Cholinergic regulation of morphine release from human white blood cells: evidence for a novel nicotinic receptor via pharmacological and microarray analysis.

Recent work from our laboratory has demonstrated that human white blood cells make morphine and that substances of abuse, i.e. 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+/-0.19%, 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 microg/ml and at 10 microg/ml), which preferentially blocks nicotinic receptors at autonomic ganglia, with nicotine, exerted a very weak inhibitory effect. Co-incubation of alpha-BuTx or atropine or chlorisondamine diiodide or dihydro-Beta-erythroidine hydrobromide, an alpha4Beta2 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.

Morphine enhances nitric oxide release in the mammalian gastrointestinal tract via the micro(3) opiate receptor subtype: a hormonal role for endogenous morphine.

Studies from our laboratory have revealed a novel micro opiate receptor, micro(3), which is expressed in both human vascular tissues and leukocytes. The micro(3) receptor is selective for opiate alkaloids, insensitive to opioid peptides and is coupled to constitutive nitric oxide (cNO) release. We now identify the micro(3) receptor characteristics in mammalian gut tissues. It appears that the various regions of the mouse gut release low levels of NO (0.02 to 4.6 nM ) in a pulsatile manner. We demonstrate that morphine stimulates cNO release (peak level 17 nM) in the mouse stomach, small intestine and large intestine in a naloxone and L-NAME antagonizable manner. Opioid peptides do not exhibit cNO-stimulating capabilities in these tissues. Taken together, we surmise morphine acts as a hormone to limit gut activity via micro(3) coupled to NO release since micro opiate receptors are found in the gut and endogenous morphine is not but is found in blood.

Hippocampal nitric oxide upregulation precedes memory loss and A beta 1-40 accumulation after chronic brain hypoperfusion in rats.

Chronic brain hypoperfusion (CBH) using permanent occlusion of both common carotid arteries in an aging rat model, has been shown to mimic human mild cognitive impairment (MCI), an acknowledged high risk condition that often converts to Alzheimer's disease. An aging rat model was used to determine whether hippocampal nitric oxide (NO) is abnormally expressed following CBH for two or eight weeks. At each time point, spatial memory was measured with the Morris water maze and hippocampal A beta 1-40/1-42 concentrations were obtained using sandwich ELISA. Real-time amperometric measures of NO representing the constitutive isoforms of neuronal nitric oxide synthase (nNOS) and endothelial (e)NOS were also taken at each time point to ascertain whether NO levels changed as a result of CBH, and if so, whether such NO changes preceded or followed any memory or amyloid-beta pathology. We found that two weeks after CBH, NO hippocampal levels were upregulated nearly four-fold when compared to nonoccluded rats but no alteration in spatial memory of A beta products were observed at this time point. By contrast, NO concentration had declined to control levels by eight weeks but spatial memory was found significantly impaired and A beta 1-40 (but not A beta 1-42) had increased in the CBH group when compared to control rats. Since changes in shear stress are known to upregulate eNOS but generally not nNOS, these results suggest that shear stress induced by CBH hyperactivated vascular NO derived from eNOS in the first two weeks as a reaction by the capillary endothelium to maintain homeostasis of local cerebral blood flow. The return of vascular NO to basal levels after eight weeks of CBH may have triggered metabolic changes within hippocampal cells resulting in hippocampal dysfunction as reflected by spatial memory impairment and by accumulation of A beta 1-40 peptide. In conclusion, our study shows that CBH initiates spatial memory loss in aging rats thus mimicking human MCI and also increases A beta 1-40 in the hippocampus. The memory and amyloid changes are preceded by NO upregulation in the hippocampus. These preliminary findings may be important in understanding, at least in part, the molecular mechanisms that precede memory impairment during chronic brain ischemia and as such, the pre-clinical stage leading to Alzheimer's disease.

Morphine 6 glucuronide stimulates nitric oxide release in mussel neural tissues: evidence for a morphine 6 glucuronide opiate receptor subtype.

We have previously demonstrated that Mytilus edulis pedal ganglia contain opiate alkaloids, i.e., morphine and morphine 6 glucuronide (M6G), as well as mu opiate receptor subtype fragments exhibiting high sequence similarity to those found in mammals. Now we demonstrate that M6G stimulates pedal ganglia constitutive nitric oxide (NO) synthase (cNOS)-derived NO release at identical concentrations and to similar peak levels as morphine. However, the classic opiate antagonist, naloxone, only blocked the ability of morphine to stimulate cNOS-derived NO release and not that of M6G. CTOP, a mu-specific antagonist, blocked the ability of M6G to induce cNOS-derived NO release as well as that of morphine, suggesting that a novel mu opiate receptor was present and selective toward M6G. In examining a receptor displacement analysis, both opiate alkaloids displaced [3H]-dihydromorphine binding to the mu opiate receptor subtype. However, morphine exhibited a twofold higher affinity, again suggesting that a novel mu opiate receptor may be present.

Real-time RT-PCR measurement of the modulation of Mu opiate receptor expression by nitric oxide in human mononuclear cells.

BACKGROUND: In previous studies, we have attributed opiate alkaloid selectivity to a subtype of the neuronal mu receptor known as mu3, expressed on human blood cells. Opiate alkaloid activation of this receptor subtype leads to the release of constitutively derived nitric oxide. In this report, we show by real-time RT-PCR that the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) initiates the down regulation of mu receptor gene expression in human mononuclear cells after 30 minutes. Superoxide dismutase, a free radical scavenger, blocks the effect of SNAP. MATERIAL AND METHODS: Human mononuclear cells isolated from whole blood were treated with SNAP (100 microM), and also with SNAP plus superoxide dismutase (100 U/ml) at different time points. Real-time RT-PCR with total RNA extracted from the cells was used to analyze expression of the mu opiate receptor. RESULTS: Mu opiate receptor gene expression was significantly down regulated in cells treated with SNAP at 30 min, and superoxide dismutase blocked the effect of SNAP. At 2 and 6 hours, a rebound effect was observed as noted by an increase in mu receptor expression, and at 24 hours mu receptor expression returned to control levels in the SNAP-treated cells. CONCLUSIONS: This study confirms that human mononuclear cells express the mu opiate receptor transcript and demonstrates that nitric oxide is involved in regulation of its expression.

HIV gp120 and morphine alter mu opiate receptor expression in human vascular endothelium.

We find that chronic exposure of human saphenous vein, atria and internal thoracic artery endothelium to the human immunodeficiency virus surface glycoprotein gp120, results in an increase in endothelial mu opioid receptor expression (52%). gp120 acts, in this regard, as a proinflammatory cytokine (e.g. interleukin-1-alpha) by increasing endothelial mu opioid receptor expression. In contrast, morphine decreases mu opioid receptor expression by 90% in a dose dependent fashion. Pretreatment of these tissues with the respective antagonists e.g., naloxone and anti-gp120 blocks the opiate decrease and increase gp120 induced increase in mu expression, respectively. Further, pretreatment of these endothelia with morphine inhibits gp120-stimulated mu transcript expression. Therefore, the immune down-regulating action of morphine may prevent viral replication because this process requires immune activation that can, in part, be provided for by gp120 proinflammatory actions.