PubMed Health⌕ Search

Biomedical subjects

Patrick Cadet

Publications and source records attributed to Patrick Cadet.

At least 19 recordsLinked to original sources
PubMed2006

Alcohol-, nicotine-, and cocaine-evoked release of morphine from invertebrate ganglia: model system for screening drugs of abuse.

BACKGROUND: Invertebrates express regulatory receptors, transporters, and channels responsive to established drugs of abuse, many of which mediate their effects through catecholamine pathways. We hypothesized that invertebrate neural systems may serve as models by which to evaluate the interactive pharmacological effects of these agents. MATERIAL AND METHODS: Ex vivo pharmacological trials determined the effects of saturating levels of ethanol on morphine levels in pooled Mytilus edulis ganglia via HPLC coupled to electrochemical detection and/or HPLC/RIA analyses. Additional trials evaluated the ability of ethanol, nicotine, and cocaine, to promote evoked release of 125I-labeled morphine from neural tissues, because intrinsically low levels of morphine did not allow direct quantification of its release. RESULTS: Incubation of pooled M. edulis pedal ganglia with 200 mM ethanol (approximately 1% ethanol v/v) resulted in a two-fold increase in morphine concentration at 15 min, return to baseline at 30 min, and a 50% decrease in morphine concentration at 60 min. Separate incubations of pooled M. edulis pedal ganglia and H. americanus nerve cord with ethanol, cocaine, and nicotine resulted in a statistically significant enhancement of 125I-trace labeled morphine release. CONCLUSIONS: The stimulatory effects of ethanol, nicotine, and cocaine on cellular expression and release of endogenous morphine suggest convergent mechanisms underlying the reinforcing and addictive properties for a variety of drugs of abuse. The evolutionary conservation of L-tyrosine as a common precursor to catecholamine and opiate/opioid signaling systems may define a functional triad involving endogenous morphine, dopamine, and other classes of addictive drugs.

Animals↗
PubMed2005

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 mu3 opiate receptor subtype.

Base Sequence↗
PubMed2005

Morphine via nitric oxide modulates beta-amyloid metabolism: a novel protective mechanism for Alzheimer's disease.

BACKGROUND: The deposition of intracellular and extracellular beta-amyloid peptide (Abeta) in the brain is a pathologic feature of Alzheimer's disease (AD), a prevalent neurodegenerative disorder. However, the exact role of the Abeta peptide in causing AD's symptoms is unclear. MATERIAL/METHODS: CRL-2266 SH-SY5Y human neuroblastoma cells (ATCC, USA) and HTB-11 human neuroblastoma cells (ATCC, USA) were cultured. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to analyze the effects of beta25-35, morphine, and SNAP treatments upon BACE-1 and BACE-2 mRNA expression semi-quantitative RT-PCR. The production of NO in SH-SY5Y cells was detected using the Apollo 4000 Free Radical Analyzer (World Precision Instruments). RESULTS: Untreated HTB-11 neuroblastoma cells constitutively express BACE-1 and BACE-2 mRNA. Morphine down regulates the expression of BACE-1 and up regulates the expression of BACE-2 in a naloxone antagonizable manner. When HTB-11 cells were treated with L-NAME, a cNOS inhibitor; the effects of morphine were blocked. SNAP (a NO donor) mimicked the effect of morphine. In SH-SY5Y cells, Abeta treated cells show a dose-dependent decrease in NO release, demonstrating that Ab is dose-dependently inhibiting the release of constitutive NO. CONCLUSIONS: Ab and morphine/NO each inhibit the production of the other. This suggests that a deficiency of basal NO or endogenous morphine may trigger drastically reduced levels of basal NO. The outcome is chronic vasoconstriction and brain hypoperfusion and eventual neuronal death. This novel theorized mechanism for AD supports an increasingly-accepted vascular pathological hypothesis for the disease.

Alzheimer Disease↗
PubMed2005

Regulation of various genes in human leukocytes acutely exposed to morphine: expression microarray analysis.

BACKGROUND: Previous studies from our laboratory have identified a novel mu opiate receptor micro(3), which is expressed in several different cell types and tissues including human vascular endothelial cells, leukocytes and neural tissues. This novel micro receptor is selective for the opiate alkaloid morphine, since this receptor does not bind other opioid peptides. MATERIAL/METHODS: This report details the acute affects of morphine exposure (1 microg/ml) to human leukocytes by analyzing gene expression using microarrays (Applied Biosystems). Robust estimation of the median fold change was used to identify candidates for significantly differentially expressed genes. An independent experiment using four same sample arrays was used to test the algorithm and to confirm the calculated percentage of falsely significant genes. RESULTS: Data obtained from this study demonstrate that acute morphine exposure differentially affected genes that are involved in immune function, signal transduction, cell adhesion, and apoptosis. CONCLUSIONS: Acute morphine exposure to human leukocytes results in specific and significant alterations in gene expression.

Algorithms↗
PubMed2005

Endogenous morphine: opening new doors for the treatment of pain and addiction.

Nitric oxide (NO) signalling is at the forefront of intense research interest because its many effects remain controversial and seemingly contradictory. This paper examines its role as a potential mediator of pain and tolerance. Within this context discussion covers endogenous morphine, documenting its ability to be made in animal tissues, including nervous tissue, and in diverse animal phyla. Supporting morphine as an endogenous signalling molecule is the presence of the newly cloned mu3 opiate receptor subtype found in animal (including human) immune, vascular and neural tissues, which is coupled to NO release. Importantly, this mu opiate receptor subtype is morphine-selective and opioid peptide-insensitive, further highlighting the presence of morphinergic signalling coupled to NO release. These findings provide novel insights into pain and tolerance as morphinergic signalling exhibits many similarities with NO actions. Taken together, a select morphinergic signalling system utilising NO opens the gate for the development of novel pharmaceuticals and/or the use of old pharmaceuticals in new ways.

Analgesics↗
PubMed2005

Morphine modulation of the ubiquitin-proteasome complex is neuroprotective.

BACKGROUND: Over the past several decades, there is a growing need for the development of neuroprotective compounds, e.g, those that can prevent neural death. It was proposed that nitric oxide (NO), when induced by morphine, would produce neuroprotection in a human neuroblastoma cell line when tested concomitantly with compounds that produce intracellular oxidative stress and neuroinflammation. MATERIAL/METHODS: NO involvement in intracellular protein degradation controlled by the ubiquitin-proteasome complex was examined. Experiments were performed examining the following: a) neural cell viability and morphology; b) gene specific mRNA levels via semi-quantitative RT-PCR; c) protein levels via Western blotting; d) enzymatic activity via fluorogenic substrate-cleaving assays; and lastly, NO release via the Apollo 4000 real-time amperometric detector. RESULTS: Morphine induces the production of NO in human neuroblastoma cells, which can be blocked by naloxone and the cNOS inhibitor L-NAME. Rotenone, which induces oxidative stress and increases the expression of the proteasomal catalytic X subunit, causes the cells to die and morphine inhibits this process via NO. Rotenone also increases the activity of the 20S proteasome, whereas morphine alone or in the presence of rotenone caused a decrease in the activity of the 20S proteasome. Morphine decreases the expression of the immunoproteasome catalytic subunit LMP7 in response to inflammatory stimulation, demonstrating that morphine's neuroprotective action does not apply to only oxidative stress. Morphine significantly increases free ubiquitin, suggesting that morphine is inducing neuroprotection by reducing the amount of oxidized proteins targeted for degradation. CONCLUSIONS: Significant neuroprotection on the cellular and molecular levels was demonstrated and serves as a foundation for future work concerning the development of novel ligands for morphine's mu3 opiate receptor in an effort to prevent cellular death associated with neurodegenerative diseases.

Enzyme Inhibitors↗
PubMed2005

Tyrosine and tyramine increase endogenous ganglionic morphine and dopamine levels in vitro and in vivo: cyp2d6 and tyrosine hydroxylase modulation demonstrates a dopamine coupling.

BACKGROUND: The ability of animals to make morphine has been in question for the last 30 years. Studies have demonstrated that animals do contain morphine precursors and metabolites, as well as the ability to use some morphine precursors to make morphine. MATERIAL/METHODS: The present study uses excised ganglia from the marine invertebrate Mytilus edulis as well as whole animals. Morphine and dopamine levels were determined by high performance liquid chromatography coupled to electrochemical detection and radioimmunoassay. Tissues and whole animals were also exposed to morphine precursors and exposed to the CYP2D6 inhibitor quinidine and the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (AMPT). Additionally, via RT-PCR, a cDNA fragment of the CYP2D6 enzyme in the ganglia of M. edulis was identified. RESULTS: Pedal ganglia incubated with either tyramine or tyrosine, or whole animals receiving injections, exhibited a statistically significant concentration- and time-dependent increase in their endogenous morphine and dopamine levels (2.51 +/- 0.76 ng/g for tyrosine and 2.39 +/- 0.64 ng/g for tyramine compared to approximately 1.0 ng/g morphine wet weight). Incubation with quinidine and/or AMPT diminished ganglionic morphine and dopamine synthesis at various steps in the synthesis process. We also demonstrated that CYP2D6 mediates the tyramine to dopamine step in this process, as did tyrosine hydroxylase in the step from tyrosine to L-DOPA. Furthermore, via RT-PCR, we identified a cDNA fragment of the CYP2D6 enzyme in the ganglia, which exhibits 94% sequence identity with its human counterpart. Evidence that tyrosine and tyramine were, in part, being converted to dopamine then morphine, and that this process can be inhibited by altering either or both CYP2D6 or tyrosine hydroxylase, is also provided. CONCLUSIONS: It appears that animals have the ability to make morphine. This process also appears to be dynamic in that the inhibition of one pathway allows the other to continue with morphine synthesis. Moreover, dopamine and morphine synthesis were coupled.

Animals↗
PubMed2004

Differential expression of the human mu opiate receptor from different primary vascular endothelial cells.

BACKGROUND: Studies from our laboratory have identified a novel mu opiate receptor, mu3, which is expressed in several tissues, such as human vascular endothelial cells, leukocytes and invertebrate neural tissues. This novel mu receptor has been shown to be selective for opiate alkaloids, insensitive to opioid peptides, and also is coupled to constitutive nitric oxide release. MATERIAL/METHODS: In this study, we compare the mu3 receptor gene expression from three different vascular endothelial primary cell lines at the molecular level using a Taqman probe for the mu opiate receptor. RESULTS: Results from this study demonstrate that human umbilical vein endothelial cells (2.0, relative gene expression) and human pulmonary artery endothelial cells (1.1, relative gene expression) expressed more of the mu opiate receptor as compared to human arteriole endothelial cells (0.82, relative gene expression). CONCLUSIONS: The individual variations in mu receptor expression in these vascular tissues may explain the large variance in graft survival using saphenous veins for coronary artery bypass surgery.

Base Sequence↗
PubMed2004

Endogenous morphinergic signaling and tumor growth.

The mu3 opiate receptor subtype has been characterized by various binding assays as opiate alkaloid selective (e.g. morphine) and opioid peptide (e.g. methionine enkephalin) insensitive. This opiate receptor subtype has been found on human, including cancer cell lines, and invertebrate tissues, demonstrating that it has been conserved during evolution. Furthermore, in numerous reports, this receptor is coupled to constitutive nitric oxide release. In this regard, for example, morphine immune down regulating activities parallels those actions formerly attributed to nitric oxide. We have now identified the mu3 receptor at the molecular level and sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the mu opiate receptor gene (MOR). Furthermore, using Northern blot, reverse transcription coupled to polymerase chain reaction (RT-PCR) and sequence analysis, we have demonstrated the expression of this new mu variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells. The presence of this mu splice variant, adds to the growing body of evidence supporting the hypothesis that morphine is an endogenous signaling molecule in neural, immune and vascular systems. In addition to their use in the treatment of pain, opioid peptides appear to be important in the growth regulation of normal and neoplastic tissue. This review will focus on the influence of opiate alkaloids, e.g., morphine, on tumor growth, with emphasis on immuno-regulatory and antiproliferative mechanisms.

Alternative Splicing↗
PubMed2004

Music alters constitutively expressed opiate and cytokine processes in listeners.

BACKGROUND: Listening to music as a means of inducing a sense of calm and relaxation has been known for some time. Although these effects are robust, little research has been conducted into exploring the underlying neurochemical changes, which must occur to provide an individual with these objective sensations. MATERIAL/METHODS: In the current report we utilize an AB design to explore observed differences in blood plasma signaling molecules in pre- and post music listening groups when compared with controls. We focus chiefly on cytokines, as well as peripheral blood mononuclear cells and polymorphonuclear cells. We utilized reverse transcription followed by real-time polymerase chain reactions to determine relative mRNA expression for the mu opiate receptor gene. Using high pressure liquid chromatography coupled to electrochemical detection as well as nano electrospray ionization double quadrupole orthogonal acceleration time of flight-mass spectrometry we determined opiate alkaloid levels. RESULTS: Our findings are two-fold: with regard to mu opiate receptor expression, mononuclear cells showed a statistically significant increase in subjects in the music group compared to the control. Plasma morphine levels were found to be non-significantly lower in subjects after listening to music when compared to control subjects whereas morphine 6 glucuronide levels increased slightly, suggesting morphine's conversion to morphine 6 glucuronide. IL- 6 levels were significantly lower as well whereas IL-1b, IL-10 and cortisol values were unchanged. CONCLUSIONS: Taken together, it appears that music-listeners exhibit plasma signal molecule changes consistent with the physiological changes associated with the reported actions of music, i.e, lower blood pressure.

Adolescent↗
PubMed2004

Mu opiate receptor subtypes.

The . opiate receptor gene (MOR) has at least 14 exons that can generate 15 different splice variants. Recently, two new human MOR splice variants (hMOR-1O and hMOR-1X) have been identified and characterized. The two variants containing human MOR exons 1, 2, and 3 and a fourth alternative exon O, or exon X, are expressed in human brain tissue, and are selective for mu opioid binding in transfected cells. It is unclear; however, what the biologic role of these two novel human splice variants is in vivo. The mu3 opiate receptor subtype found in various human tissues where it is coupled to constitutive nitric oxide synthase derived nitric oxide release is characterized by its opiate alkaloid selectivity and its insensitivity to opioid peptides. The mu3 clone exhibits 100% identity to the mu1 receptor subtype in the center and conserved region, but with a truncated 5'-end (position 503 of mu1 mRNA) (missing several hundred nucleotides). In addition, the 3'-end of the new clone contains the 3'-end of the mu1 receptor, followed by a new fragment of 263 bases, and then by a 202 bp fragment of the 3'-end of the mu1 gene untranslated region. When mu3 is expressed in a heterologous system, the protein produced from this cDNA exhibits all of the expected biochemical characteristics of the mu3 receptor. The isolation of this novel splice variant adds support to the presence of morphinergic signaling in animals.

Alternative Splicing↗
PubMed2004

Opiate alkaloids and nitric oxide production in the nematode Ascaris suum.

The tissue distribution, course of secretion, and sex differences of morphine were delineated in Ascaris suum. Nitric oxide (NO) release in various tissues in response to morphine and its metabolite morphine-6-glucuronide (M6G) were also examined. Ascaris suum of both sexes along with their incubation fluid were analyzed for morphine concentrations by high-performance liquid chromatography (HPLC) over a 5-day period. Various tissues were also dissected for HPLC and NO analysis. Morphine was found to be most prevalent in the muscle tissue, and there is significantly more morphine in females than males, probably because of the large amounts present in the female uterus. Morphine (10(-9) M) and M6G (10(-9) M) stimulated the release of NO from muscles. Naloxone (10(-7) M) and N-nitro-L-arginine methyl ester (10(-6) M) blocked (P < 0.005) morphine-stimulated NO release from A. suum muscle tissue. D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH2 (CTOP) (10(-7) M) did not block morphine's NO release. However, naloxone could not block M6G-stimulated NO release by muscles, whereas CTOP (10(-7) M) blocked its release. These findings were in seeming contradiction to our earlier inability to isolate a mu opiate receptor messenger RNA by reverse transcriptase-polymerase chain reaction using a human mu primer. This suggests that a novel mu opiate receptor was possibly present and selective toward M6G.

Animals↗
PubMed2004

Nitric oxide modulates microglial activation.

BACKGROUND: Nitric oxide (NO) has important physiological regulatory roles, i.e, vasodilation, neurotransmitter release, etc. Little is known about the processes in neural tissues, which stabilize microglia. This study attempts to answer this question by demonstrating a role for basal NO in maintaining microglia juxtaposed to neurons. MATERIAL/METHODS: Mytilus edulis (a marine bivalve), were used to examine microglia egress from excised pedal ganglia microscopically. Nitric oxide is measured in excised pedal ganglia amperometrically in real-time. RESULTS: Pedal ganglia exhibit basal NO release (1 nM range). Inhibition of basal NO release by L-NAME results in greater numbers of microglia in the incubation medium. This process appears to involve two phases of egress. The first involves a slow egress of microglia, whereas the second, occurring 18 hours later, involves a more rapid release of these cells. Low levels of the NO donor SNAP (1 nM) does not interrupt microglial egress, whereas in the presence of L-NAME it does. Exposing the ganglia to high NO levels for a short period of time inhibits their egress. CONCLUSIONS: Spontaneous ganglionic NO release maintains/stabilizes microglia juxtaposed to neurons. Excised ganglia at the various observation periods reveals a transition of constitutive nitric oxide synthase (NOS) to inducible NOS derived NO. It also appears that the microglia in some unknown manner become insensitive to iNOS derived NO since they exhibit enhanced migration during this last phase of the ganglionic NO response. Taken together, NO is involved with regulating microglial activation.

Animals↗
PubMed2004

Nitric oxide modulates the physiological control of ciliary activity in the marine mussel Mytilus edulis via morphine: novel mu opiate receptor splice variants.

OBJECTIVES: The study sought to determine how dopamine controls ganglionic processes involved with modulating lateral cilia beating via the peripheral branchial nerve. METHODS: The lateral cilia found on the gill filaments exhibit metachronal ciliary beating determined stroboscopically. Novel opiate receptors were determined pharmacologically and demonstrated by RT-PCR and sequence analysis of total RNA from Mytilus edulis visceral ganglia. RESULTS: Dopamine applied to the visceral ganglion inhibits the activity of lateral cilia in a concentration and haloperidol sensitive manner. Morphine or DAMGO significantly enhances ciliary beating in a naloxone sensitive manner, whereas L-NAME, a nitric oxide synthase inhibitor, only antagonized morphine's action. SNAP, a nitric oxide donor, also enhanced lateral ciliary beating rates. Supporting the observation, i.e., morphine sensitive nitric oxide enhancement of ciliary beating and DAMGO insensitive, that two different mu opiate receptors are present in this tissue, a 602 bp fragment of the human micro 3 opiate receptor and a 935 bp fragment, designated micro 4 have been demonstrated. CONCLUSIONS: The lateral epithelium of the gill is innervated by serotonergic, cilioexcitatory neurons and dopaminergic, cilioinhibitory neurons, originating in the visceral ganglion. This data supports previous reports that demonstrate inhibiting ganglionic dopamine release allows the serotonin signals to prevail uncontrolled, enhancing ciliary rates. Supporting the observation that two different mu opiate receptors are present in this tissue, evidence is presented that identifies a 602 bp fragment of the human micro 3 opiate receptor and a 935 bp fragment, designated micro 4. Overall, the data strongly suggests that the two alternatively spliced mu opiate receptors may be involved in the physiological regulation of lateral ciliary activity in the visceral ganglia via dopamine and nitric oxide.

Alternative Splicing↗
PubMed2003

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.

Alkaloids↗
PubMed2003

Molecular identification and functional expression of mu 3, a novel alternatively spliced variant of the human mu opiate receptor gene.

Studies from our laboratory have revealed a novel mu opiate receptor, mu 3, which is expressed in both vascular tissues and leukocytes. The mu 3 receptor is selective for opiate alkaloids and is insensitive to opioid peptides. We now identify the mu 3 receptor at the molecular level using a 441-bp conserved region of the mu 1 receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the mu opiate receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the mu 3 receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the mu 3 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 [(3)H]dihydromorphine further supports the opiate alkaloid selectivity and opioid peptide insensitivity of this receptor. These data suggest that this new mu opiate receptor cDNA encodes the mu 3 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 mu variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.

Alternative Splicing↗
PubMed2003

Estrogen signaling at the cell surface coupled to nitric oxide release in Mytilus edulis nervous system.

In previous studies we have demonstrated release of nitric oxide (NO) in human tissues following exposure to estrogen. We now designed experiments to determine whether estrogen is present in the neural tissue of Mytilus edulis, a marine mollusk, and whether, as in vertebrates, it stimulates constitutive NO synthase activity. After HPLC purification of 17beta-estradiol (17beta-E(2)) from M. edulis ganglionic tissue, we confirmed the presence of 17beta-E(2) by RIA and ES-Q-TOF-MS analysis. We further found that when either exogenous or endogenous (purified HPLC fraction) 17beta-E(2) was added to pedal ganglia, there was immediate concentration-dependent NO release. Furthermore, 17beta-E(2) conjugated to BSA also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor antagonist, inhibited the action of both 17beta-E(2) and 17beta-E(2) conjugated to BSA, further supporting the presence of an estrogen receptor. In addition, by Western blot analysis with anti-ER-beta antibodies, we observed a 55-kDa protein in both the membrane and cytosolic fractions in pedal ganglia as well as in human leukocytes (that have been previously shown to express ER-beta). In summary, our results suggest that a physiological dose of estrogen acutely stimulates NO release within pedal ganglia via an estrogen cell surface receptor.

Animals↗