Cholinergic Regulation of Morphine Release from Human White Blood Cells: Evidence for a Novel Nicotinic Receptor via Pharmacological and Micro Array Analysis

Abstract

Recent work from our laboratory has demonstrated that human white blood cells make morphine and that substances of abuse, Le, nicotine, alcohol and cocaine have the ability to release this endogenous substance, suggesting a common mechanism of action. We now demonstrate that the nicotinic process is more complex than formerly envisioned. The incorporation rate of 125I-labeled morphine into PMN and MN are 7.85±0.36%, 1.42±O.19%, respectfully, suggesting in MN this process is of low activity. Separate incubations of PMN with varying concentrations of nicotine or the nicotine agonist epibatidine resulted in a statistically significant enhancement of 125I-trace labeled morphine released into the extracellular medium. In order to ascertain the specificity of the nicotine stimulated morphine release the following experiments were performed. Co-incubation of hexamethonium dichloride (5 Jig/ml and at 10 Jig/ml), which preferentially blocks nicotinic receptors at autonomic ganglia, with nicotine, exerted a very weak inhibitory effect Coincubation of u-BuTx or atropine or chlorisondamine diiodide or dlhydro-jl-erythroidine hydrobromide, an a4p2 receptor antagonist, did not block nicotine induced morphine release alone or in combination, suggesting either the response was not specific or it was mediated by a novel nicotinic receptor. Human leukocyte total RNA isolated from whole blood were analyzed, using the Human Genome Survey microarray (Applied Biosystems), for cholinergic receptor expression. PMN nicotinic receptor gene expression was present and contained numerous variants (eight). The number of variants suggests that indeed a novel nicotinic receptor may be mediating this effect, while simultaneously demonstrating the significance of the cholinergic receptor expression in these immune cells.Recently, it was demonstrated that normal human white blood cells (WBC) contain and have the ability to synthesize morphine and release it into the microenvironment (I). Similar findings were reported in invertebrate ganglia (2). Taken together, these studies provide evidence that the synthesis of morphine by various tissues and diverse animals is more widespread than previously thought and now includes human immune cells.Importantly, in separate incubations the pooling of Mytilus edulis (invertebrate marine bivalve) pedal ganglia and Homarus americanus (lobster) nerve cord with ethanol, cocaine, or nicotine resulted in a statistically significant enhancement of 125I-trace

Endogenous Morphine Levels Are Increased in Sepsis: A Partial Implication of Neutrophils

“…Atypical cholinergic regulation of morphine secretion from human white blood cells has been demonstrated before [22]. Our present study shows that morphine is present in secondary granules and is secreted from neutrophils in response to LPS and IL-8 exposure, suggesting that at least part of the circulating morphine in the blood of septic patients stems from PMN.…”

“…Stimuli such as alcohol, nicotine, and cocaine induce morphine release from human white blood cells in vitro [21]. Furthermore, a nonclassical cholinergic regulation of morphine release from human white blood cells was demonstrated [22]. Leukocytes play an important role in innate immune responses and represent a major defense mechanism against infection.…”

Endogenous Morphine in SH-SY5Y Cells and the Mouse Cerebellum

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“…Morphine is known to pass the blood brain barrier and to penetrate into the CNS more actively than M6G, probably because it is more hydrophilic [62]. Uptake mechanisms for morphine also appear to exist in non-neuronal cells, as shown by studies with radioactive morphine and human primary white blood cells [22], [35], [36], [37].…”

The relationship between Bcl-2 gene expression and learning & memory impairment in chronic aluminum-exposed rats

“…Under normal conditions, Bcl-2 regulates cellular apopto-sis, in opposition to the pro-apototic cellular protein Bax (Cadoni et al, 2007;Deepak et aL, 2007), thereby preserving cellular function (Lanzilli et al, 2006;Lazarova et al, 2006;Staibano et aL, 2006). This suggests that A1 may lead to extensive toxic effects which accelerate neuronal apoptosis, known to associated with Ca 2+ overload and lipid peroxidation (Raft, 1992;D'Alimonte et al, 2007;di Lorenzo et al, 2007;Zhu et al, 2007). However, when Bcl-2 is over-expressed, the regulation of cell apoptosis may change.…”