Patrick Cadet

Cell-Surface Estrogen Receptors Mediate Calcium-Dependent Nitric Oxide Release in Human Endothelia

BACKGROUND Although estrogen replacement therapy has been associated with reduction of cardiovascular events in postmenopausal women, the mechanism for this benefit remains unclear. Because nitric oxide (NO) is considered an important endothelium-derived relaxing factor and may function to protect blood vessels against atherosclerotic development, we investigated the acute effects of physiological levels of estrogen on NO release from human internal thoracic artery endothelia and human arterial endothelia in culture. METHODS AND RESULTS We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase activity in human endothelial cells by acting on a cell-surface receptor. NO release was measured in real time with an amperometric probe. 17beta-Estradiol exposure to internal thoracic artery endothelia and human arterial endothelia in culture stimulated NO release within seconds in a concentration-dependent manner. 17beta-Estradiol conjugated to bovine serum albumin also stimulated NO release, suggesting action through a cell-surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized this action. We further showed with the use of dual emission microfluorometry that 17beta-estradiol-stimulated release of endothelial NO was dependent on the initial stimulation of intracellular calcium transients. CONCLUSIONS Physiological doses of estrogen immediately stimulate NO release from human endothelial cells through activation of a cell-surface estrogen receptor that is coupled to increases in intracellular calcium.

Molecular Identification and Functional Expression of μ3, a Novel Alternatively Spliced Variant of the Human μ Opiate Receptor Gene

Studies from our laboratory have revealed a novel μ opiate receptor, μ3, which is expressed in both vascular tissues and leukocytes. The μ3 receptor is selective for opiate alkaloids and is insensitive to opioid peptides. We now identify the μ3 receptor at the molecular level using a 441-bp conserved region of the μ1 receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the μ opiate receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the μ3 receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the μ3 receptor cDNA exhibited dose-dependent release of NO following treatment with morphine, but not opioid peptides (i.e., Met-enkephalin). Naloxone was able to block the effect of morphine on COS-1 transfected cells. Nontransfected COS-1 cells did not produce NO in the presence of morphine or the opioid peptides at similar concentrations. Receptor binding analysis with [3H]dihydromorphine further supports the opiate alkaloid selectivity and opioid peptide insensitivity of this receptor. These data suggest that this new μ opiate receptor cDNA encodes the μ3 opiate receptor, since it exhibits biochemical characteristics known to be unique to this receptor (opiate alkaloid selective and opioid peptide insensitive). Furthermore, using Northern blot, RT-PCR, and sequence analysis, we have demonstrated the expression of this new μ variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.

Evidence for nitric oxide production and utilization as a bacteriocidal agent by invertebrate immunocytes

The present study demonstrates that molluscan immunocytes are able to produce a chemical bacteriocidal substance which can be indirectly identified as nitric oxide (NO). The cells were analyzed in vitro on slides using computer-assisted microscopic image analysis to detect changes in cell conformation as well as to quantify the number of bacteria present. Sodium nitroprusside yields NO in solution causing bacterial clumping. The same phenomenon occurs in the presence of invertebrate immunocytes. Escherichia coli lipopolysaccharide also increases the number of bacteria found around the immunocytes, but this effect is selectively prevented by the addition of inhibitors of nitric oxide synthase, suggesting that this bacterial clumping is caused by the cells liberating NO. Interestingly the cells presumably producing NO maintain a round morphology. These findings suggest that immunocytes are able to kill bacteria by two mechanisms, i.e., phagocytosis and NO production.

IL-10 as a mediator in the HPA axis and brain.

Certain functional interactions between the nervous, endocrine, and immune systems are mediated by cytokines. The pro-inflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor (TNF) were among the first to be recognized in this regard. A modulator of these cytokines, IL-10, has been shown to have a wide range of activities in the immune system; in this review, we describe its production and actions in the hypothalamic-pituitary-adrenal (HPA) axis. IL-10 is produced in pituitary, hypothalamic, and neural tissues in addition to lymphocytes. IL-10 enhances corticotropin releasing factor (CRF) and corticotropin (ACTH) production in hypothalamic and pituitary tissues, respectively. Further downstream in the HPA axis endogenous IL-10 has the potential to contribute to regulation of glucocorticosteroid production both tonically and following stressors. Our studies and those of others reviewed here indicate that IL-10 may be an important endogenous regulator in HPA axis activity and in CNS pathologies such as multiple sclerosis. Thus, in addition to its more widely recognized role in immunity, IL-10s neuroendocrine activities described here point to its role as an important regulator in communication between the immune and neuroendocrine systems.

Morphine inhibits NF-κB nuclear binding in human neutrophils and monocytes by a nitric oxide-dependent mechanism

Background The transcription factor NF-&kgr;B plays a pivotal role in gene expression of inflammatory mediators such as cytokines or adhesion molecules. NF-&kgr;B–mediated transcriptional activation of these genes is inhibited by nitric oxide (NO) in a variety of cells, including monocytes. Morphine mediates NO release in a naloxone antagonizable manner in monocytes and neutrophils. Methods The influence of morphine on NF-&kgr;B activation was investigated in a whole-blood flow cytometric assay. A specific antibody against the p65 subunit of NF-&kgr;B was used and detected by fluoresceine-isothiocyanate–labeled anti–immunoglobulin G. Nuclei were stained with propidium iodide. Leukocyte subpopulations were evaluated by gating on neutrophils and monocytes. The median fluorescence channel was determined. Different morphine concentrations (50 nm, 50 &mgr;m, 1 mm) and incubation intervals (10–150 min) were used. Results Morphine inhibits lipopolysaccharide-induced NF-&kgr;B nuclear binding in human blood neutrophils and monocytes in a time-, concentration-, and naloxone-sensitive–dependent manner. Similar effects were achieved with the NO donor S-nitroso-N-acetyl-pencillamine and the antioxidant N-acetyl-cysteine. The NO synthase inhibitors N&ohgr;-nitro-l-arginine-methyl-esther and N&ohgr;-nitro-l-arginine completely abolished the morphine-induced attenuation of NF-&kgr;B nuclear binding, demonstrating that the inhibitory action is mediated by NO release. Conclusion Morphine causes immunosuppression, at least in part, via the NO-stimulated depression of NF-&kgr;B nuclear binding.

Interleukin-10 messenger ribonucleic acid in human placenta: implications of a role for interleukin-10 in fetal allograft protection.

OBJECTIVE Our purpose was to determine whether interleukin-10 is expressed in human placental tissue, which might imply a role for it in fetal allograft protection. STUDY DESIGN Detection of interleukin-10 messenger ribonucleic acid in human placental tissue and in human placental JAR cells by reverse transcription-coupled polymerase chain reaction was studied. RESULTS Interleukin-10 messenger ribonucleic acid was detected in human placental tissue from term mothers and in human placental JAR cells. Sequence analysis of the expected interleukin-10 complementary deoxyribonucleic acid fragment revealed 100% homology to authentic interleukin-10 complementary deoxyribonucleic acid. CONCLUSION Our results indicated that human placental tissue from term mothers expressed high levels of interleukin-10 messenger ribonucleic acid, suggesting that cells that produce interleukin-10 and that are associated with the placenta may play a role in preventing rejection of the fetal allograft by the mother.

Human White Blood Cells Synthesize Morphine: CYP2D6 Modulation

Human plasma contains low, but physiologically significant, concentrations of morphine that can increase following trauma or exercise. We now demonstrate that normal, human white blood cells (WBC), specifically polymorphonuclear cells, contain and have the ability to synthesize morphine. We also show that WBC express CYP2D6, an enzyme capable of synthesizing morphine from tyramine, norlaudanosoline, and codeine. Significantly, we also show that morphine can be synthesized by another pathway via l-3,4-dihydroxyphenylalanine (l-DOPA). Finally, we show that WBC release morphine into their environment. These studies provide evidence that 1) the synthesis of morphine by various animal tissues is more widespread than previously thought and now includes human immune cells. 2) Moreover, another pathway for morphine synthesis exists, via l-DOPA, demonstrating an intersection between dopamine and morphine pathways. 3) WBC can release morphine into the environment to regulate themselves and other cells, suggesting involvement in autocrine signaling since these cells express the μ3 opiate receptor subtype.

Human Vascular and Cardiac Endothelia Express Mu Opiate Receptor Transcripts

Pharmacologic and immunologic evidence suggests that nitric oxide-coupled mu-subtype opiate receptors are expressed in human vascular endothelium. In this study, we present molecular evidence of mu opiate receptor expression. Using primers derived from the human neuronal mu1 opiate receptor, we used RT-PCR to detect expression of mu transcripts from human endothelia. Sequence analysis of the RT-PCR products revealed 100% identity with the neuronal human mu1 receptor. We further show that pretreatment of human internal thoracic artery and cardiac atrial endothelium with the proinflammatory cytokines interleukin-1-alpha and -beta led to a significant increase in both the expression of the mu transcript and in morphine-stimulated nitric oxide release measured amperometrically. Taken together, these studies provide molecular evidence that mu-type opiate receptors are expressed in human vascular endothelia and that their expression can be upregulated by proinflammatory cytokines.

Presence of reticuline in rat brain: a pathway for morphine biosynthesis.

We demonstrate the presence of reticuline, an isoquinoline alkaloid that was purified and identified in the rat brain. This was achieved by high-performance liquid chromatography coupled with electrochemical detection. This material was finally identified by nano-electrospray ionization quadrupole time-of-flight tandem mass spectrometry. The expression of this tetrahydroisoquinoline alkaloid in rat brain is at 12.7+/-5.4 ng/g wet tissue. Furthermore, rat chow, rat small and large intestine and bacteria cultured from these tissues did not contain either morphine or reticuline, eliminating the possibility of contamination or an exogenous source of these compounds. This finding adds information which suggests that morphine biosynthesis may occur in rat neural tissues, and that its biosynthesis pathway may be similar to that reported in the poppy plant.

Ascaris suum, an Intestinal Parasite, Produces Morphine

The parasitic worm Ascaris suum contains the opiate alkaloid morphine as determined by HPLC coupled to electrochemical detection and by gas chromatography/mass spectrometry. The level of this material is 1168 ± 278 ng/g worm wet weight. Furthermore, Ascaris maintained for 5 days contained a significant amount of morphine, as did their medium, demonstrating their ability to synthesize the opiate alkaloid. To determine whether the morphine was active, we exposed human monocytes to the material, and they immediately released nitric oxide in a naloxone-reversible manner. The anatomic distribution of morphine immunoreactivity reveals that the material is in the subcuticle layers and in the animals’ nerve chords. Furthermore, as determined by RT-PCR, Ascaris does not express the transcript of the neuronal μ receptor. Failure to demonstrate the expression of this opioid receptor, as well as the morphine-like tissue localization in Ascaris, suggests that the endogenous morphine is intended for secretion into the microenvironment.