Life Sciences Vol . 16, pp . 1895-1900 Printed in the II .S .l1.
Perganon Press
EFFECT OF CYCLIC NUCLEOTIDES AND PHOSPHODIESTERASE INHIBITION ON MORPHIIdE TOLERANCE AND PHYSICAL DEPENDENCE I. K. Ho, H. H. Loh, H. N. Bhargava and E. Leong Wsy Departmart of Pharmacology and Langley Porter Neuropsychiatrie Institute, University of California, San Francisco, California 94143. (Received
in final forn May 24, 1975)
Summary The effects of cyclic nucleotides and theophylline were assessed in mice rardered tolerant to and physically depardart on morphine by the pellet implantation procedure. Tolerance was quantified by the increase in amount of morphine to produce analgesia and depardarce by the decrease in amount of naloxone to precipitate withdrawal jumping. By these criteria, pretreatment with a single intravarous injection of cyclic 3', 5'-adenosine monophosphate (CAMP) was found to enhance markedly tolerance and dependence developmart. Repeated injections of theophylline were also effective. Cycloheximide and beta-adrarergic Mockers prevented the accelerating effect of cAMP and with more frequent administration also decreased the developmart of tolerance and dependence . It is concluded thatcAMPmayhave a role in morphine tolerance and depardarce developmart. Ia recent years, considerable evidarce has been obtained on the biochemicalprocessesthatmaybe cancernedinthedevelopment of tolerance to and physical dependence on morphine . Ia our laboratory, we have found that the development of morphine tolerance and physical depardarce can Either syndrome may be be altered by pharmacologic manipulations. inhibited or accelerated by selection of compounds which act by differart mechanisms . These changes can be achieved with a dose of an agart that may or may not change morphine responses acutely. These studies have been summarized in a review (1). Ia brief, evidarce has been obtained suggesting that serotonin gamma-amino-butyric acid, and adenosine 3', 5'-cyclic monophosphate (CAMP) may play some associated role in morphine tolerance and dependence development. The possibility that cAMP might interact with morphine is suggested bY numerous reports indicating that morphine can alter the functional state of the biogariç amines which have bear repeatedly demonstrated to have the ability to modify morphine action (2) . Moreover, morphine was found to activate cerebral adenyl cyclase (3~ tin should like to summarize our previous findings (4, 5, 6) on the effects of cAMP on morphine tolerance and dependence and also presart some more recent data . Supported by grants DA-0037 and DA-00563 from the National Institute of I. K. Ho is the recipfart of a Faculty Development Award Drug Abuse. from the Pharmaceutical Manufacturer Association Foundation in Basic Pharmacology . Contribution number 75-4 .
18%
Cyclic Nucleotides and 1~lorphine Tolerance
Vol . 16, No . 12
Experimental Procedures The methods for assessment of tolerance and physical dependence were similar to those previously reported (7). Mice were rendered tolerant to and physically dependent on morphine by the s . c. implantation of a specially formulated morphine pellet for 3 days . On the 4th day, the pellet was removed and 6 hours Later the degree of tolerance and physical dependence development was assessed . Tolerance was quantified by determining the increase in the median analgetic dose of morphine (AD50) using the tail flick procedure. Physical dependence was measured by determining the amount of aaloxone (ED50) to precipitate withdrawal jumping. An inverse relationship exists between the two parameters ; a decrease in the naloxoae ED50 is indicative of increased physical dependence . Various cAMP agoniste and antagonists were tested for their effects on tolerance and physical dependence development. Usually, a single injec tion of a drug was made rp for to implantation of a morphine or a placebo pellet and the effect of this maneuver on the morphine AD50 and thenaloxone ED50 was made on the 4th day 6 hours after pellet removal. Thus, the assessment of the effect of the agent was made 80 hours after its administration. With shorter acting compounds, it was sometimes necessary to repeat with an additional dose at daily intervals for two additional days . Under these conditions, the assessment for the drug effect was made 30 hours after its last administration . The specific conditions for each chemical agent will be described in Results . Results Effect of cAMP on the development of tolerance. Under conditions where pretreatment with cAMP did not alter the acute response to morphine, cAMP markedly accelerated tolerance development (6 ) . In mice receiving one single injection of cAMP LO mg/kg i, v. , two hours prior to morphine pellet implantation, the AD50 of morphine was found to be considerably higher than that of controls . Ia saline-treated animals, morphine pellet implantation effected a 10-fold increase in the AD50 (from 7 . 5 to 76 mg/kg) , In the CAMP group, the AD50 was increased more than 3-fold (from 8 to 270 mg/kg) , Based on the relative increase in their respective AD50 values, the cAMP group was more than 3 times as tolerant as the control group. Similar experiments with a 2. 5 mg/kg dose of cAMP resulted in a doubling of the morphine AD50 over that of saline-treated mice . Cycloheximide prevented the accelerating effect of cAMP on morphine tolerance development. As shown on the right in Figure 1, in mice rendered tolerant to morphine by pellet implantation, a single injection of cAMP two hours before implanting a morphine pellet resulted in a morphine AD50 3 times that of a similarly implanted group receiving saline instead of cAMP . In a third group of mice receiving cycloheximide daily in addition to cAMP, the CAMP effect was prevented; the increase in the morphine AD50 noted after pellet implantation in the cAMP group was no greater than that in the animals given the vehicle. Likewise, the group receiving cycloheximide alone exhibited an AD50 nearly identical with the group given the vehicle . Thus, cycloheximide, under conditions which did not affect the morphine AD50 or mortality, blocked the accelerating effect of cAMP on tolerance development. With high doses and more frequent administrations, cycloheximide will also block the development of tolerance to morphine (8) but it is difficult to prevent deaths under such conditions .
Vol. 16, No. 12
grclic Nucleotides and Norpbine Tolerance
1897
Figure 1 aoo
seo
wiw
Effect of cycloheximide on cAMP enhancement of morphine dependence and tolerance development. The naloxone ED50 and morphine AD50 plus 95°}6 confidence limits were determined 6 hours after removal of a morphine pellet implanted for 72 hours . S denotes saline ; CY, cycloheximide. The doses were CY 20 mg/kg i. p. ; cAMP LO mg/kg i. v. From Ho et al . , (6 ) Effe ct of cAMP on the development of morphine physical dependence The injection of cAMP prior to morphine pellet implantation also accelerated the development of physical dependence (6). Enhancement of dependence development by cAMP was indicated by adecrease in the amount of naloxoae needed to induce precipitated withdrawal jumping . Is mice previously rendered dependent by pellet implantation, the administration of cAMP immediately after pellet removal dfd not alter the naloxose ED50 significantly . Ia contrast, in implanted mice pretreated with cAMP, a higher degree of physical dependence was evidenced by a decrease in the naloaone ED50 to one-fourth that of the. group receiving the vehicle . Enhancementof morphine physical dependence development by cAMP also was shown after abrupt withdrawal ; the body weight loss, which occurred after removal of the morphine pellet, was greater with cAMP pretreatment . Cycloheaimide prevented the accelerating effect of cAMP on the development of physical dependent on morphine . As shown oa the left is Fig. 1, in mice pretreated with a single injection of cAMP two hours before morphine pellet implantation, the naloxone ED50 was one-third that of morphine pellet-implanted animals treated with saline . The administration of cycloheximide once daily at a dose which did not significantly alter the naloxone precipitated withdrawal response reduced the enhancing effect of cAMP . The nalozose ED50 of the cAMP-plus-cycloheaimide-treated group was about twice that of the cAMP-treated group and not significantly different from morphine implanted animal treated with Balise .
1898
Cyclic
Nucleotides and Morphine Tolerance
Vol . 16, No . 12
More frequent administration of cycloheximide not only blocked the accelerating effect of cAMP on morphine dependence development but also reduced the development of physical dependence in morphine pellet implanted mice . The latter observation is consistent with an earlier study that dependence development, resulting from repeated injections of morphine for three weeks, is blocked by the daily administration of cycloheximide (8), Two other nucleotides, adenosine-2', 3'-cyclic monophosphate (7.', 3' CAMP) and guanosine-3', 5'-cyclic monophosphate (3', 5'-cGMP) were also assessed for their effect on morphine tolerance and physical dependence . As shown in Table 1, following pretreatment with a dose similar to that for cAMP (10 mg/kg i, v, ) neither compound altered the morphine AD50 or naloxone ED50 of animals of implanted with a morphine pellet, Table 1 EFFECT OF 2', 3'-CAMP AND 3', 5'-cGMP ON THE DEVELOPMENT OF MORPHINE TOLERANCE AND DEPENDENCE . Compound
Niôrphine Naloxoae AD50* ED50~ Vehicle 0 (4 - 78) 0, 53 (0, 4 -0 . 2) 2',3'-CAMP 90 (63 -128) 0.48 (0 .28-0 .83) 3', 5' -cGMP 84 (14 -171) 0, 45 (0, 32-0 . 63) 3' 5'-CAMP+ 270 200-360 0, 08 0. 05-0 .12 #determine after subcutaneous administration (mg kg) hours after removal of a morphine pellet implanted for 3 days ; parentheses include the 95% confidence limits, +From Ho et al, , (6)
Effect of theophylline on tolerance and dependence The phosphodiesterase inhibitor, theophylline, enhanced tolerance and physical dependence development, The compound was administered at dose of 100 mg/kg intraperitoneally commencing 2 hours before mor phine pellet implantation and repeated every 24 hours for 2 additional days . A control group received the vehicle instead of theophylline, The morphine AD50 and naloxone ED50 were determined in the usual manner 6 hours after pellet removal . As shown on the left of Figure 2, theophylliae treated animals were about 3 times more tolerant than the control group. The morphine AD50 of the theophylline pretreated group was 235 mg/kg whereas it was 78 mg/kg for the control group receiving the vehicle. The development of a higher degree of physical dependence after theophylline pretreatment ie shown by the data s++"+~+T+~+ arized on the right of Figure 2, The naloxone ED50 of the theophylline group (0, 29 mg/kg) was one-third that of the control group (0, 76 mg/kg) . Effect of beta ad renergic blockers The enhancing effect of cAMP on tolerance and physical dependence development was blocked by pretreatment with the beta-adrenergic blockers . A single injection of dichloroisoprotereaol (DCI, 40Wg) pronethalol(70 Wg), and propanolol (70 Wg) intracerebrally 20 minutes prior to cAMP, prevented the accelerating effect of cAMP on tolerance and physical dependence development, Under these conditions and physical dependence development
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was not significantly altered by the individual betablockere . In time course studies conducted with propanolol, the compound was found to be largely When given 20 ineffective when given 24 and 48 hours after cAMP . minutes or 4 hours after cAMP its action was greatly reduced (Loh _et _al. , unpublished) . ro
oe
Mgure 2
Q2
V 3a
T 3a
V &
T 5t
V 3a
T 3a
Effect of theophylline on the development of tolerance and physical dependence . V denotes the vehicle and T, theophyüine 100 mg/kg i. p, , administered at daily intervals for 3 days . Administration of DCI for three days intracerebrally, resulted in a decrease in tolerance development . As shown in Table 2, an ü-fold tolerance developed in morphine implanted animals treated with saline whereas with DCI treatment, only a 4-fold tolerance developed, The three daily injections of DCI in the placebo pellet implanted animals did not alter the response to morphine (9) . Similar results were obtained with experiments oa propanolol . Repeated administration of DCI also inhibited the development of physical dependence . When DCI was given prior to and during the development of dependence, the naloxone ED50 increased 8-fold as shown in Table 2: The acute administration of DCI to the dependent animals did not alter withdrawal response as evidenced by the fact that the naloxone ED50 of saline and DCI treated were nearly identical. Also, acute or chronic treatment with saline in morphine implanted animals did not alter the naloxone ED50 . Table 2 EFFECT OF SINGLE AND REPEATED INJECTIONS OF DCI ON MORPHINE AD 50 AND NALOXONE ED50 ins DCI 3X
Morphine AD50 # M P 4.5+0.9 50 .0+9 .0 22 . 0 + 4. 5 5. 0 + 1 . 0
Naloxone ED50* M 0.15+0.03 1. 20 + 0. 20
~°mg kg + S. E,M.P~eâotes a placebo pellet implant; M a morphine pellet. From Bhargsva et al ., (9).
1900
Gyc11c Nucleotides and Morphine Tolerance
Vol. 16, No . 12
Die cueeion The results confirm our previous findings that cAMP may be involved with processes concerned with morphine tolerance and dependence development (6), Although cAMP was administered intravenously, it ie also effective at much lower doses after intracerebral administration . The striking finding ie that a single injection of the cAMP produces a marked effect that is measurable 80 hours after its administration . Some degree of selectivity in action must exist, since two other nucleotides tested were inactive, The inhibition of tolerance and dependence by the beta adrenergic blockere, DCI and propaaolol are also compatible with an action that may be mediated by cAMP, The effects noted with theophylline were lees dramatic but consistent with the above findings . Although enhancement of tolerance and dependence developed was observed, it was necessary to repeat the theophylline administration, We can only presume that the degree of phosphodiesterase inhibition was insufficient or that theophylline possesses other pharmacologic effects that may be opposite to those elicited by cAMP, Since the accelerating effect of cAMP on morphine tolerance and dependence development is blocked by cycloheximide, it appears that cAMP ie participating in processes that may be initiating or accelerating selective synthesis of a protein or other macromolecule, Furthermore, the mechanism can be turned off as evidenced by the fact that the beta blockers completely prevented the accelerating effect of cAMP when they were administered before cAMP, This effecwae obtained under conditions where the beta blocker ear se did not affect tolerance and physical dependence . The mechanism by which blockade of cAMP action is elicited ie not clear since the site of action of cAMP should be distal to the site of blockade, if one considers the beta-receptor to be a eubunit of adenyl cyclase in the cellular membrane (10) . However, since the enhancing effect of cAMP on tolerance and dependence was obtained with cAMP administered exogenously, an action at other sites can not be excluded and only further studies can provide an answer . References 1. 2. 3. 4. 5. 6. 7. 8, 9. 10 .
WAY, E. L. , Proc, 5th Int, Congr . Pharmacology, San Francisco 1972, vol, 1, p. 77-94 Karger, Basel (1973) . WAY, E, L. and F. H, SHEN, in Narcotic Drugs, Biochemical Pharmacology (D, H, Clouet, ed), p. 229-253, Plenum Press, New York, (1971) . CHOU, W. S. , A, K. S. HO, and H, H. LOH, Proc . West, Pharmacol. Soc, 14, 42-45 (1971) . HO, I, K, , S. E. LU, H. H. LOH, and E, L. WAY. Nature (London) 328, 397-8 (1972), HO, I. K, , H. H. LOH, and E. L. WAY. J. Pharmacol . exp, Ther, 185, 336-346 (1973) . HO, I, K. , H. H. LOH, and E, L. WAY . J. Pharmacol, exp . Ther . 185, 347-357 (1973) . WAY, E. L, , H, H. LOH and F, H, 5HEN . J. Pharmacol, exp, Ther, 167, 1-8 (1968) . LOH, H. H. , F, SHEN and E, L. WAY. Biochem. Pharmacol. 18, 2711-2721 (1969) . BHARGAVA, H, N. , S. L, CHAN and E, L, WAY. Proc . West . Pharmacol. Soc, 15, 4-7 (1972), ROBISON, G. A. , R. W, BUTCHFR and E. W, SUTHERLAND . Ann N, Y. Acad . Sci, 139, 703-723 (1967) .
Perganon Press
EFFECT OF CYCLIC NUCLEOTIDES AND PHOSPHODIESTERASE INHIBITION ON MORPHIIdE TOLERANCE AND PHYSICAL DEPENDENCE I. K. Ho, H. H. Loh, H. N. Bhargava and E. Leong Wsy Departmart of Pharmacology and Langley Porter Neuropsychiatrie Institute, University of California, San Francisco, California 94143. (Received
in final forn May 24, 1975)
Summary The effects of cyclic nucleotides and theophylline were assessed in mice rardered tolerant to and physically depardart on morphine by the pellet implantation procedure. Tolerance was quantified by the increase in amount of morphine to produce analgesia and depardarce by the decrease in amount of naloxone to precipitate withdrawal jumping. By these criteria, pretreatment with a single intravarous injection of cyclic 3', 5'-adenosine monophosphate (CAMP) was found to enhance markedly tolerance and dependence developmart. Repeated injections of theophylline were also effective. Cycloheximide and beta-adrarergic Mockers prevented the accelerating effect of cAMP and with more frequent administration also decreased the developmart of tolerance and dependence . It is concluded thatcAMPmayhave a role in morphine tolerance and depardarce developmart. Ia recent years, considerable evidarce has been obtained on the biochemicalprocessesthatmaybe cancernedinthedevelopment of tolerance to and physical dependence on morphine . Ia our laboratory, we have found that the development of morphine tolerance and physical depardarce can Either syndrome may be be altered by pharmacologic manipulations. inhibited or accelerated by selection of compounds which act by differart mechanisms . These changes can be achieved with a dose of an agart that may or may not change morphine responses acutely. These studies have been summarized in a review (1). Ia brief, evidarce has been obtained suggesting that serotonin gamma-amino-butyric acid, and adenosine 3', 5'-cyclic monophosphate (CAMP) may play some associated role in morphine tolerance and dependence development. The possibility that cAMP might interact with morphine is suggested bY numerous reports indicating that morphine can alter the functional state of the biogariç amines which have bear repeatedly demonstrated to have the ability to modify morphine action (2) . Moreover, morphine was found to activate cerebral adenyl cyclase (3~ tin should like to summarize our previous findings (4, 5, 6) on the effects of cAMP on morphine tolerance and dependence and also presart some more recent data . Supported by grants DA-0037 and DA-00563 from the National Institute of I. K. Ho is the recipfart of a Faculty Development Award Drug Abuse. from the Pharmaceutical Manufacturer Association Foundation in Basic Pharmacology . Contribution number 75-4 .
18%
Cyclic Nucleotides and 1~lorphine Tolerance
Vol . 16, No . 12
Experimental Procedures The methods for assessment of tolerance and physical dependence were similar to those previously reported (7). Mice were rendered tolerant to and physically dependent on morphine by the s . c. implantation of a specially formulated morphine pellet for 3 days . On the 4th day, the pellet was removed and 6 hours Later the degree of tolerance and physical dependence development was assessed . Tolerance was quantified by determining the increase in the median analgetic dose of morphine (AD50) using the tail flick procedure. Physical dependence was measured by determining the amount of aaloxone (ED50) to precipitate withdrawal jumping. An inverse relationship exists between the two parameters ; a decrease in the naloxoae ED50 is indicative of increased physical dependence . Various cAMP agoniste and antagonists were tested for their effects on tolerance and physical dependence development. Usually, a single injec tion of a drug was made rp for to implantation of a morphine or a placebo pellet and the effect of this maneuver on the morphine AD50 and thenaloxone ED50 was made on the 4th day 6 hours after pellet removal. Thus, the assessment of the effect of the agent was made 80 hours after its administration. With shorter acting compounds, it was sometimes necessary to repeat with an additional dose at daily intervals for two additional days . Under these conditions, the assessment for the drug effect was made 30 hours after its last administration . The specific conditions for each chemical agent will be described in Results . Results Effect of cAMP on the development of tolerance. Under conditions where pretreatment with cAMP did not alter the acute response to morphine, cAMP markedly accelerated tolerance development (6 ) . In mice receiving one single injection of cAMP LO mg/kg i, v. , two hours prior to morphine pellet implantation, the AD50 of morphine was found to be considerably higher than that of controls . Ia saline-treated animals, morphine pellet implantation effected a 10-fold increase in the AD50 (from 7 . 5 to 76 mg/kg) , In the CAMP group, the AD50 was increased more than 3-fold (from 8 to 270 mg/kg) , Based on the relative increase in their respective AD50 values, the cAMP group was more than 3 times as tolerant as the control group. Similar experiments with a 2. 5 mg/kg dose of cAMP resulted in a doubling of the morphine AD50 over that of saline-treated mice . Cycloheximide prevented the accelerating effect of cAMP on morphine tolerance development. As shown on the right in Figure 1, in mice rendered tolerant to morphine by pellet implantation, a single injection of cAMP two hours before implanting a morphine pellet resulted in a morphine AD50 3 times that of a similarly implanted group receiving saline instead of cAMP . In a third group of mice receiving cycloheximide daily in addition to cAMP, the CAMP effect was prevented; the increase in the morphine AD50 noted after pellet implantation in the cAMP group was no greater than that in the animals given the vehicle. Likewise, the group receiving cycloheximide alone exhibited an AD50 nearly identical with the group given the vehicle . Thus, cycloheximide, under conditions which did not affect the morphine AD50 or mortality, blocked the accelerating effect of cAMP on tolerance development. With high doses and more frequent administrations, cycloheximide will also block the development of tolerance to morphine (8) but it is difficult to prevent deaths under such conditions .
Vol. 16, No. 12
grclic Nucleotides and Norpbine Tolerance
1897
Figure 1 aoo
seo
wiw
Effect of cycloheximide on cAMP enhancement of morphine dependence and tolerance development. The naloxone ED50 and morphine AD50 plus 95°}6 confidence limits were determined 6 hours after removal of a morphine pellet implanted for 72 hours . S denotes saline ; CY, cycloheximide. The doses were CY 20 mg/kg i. p. ; cAMP LO mg/kg i. v. From Ho et al . , (6 ) Effe ct of cAMP on the development of morphine physical dependence The injection of cAMP prior to morphine pellet implantation also accelerated the development of physical dependence (6). Enhancement of dependence development by cAMP was indicated by adecrease in the amount of naloxoae needed to induce precipitated withdrawal jumping . Is mice previously rendered dependent by pellet implantation, the administration of cAMP immediately after pellet removal dfd not alter the naloxose ED50 significantly . Ia contrast, in implanted mice pretreated with cAMP, a higher degree of physical dependence was evidenced by a decrease in the naloaone ED50 to one-fourth that of the. group receiving the vehicle . Enhancementof morphine physical dependence development by cAMP also was shown after abrupt withdrawal ; the body weight loss, which occurred after removal of the morphine pellet, was greater with cAMP pretreatment . Cycloheaimide prevented the accelerating effect of cAMP on the development of physical dependent on morphine . As shown oa the left is Fig. 1, in mice pretreated with a single injection of cAMP two hours before morphine pellet implantation, the naloxone ED50 was one-third that of morphine pellet-implanted animals treated with saline . The administration of cycloheximide once daily at a dose which did not significantly alter the naloxone precipitated withdrawal response reduced the enhancing effect of cAMP . The nalozose ED50 of the cAMP-plus-cycloheaimide-treated group was about twice that of the cAMP-treated group and not significantly different from morphine implanted animal treated with Balise .
1898
Cyclic
Nucleotides and Morphine Tolerance
Vol . 16, No . 12
More frequent administration of cycloheximide not only blocked the accelerating effect of cAMP on morphine dependence development but also reduced the development of physical dependence in morphine pellet implanted mice . The latter observation is consistent with an earlier study that dependence development, resulting from repeated injections of morphine for three weeks, is blocked by the daily administration of cycloheximide (8), Two other nucleotides, adenosine-2', 3'-cyclic monophosphate (7.', 3' CAMP) and guanosine-3', 5'-cyclic monophosphate (3', 5'-cGMP) were also assessed for their effect on morphine tolerance and physical dependence . As shown in Table 1, following pretreatment with a dose similar to that for cAMP (10 mg/kg i, v, ) neither compound altered the morphine AD50 or naloxone ED50 of animals of implanted with a morphine pellet, Table 1 EFFECT OF 2', 3'-CAMP AND 3', 5'-cGMP ON THE DEVELOPMENT OF MORPHINE TOLERANCE AND DEPENDENCE . Compound
Niôrphine Naloxoae AD50* ED50~ Vehicle 0 (4 - 78) 0, 53 (0, 4 -0 . 2) 2',3'-CAMP 90 (63 -128) 0.48 (0 .28-0 .83) 3', 5' -cGMP 84 (14 -171) 0, 45 (0, 32-0 . 63) 3' 5'-CAMP+ 270 200-360 0, 08 0. 05-0 .12 #determine after subcutaneous administration (mg kg) hours after removal of a morphine pellet implanted for 3 days ; parentheses include the 95% confidence limits, +From Ho et al, , (6)
Effect of theophylline on tolerance and dependence The phosphodiesterase inhibitor, theophylline, enhanced tolerance and physical dependence development, The compound was administered at dose of 100 mg/kg intraperitoneally commencing 2 hours before mor phine pellet implantation and repeated every 24 hours for 2 additional days . A control group received the vehicle instead of theophylline, The morphine AD50 and naloxone ED50 were determined in the usual manner 6 hours after pellet removal . As shown on the left of Figure 2, theophylliae treated animals were about 3 times more tolerant than the control group. The morphine AD50 of the theophylline pretreated group was 235 mg/kg whereas it was 78 mg/kg for the control group receiving the vehicle. The development of a higher degree of physical dependence after theophylline pretreatment ie shown by the data s++"+~+T+~+ arized on the right of Figure 2, The naloxone ED50 of the theophylline group (0, 29 mg/kg) was one-third that of the control group (0, 76 mg/kg) . Effect of beta ad renergic blockers The enhancing effect of cAMP on tolerance and physical dependence development was blocked by pretreatment with the beta-adrenergic blockers . A single injection of dichloroisoprotereaol (DCI, 40Wg) pronethalol(70 Wg), and propanolol (70 Wg) intracerebrally 20 minutes prior to cAMP, prevented the accelerating effect of cAMP on tolerance and physical dependence development, Under these conditions and physical dependence development
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was not significantly altered by the individual betablockere . In time course studies conducted with propanolol, the compound was found to be largely When given 20 ineffective when given 24 and 48 hours after cAMP . minutes or 4 hours after cAMP its action was greatly reduced (Loh _et _al. , unpublished) . ro
oe
Mgure 2
Q2
V 3a
T 3a
V &
T 5t
V 3a
T 3a
Effect of theophylline on the development of tolerance and physical dependence . V denotes the vehicle and T, theophyüine 100 mg/kg i. p, , administered at daily intervals for 3 days . Administration of DCI for three days intracerebrally, resulted in a decrease in tolerance development . As shown in Table 2, an ü-fold tolerance developed in morphine implanted animals treated with saline whereas with DCI treatment, only a 4-fold tolerance developed, The three daily injections of DCI in the placebo pellet implanted animals did not alter the response to morphine (9) . Similar results were obtained with experiments oa propanolol . Repeated administration of DCI also inhibited the development of physical dependence . When DCI was given prior to and during the development of dependence, the naloxone ED50 increased 8-fold as shown in Table 2: The acute administration of DCI to the dependent animals did not alter withdrawal response as evidenced by the fact that the naloxone ED50 of saline and DCI treated were nearly identical. Also, acute or chronic treatment with saline in morphine implanted animals did not alter the naloxone ED50 . Table 2 EFFECT OF SINGLE AND REPEATED INJECTIONS OF DCI ON MORPHINE AD 50 AND NALOXONE ED50 ins DCI 3X
Morphine AD50 # M P 4.5+0.9 50 .0+9 .0 22 . 0 + 4. 5 5. 0 + 1 . 0
Naloxone ED50* M 0.15+0.03 1. 20 + 0. 20
~°mg kg + S. E,M.P~eâotes a placebo pellet implant; M a morphine pellet. From Bhargsva et al ., (9).
1900
Gyc11c Nucleotides and Morphine Tolerance
Vol. 16, No . 12
Die cueeion The results confirm our previous findings that cAMP may be involved with processes concerned with morphine tolerance and dependence development (6), Although cAMP was administered intravenously, it ie also effective at much lower doses after intracerebral administration . The striking finding ie that a single injection of the cAMP produces a marked effect that is measurable 80 hours after its administration . Some degree of selectivity in action must exist, since two other nucleotides tested were inactive, The inhibition of tolerance and dependence by the beta adrenergic blockere, DCI and propaaolol are also compatible with an action that may be mediated by cAMP, The effects noted with theophylline were lees dramatic but consistent with the above findings . Although enhancement of tolerance and dependence developed was observed, it was necessary to repeat the theophylline administration, We can only presume that the degree of phosphodiesterase inhibition was insufficient or that theophylline possesses other pharmacologic effects that may be opposite to those elicited by cAMP, Since the accelerating effect of cAMP on morphine tolerance and dependence development is blocked by cycloheximide, it appears that cAMP ie participating in processes that may be initiating or accelerating selective synthesis of a protein or other macromolecule, Furthermore, the mechanism can be turned off as evidenced by the fact that the beta blockers completely prevented the accelerating effect of cAMP when they were administered before cAMP, This effecwae obtained under conditions where the beta blocker ear se did not affect tolerance and physical dependence . The mechanism by which blockade of cAMP action is elicited ie not clear since the site of action of cAMP should be distal to the site of blockade, if one considers the beta-receptor to be a eubunit of adenyl cyclase in the cellular membrane (10) . However, since the enhancing effect of cAMP on tolerance and dependence was obtained with cAMP administered exogenously, an action at other sites can not be excluded and only further studies can provide an answer . References 1. 2. 3. 4. 5. 6. 7. 8, 9. 10 .
WAY, E. L. , Proc, 5th Int, Congr . Pharmacology, San Francisco 1972, vol, 1, p. 77-94 Karger, Basel (1973) . WAY, E, L. and F. H, SHEN, in Narcotic Drugs, Biochemical Pharmacology (D, H, Clouet, ed), p. 229-253, Plenum Press, New York, (1971) . CHOU, W. S. , A, K. S. HO, and H, H. LOH, Proc . West, Pharmacol. Soc, 14, 42-45 (1971) . HO, I, K, , S. E. LU, H. H. LOH, and E, L. WAY. Nature (London) 328, 397-8 (1972), HO, I. K, , H. H. LOH, and E. L. WAY. J. Pharmacol . exp, Ther, 185, 336-346 (1973) . HO, I, K. , H. H. LOH, and E, L. WAY . J. Pharmacol, exp . Ther . 185, 347-357 (1973) . WAY, E. L, , H, H. LOH and F, H, 5HEN . J. Pharmacol, exp, Ther, 167, 1-8 (1968) . LOH, H. H. , F, SHEN and E, L. WAY. Biochem. Pharmacol. 18, 2711-2721 (1969) . BHARGAVA, H, N. , S. L, CHAN and E, L, WAY. Proc . West . Pharmacol. Soc, 15, 4-7 (1972), ROBISON, G. A. , R. W, BUTCHFR and E. W, SUTHERLAND . Ann N, Y. Acad . Sci, 139, 703-723 (1967) .
