Withdrawal-induced Withdrawal-induced Inhibition of Inhibition of

Phagocytosis in Phagocytosis in Morphine Tolerant Morphine Tolerant

Macrophages may be Macrophages may be Due to an Increase in Due to an Increase in

cAMPcAMP Cristhian J. Ildefonso¹, Fernando L. Renaud¹ and Ana M. Lugo-Chinchilla²¹Biology Department, University of Puerto Rico, Rio Piedras Campus, and

²Biology Department, Interamerican University, Bayamón Campus.

AbstractAbstractWe have previously demonstrated that acute exposure to 100nM We have previously demonstrated that acute exposure to 100nM

morphine inhibits phagocytosis by murine peritoneal macrophages, morphine inhibits phagocytosis by murine peritoneal macrophages, whereas chronic treatment induces a state of putative tolerance. The whereas chronic treatment induces a state of putative tolerance. The tolerant state is also accompanied by putative dependence, since tolerant state is also accompanied by putative dependence, since withdrawal from tolerant cells inhibits phagocytosis. Tolerant and withdrawal from tolerant cells inhibits phagocytosis. Tolerant and dependence to morphine in the nervous system are based at least in part dependence to morphine in the nervous system are based at least in part on an upregulation of the cAMP pathway. In the present study the role of on an upregulation of the cAMP pathway. In the present study the role of this pathway in tolerant and dependent macrophages was investigated. this pathway in tolerant and dependent macrophages was investigated. We found that morphine does not change basal levels of cAMP during We found that morphine does not change basal levels of cAMP during acute and chronic treatment. On the other hand, drug withdrawal from acute and chronic treatment. On the other hand, drug withdrawal from tolerant cells results in a transient increase in cAMP levels, which suggests tolerant cells results in a transient increase in cAMP levels, which suggests that this cyclic nucleotide could be involved in the inhibition of that this cyclic nucleotide could be involved in the inhibition of phagocytosis. This was confirmed by the fact that artificially increasing phagocytosis. This was confirmed by the fact that artificially increasing levels of cAMP in tolerant cells with dibutyryl cAMP also resulted in levels of cAMP in tolerant cells with dibutyryl cAMP also resulted in inhibition. Furthermore, addition of an inhibitor of protein kinase A (PKA), inhibition. Furthermore, addition of an inhibitor of protein kinase A (PKA), Rp-Adenosine 3’,5’-cyclic monophosphorothioate triethylammonium salt, Rp-Adenosine 3’,5’-cyclic monophosphorothioate triethylammonium salt, prior to drug withdrawal from tolerant cells, abrogates withdrawal-induced prior to drug withdrawal from tolerant cells, abrogates withdrawal-induced inhibition of phagocytosis, thus suggesting that activation of PKA by the inhibition of phagocytosis, thus suggesting that activation of PKA by the increase in cAMP is necessary for inhibition to take place. We conclude increase in cAMP is necessary for inhibition to take place. We conclude that, as in the nervous system, morphine exerts its effects in that, as in the nervous system, morphine exerts its effects in macrophages via the cAMP pathway. ( Sponsored by NIH Grant macrophages via the cAMP pathway. ( Sponsored by NIH Grant SO6GM08102, the FIPI Program of the University of Puerto Rico, and by SO6GM08102, the FIPI Program of the University of Puerto Rico, and by NIH-MARC Honors Program, Grant number 078210)NIH-MARC Honors Program, Grant number 078210)

Macrophages at work Macrophages at work

Macrophages are cells of the immune system that have Macrophages are cells of the immune system that have the capability to phagocyte any foreign body. They are the capability to phagocyte any foreign body. They are a differentiated form of blood monocytes that reside in a differentiated form of blood monocytes that reside in a specific region of the body of an organism.a specific region of the body of an organism.

Effects of 100 nM Morphine on Effects of 100 nM Morphine on Phagocytosis by Murine Phagocytosis by Murine Peritoneal MacrophagesPeritoneal Macrophages

“*” Significant difference (P<0.05) between this value and that of the control group.

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Time of exposure (hrs)

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Specific Research AimsSpecific Research Aims

• Study the pathway utilized by Study the pathway utilized by morphine to exerts its effects on morphine to exerts its effects on macrophages.macrophages.

• Study the cellular basis by which Study the cellular basis by which morphine withdrawal from tolerant morphine withdrawal from tolerant macrophages inhibits phagocytosis. macrophages inhibits phagocytosis.

Effects of 100nM Morphine on Effects of 100nM Morphine on the cAMP levels of the cAMP levels of

MacrophagesMacrophages

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Time of exposure to 100nM Morphine

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Cells were cultured with 100nM Morphine for different times and one group was exposed also to Prostaglandin 1. This was followed by a radioimmunoassay to measure the cAMP levels. There were no significant difference (P<0.05) between any group value and that of the control group.

Effect of 100nM Morphine Effect of 100nM Morphine Withdrawal on cAMP levels Withdrawal on cAMP levels

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Macrophages were cultured with 100nM Morphine (M) for 8 hr before drug withdrawal (Wd). cAMP levels were determined at different times after M Wd by a radioimmunoassay (RIA). “*”Significant difference (P<0.05) between value and that of the control cells.

Effect of Dibutyryl cAMP on Effect of Dibutyryl cAMP on Phagocytosis by Opiate-naïve CellsPhagocytosis by Opiate-naïve Cells

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Macrophages were cultured in RPMI and exposed to different concentrations of dibutyryl cAMP, followed by a phagocytosis assay. “*”Significant difference (P<0.05) between the value and that of the control group.

Effect of Dibutyryl cAMP on Effect of Dibutyryl cAMP on Phagocytosis by Morphine Tolerant Phagocytosis by Morphine Tolerant

MacrophagesMacrophages

Macrophages were cultured with 100nM Morphine for 8hrs and then exposed to different concentrations of dibutyryl cAMP, followed by a phagocytosis assay. “*” Significant difference (P<0.05) between the value and that of the control group.

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Summary of Effects of Dibutyryl Summary of Effects of Dibutyryl cAMP and of Drug withdrawal cAMP and of Drug withdrawal

on Phagocytosis by Macrophageson Phagocytosis by Macrophages

Withdrawal and dibutyril cAMP (Db) groups were cultivated with 100nM Morphine and then exposed for 30mins. to Db or drug withdrawal with RPMI. “*” Significant difference (P<0.05) between the value and that of the control group.

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Effect of 20nM Butyrate on Effect of 20nM Butyrate on Phagocytosis of Tolerant Phagocytosis of Tolerant

MacrophagesMacrophages

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“*” Significant difference (P<0.05) between the value and that of control group.

Effects of Rp-Adenosine 3’,5’-Effects of Rp-Adenosine 3’,5’-cyclic monophosphorothioate cyclic monophosphorothioate

on Morphine Withdrawal on Morphine Withdrawal

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Treatment

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1= Control cells; 2= Morphine (M) 0.5 hr; 3= M 24hrs; 4= M 24hr +Withdrawal (Wd) 1hr; 5= M 24hr+ Rp-Adenosine 2hr + Wd 1hr.“*”Significant difference (P<0.05) between this value and that of the control group.

ConclusionsConclusionsThe present work has let us to conclude The present work has let us to conclude

that chronic morphine, as in the nervous that chronic morphine, as in the nervous system, exerts its effects on macrophage system, exerts its effects on macrophage phagocytosis through the cAMP pathway. phagocytosis through the cAMP pathway. This information should be taken into This information should be taken into account when using opioids as analgesics, or account when using opioids as analgesics, or in the therapy of drug addiction.in the therapy of drug addiction.

Pathway utilized by Morphine in Pathway utilized by Morphine in

MacrophagesMacrophages

AcknowledgmentsAcknowledgments

This project was sponsored by NIH This project was sponsored by NIH Grant SO6GM08102, the FIPI Grant SO6GM08102, the FIPI

Program of the University of Puerto Program of the University of Puerto Rico, and by NIH-MARC U*STAR Grant Rico, and by NIH-MARC U*STAR Grant

Number 5T34GM07821-23.Number 5T34GM07821-23.