Reversibility of morphine effects on phagocytosis by murine macrophages.

Proneness of addicts to infections may be partially due to opiate effects on immune cells. We find that acute morphine inhibits phagocytosis in murine peritoneal macrophages in vitro with apparent desensitization at high concentrations, whereas chronic exposure results in a state akin to tolerance/dependence where macrophages seem to require morphine to phagocytize at a control level. However, both putative desensitization and tolerance/dependence are reversible, since drug re-addition several hours after withdrawal results in inhibition, as in acute exposure. This shows that opiate effects on immune cells are variably related to the experimental context in which they are administered, which is of relevance for understanding their potential role in immunosuppression.

Effect of morphine on Fc-mediated phagocytosis by murine macrophages in vitro.

Acute exposure to morphine has been shown to inhibit phagocytosis in murine macrophages, whereas chronic exposure results in apparent desensitization. We now show that morphine may be either inhibitory or stimulatory depending on concentration and exposure time. Furthermore, under some conditions drug withdrawal from putatively desensitized cells will result in inhibition of phagocytosis, suggesting that a state akin to dependence has developed. Desensitization can also develop with intermittent exposures if the opiate-free period between drug exposures is shorter than 4 h. These effects of morphine on macrophages are important in understanding the role of this drug as an immunomodulatory agent.

A novel opioid mechanism seems to modulate phagocytosis in Tetrahymena.

We have previously reported that a beta-endorphin-like substance inhibits phagocytosis in Tetrahymena perhaps by a mu-like opioid receptor. We now report a further characterization of the elements involved in the signal transduction mechanism of this opioid. Affinity chromatography followed by immunoblots of both intracellular extracts and extracellular medium reveal the presence of two main proteins of 64 and 75 kDa. These molecular weights are much higher than that of any known opioid peptide or precursor protein and suggest that we may be dealing with either a novel opioid or with proteins that by chance cross-react with anti-beta-endorphin antibody. Nevertheless, when the biological activity of these proteins was tested it was found that they had an effect similar to that of mammalian beta-endorphin, namely inhibition of phagocytosis by a naloxone-reversible mechanism. We have probed a size-selected Tetrahymena library with a pro-opiomelanocortin probe and have obtained several positive clones; the sequencing of their inserts should establish whether we are dealing with a bona fide member of the opioid family. Another aspect we have been studying is the G-proteins which appear to be involved in the modulation of phagocytosis. We have found, by means of Western blotting (using an antibody against the conserved GTP-binding region of the alpha-subunit), two bands of 51 and 59 kDa; no alpha-subunit of 59 kDa had been reported previously and may represent a novel G-protein. In spite of these differences, the opioid signal transduction mechanism appears to remarkably resemble that present in more complex organisms.

Pharmacological characterization of an opioid receptor in the ciliate Tetrahymena.

A pharmacological characterization has been performed of the opioid receptor involved in modulation of phagocytosis in the protozoan ciliate Tetrahymena. Studies on inhibition of phagocytosis by mammalian prototypic opioid agonists revealed that morphine and beta-endorphin have the highest intrinsic activity, whereas all the other opioids tested can only be considered partial agonists. However, morphine (a mu-receptor agonist) is twice as potent as beta-endorphin (a delta-receptor agonist). Furthermore, the sensitivity for the opioid antagonist naloxone, determined in the presence of morphine and beta-endorphin, is very similar to the sensitivity exhibited by mammalian tissues rich in mu-opioid receptors. We suggest that the opioid receptor coupled to phagocytosis in Tetrahymena is mu-like in some of its pharmacological characteristics and may serve as a model system for studies on opioid receptor function and evolution.

Inhibition by opioids of phagocytosis in peritoneal macrophages.

We have tested the effect of prototypic opioid agonists on phagocytosis of sheep erythrocytes by mouse peritoneal macrophages. It was found that morphine and all the opioid peptides tested inhibited phagocytosis by a biphasic, naloxone-reversible mechanism. Delta agonists were the most effective inhibitors, suggesting that the response is mediated by a delta receptor. Chronic exposure to morphine apparently results in the development of tolerance since under these conditions the inhibitory effect of the opiate is abolished. These results are similar to previously reported effects of opioids on endocytosis in other systems, which suggests that this inhibition is part of a basic regulatory mechanism that has been conserved in evolution.

Effect of chronic opioid treatment on phagocytosis in Tetrahymena.

Opioid inhibition of phagocytosis in the protozoan ciliate Tetrahymena is antagonized by naloxone and this antagonism can be surmounted by increasing agonist concentration, which suggests a receptor-mediated mechanism. Desensitization of the opioid effect is time dependent in addition to concentration dependent. Chronic exposure to opioids results in the development of tolerance to the inhibitory effect of the agonists, and withdrawal of the latter results in a decrease in phagocytic capacity, which suggests that a state akin to dependence has been developed in these cells. Naloxone appears to behave as a partial agonist in tolerant cells, and there seems to exist cross-tolerance to mu and delta agonists.

Phagocytosis in Tetrahymena thermophila: naloxone-reversible inhibition by opiates.

1. Nanomolar concentrations of opiates inhibit phagocytosis in the ciliated protozoan Tetrahymena thermophila. 2. Naloxone and naltrexone counteract the effect of the opiate agonists tested. 3. The dose-response curves are U-shaped, with no detectable effect at low or high concentrations. 4. An increase in extracellular calcium and dopamine counteract the inhibition caused by metenkephalin. 5. The recognition mechanism for opiates in Tetrahymena cannot be classified as belonging to any of the mammalian opiate receptor subtypes and is perhaps a primitive receptor.

Calcium regulates the regeneration of cilia in Tetrahymena thermophila.

A study of calcium metabolism in Tetrahymena during the regeneration of cilia evidenced that the process is inhibited by nifedipine and trifluoperazine. This suggests that calcium ions play an important regulatory role in this process. This was confirmed by studies on calcium uptake and efflux which showed that there was a net increase in calcium uptake prior to the reinitiation of motility. The increase coincided with a period of sensitivity to the calcium antagonist TMB-8 and with an increase in the intracellular level of cGMP. The process was also inhibited by neomycin and stimulated by phorbol esters, which suggests that hydrolysis of phosphatidylinositol phosphates may take place as part of the calcium regulatory network during the regeneration of cilia.

Biogenic amines stimulate regeneration of cilia in Tetrahymena thermophila.

Serotonin and catecholamines affect the regeneration of cilia in Tetrahymena thermophila in a dose-dependent manner: micromolar concentrations are stimulatory, whereas millimolar concentrations have little or no effect. This conclusion is based on motility measurements in regenerating cells and on ciliary counts in scanning electron micrographs. In addition, the recognition mechanism for each hormone appears to be specific and independent. Our results suggest an evolutionary link with hormonal mechanisms in multicellular eukaryotes.

Effect of biogenic amines on phagocytosis in Tetrahymena thermophila.

Stimulation of phagocytosis by serotonin and catecholamines in Tetrahymena grown in proteose-peptone medium proved to be concentration dependent, the optimal concentrations being approximately 0.1 to 1.0 microM. The serotonergic antagonists, spiperone, and metergoline, also stimulated the process, whereas the beta- and alpha-adrenergic antagonists, propranolol, alprenolol, and ergocryptine, had no effect or inhibited phagocytosis. A wide variety of derivatives of the biogenic amines had no effect on phagocytosis, demonstrating the specificity of recognition mechanism for neurohormones in Tetrahymena. Such hormones act by at least two independent mechanisms, one for adrenergic agonists, another for dopamine. Presumably, recognition mechanisms for hormones in protozoa resemble in some respects those in multicellular organisms, therefore bespeaking a common origin.

On the possible role of serotonin in the regulation of regeneration of cilia.

A study was made of the interrelationship of serotonin, cAMP, and calcium ions in the regulation of regeneration of cilia by Tetrahymena pyriformis. All these compounds stimulated the regeneration, whereas a blocker of serotonin synthesis, p-chlorophenylalanine, and a calcium chelator, EGTA, inhibited the process. This inhibition could be overcome by the addition of any of the stimulatory compounds. cAMP was also found to be inhibitory at high concentrations. The intracellular concentration of this nucleotide was found to increase during the regeneration, and this increase occurred precociously in the presence of serotonin. It was concluded that serotonin may regulate ciliary regeneration by a mechanism involving cAMP And calcium ions, but that the causal relationships among these compounds still need to be established.