILSON, G . W. PASTERNAR, and S . H. SNYDER, Nature 253 448 (1975) . E . L . WAY H . H. LOH, and R. H. SHEN, J . Pharmacol . exp . Ther . 16 7 1 (169) . R. J . HIT2MAIai, B . A . HITZMANN, and H . H. LOH, Life Sci . 14 2392 (1974) . A, E . TAREMORI, G . HAYASRI, and S . E . SMITS, Eur . J . Pharmacol. 20 85 (1972) . R . TANAHA and R . P . STRICRIAND, Arch . Biochem . Biovhvs . 111 583 (1965) . S . J . MULE, Biochem. Pharmacol . 19 581 (1970) . S . R . LEVINS an S, Biochem, Biophva . Acta . 288 241 (1972) . H . H . LOH, T . M. CHO, Y . C . WU, and E . L . WAY, Life Sciences 14 2231 (1974) . B . LIBET and T . TOSARA, Proc . U . S . Nat . Acad . Sci. 6 7 667 (1970) . H. 0 . J . COLLIER, Nature 205 181 (1965) . J . R . HODGSON, R. L-81fISTOW, and T . R. CASTLES, Nature 248 671 (1974) .