Brain Research, 544 (1991) 191-195 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0006-8993/91/$03.50 ADONIS 000689939116431R
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BRES 16431
Modification of morphine-induced analgesia and toxicity by pertussis toxin Kabirullah Lutfy, Shu-Chin Janet Lee Chang, Joseph Candido, Young Jang, Victor Sierra and Byron C. Yoburn College of Pharmacy and Allied Health Professions, St. John's University, Queens, NY 11439 (U.S.A.)
(Accepted 16 October 1990) Key words: Morphine; Analgesia; Lethality; Pentobarbital; Naloxone; Guanine nucleotide binding protein, Gi, Gs, Go; Adenylate cyclase; Opioid receptor
The present study evaluates the effect of pertussis toxin (PTX) on morphine-induced analgesia and lethality. Mice were injected with 0.2 ~g PTX intracerebroventricularly (i.c.v.) and 0.2/~g PTX intrathecally (i.t.) or saline. Mice were tested for morphine-induced analgesia (tall flick) and lethality 16 days later; mice were also examined for pentobarbitaMnduced mortality. Morphine analgesic potency was decreased by approximately 4-fold in PTX-treated mice compared to controls. Conversely, the lethal potency of morphine was increased by 10-fold in PTX-treated mice compared to controls. PTX treatment did not alter the lethal potency of pentobarbital. Morphine-induced analgesia and lethality were dose-dependently antagonized by naloxone in both PTX and saline-treated groups. The results of this study suggest that morphine analgesia is mediated through PTX-sensitive G proteins. On the other hand, morphine-induced lethality appears to be limited by PTX-sensitive factor(s) since PTX treatment enhanced morphine's lethal potency. The increase in lethal potency of morphine may be due to unmasking of an excitatory opioid receptor mediated effect by PTX. INTRODUCTION It is generally accepted that receptors for different hormones and neurotransmitters are coupled to adenylate cyclase (AC) via guanine nucleotide binding proteins, known as G or N proteins s'n-13. Based on both functional and structural criteria, 4 classes of these proteins, termed Gi, Gs, G o and G t have been described s' 11-13
One of the intracellular events believed to mediate opioid agonist effects is the inhibition of AC 3'4'31'32. It has been demonstrated that opioids interact with stereospecific receptors that are coupled to AC through an inhibitory GTP-binding protein (Gi) which can inhibit activated A C and reduce the level of cyclic adenosine 3", 5"-monophosphate (cAMP) 3'4'6'19'31'32. In addition, based on in vitro studies it has been shown that opioid receptors might couple to AC via an excitatory guanine nucleotide regulatory protein (Gs) which stimulates cAMP production 7,33. In addition to G i, another GTPbinding protein G o is present in neuronal tissues 8'11-13. Although, its function is not dear, it is believed to be involved in regulation of ion fluxes by opioids 1,s'H13,24,25
The nature of coupling of the receptor to AC can be probed by taking advantage of bacterial toxins that interfere with signal transduction 6'1°'19'35. Specifically, signals initiated at the level of the receptor can be interrupted by pertussis toxin (PTX) and cholera toxin (CTX) which ADP-ribosylate G i and G s, respectively6" 10,13,21,29,35 The ultimate result of the ADP-ribosylation by these bacterial toxins appears to be an increase in cAMP leve16'l°'22'23; although the toxins produce their effects by acting on two distinct substrates. It has been proposed that CTX activates AC by persistent activation of Gs23. In contrast, PTX acts by increasing AC activity by interfering with the action of Gi 8'10'14'16'17'21'35. It has been demonstrated that PTX treatment modifies the pharmacologic effects of opioids 16'21'26'27'29'34 in a time-dependent manner 4'26'2s'33. In in vivo studies PTX will dramatically limit the analgesic effects of opioid agonists 14'26-29. In some studies, PTX treatment that can abolish inhibitory effects of opioids has been shown to concomitantly enhance excitatory effects of opioids 22' 33,37 Further, informal observations in our laboratory indicated that PTX-treated mice were agitated following doses of morphine that had no such effect in controls. Taken together, these findings raised the possibility that
Correspondence: B.C. Yoburn, Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John's University, Queens, NY 11439, U.S.A.
192 PTX treatment may simultaneously inhibit analgesic effects while potentiating other opioid-mediated effects such as seizures and toxicity. The present study evaluated the effect of PTX in vivo on morphine-induced analgesia and lethality.
MATERIALS AND METHODS
Subjecls Male, Swiss Webster mice (22-24 g) obtained from Taconic Farms (Germantown, NY) were employed in all experiments. Mice were maintained 5 per cage with free access to food and water. Mice were housed for at least 24 h prior to experimentation and used only once.
Effect of PTX on morphine-induced analgesia and lethality Mice were injected with 0.2/zg PTX i.c.v, and 0.2/zg PTX i.t. Controls were injected with saline. The PTX dose was selected based on previous reports 14'26-29 and preliminary studies in our laboratory 33. I.t. drug administration was by the method of Hylden and Wilcox 15. I.c.v. injections were made by a modification of a previously described method 3a. Sixteen days later, mice were weighed and a baseline tail flick (see below) latency was determined for each mouse. Mice were then injected with morphine (2.0-12.5 mg/kg, s.c.; n = 4-9/dose) and tested for analgesia 30 min later; or mice were injected with morphine (20.0-500.0 mg/kg, s.c.; n = 6/dose) and observed at 1, 2 and 24 h. At the end of 24 h, cumulative lethality was determined.
and 95% confidence limits. The X2-test and Student's t-test were used to evaluate differences among various groups.
RESULTS
Effect o f P T X on morphine-induced analgesia and lethality PTX treatment did not alter baseline tail flick latencies (P > 0.05) (saline-treated = 2.18 + 0.55 s; PTX-treated = 1.98 + 0.33 s, n /> 33/group). However, body weight was significantly (P < 0.01) decreased in PTX-treated mice compared to controls in morphine-treated groups (saline-treated = 35.1 + 2.6 g; PTX-treated = 30.6 + 3.6 g, n /> 8 1 / g r o u p ) , a n d in p e n t o b a r b i t a l - t r e a t e d
groups
( s a l i n e - t r e a t e d = 37.5 + 2.6 g; P T X - t r e a t e d = 33~6 + 3.2 g, n /> 2 0 / g r o u p ) . T r e a t m e n t w i t h P T X suppressed morphine antinoci-
analgesic dose-response curve was significantly shifted to the right in the PTX-treated group compared to the saline-treated group (Fig. 1, bottom). The relative potency of morphine was decreased in PTX-treated mice by approximately 4-fold compared to controls (Table I). Conversely, treatment with PTX significantly enhanced the lethal potency of ception. The morphine
Effect of PTX on pentobarbital-induced lethality. Mice were injected i.c.v, and i.t. with PTX or saline as described. Sixteen days later, mice were weighed and injected with pentobarbital (100-150 mg/kg, s.c.; n = 5-6/dose) and observed at 1, 2 and 24 h. Cumulative lethality at 24 h was determined.
Effect of naloxone on morphine-induced analgesia and lethality in PTX-treated mice Mice were injected i.c.v, and i.t. with PTX or saline as described. Sixteen days later, mice were weighed. Baseline tail flick latencies were measured for only those mice used for analgesia studies. All mice were then treated with either saline or naloxone (0.1-10.0 mg/kg, s.c.; n = 6--7 mice/dose). Five minutes after naloxone, mice were injected with a single dose of morphine (4.0 or 400 mg/kg in controls; 7.5 or 100 mg/kg in PTX-treated group) and were examined for analgesia or lethality. The morphine doses were based on the previous dose-response studies.
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191
BRES 16431
Modification of morphine-induced analgesia and toxicity by pertussis toxin Kabirullah Lutfy, Shu-Chin Janet Lee Chang, Joseph Candido, Young Jang, Victor Sierra and Byron C. Yoburn College of Pharmacy and Allied Health Professions, St. John's University, Queens, NY 11439 (U.S.A.)
(Accepted 16 October 1990) Key words: Morphine; Analgesia; Lethality; Pentobarbital; Naloxone; Guanine nucleotide binding protein, Gi, Gs, Go; Adenylate cyclase; Opioid receptor
The present study evaluates the effect of pertussis toxin (PTX) on morphine-induced analgesia and lethality. Mice were injected with 0.2 ~g PTX intracerebroventricularly (i.c.v.) and 0.2/~g PTX intrathecally (i.t.) or saline. Mice were tested for morphine-induced analgesia (tall flick) and lethality 16 days later; mice were also examined for pentobarbitaMnduced mortality. Morphine analgesic potency was decreased by approximately 4-fold in PTX-treated mice compared to controls. Conversely, the lethal potency of morphine was increased by 10-fold in PTX-treated mice compared to controls. PTX treatment did not alter the lethal potency of pentobarbital. Morphine-induced analgesia and lethality were dose-dependently antagonized by naloxone in both PTX and saline-treated groups. The results of this study suggest that morphine analgesia is mediated through PTX-sensitive G proteins. On the other hand, morphine-induced lethality appears to be limited by PTX-sensitive factor(s) since PTX treatment enhanced morphine's lethal potency. The increase in lethal potency of morphine may be due to unmasking of an excitatory opioid receptor mediated effect by PTX. INTRODUCTION It is generally accepted that receptors for different hormones and neurotransmitters are coupled to adenylate cyclase (AC) via guanine nucleotide binding proteins, known as G or N proteins s'n-13. Based on both functional and structural criteria, 4 classes of these proteins, termed Gi, Gs, G o and G t have been described s' 11-13
One of the intracellular events believed to mediate opioid agonist effects is the inhibition of AC 3'4'31'32. It has been demonstrated that opioids interact with stereospecific receptors that are coupled to AC through an inhibitory GTP-binding protein (Gi) which can inhibit activated A C and reduce the level of cyclic adenosine 3", 5"-monophosphate (cAMP) 3'4'6'19'31'32. In addition, based on in vitro studies it has been shown that opioid receptors might couple to AC via an excitatory guanine nucleotide regulatory protein (Gs) which stimulates cAMP production 7,33. In addition to G i, another GTPbinding protein G o is present in neuronal tissues 8'11-13. Although, its function is not dear, it is believed to be involved in regulation of ion fluxes by opioids 1,s'H13,24,25
The nature of coupling of the receptor to AC can be probed by taking advantage of bacterial toxins that interfere with signal transduction 6'1°'19'35. Specifically, signals initiated at the level of the receptor can be interrupted by pertussis toxin (PTX) and cholera toxin (CTX) which ADP-ribosylate G i and G s, respectively6" 10,13,21,29,35 The ultimate result of the ADP-ribosylation by these bacterial toxins appears to be an increase in cAMP leve16'l°'22'23; although the toxins produce their effects by acting on two distinct substrates. It has been proposed that CTX activates AC by persistent activation of Gs23. In contrast, PTX acts by increasing AC activity by interfering with the action of Gi 8'10'14'16'17'21'35. It has been demonstrated that PTX treatment modifies the pharmacologic effects of opioids 16'21'26'27'29'34 in a time-dependent manner 4'26'2s'33. In in vivo studies PTX will dramatically limit the analgesic effects of opioid agonists 14'26-29. In some studies, PTX treatment that can abolish inhibitory effects of opioids has been shown to concomitantly enhance excitatory effects of opioids 22' 33,37 Further, informal observations in our laboratory indicated that PTX-treated mice were agitated following doses of morphine that had no such effect in controls. Taken together, these findings raised the possibility that
Correspondence: B.C. Yoburn, Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John's University, Queens, NY 11439, U.S.A.
192 PTX treatment may simultaneously inhibit analgesic effects while potentiating other opioid-mediated effects such as seizures and toxicity. The present study evaluated the effect of PTX in vivo on morphine-induced analgesia and lethality.
MATERIALS AND METHODS
Subjecls Male, Swiss Webster mice (22-24 g) obtained from Taconic Farms (Germantown, NY) were employed in all experiments. Mice were maintained 5 per cage with free access to food and water. Mice were housed for at least 24 h prior to experimentation and used only once.
Effect of PTX on morphine-induced analgesia and lethality Mice were injected with 0.2/zg PTX i.c.v, and 0.2/zg PTX i.t. Controls were injected with saline. The PTX dose was selected based on previous reports 14'26-29 and preliminary studies in our laboratory 33. I.t. drug administration was by the method of Hylden and Wilcox 15. I.c.v. injections were made by a modification of a previously described method 3a. Sixteen days later, mice were weighed and a baseline tail flick (see below) latency was determined for each mouse. Mice were then injected with morphine (2.0-12.5 mg/kg, s.c.; n = 4-9/dose) and tested for analgesia 30 min later; or mice were injected with morphine (20.0-500.0 mg/kg, s.c.; n = 6/dose) and observed at 1, 2 and 24 h. At the end of 24 h, cumulative lethality was determined.
and 95% confidence limits. The X2-test and Student's t-test were used to evaluate differences among various groups.
RESULTS
Effect o f P T X on morphine-induced analgesia and lethality PTX treatment did not alter baseline tail flick latencies (P > 0.05) (saline-treated = 2.18 + 0.55 s; PTX-treated = 1.98 + 0.33 s, n /> 33/group). However, body weight was significantly (P < 0.01) decreased in PTX-treated mice compared to controls in morphine-treated groups (saline-treated = 35.1 + 2.6 g; PTX-treated = 30.6 + 3.6 g, n /> 8 1 / g r o u p ) , a n d in p e n t o b a r b i t a l - t r e a t e d
groups
( s a l i n e - t r e a t e d = 37.5 + 2.6 g; P T X - t r e a t e d = 33~6 + 3.2 g, n /> 2 0 / g r o u p ) . T r e a t m e n t w i t h P T X suppressed morphine antinoci-
analgesic dose-response curve was significantly shifted to the right in the PTX-treated group compared to the saline-treated group (Fig. 1, bottom). The relative potency of morphine was decreased in PTX-treated mice by approximately 4-fold compared to controls (Table I). Conversely, treatment with PTX significantly enhanced the lethal potency of ception. The morphine
Effect of PTX on pentobarbital-induced lethality. Mice were injected i.c.v, and i.t. with PTX or saline as described. Sixteen days later, mice were weighed and injected with pentobarbital (100-150 mg/kg, s.c.; n = 5-6/dose) and observed at 1, 2 and 24 h. Cumulative lethality at 24 h was determined.
Effect of naloxone on morphine-induced analgesia and lethality in PTX-treated mice Mice were injected i.c.v, and i.t. with PTX or saline as described. Sixteen days later, mice were weighed. Baseline tail flick latencies were measured for only those mice used for analgesia studies. All mice were then treated with either saline or naloxone (0.1-10.0 mg/kg, s.c.; n = 6--7 mice/dose). Five minutes after naloxone, mice were injected with a single dose of morphine (4.0 or 400 mg/kg in controls; 7.5 or 100 mg/kg in PTX-treated group) and were examined for analgesia or lethality. The morphine doses were based on the previous dose-response studies.
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