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Novel peptides and mechanisms of opiate tolerance
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NOVEL PEPTIDES AND MECHANISMS OF OPIATE TOLERANCE. James E. Zadina, Abba J. Kastin, Lin-Jun Ge, and Sulie L. Chang 1. VA Med. Center and Tulane Univ. Scit of Med. and ILSU Med. Center New Orleans, LA USA 70146
Summary. We have used two diverse approaches for studying cellular and tissue effects of chronic exposure to opiates. First, receptor regulation by agonists and antagonists was characterized in a human neuroblastoma cell line, SH-SY5Y. Morphine down-regulated mu and delta opiate receptors. Mu receptor down-regulation was selectively blocked by the mu antagonist CTOP and delta receptor changes by the delta antagonist ICI 174864. Naloxone upregulated opiate receptors in these cells, as it does in brain. Thus, SH-SY5Y cells provided a neuronal model for studying selective mu and delta receptor regulation in response to agonists and antagonists. In a second approach, we have shown that peptides recently isolated from brain in our laboratory (Tyr-MIF-1 and Tyr-W-MIF-1) can act as opiate agonists in several test paradigms, but as antagonists under specific circumstances in these test systems. These include chronic exposure to morphine or reduction of receptor reserve by alkylating agents. These peptides may serve as prototypes for endogenous compounds that act as agonists in the naive state and as antagonists in the tolerant state. Tolerance and dependence are adaptations to opiate agonist exposure that are likely to involve numerous molecular processes. Two of several potential contributing mechanisms that we have explored include (a) receptor regulation, which we here decribe in SH-SY5Y cells, and (b) a potential role for peptides with properties described as "antiopiate", a term used in initial characterization of the peptides to refer to their ability to antagonize opiate agonists without the connotation of specific mechanisms implied by terms such as "antagonist", or "mixed agonist/antagonist". These compounds could modulate opiate activity by effects on homologous (opiate) or heterologous (non-opiate) receptors or by post-receptor events. Opiate receptor regulation by morphine in adult brain has been controversial. Early negative reports led many investigators to conclude that receptor regulation does not play a critical role in adaptations to opiate exposure, including tolerance. Nevertheless, regulation of receptor number after chronic exposure to an agonist is typical of many receptor systems. Also, there are many demonstrations that opiates can regulate receptor number particularly in cell culture, in developing animals, and with the use of highly efficacious or selective ligands. We used human neuroblastoma SH-SY5Y cells to demonstrate morphine-induced downregulation of mu opiate receptors in a neuronal cell line (1). The down-regulation was dependent on dose, time, and temperature. Naloxone and the mu-selective antagonist CTOP blocked the down-regulation, while naloxone alone up-regulated mu receptors. Partial agonists did not induce down-regulation, implicating efficacy as a factor in the regulation. The downregulation was homologous for opiate receptors since muscarinic receptors were unaffected. In contrast to NG-108 cells, which contain only delta sites that are down-regulated by delta receptor ligands but not by morphine, our results show that morphine can down-regulate the mu receptor in a neuronal cell line (1). In subsequent studies we have found that morphine can also downregulate delta receptors, which are present in SH-SY5Y cells. Mu (CTAP) and delta (ICI174864) antagonists selectively blocked the morphine-induced down-regulation of their respective receptors. Selective mu (PL017) and delta (DPDPE) agonists were also able to down-regulate, in a specific manner, their respective receptors. Although the diffarentiating agent retinoic acid upregulated both mu and delta receptors, the phorbol agent TPA increased mu but not delta receptors. Taken together, these results show that mu and delta opiate receptors can be
S196
separately regulated in SH-SY5Y cells, providing a model for study of their relative roles in the cellular responses to agonist and antagonist exposure. A second approach that we have used to study mechanisms of tolerance involved characterization of the actions of peptides recently isolated in our laboratory from brain, including Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2). These peptides bind to opiate receptors, with a high selectivity for mu over delta and kappa receptors, and to a non-opiate site labeled by Tyr-MIF-1. The peptides induced opiate agonist-like effects in several paradigms, but antagonist properties were revealed under special conditions within these paradigms. In the guinea pig ileum, Tyr-MIF-1 and Tyr-W-MIF-1 showed an opiate agonist-like inhibition of electrically-induced contractions of the guinea pig ileum (2,3). These actions were blocked by naloxone and the mu selective antagonist CTOP, but not by the kappa antagonist nor-binaltorphamine (nor-BNI). In preparations of ileum from guinea pigs that had been implanted with morphine pellets for several days, however, Tyr-MIF-1, Tyr-W-MIF-1, and several fragments and analogs of this family of peptides were able to antagonize the actions of morphine and the mu agonist DAMGO (2-4). Similar results were observed after reduction of receptor reserve by alkylation with 13-CNA. This indicates that at least some of the antagonist actions of this group of peptides could be due to occupation of opiate receptors with lower efficacy than other agonists, attenuating the actions of these agonists. This was particularly true for a related peptide, hemorphin (Tyr-Pro-Trp-Thr), which antagonized DAMGO and showed relatively low efficacy in Furchgott analyses of experiments in the ileum after treatment with 13CNA (2). In the following model, chronic exposure to morphine reduces receptor reserve while "antiopiate" peptides act either through their own non-opiate sites or as partial agonists at opiate sites to contribute to the loss of opiate sensitivity. Thus, several processes act together to produce a wide potential range of tolerance. Chronic Morphine Reduced opiate receptor reserve Increased "antiopiate" peptides j ~
partial agonist/antagonist-
J ~(functional antagonism)
Increased requirement of opiate for similar effect (tolerance) Opiate agonist and antagonist properties of the novel peptides were also observed in vivo. Central (ICV) administration of Tyr-W-MIF-1 to opiate naive animals induced a prolonged analgesia that was reversed by naloxone (5). Central administration of Tyr-MIF-1 in morphinedependent rats, however, precipitated withdrawal signs (6). Thus, a family of novel brain peptides, including Tyr-MIF-1 and Tyr-W-MIF-1, may serve as prototypical compounds with a unique profile of activity, acting as agonists in the opiate naive state and antagonists in the tolerant state. REFERENCES
1. J.E. Zadina, S.L. Chang, L.-J. Ge and A.J. Kastin (1993) J.Pharm.Exp.Ther. 265:254-262. 2. J.E. Zadina, A.J. Kastin, D.Kersh, and A. Wyatt (1992) Life Sci. 51:869-885. 3. J. Erchegyi, A.J. Kastin, J.E. Zadina (1992) Peptides 13:623-631. 4. J. Erchegyi, J.E. Zadina, X.-D.Qiu, D.C.Kersh, L.-J.Ge, M.M. Brown and A.J. Kastin (1993) Peptide Res. 6:31-38. 5. J.E. Zadina, V. Kenigs, C.Bruno, L. Hackler, and A.J. Kastin (1993) Neurosci. Lett. 6. D.R. Malin, J.E. Zadina, J.R. Lake, R.B. Rogilio, J.E. Leyva, T.M. Benson, L.S. Corriere, B.P. Handunge and A.J. Kastin (1993) Brain Res. 610:169-171.
NOVEL PEPTIDES AND MECHANISMS OF OPIATE TOLERANCE. James E. Zadina, Abba J. Kastin, Lin-Jun Ge, and Sulie L. Chang 1. VA Med. Center and Tulane Univ. Scit of Med. and ILSU Med. Center New Orleans, LA USA 70146
Summary. We have used two diverse approaches for studying cellular and tissue effects of chronic exposure to opiates. First, receptor regulation by agonists and antagonists was characterized in a human neuroblastoma cell line, SH-SY5Y. Morphine down-regulated mu and delta opiate receptors. Mu receptor down-regulation was selectively blocked by the mu antagonist CTOP and delta receptor changes by the delta antagonist ICI 174864. Naloxone upregulated opiate receptors in these cells, as it does in brain. Thus, SH-SY5Y cells provided a neuronal model for studying selective mu and delta receptor regulation in response to agonists and antagonists. In a second approach, we have shown that peptides recently isolated from brain in our laboratory (Tyr-MIF-1 and Tyr-W-MIF-1) can act as opiate agonists in several test paradigms, but as antagonists under specific circumstances in these test systems. These include chronic exposure to morphine or reduction of receptor reserve by alkylating agents. These peptides may serve as prototypes for endogenous compounds that act as agonists in the naive state and as antagonists in the tolerant state. Tolerance and dependence are adaptations to opiate agonist exposure that are likely to involve numerous molecular processes. Two of several potential contributing mechanisms that we have explored include (a) receptor regulation, which we here decribe in SH-SY5Y cells, and (b) a potential role for peptides with properties described as "antiopiate", a term used in initial characterization of the peptides to refer to their ability to antagonize opiate agonists without the connotation of specific mechanisms implied by terms such as "antagonist", or "mixed agonist/antagonist". These compounds could modulate opiate activity by effects on homologous (opiate) or heterologous (non-opiate) receptors or by post-receptor events. Opiate receptor regulation by morphine in adult brain has been controversial. Early negative reports led many investigators to conclude that receptor regulation does not play a critical role in adaptations to opiate exposure, including tolerance. Nevertheless, regulation of receptor number after chronic exposure to an agonist is typical of many receptor systems. Also, there are many demonstrations that opiates can regulate receptor number particularly in cell culture, in developing animals, and with the use of highly efficacious or selective ligands. We used human neuroblastoma SH-SY5Y cells to demonstrate morphine-induced downregulation of mu opiate receptors in a neuronal cell line (1). The down-regulation was dependent on dose, time, and temperature. Naloxone and the mu-selective antagonist CTOP blocked the down-regulation, while naloxone alone up-regulated mu receptors. Partial agonists did not induce down-regulation, implicating efficacy as a factor in the regulation. The downregulation was homologous for opiate receptors since muscarinic receptors were unaffected. In contrast to NG-108 cells, which contain only delta sites that are down-regulated by delta receptor ligands but not by morphine, our results show that morphine can down-regulate the mu receptor in a neuronal cell line (1). In subsequent studies we have found that morphine can also downregulate delta receptors, which are present in SH-SY5Y cells. Mu (CTAP) and delta (ICI174864) antagonists selectively blocked the morphine-induced down-regulation of their respective receptors. Selective mu (PL017) and delta (DPDPE) agonists were also able to down-regulate, in a specific manner, their respective receptors. Although the diffarentiating agent retinoic acid upregulated both mu and delta receptors, the phorbol agent TPA increased mu but not delta receptors. Taken together, these results show that mu and delta opiate receptors can be
S196
separately regulated in SH-SY5Y cells, providing a model for study of their relative roles in the cellular responses to agonist and antagonist exposure. A second approach that we have used to study mechanisms of tolerance involved characterization of the actions of peptides recently isolated in our laboratory from brain, including Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2). These peptides bind to opiate receptors, with a high selectivity for mu over delta and kappa receptors, and to a non-opiate site labeled by Tyr-MIF-1. The peptides induced opiate agonist-like effects in several paradigms, but antagonist properties were revealed under special conditions within these paradigms. In the guinea pig ileum, Tyr-MIF-1 and Tyr-W-MIF-1 showed an opiate agonist-like inhibition of electrically-induced contractions of the guinea pig ileum (2,3). These actions were blocked by naloxone and the mu selective antagonist CTOP, but not by the kappa antagonist nor-binaltorphamine (nor-BNI). In preparations of ileum from guinea pigs that had been implanted with morphine pellets for several days, however, Tyr-MIF-1, Tyr-W-MIF-1, and several fragments and analogs of this family of peptides were able to antagonize the actions of morphine and the mu agonist DAMGO (2-4). Similar results were observed after reduction of receptor reserve by alkylation with 13-CNA. This indicates that at least some of the antagonist actions of this group of peptides could be due to occupation of opiate receptors with lower efficacy than other agonists, attenuating the actions of these agonists. This was particularly true for a related peptide, hemorphin (Tyr-Pro-Trp-Thr), which antagonized DAMGO and showed relatively low efficacy in Furchgott analyses of experiments in the ileum after treatment with 13CNA (2). In the following model, chronic exposure to morphine reduces receptor reserve while "antiopiate" peptides act either through their own non-opiate sites or as partial agonists at opiate sites to contribute to the loss of opiate sensitivity. Thus, several processes act together to produce a wide potential range of tolerance. Chronic Morphine Reduced opiate receptor reserve Increased "antiopiate" peptides j ~
partial agonist/antagonist-
J ~(functional antagonism)
Increased requirement of opiate for similar effect (tolerance) Opiate agonist and antagonist properties of the novel peptides were also observed in vivo. Central (ICV) administration of Tyr-W-MIF-1 to opiate naive animals induced a prolonged analgesia that was reversed by naloxone (5). Central administration of Tyr-MIF-1 in morphinedependent rats, however, precipitated withdrawal signs (6). Thus, a family of novel brain peptides, including Tyr-MIF-1 and Tyr-W-MIF-1, may serve as prototypical compounds with a unique profile of activity, acting as agonists in the opiate naive state and antagonists in the tolerant state. REFERENCES
1. J.E. Zadina, S.L. Chang, L.-J. Ge and A.J. Kastin (1993) J.Pharm.Exp.Ther. 265:254-262. 2. J.E. Zadina, A.J. Kastin, D.Kersh, and A. Wyatt (1992) Life Sci. 51:869-885. 3. J. Erchegyi, A.J. Kastin, J.E. Zadina (1992) Peptides 13:623-631. 4. J. Erchegyi, J.E. Zadina, X.-D.Qiu, D.C.Kersh, L.-J.Ge, M.M. Brown and A.J. Kastin (1993) Peptide Res. 6:31-38. 5. J.E. Zadina, V. Kenigs, C.Bruno, L. Hackler, and A.J. Kastin (1993) Neurosci. Lett. 6. D.R. Malin, J.E. Zadina, J.R. Lake, R.B. Rogilio, J.E. Leyva, T.M. Benson, L.S. Corriere, B.P. Handunge and A.J. Kastin (1993) Brain Res. 610:169-171.